WO2012075318A2 - Fn1 and alk gene translocations in cancer and alk kinase expression in ovarian cancer - Google Patents

Abstract

The invention provides the identification of the presence of polypeptides with ALK kinase activity in human cancer, such as ovarian cancer. In some embodiments, the polypeptide with ALK kinase activity is the result of a fusion between an ALK-encoding polynucleotide and an FN1 -encoding polynucleotide. Six different fusion polypeptides are described, three that include the transmembrane and kinase domains of ALK fused to the extracellular domain of the FN1 protein, and three that include the kinase domain (but not the transmembrane domain) of ALK fused to the extracellular domain of the FN1 protein. The invention also provides the discovery of full length ALK expression in ovarian cancer. In some embodiments, this aberrant expression of full length ALK in ovarian cancer results from an amplified ALK gene copy number in the ovarian cancer. The invention, in part, also provides a truncated ALK polypeptide which contains the transmembrane and the kinase domains of ALK but lacks the extracellular domain of ALK protein. The invention therefore provides, in part, isolated polynucleotides and vectors encoding the disclosed polypeptides (e.g., a FN1-ALKvariantl fusion polypeptide), probes for detecting the polynucleotides, isolated polypeptides, recombinant polypeptides, and reagents for detecting the polypeptides and polynucleotides. In some embodiments, the invention enables new methods for determining the presence of a polypeptide with ALK kinase activity in a biological sample, methods for screening for compounds that inhibit the proteins, and methods for inhibiting the progression of a cancer (e.g., an ovarian cancer).

Description

FN1 AND ALK GENE TRANSLOCATIONS IN CANCER

AND ALK KINASE EXPRESSION IN OVARIAN CANCER CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority benefit from U.S. Provisional Patent Application No. 61/418,535 filed December 1, 2010, the entire contents of which is hereby incorporated by reference.

FIELD OF THE INVENTION

The invention relates generally to proteins and genes involved in cancer (e.g., human cancer), and to the detection, diagnosis and treatment of cancer.

BACKGROUND OF THE INVENTION

Many cancers are characterized by disruptions in cellular signaling pathways that lead to aberrant control of cellular processes, or to uncontrolled growth and proliferation of cells. These disruptions are often caused by changes in the activity of particular signaling proteins, such as kinases.

Aberrant expression of protein kinase proteins can be the causative agent of (and the driver of) cancer. Aberrant expression can be caused by the fusion of the protein (or kinase portion thereof) with a secondary protein (or portion there), expression of a truncated portion of the protein, or by abnormal regulation of expression of the full-length protein.

It is known that gene translocations resulting in kinase fusion proteins with aberrant signaling activity can directly lead to certain cancers (see, e.g., Mitelman et al., Nature Reviews Cancer 7: 233-245, 2007, Futreal et al, Nat Rev Cancer 4(3): 177-183 (2004), and Falini et al, Blood 99(2): 409-426 (2002). For example, it has been shown that the BCR-ABL oncoprotein, a tyrosine kinase fusion protein, is the causative agent and drives human chronic myeloid leukemia (CML). The BCR-ABL oncoprotein, which is found in at least 90-95% of CML cases, is generated by the translocation of gene sequences from the c-ABL protein tyrosine kinase on chromosome 9 into BCR sequences on chromosome 22, producing the so-called Philadelphia chromosome. See, e.g. Kurzock et al, N. Engl. J. Med. 319: 990-998 (1988). The translocation is also observed in acute lymphocytic leukemia (ALL) and acute myeloid leukemia (AML) cases. These discoveries spurred FDA approval of imatinib mesylate (sold under the trademark Gleevec® by Novartis) and dasatinig (sold by Bristol-Mysers Squibb under the trademark

Sprycel), small molecule inhibitors of the ABL kinase, for the treatment of CML and ALL. These drugs are examples of drugs that are designed to interfere with the signaling pathways that drive the growth of tumor cells. The development of such drugs represents a significant advance over the conventional therapies for CML and ALL, chemotherapy and radiation, which are plagued by well known side-effects and are often of limited effect since they fail to specifically target the underlying causes of the malignancies.

Thus, it would be useful to identify proteins that drive cancers in order to detect cancers at an early stage, when they are more likely to respond to therapy. Additionally, identification of such proteins will, among other things, desirably enable new methods for selecting patients for targeted therapies, as well as for the screening and development of new drugs that inhibit such proteins and, thus, treat cancer.

The oncogenic role of receptor tyrosine kinases (RTKs) have been implicated in many types of solid tumors, including ovarian cancer. Somatic mutations in Her2, Her3 and EphBl, over-expression of EGFR, Her2, Her3, PDGFR and EphA2 are found to be associated with ovarian cancer. Ovarian cancer is the seventh most common cancer in women in the U.S., and the fifth leading cause of cancer-related deaths among women following lung cancer, breast cancer, colon cancer, and pancreatic cancer. Ovarian cancer can be classified into three types based on the origin of the disease. Fewer than 2% of ovarian tumors are derived from germ cells that form ova or eggs. Teratoma and dysgerminoma are most common germ cell tumors. Stromal tumors account for 1% of ovarian cancer. They are derived from structural tissue cells that hold the ovary together and produce female hormones. Examples of stromal tumors include granulosa cell tumor, thecoma, fibroma and sarcoma.

More than 90% of the ovarian cancer cases are epithelial tumors, which are derived from cells that form the lining of the ovary and which represents a series of molecularly and

etiologically distinct diseases (Vaughan et al., Nat Rev Cancer 11 :719-25). Epithelial tumors include serous tumor, endometrioid tumor, mucinous tumor and clear cell tumor. Epithelial ovarian cancer can be grouped into 2 types. Type I tumors including low-grade serous, low-grade endometrioid, clear cell, mucinous and Brenner carcinomas are confined to the ovary at presentation and genetically stable. 90% of type I tumors are curable. In contrast, type II epithelial ovarian cancer tumors, which include high-grade serous carcinoma, undifferentiated carcinoma and malignant mixed mesodermal tumors, are highly aggressive and present in advanced stage (Kurman et al, Am J Surg Pathol 34:433-443, 2010). Some type II tumors bear the TP53 tumor suppressor gene and may share no genetic alterations found in type I tumors (Kurman et al, supra; Bell et al, Mod Pathol 18 Suppl 2:S19-32, 2005). High-grade serous carcinoma stands out from other subtypes since it accounts for 70% of cases and most deaths from ovarian cancer. Recent studies have shown that high-grade serous carcinoma may arise from fallopian tube epithelium other than ovary itself (Vaughan et al., supra; Karst et al, Proc Natl Acad Sci U S A 108:7547-52, 2011). Due to the subtle nature of symptoms and inadequate screening tools, most high-grade serous ovarian carcinoma patients present at advanced stages with poor prognosis (Kurman et al, supra; Willmott and Fruehauf, Journal of Oncology, Vol. 2010, Article ID 740472).

Only 20% of ovarian cancer cases are found at an early stage. However, frequently, ovarian cancer does not result in symptoms until the cancer has spread extensively. Less than one-third of ovarian cancers are detected before they have spread outside of the ovaries. Currently surgery and chemotherapy remain standard procedures for initial treatment. Although response rates to these treatments are high, the disease recurs in the majority of patients and usually become resistant to chemotherapy. The five year survival rate for ovarian cancer is 46%, and the overall survaival rate for women with ovarian cancer has not changed over the last thirty years.

Thus, it would be useful to discover new ways to identify ovarian cancer at an early stage, and new ways (and new reagents) to treat ovarian cancer.

SUMMARY OF THE INVENTION In some embodiments, the invention is based upon the discovery of aberrant ALK expression and/or activity in cancer, particularly ovarian cancer. Two types of aberrant expression of ALK in mammalian ovarian cancer are disclosed herein. The first is over-expression (i.e., aberrant expression) of full length ALK in ovarian cancer, while the second is the expression of a novel mutant ALK polypeptide, namely a fusion resulting from a gene translocation involving the ALK kinase gene combined with the FN1 gene which encodes the fibronectin protein. The discovery of this new fusion has allowed prediction of several new translocations between the

FN1 and the ALK gene, some of which contain the transmembrane domain of ALK (called FN1- tmALK fusions) and some of which do not contain the transmembrane domain of ALK (called FN 1 -ALK fusions). All disclosed polypeptides (i.e., the discovered and predicted polypeptides) have active ALK kinase activity. Thus, in some embodiments, the invention also provides expression of mutant ALK kinase in cancer whereby the transmembrane and kinase domains of the full length ALK kinase are active but separated from the rest of the full-length ALK kinase (e.g., separate from the extracellular domain of the full length ALK protein). The invention also provides the first description of expression of the transmembrane and the kinase domains of the ALK polypeptide, either alone or as part of a fusion with the N-terminal regions of a second polypeptide (in this case the FN1 protein). The expression and/or activity of a mutant ALK polypeptide (e.g., a truncated ALK or an FNl-tmALK fusion polypeptide) may drive the proliferation and survival of a cancer, such as an ovarian cancer, in which a mutant ALK kinase is expressed or is active.

Accordingly, in a first aspect, the invention provides an isolated polynucleotide comprising a nucleotide sequence at least 95% identical to a sequence selected from the group consisting of: (a) a nucleotide sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 3, 5, 7, 10, 11, 12, 14, 15, 17, 18, 20, or 21; (b) a nucleotide sequence comprising the nucleotide sequence of SEQ ID NO: 4, 6, 8, 13, 16, or 19; (c) a nucleotide sequence encoding polypeptide comprising an N-terminal portion comprising amino acid residues 1-1085 of SEQ ID NO: 26 or amino acid residues 1-1116 of SEQ ID NO: 27 and a C-terminal portion comprising amino acid residues 1039-1392 of SEQ ID NO: 1; (d) a nucleotide sequence encoding a polypeptide comprising an N-terminal portion comprising amino acid residues 1-1085 of SEQ ID NO: 26 or amino acid residues 1-1116 of SEQ ID NO: 27 and a C-terminal portion comprising the amino acid sequence set forth in SEQ ID NO: 29; (e) a nucleotide sequence comprising (i) at least six contiguous nucleotides encompassing the fusion junction (nucleotides 3601-3606 of SEQ ID NO: 4, nucleotides 3793-3798 of SEQ ID NO: 6, or nucleotides 3346-3351 of SEQ ID NO: 8) or encoding at least six contiguous polypeptides encompassing the fusion junction (residues 1999- 1204 of SEQ ID NO: 3, residues 1263-1268 of SEQ ID NO: 5, or residues 1114-1119 of SEQ ID NO: 7) of an FNl-tmALK fusion polypeptide; (f) a nucleotide sequence comprising (i) at least six contiguous nucleotides encompassing the fusion junction (nucleotides 3601-3606 of SEQ ID NO: 13, nucleotides 3793-3798 of SEQ ID NO: 16, nucleotides 3346-3351 of SEQ ID NO: 19) or encoding at least six contiguous polypeptides encompassing the fusion junction (residues 1999- 1204 of SEQ ID NO: 14, residues 1263-1268 of SEQ ID NO: 17, or residues 1114-1119 of SEQ ID NO: 20) of an FN 1 -ALK fusion polypeptide; (g) a nucleotide sequence encoding a polypeptide comprising amino acid residues 1039-1392 of SEQ ID NO: 1, wherein said polypeptide does not comprise amino acid residues 19-1038 of SEQ ID NO: 1; (h) a nucleotide sequence encoding a polypeptide comprising amino acid sequence of SEQ ID NO: 29, wherein said polypeptide does not comprise amino acid residues 19-1038 of SEQ ID NO: 1; and (i) a nucleotide sequence complementary to any of the nucleotide sequences of (a)-(h). In further aspects, the invention provides a reagent (e.g., an isolated reagent) that specifically binds to the isolated polynucleotide, where the reagent does not specifically bind to a polynucleotide having a nucleotide sequence consisting of only adenine (A) residues or of only thymine (T) residues. The reagent itself may be another polynucleotide (e.g., a nucleic acid probe).

In further aspect, the invention provides an isolated reagent that specifically detects the isolated polynucleotide. In some embodiments, the reagent that specifically detects the isolated polynucleotide does not specifically bind to or hybridize to the polynucleotide or a nucleotide sequence complementary thereto. In some embodiments, the reagent comprises a primer pair, wherein each member of the primer pair hybridizes to nucleotide sequences adjacent to the polynucleotide or complement thereof and wherein the primer pair can amplify a nucleic acid molecule comprising the polynucleotide.

In some embodiments, the isolated polynucleotide or the reagent may further comprise a detectable label (e.g., a fluorescent label or an infrared label). In some embodiments, the reagent is a polymerase chain reaction (PCR) probe or a fluorescence in situ hybridization (FISH) probe.

In further aspects, the invention provides a method for producing a recombinant vector comprising inserting an isolated polynucleotide disclosed herein into a vector (e.g., a recombinant vector or a virus), and method for producing a recombinant host cell comprising introducing the recombinant vector comprising the isolated polynucleotide into a host cell. In further aspects, the invention provides a recombinant vector comprising an isolated polynucleotide, and a recombinant cell comprising the recombinant vector. In a further aspect, the invention provides a method for producing a recombinant polypeptide comprising culturing the recombinant host cell under conditions suitable for the expression of said fusion polypeptide and recovering said polypeptide. In some embodiments, the invention provides a recombinant polypeptide (e.g., a mutant ALK polypeptide) produced using the recombinant vector, the recombinant host cell of the invention, or by culturing the recombinant host cell under conditions suitable for the expression of said fusion polypeptide and recovering said polypeptide. In further aspects, the invention provides an isolated polypeptide comprising an amino acid sequence at least 95% identical to a sequence selected from the group consisting of: (a) an amino acid sequence comprising the amino acid sequence of SEQ ID NOs: 3, 5, 7, 10, 11, 12, 14, 15, 17, 18, 20, or 21; (b) an amino acid sequence comprising amino acid residues 1-1085 of SEQ ID NO: 26 and a C-terminal portion comprising amino acid residues 1039-1392 of SEQ ID NO: 1; (c) an amino acid sequence encoding a polypeptide comprising at least six contiguous amino acids encompassing the fusion junction (residues 1999-1204 of SEQ ID NO: 3, residues 1263—1268 of SEQ ID NO: 5, or residues 1114-1119 of SEQ ID NO: 7) of an FNl-tmALK fusion polypeptide;

(d) an amino acid sequence encoding a polypeptide comprising at least six contiguous amino acids encompassing the fusion junction (residues 1999-1204 of SEQ ID NO: 14, residues 1263—1268 of

SEQ ID NO: 17, or residues 1114-1119 of SEQ ID NO: 20)of an FN1-ALK fusion polypeptide;

(e) an amino acid sequence comprising amino acid residues 1039-1392 of SEQ ID NO: 1, wherein said polypeptide does not comprise amino acid residues 19-1038 of SEQ ID NO: 1; and (f) an amino acid sequence comprising amino acid sequence of SEQ ID NO: 29, wherein said polypeptide does not comprise amino acid residues 19-1038 of SEQ ID NO: 1.

In certain aspects, the invention provides a reagent (e.g., an isolated reagent) that specifically binds to a polypeptide disclosed herein (e.g., a mutant ALK polypeptide). In some embodiments, the reagent does not specifically bind to either full-length FNl protein or full-length ALK protein. In some embodiments, the reagent is an antibody, such as a mouse antibody, a rabbit antibody, a humanized antibody, a chimeric antibody, a polyclonal antibody, or a monoclonal antibody). In some embodiments, the reagent is a heavy-isotope labeled (AQUA) peptide. In some embodiments, the AQUA peptide comprises the amino acid sequence of the fusion junction of an FNl -ALK fusion polypeptide or an FNl-tmALK fusion polypeptide.

In another aspect, the invention provides a method for detecting the presence of a mutant ALK polypeptide or an FNl -ALK fusion polypeptide in a biological sample from a mammalian cancer or a suspected mammalian cancer, said method comprising the steps of: (a) obtaining a biological sample (e.g., a biological sample containing at least one polypeptide) from a mammalian cancer or suspected ovarian cancer; and (b) utilizing at least one reagent that specifically binds to a mutant ALK polypeptide or an FNl -ALK fusion polypeptide to determine whether said mutant ALK polypeptide is present in said biological sample, wherein specific binding of said reagent to said biological sample indicates said mutant ALK polypeptide or said FNl -ALK fusion polypeptide is present in said biological sample. In some embodiments, the mammalian cancer is mammalian ovarian cancer (e.g., from a human). In some embodiments, the mutant ALK polypeptide is truncated ALK polypeptide. In some embodiments, the mutant ALK polypeptide is an FNl-tmALK fusion polypeptide (e.g., an FNl-tmALK fusion polypeptide comprises an amino acid sequence of SEQ ID NO: 3, 5, 7, 10, 11, or 12). In some embodiments, the FN 1 -ALK fusion polypeptide comprises an amino acid sequence of SEQ ID NO: 14, 15, 17, 18, 20, or 21.

In another aspect, the invention provides a method for detecting the presence of a polypeptide with ALK kinase activity in a biological sample from a mammalian ovarian cancer or suspected mammalian ovarian cancer, said method comprising the steps of: (a) obtaining a biological sample from a mammalian ovarian cancer or suspected mammalian ovarian cancer and (b) utilizing a reagent that specifically binds said polypeptide with ALK kinase activity to determine whether said polypeptide with ALK kinase activity is present in said biological sample, wherein specific binding of said reagent to said biological sample indicates said polypeptide with ALK kinase activity is present in said biological sample. In some embodiments, the polypeptide is aberrantly expressed full-length ALK protein. In some embodiments, the polypeptide is a mutant ALK polypeptide, such as a truncated ALK polypeptide or an FNl-tmALK fusion polypeptide (e.g., comprising the amino acid sequence of SEQ ID NO: 3, 5, 7, 10, 11, or 12). In some embodiments, the polypeptide is an ALK fusion polypeptide (e.g., an NPM-ALK fusion polypeptide, an AL017-ALK fusion polypeptide, an TFG-ALK fusion polypeptide, an MSN- ALK fusion polypeptide, an TPM3-ALK fusion polypeptide, an TPM4-ALK fusion polypeptide, an ATIC-ALK fusion polypeptide, a KIF5B-ALK fusion polypeptide, an MYH9-ALK fusion polypeptide, an CLTC-ALK fusion polypeptide, an SEC31L1-ALK fusion polypeptide, an RANBP2-ALK fusion polypeptide, an CARS-ALK fusion polypeptide, an EML4-ALK fusion polypeptide, and an TFG-ALK fusion polypeptide). In some embodiments, the polypeptide is an FN1-ALK fusion polypeptide (e.g., comprising the amino acid sequence selected from the group consisting of SEQ ID NO: 14, 15, 17, 18, 20, or 21).

In various embodiments of the methods of the invention, the reagent is an antibody. In some embodiments, the reagent (e.g., the antibody) specifically binds to a full length ALK polypeptide or to a full length FN1 polypeptide. In some embodiments, the reagent (e.g., the antibody) specifically binds to an FN 1 -ALK fusion polypeptide and does not specifically bind to either full-length FN1 polypeptide or full-length ALK polypeptide. In some embodiments, the method is implemented in a format selected from the group consisting of a flow cytometry assay, an immunohistochemistry (IHC) assay, an immunofluorescence (IF) assay, an Enzyme-linked immunosorbent assay (ELISA) assay, and a Western blotting analysis assay. In some

embodiments, the reagent is a heavy-isotope labeled (AQUA) peptide. In some embodiments, the AQUA peptide comprises an amino acid sequence comprising a fusion junction of an FNl-ALK fusion polypeptide or of an FNl-tmALK fusion polypeptide. In some embodiments, the method is implemented using mass spectrometry analysis.

In further aspects, the invention provides a method for detecting the presence of a mutant ALK polynucleotide or an or an FNl-ALK fusion polynucleotide in a biological sample from a mammalian cancer or suspected mammalian cancer, said method comprising the steps of: (a) obtaining a biological sample from said mammalian cancer or suspected mammalian cancer; and (b) utilizing at least one reagent that specifically binds to a mutant ALK polynucleotide or to an FNl-ALK fusion polynucleotide to determine whether said mutant ALK polynucleotide or said or said FNl-ALK fusion polynucleotide is present in said biological sample, wherein specific binding of said reagent to said biological sample indicates said mutant ALK polynucleotide is present in said biological sample. In some embodiments, the mammalian cancer is mammalian ovarian cancer (e.g., from a human). In some embodiments, the mutant ALK polynucleotide is a truncated ALK polynucleotide. In some embodiments, the mutant ALK polynucleotide is an FNl- tmALK fusion polynucleotide (e.g., a FNl-ALK fusion polynucleotide encoding a polypeptide comprising an amino acid sequence of SEQ ID NOs: 3, 5, 7, 10, 11, or 12). In some

embodiments, the FNl-tmALK fusion polynucleotide comprises a nucleotide sequence of SEQ ID NOs: 4, 6, or 8. In some embodiments, the FNl-ALK fusion polynucleotide comprises a nucleotide sequence of SEQ ID NO: 13, 16, or 19. In some embodiments, the FNl-ALK fusion polynucleotide encodes a polypeptide comprising an amino acid sequence of SEQ ID NO: 14, 15, 17, 18, 20, or 21. In some embodiments, the mammalian cancer or suspected mammalian cancer is mammalian ovarian cancer or suspected mammalian ovarian cancer.

In yet further aspects, the invention provides a method for detecting the presence of a polynucleotide encoding a polypeptide with ALK kinase activity in a biological sample from a mammalian ovarian cancer or suspected mammalian ovarian cancer, said method comprising the steps of: (a) obtaining a biological sample from a mammalian ovarian cancer or suspected mammalian ovarian cancer and (b) utilizing a reagent that specifically binds to said polynucleotide encoding said polypeptide with ALK kinase activity to determine whether said polynucleotide is present in said biological sample, wherein specific binding of said reagent to said biological sample indicates said polynucleotide encoding said polypeptide with ALK kinase activity is present in said biological sample. In various embodiments, the polypeptide is aberrantly expressed full-length ALK polypeptide (e.g., aberrantly expressed in mammalian ovarian cancer or suspected mammalian ovarian cancer). In some embodiments, the polypeptide is a mutant ALK polypeptide, such as a truncated ALK polypeptide or an FNl-tmALK fusion polypeptide (e.g., comprising an amino acid sequence of SEQ ID NO: 3, 5, 7, 10, 11, or 12). In some embodiments, the polynucleotide comprises a nucleotide sequence of SEQ ID NO: 4, 6, or 8. In some embodiments, the polypeptide is an ALK fusion polypeptide (e.g., an NPM-ALK fusion polypeptide, an AL017-ALK fusion polypeptide, an TFG-ALK fusion polypeptide, an MSN- ALK fusion polypeptide, an TPM3-ALK fusion polypeptide, an TPM4-ALK fusion polypeptide, an ATIC-ALK fusion polypeptide, an MYH9-ALK fusion polypeptide, an CLTC-ALK fusion polypeptide, an SEC31L1-ALK fusion polypeptide, an RANBP2-ALK fusion polypeptide, an CARS-ALK fusion polypeptide, an EML4-ALK fusion polypeptide, an KIF5B-ALK fusion polypeptide, and an TFG-ALK fusion polypeptide). In some embodiments, the polypeptide is an FN1-ALK fusion polypeptide (e.g., comprising the amino acid sequence selected from the group consisting of SEQ ID NO: 14, 15, 17, 18, 20, or 21).

In various embodiments, the reagent used in the methods disclosed herein is a nucleic acid probe. In some embodiments, the reagent disclosed herein specifically detects the isolated polynucleotide. For example, the the reagent that specifically detects the isolated polynucleotide may not not specifically bind to or hybridize to the polynucleotide or a nucleotide sequence complementary thereto. In some embodiments, the reagent comprises a primer pair, wherein each member of the primer pair hybridizes to nucleotide sequences adjacent to the polynucleotide or complement thereof and wherein the primer pair can amplify a nucleic acid molecule comprising the polynucleotide.

In some embodiments, the reagent comprises a detectable label. In some embodiments, the reagent is a fluorescence in-situ hybridization (FISH) probe and said method is implemented in a FISH assay. In some embodiments, the reagent is a polymerase chain reaction (PCR) probe and said method is implemented in a PCR assay.

In various embodiments of the methods disclosed herein, the mammalian ovarian cancer or suspected mammalian ovarian cancer is a stromal tumor or a clear cell carcinoma. In various embodiments, mammalian ovarian cancer or suspected mammalian ovarian cancer is from a human. In various embodiments, the biological sample is a circulating tumor cell from a mammalian ovarian cancer or suspected mammalian ovarian cancer.

In various embodiments of the methods disclosed herein, the activity of said polypeptide is detected. In various embodiments, the expression of said polypeptide is detected. In various embodiments, the mammalian ovarian cancer or suspected mammalian ovarian cancer from which the biological sample was obtained and to which the reagent specifically binds is a mammalian ovarian cancer or suspected mammalian ovarian cancer likely to respond to an ALK-inhibiting therapeutic. One non-limiting example of an ALK-inhibiting therapeutic is crizotinib (also known as PF-02341066). Additional non-limiting examples of ALK-inhibiting therapeutics include NVT TAE-684, AP26113, CEP-14083, CEP-14513, CEP11988, WHI-P131 and WHI- P154.

In various embodiments, the patient from whom said biological sample is obtained, where the reagent specifically binds to the biological sample, is diagnosed as having a mammalian ovarian cancer or suspected mammalian ovarian cancer driven by mutant ALK polynucleotide or mutant ALK polypeptide, or is diagnosed as having a mammalian ovarian cancer or suspected mammalian ovarian cancer driven by aberrant expression of a polypeptide with ALK activity.

In yet another aspect, the invention provides a method for determining whether a compound inhibits the progression of a mammalian cancer characterized by the expression of a mutant ALK polynucleotide or a FN 1 -ALK polynucleotide, said method comprising the step of determining whether said compound inhibits the expression of said mutant ALK polynucleotide as a mutant ALK polypeptide or inhibits the expression of said FN 1 -ALK polynucleotide as a FN1- ALK polypeptide in said cancer. In another aspect, the invention provides a method for determining whether a compound inhibits the progression of a mammalian cancer characterized by the expression of a mutant ALK polypeptide or an FN 1 -ALK polypeptide, said method comprising the step of determining whether said compound inhibits the expression and/or activity of said mutant ALK polypeptide or said FN 1 -ALK polypeptide in said cancer. In various embodiments, the mutant ALK polypeptide is an FNl-ALKvariantl polypeptide, an FN1- ALKvariant3 polypeptide, an FNl-ALKvariant5 polypeptide, or a truncated ALK polypeptide. In various embodiments, the FN 1 -ALK polypeptide is an FNl-ALKvariant2 polypeptide, an FN1- ALKvariant4 polypeptide, or an FNl-ALKvariant6 polypeptide.

In another aspect, the invention provides a method for inhibiting the progression of a mammalian cancer or suspected mammalian cancer that expresses a mutant ALK polypeptide or an FN 1 -ALK polypeptide, said method comprising the step of inhibiting the expression and/or activity of said polypeptide in said mammalian cancer or suspected mammalian cancer.

In further aspects, the invention provides a method for determining whether a compound inhibits the progression of a mammalian ovarian cancer or suspected mammalian ovarian cancer characterized by the expression of a polypeptide with ALK activity, said method comprising the step of determining whether said compound inhibits the expression of said polypeptide in said cancer. In another aspect, the invention provides a method for inhibiting the progression of a mammalian cancer or suspected mammalian cancer characterized by the expression of a polypeptide with ALK activity, said method comprising the step of inhibiting the expression and/or activity of said polypeptide in said mammalian ovarian cancer or suspected mammalian ovarian cancer. In some embodiments, the cancer is from a human.

In various embodiments, the polypeptide with ALK activity is aberrantly expressed full length ALK polypeptide, an FNl-tm ALK polypeptide, a truncated ALK polypeptide, or an ALK fusion polypeptide (e.g., an NPM-ALK fusion polypeptide, an AL017-ALK fusion polypeptide, an TFG-ALK fusion polypeptide, an MSN- ALK fusion polypeptide, an TPM3-ALK fusion polypeptide, a TPM4-ALK fusion polypeptide, an ATIC-ALK fusion polypeptide, an MYH9- ALK fusion polypeptide, an CLTC-ALK fusion polypeptide, an SEC31L1-ALK fusion polypeptide, an RANBP2-ALK fusion polypeptide, an CARS-ALK fusion polypeptide, an EML4- ALK fusion polypeptide, an KIF5B-ALK fusion polypeptide, and an TFG-ALK fusion

polypeptide).

In various embodiments, the inhibition is determined using at least one reagent selected from the group consisting of a reagent that specifically binds to a polynucleotide disclosed herein, a reagent that specifically binds to polypeptide disclosed herein, a reagent that specifically binds to a full length ALK polynucleotide, a reagent that specifically binds to a full length ALK

polypeptide, a reagent that specifically binds to a full length FN1 polynucleotide, and a reagent that specifically binds to a full length FN1 polypeptide.

In various embodiments, the expression and/or activity of said polypeptide is inhibited with a composition comprising a therapeutic selected from the group consisting of crizotinib (also known as PF-02341066), NVT TAE-684, AP26113, CEP-14083, CEP-14513, CEP11988, WHI- P131 and WHI-P154.

BRIEF DESCRIPTION OF THE DRAWINGS Figures 1A, IB, 1C, ID, and IE are sequences and diagrams showing a fragment of the cDNA containing the fusion junction (Fig. 1A), a fragment of the polypeptide containing the fusion junction (Fig. IB), a fragment of genomic DNA containing the fusion junction (Fig. 1C), a schematic showing the FNl gene, the ALK gene, and the fusion gene (Fig. ID), and the full length amino acid sequence of the FNl-ALKvariantl (Fig. IE), all showing the breakpoint in the FNl gene and the ALK gene which give rise to the FNl-ALKvariant 1 fusion polypeptide. In Fig. 1A, the fragment of the shown cDNA sequence (SEQ ID NO: 22) contains the FNl exon 22, FNl exon 23 linked to ALK exon 19, and ALK exon 20, where alternative exons are in different colors (black or blue) and the ALK portion of the sequence is underlined. In Fig. IB, the shown protein sequence (SEQ ID NO: 23) contains the FNl exon 22, FNl exon 23 linked to ALK exon 19, and ALK exon 20 where the amino acids encoded by alternative exons are in different colors (black or blue), the ALK portion is underlined, and the amino acid amino acids at the junction of the two exons (i.e., exon 23 of FNl and exon 19 of ALK) are shown in red, and the transmembrane domain encoded by ALK exonl9 is highlighted in yellow. In Fig. 1C, the shown genomic sequence (SEQ ID NO: 24) contains a portion of the FNl exon 23 (in capital letters), a portion of the ALK exon 19 (in capital letters), and the intron between these exons which contains 1-946 bp of FNl intron 23 and 1343-1356 bp of ALK-intronl8 (a total of 960bp, shown in lower case letters), where both intron and exon sequences from ALK are underlined. In Fig. ID, the gene structure of FNl gene, ALK gene, and the FNl -ALK variant 1 fusion gene is shown schematically where the exons are shown as boxes (black boxes in the full length FNl and ALK genes) and introns are shown as lines. The location and orientation of FNl and ALK genes are also shown. Exons and joint sequences in FNl (blue) and ALK (red), positions of the novel breakpoint (Novel BP) and the common breakpoint (Common BP) are indicated. A single PCR product of ~lkb amplified from OC19 gDNA using PCR primers annealing to FNl Exon23 and ALK Exon 19 was detected by agarose electrophoresis. In Fig. IE, the predicted full length amino acid sequence of the FNl-ALKvariant 1 fusion protein is provided (SEQ ID NO: 3). Residues corresponding to FNl or ALK are indicated in blue and red, respectively. Peptide sequence encoded by ALK Exon 19 is underlined. Amino acid sequence spanning the trans-membrane domain is highlighted in yellow. Figure 2 A is a schematic drawing of the FNl-ALKvariantl fusion polypeptide, a non-limiting polypeptide of the invention . The portion of the polypeptide from FNl is shown in blue, and the portion of the polypeptide from ALK is shown in white, where the transmembrane domain from ALK is shown in black and the kinase domain from ALK is shown in red.

Figure 2B is another schematic diagram showing the FNl-ALKvariantl fusion polypeptide, in a stromal sarcoma patient, OC19. Fusion of the amino-terminal 1201 amino acids of fibronectin 1 (FNl) (comprising FN assembly, Gelatin and FN binding domains) to the carboxyl-terminal 598 amino acids transmembrane (TM) and the intracellular region containing the kinase domain of ALK. The type I, II and III modules in fibronectin 1 (FNl) are shown in rectangles, diamonds and ovals, respectively, with domains required for fibrillogenesis shaded in blue. Domains subjected to alternative splicing in various fibronectin isoforms are shaded in light yellow (B, A and V). The positions of FNl domains involved in binding to various extracellular matrix proteins are indicated. The ALK amino acid positions of the novel breakpoint (Novel BP, black arrow) in FNl -ALK and the common breakpoint (Common BP, gray arrow) are indicated.

Figure 3 is an agarose gel showing an electrophoretically resolved PCR product amplified from patient OC19 with primers annealing to FNl exon 23 and ALK exon 19. As predicted, the fused intron had a molecular mass of about 1 kb.

Figures 4A and 4B are, respectively, a schematic diagram and agarose gels showing resolved RT- PCR products from the indicated patients or cell lines. Fig. 4A shows the annealing positions of primers FNlE21f, ALKE16f, and ALKGSP3 on the ALK gene (upper) and on the FNl- ALKvariantl fusion gene (lower) used to amplify the ALK cDNA region from Exon 16 to Exon 20 and the FNl -ALK cDNA region from FNl Exon 21 to ALK Exon 20 are indicated. Fig. 4B shows the electrophoretically resolved RT-PCR products ALK exonl6-GSP3 (ALK unfused, upper), FNl exon22-ALK GSP3 (ALK fused to FNl, middle) and GAPDH (qualitative control, lower) from patients XY1-OC16, XY1-OC19, XY1-OC26, XY1-OC7 and XY1-OC8, and from three ovarian cancer cell lines, Ovamana, Ovsaho and Ovmiu.

Figure 5A is a Western blotting analysis showing the results following blotting with a full length ALK-specific antibody (which specifically binds to the ALK kinase domain) of ovarian cancer tissues from serous carcinoma tissue OC16, OC26 and OC29a or stromal sarcoma patient OC19. As a positive control, cell lysates from the EM14-ALK fusion protein expressing lung cancer cell line H2228 was also probed with this antibody. Antibody that specifically bind b-Actin was used as a loading control (i.e., to confirm equal loading of protein in all wells).

Figures 5B-5D are photographs showing the results of immunohistochemical (IHC) staining of paraffin sections of ovarian tissue from serous carcinoma patients OC29a (Fig. 5B), OC26 (Fig. 5C) and stromal sarcoma patient OC19 (Fig. 5D) with an antibody against ALK kinase domain. Images represent x40 magnification.

Figure 6 is a Western blotting analysis showing the results following blotting with a full length ALK-specific antibody of numerous ovarian cancer cell lines. Blotting with a b-actin-specific antibody was done to show equal loading of protein in all lanes. Figure 7A is a bar graph showing the results of ALK gene amplification of various ovarian cancer cell lines and tissue samples. ALK relative gene quantities are shown in blue bars and GAPDH relative gene quantities (which is always one) are shown in red.

Figure 7B is a bar graph showing ALK gene copy number variation in serous carcinoma patients. qPCR analysis was performed with genomic DNA isolated from tumor tissue OC07, OC08,

OC16, OC19, and OC26. The value of relative gene copy number of ALK (blue bars) to that of an Ultra Concservative element (UCE) (red bars) in each gDNA sample is shown. Calculations were performed to normalize a copy number of 2 to a value of 1 in the bar graph. Standard deviations were based on duplicate normalized Ct values. This result represents 3 independent experiments.

Figure 8A is a schematic diagram showing the multiple cloning site of the MSC-neo vector flanked by the upstream and downstream long terminal repeats (LTRs) and the downstream neomycine ("neo") gene which confers G418 resistance of empty vector (i.e., no insert; MSCV- neo in top diagram); inserted full length ALK cDNA (MSCV-Neo ALK in middle diagram); and inserted FNl-ALKvariantl cDNA (MSCV-Neo-FNl-ALK in bottom diagram). In the middle and bottom diagrams, ALK sequences are outlined in red, FN1 sequences are outlined in blue, the transmembrane domain from ALK is shown as a black bar, and the kinase domain from ALK is shown as a red bar.

Figure 8B shows Western blot analysis of protein lysates prepared from 3T3 cells stably transfected with Neo, ALK or FNl-ALK. Arrows from top to bottom indicate full length ALK or FNl-ALK, 140 kd cleaved form of ALK in 3T3/ALK cells and -78 kd ALK signal in 3T3/FN1- ALK cells, respectively. Bloting with an anti-b-Actin antibody was used as loading control.

Figure 8C shows the results of immunofluorescence staining of 3T3 cells stably transfected with Neo, ALK or FNl-ALK (upper panels), and results of an in vivo tumorigenicity assay of nude mice injected with the transduced 3T3 cells. Stably transfected 3T3 cells expressing neomycin (Neo), ALK or FNl-ALK were stained with antibodies against ALK (green), Keratin (red) or DRAQ5® (blue) (Fig. 8C, upper panel). These cells were injected subcutaneously into nude mice (Fig. 8C, lower panel). The tumor images, the rate of tumor growth and the average size of tumors are reported 12 days after injection. The results are representative of two independent experiments.

Figures 9A-9C are line graphs showing that ALK and FNl-ALK tumors are sensitive to an ALK inhibitor, Crizotinib. Four to six nude mice carrying 3T3 tumors expressing ALK (Fig. 9A), FN1- ALKvariantl (Fig. 9B), or SRC (Fig. 9C) were treated with vehicle (green squares) or

lOOmg/kg/day Crizotinib (blue diamonds) by oral gavage when tumors are palpable. The tumors were measured every other day until the mean tumor size of the vehicle treated mice reached 1500mm³.

Figure 9D is a Western blotting analysis using antibodies against ALK, phospho-ALK

(Y 1278/1282/1283) and β-Actin of tumor cells from mice carrying FNl-ALK tumors that were treated with vehicle or Crizotinib for 24 hours before the tumors were harvested and analyzed by western blot assay. Arrows indicate the positions of the full length FNl-ALK and the truncated ALK variant (lower arrow). Note the ALK signal with lower molecular weight (*) next to the FNl-ALK, which is likely non-phosphorylated FNl-ALK.

Figures 1 OA- IOC are line graphs showing that ALK and FNl-ALK variant 1 tumors are sensitive to TAE684 treatment._Four to six nude mice carrying 3T3 tumors expressing ALK (Fig. 10A), FNl-ALKvariantl (Fig. 10B), or SRC (Fig. IOC) were treated with vehicle (green squares) or lOmg/kg/day TAE (red triangles) by oral gavage when tumors are palpable. The tumors are measured every other day until the mean tumor size of the vehicle treated mice reached 1500mm . Figure 1 lA-1 ID are images of immunohistochemistry analyses of ovarian tissue microarrays

(TMAs). The shown images from 4 serous carcinoma specimen that are ALK +++ (Figs. 1 1 A and 1 IB) and ALK ++ (Figs. 1 1C and 1 ID), representing 40x magnification.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The invention is based upon the unexpected discovery of ALK kinase expression in human ovarian cancer. The invention also provides novel gene translocations between the FN1 gene and the ALK gene, and novel expression of the transmembrane and kinase domains of an ALK polypeptide without co-expression of the extracellular domain of ALK in cancer (such as ovarian cancer). The invention also provides the discovery of a polypeptide with ALK kinase activity in human ovarian cancer. As the mutant ALK polypeptides, including truncated ALK polypeptide and FN 1 -ALK fusion proteins (i.e., encoded by the FN 1 -ALK gene translocations), all contain the entire kinase domain of ALK, they all are expected to have ALK kinase activity. One or more of these mutant ALK polypeptides may be expressed in a subset of mammalian cancers, such as human cancers, and may drive the proliferation and survival of the cancer in which it is expressed. Such cancers may be identified (e.g., diagnosed) and/or treated in accordance with the teachings provided herein.

Based on these discoveries, a patient whose ovarian cancer (or suspected ovarian cancer) expresses a protein with ALK activity or a patient whose cancer (or suspected cancer) expressed mutant ALK (e.g., a truncated FNl-tmALK) or an FN1-ALK fusion polypeptide) where healthy patients do not express such proteins with ALK activity or mutant or FN 1 -ALK fusion

polypeptide in that tissue may respond favorably to administration of an ALK inhibitor (e.g. , the growth of the cancer may slow or stop as compared to an untreated patient suffering from the same cancer).

The published patents, patent applications, websites, company names, and scientific literature referred to herein establish the knowledge that is available to those with skill in the art and are hereby incorporated by reference in their entirety to the same extent as if each was specifically and individually indicated to be incorporated by reference. Any conflict between any reference cited herein and the specific teachings of this specification shall be resolved in favor of the latter.

The further aspects, advantages, and embodiments of the invention are described in more detail below. The patents, published applications, and scientific literature referred to herein establish the knowledge of those with skill in the art and are hereby incorporated by reference in their entirety to the same extent as if each was specifically and individually indicated to be incorporated by reference. Any conflict between any reference cited herein and the specific teachings of this specification shall be resolved in favor of the latter. Likewise, any conflict between an art-understood definition of a word or phrase and a definition of the word or phrase as specifically taught in this specification shall be resolved in favor of the latter. As used herein, the following terms have the meanings indicated. As used in this specification, the singular forms "a," "an" and "the" specifically also encompass the plural forms of the terms to which they refer, unless the content clearly dictates otherwise. The term "about" is used herein to mean

approximately, in the region of, roughly, or around. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. In general, the term "about" is used herein to modify a numerical value above and below the stated value by a variance of 20%.

Technical and scientific terms used herein have the meaning commonly understood by one of skill in the art to which the present invention pertains, unless otherwise defined. Reference is made herein to various methodologies and materials known to those of skill in the art. Standard reference works setting forth the general principles of recombinant DNA technology, all of which are incorporated herein by reference in their entirety, include Ausubel et al. CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, N.Y. (1989 and updates through September 2010), Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold

Spring Harbor Laboratory Press, New York (1989); Kaufman et al., Eds., Handbook of Molecular and Cellular Methods in Biology in Medicine, CRC Press, Boca Raton (1995); McPherson, Ed., Directed Mutagenesis: A Practical Approach, IRL Press, Oxford (1991). Standard reference works setting forth the general principles of pharmacology, all of which are incorporated herein by reference in their entirety, include Goodman and Gilman's The Pharmacological Basis of

Therapeutics, 11th Ed., McGraw Hill Companies Inc., New York (2006). Accordingly, in a first aspect, the invention provides an isolated polynucleotide comprising a nucleotide sequence at least 95% identical to a sequence selected from the group consisting of: (a) a nucleotide sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 3, 5, 7, 10, 1 1 , 12, 14, 15, 17, 18, 20, or 21 ; (b) a nucleotide sequence comprising the nucleotide sequence of SEQ ID NO: 4, 6, 8; 13, 16, or 19; (c) a nucleotide sequence encoding polypeptide comprising an N-terminal portion comprising amino acid residues 1-1085 of SEQ ID NO: 26 or amino acid residues 1-1 1 16 of SEQ ID NO: 27 and a C-terminal portion comprising amino acid residues 1039-1392 of SEQ ID NO: 1 ; (d) a nucleotide sequence encoding a polypeptide comprising an N-terminal portion comprising amino acid residues 1-1085 of SEQ ID NO: 26 or amino acid residues 1-1 1 16 of SEQ ID NO: 27 and a C-terminal portion comprising the amino acid sequence set forth in SEQ ID NO: 29; (e) a nucleotide sequence comprising (i) at least six contiguous nucleotides encompassing the fusion junction (nucleotides 3601-3606 of SEQ ID NO: 4, nucleotides 3793-3798 of SEQ ID NO: 6, or nucleotides 3346-3351 of SEQ ID NO: 8) or encoding at least six contiguous polypeptides encompassing the fusion junction (residues 1999- 1204 of SEQ ID NO : 3 , residues 1263-1268 of SEQ ID NO: 5, or residues 1 1 14-1 1 19 of SEQ ID NO: 7) of an FNl-tmALK fusion polypeptide; (f) a nucleotide sequence comprising (i) at least six contiguous nucleotides encompassing the fusion junction (nucleotides 3601-3606 of SEQ ID NO: 13, nucleotides 3793-3798 of SEQ ID NO: 16, nucleotides 3346-3351 of SEQ ID NO: 19) or encoding at least six contiguous polypeptides encompassing the fusion junction (residues 1999- 1204 of SEQ ID NO: 14, residues 1263-1268 of SEQ ID NO: 17, or residues 1 1 14-1 1 19 of SEQ ID NO: 20) of an FN1-ALK fusion polypeptide; (g) a nucleotide sequence encoding a polypeptide comprising amino acid residues 1039-1392 of SEQ ID NO: 1 , wherein said polypeptide does not comprise amino acid residues 19-1038 of SEQ ID NO: 1 ; (h) a nucleotide sequence encoding a polypeptide comprising amino acid sequence of SEQ ID NO: 29, wherein said polypeptide does not comprise amino acid residues 19-1038 of SEQ ID NO: 1 ; and (i) a nucleotide sequence complementary to any of the nucleotide sequences of (a)-(h).

As used herein, by "purified" (or "isolated"") refers to a polynucleotide (or nucleotide sequence or nucleic acid molecule) or polypeptide (or protein or an amino acid sequence) that is removed or separated from other components present in its natural environment. For example, a purified FN1-ALK fusion polypeptide is one that is separated from other components of a cell (e.g., the endoplasmic reticulum, cytoplasmic proteins and/or RNA). A purified FN1-ALK polynucleotide is one that is separated from other nuclear components (e.g., histones) and/or from upstream or downstream nucleic acid sequences (e.g., a purified FN 1 -ALK polynucleotide is separated from the endogenous FN1 gene promoter). A purified nucleic acid sequence or amino acid sequence is at least 60% free, or at least 75% free, or at least 90%> free, or at least 95% free from other components present in natural environment of the indicated nucleic acid sequence or amino acid sequence.

As used herein, by "polynucleotide" (or "nucleotide sequence" or "nucleic acid molecule") refers to an oligonucleotide, nucleotide, or polynucleotide, and fragments or portions thereof, and to DNA or RNA of genomic or synthetic origin, which may be single- or double-stranded, and represent the sense or anti-sense strand. A polynucleotide with a gene name in front of it means that the indicated polynucleotide comprises all or part (e.g., an mRNA or cDNA of a gene) of the indicated gene and may encode all or a portion of the indicated gene product (e.g., a polypeptide). For example, an FN1 polynucleotide is all or part of the FN1 gene and may encode all or a portion of a FN1 polypeptide. It should be noted that not all nucleotides in a polynucleotide need to encode amino acid residues. For example, the FNl-ALKvariantl fusion polynucleotide may comprise portions of intron sequences that do not encode any amino acids in the resulting FNl- ALKvariantl fusion polypeptide.

ALK (anaplastic lymphoma kinase) is a 1620 amino acid long receptor tyrosine kinase that is prone to aberrant expression leading to cancer. A description of full length human ALK kinase (with the amino acid sequence of the human ALK protein) can be found at UniProt Accession No. Q9UM73 (see also U.S. Pat. No. 5,770,421 , entitled "Human ALK Protein Tyrosine Kinase"). As shown in Table 1 , the signal peptide, extracellular, transmembrane, and kinase domains of ALK are found at the following amino acid residues in SEQ ID NO: 1 :

Table 1

The polypeptide sequence of exon 20 onward of the ALK protein is included herein as SEQ ID NO: 30. The polypeptide sequence of exon 19 onward of the ALK protein is included herein as SEQ ID NO: 29. The polypeptide sequence of the ALK protein starting with the transmembane domain and including the rest of the C 'terminal portion of the protein is set forth in SEQ ID NO: 25.

Additionally, there are multiple known naturally-occurring variants of ALK (see, e.g., Greenman et al., Nature 446: 153-158, 2007). The nucleotide and amino acid sequences of murine full-length ALK are known (see Iawahara et al., Oncogene 14(4): 439-449, 1997). Using routine experimentation, the ordinarily skilled biologist would be readily able to determine corresponding sequences in non-human mammalian ALK homologues.

By "wild-type" ALK is meant the expression and/or activation of full length ALK kinase (i.e., 1620 amino acid long polypeptide or 1602 amino acid long polypeptide following removal of the signal peptide sequence) in healthy (or normal) tissue (e.g., non-cancerous tissue) of a normal individual (e.g., a normal individual who is not suffering from cancer). Pulford et al., Journal of Cellular Physiology, 199:330-358, 2004 provides a comprehensive review relating to ALK and fusion polypeptides that include portions of the full length ALK polyepeptide. In normal humans, full-length ALK expression has been detected in the brain and central nervous system, and has been reported in the small intestine and testis (see, e.g., Morris et al, Oncogene 14:2175-2188,

1997). However, ALK kinase (full length or truncated) does not appear to be expressed in normal ovarian tissue in humans, a finding which the inventors have confirmed using various

commercially available ALK-specific antibodies (e.g., Catalog Nos. 3791 and 3333 from Cell Signaling Technology, Inc., Danvers, MA). However, using the methods described in the below Examples, the inventors have made the surprising discovery of ALK kinase expression in ovarian cancer. Such expression in an atypical cell (in this case a cancerous cell) where no expression is seen in a typical cell (e.g., a non-cancerous ovarian cell) is aberrant.

Numerous examples of aberrantly expressed ALK kinase have been found in other cancers. For example, point mutations within the kinase domain have been found in neuroblastoma, overexpression of ALK has been found in numerous cancers (including, e.g., retinoblastoma, breast cancer, and melanoma), and fusion proteins comprising the kinase domain (but not the transmembrane domain) of ALK fused to all or a portion of a second protein have been discovered in various cancers including non-small cell lung cancer (NSCLC) in inflammatory myofibroblastic tumor. See review in Palmer et al., Biochem. J. 420(3): 345-361 (May 2009), herein incorporated by reference in its entirety.

In addition, in accordance with the invention, aberrant expression (e.g., overexpression) of full length ALK in a cancer (e.g., an ovarian cancer) may be the result of amplification of the ALK gene in the cancer cell's genome. As shown in the Examples below, some of the ovarian cancers that aberrantly express full length ALK have more copies of the ALK gene (as compared to copies of GAPDH, an essential house-keeping gene) as compared to cells that do not express full length ALK.

Accordingly, as used herein, the term "ALK fusion" refers to a portion of the ALK polypeptide comprising the kinase domain but not the transmembrane domain of ALK

(polynucleotide encoding the same) fused to all or a portion of another polypeptide (or

polynucleotide encoding the same), where the name of that second polypeptide or polynucleotide is named in the fusion. (The term "fusion" simply means all or a portion of a polypeptide or polynucleotide from first gene fused to all or a portion of a polypeptide or a polynucleotide from a second gene). For example, an NPM-ALK fusion is a fusion between a portion of the NPM polypeptide or polynucleotide and a portion of the ALK polypeptide (or polynucleotide encoding the same) comprising the kinase domain but not the transmembrane domain of ALK. An ALK fusion often results from a chromosomal translocation or inversion. There are numerous known ALK fusions, all of which are ALK fusions and include, without limitation, NPM-ALK, AL017- ALK, TFG-ALK, MSN-ALK, TPM3-ALK, TPM4-ALK, ATIC-ALK, MYH9-ALK, CLTC-ALK, SEC31L1-ALK, RANBP2-ALK, CARS-ALK, EML4-ALK, KIF5B-ALK, and TFG-ALK (see, e.g., Palmer et al., Biochem. J. 420(3): 345-361, 2009 (and the articles cited therein), Rikova et al., Cell 131 : 1190-1203, 2007; Soda et al, Nature 448: 561-566, 2007; Morris et al, Science 263: 1281-1284, 1994; Du et al, J. Mol. Med 84: 863-875, 2007; Panagopoulos et al, Int. J. Cancer

118: 1181-1186, 2006; Cools et al, Genes Chromosomes Cancer 34: 354-362, 2002; Debelenko et al, Lab. Invest. 83: 1255-1265, 2003; Ma et al, Genes Chromosomes Cancer 37: 98-105, 2003; Lawrence et al, Am. J. Pathol. 157: 377-384, 1995; Hernandez et al, Blood 94: 3265-3268, 1999; Takeuchi K., Clin Cancer Res. 15(9):3143-3149, 2009; Tort et al, Lab. Invest. 81 : 419-426, 2001; Trinei et al, Cancer Res. 60: 793-798, 2000; and Touriol et al, Blood 95: 3204-3207, 2000. Some of these ALK fusions have multiple variants, all of which are considered ALK fusions and, thus, are included in the definition of a mutant ALK. For example, there are multiple variants of TFG- ALK (see, e.g., Hernandez et al., Amer. J. Pathol. 160: 1487-1494, 2002) and at least nine known variants of EML4-ALK (see, e.g., Horn et al, J. of Clinical Oncology 27(26): 4232-4235, 2009, U.S. Patent No. 7,700,330 and EP Patent No. 1 914 240).

The present invention stems, in part, from the discovery of a new gene translocation involving the ALK gene. As described below in the Examples, the human gene translocations (and resultant fusion polypeptides) between the FNl gene and the ALK gene are identified using global phosphopeptide profiling in ovarian cancer samples taken from human patients (see Examples below). Disclosed herein are several gene translocations between the ALK gene and the FNl gene (which encodes fibronectin). Altogether, six translocations are disclosed.

Table 2 provides a description of the six fusions between the FNl gene and the ALK gene and their cDNA and encoded protein sequences with and without the 31 amino acid long signal peptide sequence from the FNl protein.

Table 2

Note that the signal peptide sequence has the following sequence:

MLRGPGPGLLLLAVQCLGTAVPSTGASKSKR (SEQ ID NO: 9). Note that the genomic sequence showing the breakpoint between the FNl gene and the ALK gene in variant is provided in Fig. 1C (SEQ ID NO: 24).

It should be noted that in all of the ALK fusion proteins described herein (e.g., the FN1- ALK fusion proteins, the NPM-ALK fusion proteins, the EML4-ALK fusion proteins, etc...) and in the mutant ALK proteins described herein (e.g., the FNl-tmALK fusions), the amino acid at the fusion junction (regardless of the numbering) may appear in either full-length protein member of the fusion (e.g., the amino acid at the fusion junction in a FNl-ALKvariantl fusion polypeptide may appear in either full-length FNl protein or full-length ALK protein), or the amino acid, being created by a codon with nucleotides from fused exons of both protein members, may be unique to the fusion polypeptide and not appear in either full-length protein member of the fusion.

The gene translocations between the FNl gene and the ALK gene occur on human chromosome 2 and will result in expression of fusion polypeptides that combine the N-terminus of FNl with the kinase domain of ALK. Some of the gene translocations between the FNl and the ALK genes result in polypeptides containing the transmembrane domain of the ALK polypeptide. These fusions are referred to herein as FNl-tmALK polypeptides (or FNl-tmALK fusion polypeptides) and are encoded by FNl-tmALK polynucleotides (or FNl-tmALK fusion polynucleotides). Those fusions that do not contain the transmembrane domain of ALK are referred to as FNl -ALK polypeptides (or FNl -ALK fusion polypeptides) and are encoded by FNl -ALK polynucleotides (or FNl -ALK fusion polynucleotides). As shown above, only FN1- ALKvariant2, FNl-ALKvariant4, and FNl-ALKvariant6 are included within the definition of "ALK fusion" as used herein (see full definition elsewhere herein).

Unlike the other known ALK fusions, the FNl-tmALK fusions (i.e., FNl-ALKvariantl, FNl-ALKvariant3, and FNl-ALKvariant5) are the only ALK fusions described thus far that include the transmembrane domain of ALK as well as the kinase domain of ALK. These three fusions (i.e., FNl-ALKvariantl, FNl-ALKvariant3, and FNl-ALKvariant5) are included within the definition of "mutant ALK" (see full definition elsewhere herein).

The fusions between the FNl gene and the ALK gene described herein (i.e., all six variants) can obtained from any species, particularly mammalian, including bovine, ovine, porcine, murine, equine, primate, and human, from any source whether natural, synthetic, semi- synthetic, or recombinant. In some embodiments, the fusion between the FNl gene and the ALK gene encode a FNl-tmALK fusion polypeptide comprising a N-terminal portion comprising amino acid residues 1-1085 of SEQ ID NO: 26 and a C-terminal portion comprising amino acid residues 1039-1392 of SEQ ID NO: 1 , a C-terminal portion comprising the amino acid residues of SEQ ID NO: 25 or a C-terminal portion comprising the amino acid residues of SEQ ID NO: 29. In some embodiments, the fusion between the FN1 gene and the ALK gene encodes a FNl -tmALK fusion polypeptide comprising a N-terminal portion comprising amino acid residues 1-1 1 16 of SEQ ID NO: 27 and C-terminal portion comprising amino acid residues 1039-1392 of SEQ ID NO: 1 , C- terminal portion comprising the amino acid residues of SEQ ID NO: 25 or a C-terminal portion comprising the amino acid residues of SEQ ID NO: 29.

In some embodiments, the fusion between the FN1 gene and the ALK gene encodes a FN 1 -ALK fusion polypeptide comprising a N-terminal portion comprising amino acid residues 1-1085 of SEQ ID NO: 26 and a C-terminal portion comprising amino acid residues 1 1 16-1392 of SEQ ID NO: 1 or a C-terminal portion comprising the amino acid residues of SEQ ID NO: 30. In some embodiments, the fusion between the FN1 gene and the ALK gene encodes a FN 1 -ALK fusion polypeptide comprising a N-terminal portion comprising amino acid residues 1-1 1 16 of SEQ ID NO: 27 and C-terminal portion comprising amino acid residues 1 1 16-1392 of SEQ ID NO: 1 or a C-terminal portion comprising the amino acid residues of SEQ ID NO: 30.

As used herein, the term "mutant ALK" polypeptide or polynucleotide means the transmembrane and kinase domains of an ALK polypeptide (e.g., detectable by expression by, for example, Western blot, IHC, or mass spectrometry analysis or detectable by activity by, for example, an in vitro kinase assay) or a polynucleotide encoding the transmembrane and kinase domains of a ALK kinase (e.g., detectable by PCR, FISH, or Southern blotting analysis), where the transmembrane and kinase domains are present without the extracellular domain of full length ALK. The transmembrane and kinase domains (or nucleotide sequences encoding the same) of the mutant ALK may be either alone (also referred to as "truncated ALK", see description below) or may be fused (e.g., a fusion polypeptide via a peptide bond or a fusion polynucleotide via a phosphodiester bond) to all or a portion of a second polypeptide or polynucleotide (e.g., a FN1 polypeptide or polynucleotide). Thus included within the definition of mutant ALK are the FN1- ALKvariantl , the FNl -ALKvariant3, and the FNl-ALKvariant5 fusions described herein. As a mutant ALK comprises a functional kinase domain of ALK, all mutant ALK polypeptides are also within the definition of "polypeptide with ALK kinase activity".

As used herein, by the term "polypeptide with ALK kinase activity" is meant any polypeptide that retains the full kinase domain of ALK and thus, has ALK kinase activity. In some embodiments, the polypeptide with ALK kinase activity contains a tyrosine residue that can be phosphorylated. In some embodiments, the polypeptide with ALK kinase activity contains a phosphorylated tyrosine residue. Non-limiting polypeptides with ALK kinase activity include full length ALK, truncated ALK, ALK fusion polypeptides (e.g., NPM-ALK fusion, the various EML4-ALK fusions, ATIC-ALK fusion, CARS-ALK fusion, and the FN 1 -ALK fusions described herein, including FNl -ALKvariant2, FNl-ALKvariant4, and FNl-ALKvariant6), and mutant ALK (e.g., truncated ALK and the FNl-tmALK fusions described herein, including FN1- ALKvariantl , FNl-ALKvariant3, and FNl-ALKvariant5).

A truncated ALK comprises the kinase domain (e.g., amino acid residues 1 1 16-1392 of SEQ ID NO: 1 or nucleotide sequences encoding the same) with the transmembrane domain of ALK (e.g., amino acid residues 1039-1059 of SEQ ID NO: 1 or nucleotide sequences encoding the same) such that the kinase domain and transmembrane domains of ALK are separated from the other domains (e.g., the extracellular domain) of full-length ALK kinase. The full length amino acid sequence of ALK kinase is provided in SEQ ID NO: 1. The transmembrane and kinase domain of the ALK kinase is provided in SEQ ID NO: 25; however the term "truncated ALK" also includes also those amino acid residues or nucleotide sequences which flank the ALK transmembrane and kinase domain so long as those flanking amino acid residues or nucleotide sequences do not themselves, constitute (or encode) the extracellular domain of full length ALK. The truncated ALK polypeptide of the invention may have an amino acid sequence consisting essentially of amino acids 1039-1059 and 1 1 16-1392 from SEQ ID NO: 1 , or may consist essentially of amino acids 1039-1392 of SEQ ID NO: 1 , or may consist essentially of amino acids 1039-1620 of SEQ ID NO: 1 (this is SEQ ID NO: 25). In some embodiments, the truncated ALK polypeptide does not include amino acid residues 19-1038 of SEQ ID NO: 1.

The FN1 polynucleotide sequence is provided herein as SEQ ID NO: 28. The FN1 protein sequence minus signal sequence is provided herein as SEQ ID NO: 26. The FN1 protein sequence including the signal sequence is provided herein as SEQ ID NO: 27.

Where the mutant ALK is a transmembrane and kinase domains (or nucleotide sequences encoding the same) of the mutant ALK fused (e.g., a fusion polypeptide via a peptide bond or a fusion polynucleotide via a phosphodiester bond) to all or a portion of a second polypeptide or polyncuelotide (e.g., a FN1 polypeptide or polynucleotide), the ALK portion of the mutant ALK may have an amino acid sequence consisting essentially of amino acids 1039-1059 and 1 1 16-1392 from SEQ ID NO: 1 , or may consist essentially of amino acids 1039-1392 of SEQ ID NO: 1 , or may consist essentially of amino acids 1039-1620 of SEQ ID NO: 1 (this is SEQ ID NO: 25). The FNl-tmALK fusions disclosed herein (i.e., FNl-ALKvariantl, FNl-ALKvariant3, and FN1- ALKvariant5) are the first known fusions involving the ALK gene to include the transmembrane domain of ALK in addition to the kinase domain of ALK. In some embodiments, the FNl- tmALK fusion comprises the amino acid residues 1-1116 of SEQ ID NO: 27, or amino acid residues 1-1201 of SEQ ID NO: 27, or amino acid residues 1-1265 of SEQ ID NO: 27. In some embodiments, the FNl-tmALK fusion comprises the amino acid residues 1-1085 of SEQ ID NO: 26 or amino acid residues 1-1170 of SEQ ID NO: 26, or amino acid residues 1-1234 of SEQ ID NO: 26.

The present invention further relates to variants of the nucleic acid molecules of the present invention, which encode portions, analogs or derivatives of an FN 1 -ALK fusion polypeptide, a an FNl-tmALK fusion polypeptide, or a truncated ALK polypeptide disclosed herein. Variants may occur naturally, such as a natural allelic variant. By an "allelic variant" is intended one of several alternate forms of a gene occupying a given locus on a chromosome of an organism. See, e.g. GENES II, Lewin, B., ed., John Wiley & Sons, New York (1985). Non-naturally occurring variants may be produced using art-known mutagenesis techniques.

Such variants include those produced by nucleotide substitutions, deletions or additions. The substitutions, deletions or additions may involve one or more nucleotides. The variants may be altered in coding regions, non-coding regions, or both. Alterations in the coding regions may produce conservative or non-conservative amino acid substitutions, deletions or additions. Some alterations included in the invention are silent substitutions, additions and deletions, which do not alter the properties and activities (e.g. kinase activity) of the FN 1 -ALK fusion polypeptides and mutant ALK polypeptides disclosed herein.

Further embodiments of the invention include isolated polynucleotides comprising a nucleotide sequence at least 90% identical. In some embodiments of the invention the nucleotide is at least 95%>, 96%>, 97%>, 98%> or 99%> identical, to a mutant ALK polynucleotide (for example, a nucleotide sequence encoding the FNl-ALKvariantl fusion polypeptide having the amino acid sequence set forth in SEQ ID NOs: 3, or a nucleotide sequence encoding the N-terminal of FN1 and the transmembrane and kinase domains of ALK; or a nucleotide complementary to such exemplary sequences.

By a polynucleotide having a nucleotide sequence at least, for example, 95%> "identical" to a reference nucleotide sequence or by a polypeptide having an amino acid sequence at least, for example, 95% "identical" to a reference amino acid sequence is intended that the nucleotide sequence or the amino acid sequence is identical to the reference sequence except that the polynucleotide sequence may include up to five point mutations per each 100 nucleotides of the reference nucleotide sequence and the polypeptide sequence may include up to five amino acid alterations per each 100 amino acids of the reference amino acid sequence.

In other words, to obtain a polynucleotide having a nucleotide sequence at least 95% identical to a reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or a number of nucleotides up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence. These mutations of the reference sequence may occur at the 5' ' terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either

individually among nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence. Similarly, to obtain a polypeptide having an amino acid sequence at least 95% identical to a reference amino acid sequence, up to 5% of the amino acid residues in the reference sequence may be deleted or substituted with another amino acid, or a number of amino acids up to 5% of the total amino acid residues in the reference sequence may be inserted into the reference sequence. These alterations of the reference sequence may occur at the amino or carboxy terminal positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence or in one or more contiguous groups within the reference sequence.

As a practical matter, whether any particular nucleic acid sequence or amino acid sequence is at least 90%>, 95%, 96%, 97%, 98% or 99% identical to a reference nucleic acid or amino acid sequence described herein can be determined conventionally using known computer programs such as the Bestfit program or the BLAST program from the NCBI. The Bestfit program

(Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group,

University Research Park, 575 Science Drive, Madison, Wis. 53711) and the BLAST program (National Center for Biotechnology Information) be used the default settings for determining similarity. Bestfit uses the local homology algorithm of Smith and Waterman {Advances in Applied Mathematics 2: 482-489 (1981)) to find the best segment of similarity between two sequences. BLAST uses the algorithm described in (Altschul SF et al, "Basic local alignment search tool". J. Mol. Biol. 215 (3): 403-410 (1990) to find similarity between two sequences. When using Bestfit, BLAST, or any other sequence alignment program to determine whether a particular sequence is, for instance, 95% identical to a reference FNl-tmALK fusion polynucleotide or polypeptide sequence according to the present invention, the parameters are set, of course, such that the percentage of identity is calculated over the full length of the reference sequence and that gaps in homology of up to 5% of the total number of residues (i.e., nuceotides or amino acid residues) in the reference sequence are allowed.

As used herein, a "portion" or "fragment" means a sequence fragment less than the whole sequence. For example, a 50 nucleotide sequence is a portion of a 100 nucleotide long sequence. Similarly, a 50 amino acid residue long sequence is a portion of a 100 amino acid long sequence. In some embodiments, a nucleic acid fragment or portion comprises at least 20 nucleotides, or at least 30 nucleotides, or at least 45 nucleotides, or at least 60 nucleotides, or at least 70 nucleotides, or at least 90 nucleotides. In some embodiments, an amino acid fragment or portion comprises at least 6 amino acid residues, or at least 10 amino acid residues, or at least 20 amino acid residues, or at least 30 amino acid residues, or at least 45 amino acid residues, or at least 60 amino acid residues, or at least 70 amino acid residues, or at least 90 amino acid residues.

Thus, the present invention provides, in part, isolated polynucleotides that encode a FN1- ALK fusion or a FNl-tmALK polypeptide, nucleotide probes that hybridize to such

polynucleotides, and methods, vectors, and host cells for utilizing such polynucleotides to produce recombinant fusion polypeptides. Unless otherwise indicated, all nucleotide sequences determined by sequencing a DNA molecule herein were determined using an automated DNA sequencer (such as the Model 373 from Applied Biosystems, Inc.), and all amino acid sequences of polypeptides encoded by DNA molecules determined herein were determined using an automated peptide sequencer. As is known in the art for any DNA sequence determined by this automated approach, any nucleotide sequence determined herein may contain some errors. Nucleotide sequences determined by automation are typically at least about 90% identical, and more typically at least about 95% to about 99.9% identical to the actual nucleotide sequence of the sequenced DNA molecule. The actual sequence can be more precisely determined by other approaches including manual DNA sequencing methods well known in the art. As is also known in the art, a single insertion or deletion in a determined nucleotide sequence compared to the actual sequence will cause a frame shift in translation of the nucleotide sequence such that the predicted amino acid sequence encoded by a determined nucleotide sequence will be completely different from the amino acid sequence actually encoded by the sequenced nucleic acid molecule, beginning at the point of such an insertion or deletion. By "nucleotide sequence" of a nucleic acid molecule or polynucleotide is intended, for a DNA molecule or polynucleotide, a sequence of deoxyribonucleotides (abbreviated A, G, C and T), and for an RNA molecule or polynucleotide, the corresponding sequence of ribonucleotides (A, G, C and U), where each thymidine

deoxyribonucleotide (T) in the specified deoxyribonucleotide sequence is replaced by the ribonucleotide uridine (U). For instance, reference to an RNA molecule having the sequence of SEQ ID NO: 4 or set forth using deoxyribonucleotide abbreviations is intended to indicate an

RNA molecule having a sequence in which each deoxyribonucleotide A, G or C of SEQ ID NO: 4 has been replaced by the corresponding ribonucleotide A, G or C, and each deoxyribonucleotide T has been replaced by a ribonucleotide U.

The present invention includes in its scope nucleic acid molecules at least 90%, 95%, 96%>, 97%, 98% or 99% identical to the nucleic acid sequences set forth in SEQ ID NOs: 4, 6, 8, 13, 16, or 19 or nucleotides encoding the amino acid sequences set forth in SEQ ID NOs: 3, 5, 7, 10, 11, 12, 14, 15, 17, 18, 20, or 21 irrespective of whether they encode a polypeptide having ALK kinase activity. This is because even where a particular nucleic acid molecule does not encode a polypeptide having ALK kinase activity, one of skill in the art would still know how to use the nucleic acid molecule, for instance, as a hybridization probe or a polymerase chain reaction (PCR) probe (also called a PCR primer). Uses of the nucleic acid molecules of the present invention that do not encode a polypeptide having ALK kinase activity include, inter alia, (1) isolating the FN1- ALK translocation gene or allelic variants thereof in a cDNA library; (2) in situ hybridization {e.g., "FISH") to metaphase chromosomal spreads to provide precise chromosomal location of the FN1-ALK translocation gene, as described in Verma et al, HUMAN CHROMOSOMES: A MANUAL OF BASIC TECHNIQUES, Pergamon Press, New York (1988); and Northern Blot analysis for detecting FN 1 -ALK mRNA expression in specific tissues.

Within the invention are also nucleic acid molecules having sequences at least 95% identical to a nucleic acid sequence that encodes a FN 1 -ALK fusion polypeptide, an FNl-tmALK fusion polypeptide or a truncated ALK polypeptide lacking an extracellular domain of full-length ALK kinase. In some embodiments, the encoded FN 1 -ALK fusion polypeptide, FNl-tmALK fusion polypeptide, and/or truncated ALK polypeptide has kinase activity. Such activity may be similar, but not necessarily identical, to the activity of full length ALK protein, as measured in a particular biological assay. For example, the kinase activity of ALK can be examined by determining its ability to phosphorylate one or more tyrosine containing peptide substrates, for example, "Src-related peptide" (RRLIEDAEYAARG), which is a substrate for many receptor and nonreceptor tyrosine kinases. Due to the degeneracy of the genetic code, one of ordinary skill in the art will immediately recognize that a large number of the nucleic acid molecules having a sequence at least 90%, 95%, 96%o, 97%o, 98%), or 99% identical to the nucleic acid sequence of the cDNAs described herein, to the nucleic acid sequences set forth in SEQ ID NOs 4, 6, 8, 13, 16, or 19 or to nucleic acid sequences encoding the amino acid sequences set forth in SEQ ID NOs: 3, 5, 7, 10, 11, 12, 14, 15, 17, 18, 20, or 21 will encode a fusion polypeptide having ALK kinase activity. In fact, since degenerate variants of these nucleotide sequences all encode the same polypeptide, this will be clear to the skilled artisan even without performing the above described comparison assay. It will be further recognized in the art that, for such nucleic acid molecules that are not degenerate variants, a reasonable number will also encode a polypeptide having ALK kinase activity. This is because the skilled artisan is fully aware of amino acid substitutions that are either less likely or not likely to significantly effect protein function (e.g., replacing one aliphatic amino acid with a second aliphatic amino acid). For example, guidance concerning how to make phenotypically silent amino acid substitutions is provided in Bowie et al., "Deciphering the Message in Protein Sequences: Tolerance to Amino Acid Substitutions," Science 247: 1306-1310 (1990), which describes two main approaches for studying the tolerance of an amino acid sequence to change. Skilled artisans familiar with such techniques also appreciate which amino acid changes are likely to be permissive at a certain position of the protein. For example, most buried amino acid residues require nonpolar side chains, whereas few features of surface side chains are generally conserved. Other such phenotypically silent substitutions are described in Bowie et al., supra. , and the references cited therein.

Methods for DNA sequencing that are well known and generally available in the art may be used to practice any polynucleotide embodiments of the invention. The methods may employ such enzymes as the Klenow fragment of DNA polymerase I, SEQUENASE® (US Biochemical Corp, Cleveland, Ohio), Taq polymerase (Invitrogen), thermostable T7 polymerase (Amersham, Chicago, 111.), or combinations of recombinant polymerases and proofreading exonucleases such as the ELONGASE Amplification System marketed by Gibco BRL (Gaithersburg, Md.). The process may be automated with machines such as the Hamilton Micro Lab 2200 (Hamilton, Reno, Nev.), Peltier Thermal Cycler (PTC200; MJ Research, Watertown, Mass.) and the ABI 377 DNA sequencers (Applied Biosystems).

Polynucleotide sequences encoding a mutant ALK polypeptide may be extended utilizing a partial nucleotide sequence and employing various methods known in the art to detect upstream sequences such as promoters and regulatory elements. For example, one method that may be employed, "restriction-site" PCR, uses universal primers to retrieve unknown sequence adjacent to a known locus (Sarkar, G., PCR Methods Applic. 2: 318-322 (1993)). In particular, genomic DNA is first amplified in the presence of primer to linker sequence and a primer specific to the known region. Exemplary primers are those described in Example 4 herein. The amplified sequences are then subjected to a second round of PCR with the same linker primer and another specific primer internal to the first one. Products of each round of PCR are transcribed with an appropriate RNA polymerase and sequenced using reverse transcriptase.

Inverse PCR may also be used to amplify or extend sequences using divergent primers based on a known region (Triglia et ah, Nucleic Acids Res. 16: 8186 (1988)). The primers may be designed using OLIGO 4.06 Primer Analysis software (National Biosciences Inc., Plymouth, Minn.), or another appropriate program, to be 22-30 nucleotides in length, to have a GC content of 50% or more, and to anneal to the target sequence at temperatures about 68-72 °C. The method uses several restriction enzymes to generate a suitable fragment in the known region of a gene. The fragment is then circularized by intramolecular ligation and used as a PCR template.

Another method which may be used is capture PCR which involves PCR amplification of DNA fragments adjacent to a known sequence in human and yeast artificial chromosome DNA (Lagerstrom et ah, PCR Methods Applic. 1: 111-119 (1991)). In this method, multiple restriction enzyme digestions and ligations may also be used to place an engineered double-stranded sequence into an unknown portion of the DNA molecule before performing PCR. Another method which may be used to retrieve unknown sequences is that described in Parker et al. , Nucleic Acids Res. 19: 3055-3060 (1991)). Additionally, one may use PCR, nested primers, and PROMOTERFINDER® libraries to walk in genomic DNA (Clontech, Palo Alto, Calif). This process avoids the need to screen libraries and is useful in finding intron/exon junctions.

When screening for full-length cDNAs, libraries that have been size-selected to include larger cDNAs may be used or random-primed libraries, which contain more sequences that contain the 5' regions of genes. A randomly primed library is useful for situations in which an oligo d(T) library does not yield a full-length cDNA. Genomic libraries may be useful for extension of sequence into the 5' and 3' non-transcribed regulatory regions.

Capillary electrophoresis systems, which are commercially available, may be used to analyze the size or confirm the nucleotide sequence of sequencing or PCR products. In particular, capillary sequencing may employ flowable polymers for electrophoretic separation, four different fluorescent dyes (one for each nucleotide) that are laser activated, and detection of the emitted wavelengths by a charge coupled device camera. Output/light intensity may be converted to electrical signal using appropriate software (e.g., GENOTYPER™ and SEQUENCE

NAVIGATOR™, Applied Biosystems) and the entire process from loading of samples to computer analysis and electronic data display may be computer controlled. Capillary

electrophoresis is useful for the sequencing of small pieces of DNA that might be present in limited amounts in a particular sample.

The present invention also provides recombinant vectors that comprise an isolated polynucleotide of the present invention, host cells which are genetically engineered with the recombinant vectors, and the production of recombinant FNl-ALK polypeptides, recombinant FNl-tmALK polypeptides, truncated ALK polypeptides, or fragments thereof by recombinant techniques.

Using the information provided herein, such as the nucleotide sequences set forth in SEQ ID NOs: 4, 6, 8, 13, 16, and 19 a nucleic acid molecule of the present invention encoding a FN1- ALK fusion polypeptide may be obtained using standard cloning and screening procedures, such as those for cloning cDNAs using mRNA as starting material. The fusion gene can also be identified in cDNA libraries in other human cancers in which the translocation between the FN1 gene and the ALK gene occurs, or in which a deletion or alternative translocation results in expression of a truncated ALK kinase lacking the extracellular domain of full-length ALK.

The nucleotide sequences of the translocations between the FN1 gene and the ALK gene encode the FNl-ALKvariantl fusion polypeptide, the FNl-ALKvariant2 fusion polypeptide, the FNl-ALKvariant3 fusion polypeptide, the FNl-ALKvariant4 fusion polypeptide, the FN1- ALKvariant4 fusion polypeptide, and FNl-ALKvariant6 fusion polypeptide. The fusion polynucleotides comprise the portion of the nucleotide sequence of full-length FN1 that encodes the N-terminus of the fibronectin protein with the portion of the nucleotide sequence of full-length ALK that encodes the kinase domain of the ALK protein either without (in the FNl-ALK fusions) or with (in the FNl -tmALK fusions) the transmembrane domain of ALK

As indicated, the present invention provides, in part, the mature forms of the FNl -ALK and FNl-tmALK fusion proteins. According to the signal hypothesis, proteins secreted by mammalian cells have a signal or secretory leader sequence which is cleaved from the mature protein once export of the growing protein chain across the rough endoplasmic reticulum has been initiated. Most mammalian cells and even insect cells cleave secreted proteins with the same specificity. However, in some cases, cleavage of a secreted protein is not entirely uniform, which results in two or more mature forms on the protein. Further, it has long been known that the cleavage specificity of a secreted protein is ultimately determined by the primary structure of the complete protein, that is, it is inherent in the amino acid sequence of the polypeptide. Therefore, the present invention provides, in part, mature FNl-tmALK fusion polypeptides having the amino acid sequences set forth in SEQ ID NOs: 10, 11, or 12, with additional amino acid residues from the signal sequence located N'terminal to SEQ ID NOs: 10, 11, or 12. Therefore, the present invention provides, in part, mature FNl-tmALK fusion polypeptides having the amino acid sequences set forth in SEQ ID NOs: 15, 18, or 21, with additional amino acid residues from the signal sequence located N'terminal to SEQ ID NOs: 15, 18, or 21. In some embodiments, the signal sequence comprises the amino acid sequence set forth in SEQ ID NO:9. The invention also provides nucleotide sequences encoding the amino acid sequences set forth in SEQ ID NOs: 10, 11, 12, 15, 18, or 21 with additional nucleic acid residues located 5' to the 5'-terminal residues of these sequences. In some embodiments, the additional nucleic acid residues encode the amino acid sequence of SEQ ID NO: 9.

As indicated, polynucleotides of the present invention may be in the form of RNA, such as mRNA, or in the form of DNA, including, for instance, cDNA and genomic DNA obtained by cloning or produced synthetically. The DNA may be double-stranded or single-stranded. Single- stranded DNA or RNA may be the coding strand, also known as the sense strand, or it may be the non-coding strand, also referred to as the anti-sense strand.

Isolated polynucleotides are nucleic acid molecules, DNA or RNA, which have been removed from their native environment. For example, recombinant DNA molecules contained in a vector are considered isolated for the purposes of the present invention. Further examples of isolated DNA molecules include recombinant DNA molecules maintained in heterologous host cells or purified (partially or substantially) DNA molecules in solution. Isolated RNA molecules include in vivo or in vitro RNA transcripts of the DNA molecules of the present invention.

Isolated nucleic acid molecules according to the present invention further include such molecules produced synthetically.

Isolated polynucleotides disclosed herein include the nucleic acid molecules having the sequences set forth in SEQ ID NOs: 4, 6, 8, 13, 16, or 19, nucleic acid molecules comprising the coding sequence for the FN1-ALK fusion or the FNl-tmALK proteins that comprise a sequence different from those described above but which, due to the degeneracy of the genetic code, still a mutant ALK polypeptide. The genetic code is well known in the art, thus, it would be routine for one skilled in the art to generate such degenerate variants.

In another embodiment, the invention provides an isolated polynucleotide encoding the FN 1 -ALK fusion or the FNl-tmALK fusion polypeptides comprising the translocation nucleotide sequence contained in the above-described cDNA clones. In some embodiments, such nucleic acid molecule will encode the mature FNl-ALKvariantl fusion polypeptide, the mature FN1- ALKvariant2 fusion polypeptide, the mature FNl-ALKvariant3 fusion polypeptide, the mature FNl-ALKvariant4 fusion polypeptide, the mature FNl-ALKvariant5 fusion polypeptide, or the mature FNl-ALKvariant6 fusion polypeptide. In another embodiment, the invention provides an isolated nucleotide sequence encoding an FN 1 - ALK polypeptide or an FN 1 -tmALK polypeptide comprising the extracellular domain of FN1 and the transmembrane and kinase domains of ALK. In another embodiment, the invention provides an isolated nucleotide sequence encoding a FN1- ALK fusion or FNl-tmALK fusion including the kinase domain of ALK. In one embodiment, the polypeptide comprising the transmembrane and kinase domain of ALK comprises the amino acid sequence set forth in SEQ ID NO: 25. In one embodiment, the polypeptide comprising the kinase domain of ALK comprises the amino acid sequence set forth in SEQ ID NO: 30.

The invention further provides isolated polynucleotides comprising nucleotide sequences having a sequence complementary to one of the mutant ALK polypeptides disclosed herein. Such isolated molecules, particularly DNA molecules, are useful as probes for gene mapping, by in situ hybridization with chromosomes, and for detecting expression of the FN 1 -ALK fusion protein or truncated ALK kinase polypeptide in human tissue, for instance, by Northern blot analysis.

The present invention is further directed to fragments of the isolated nucleic acid molecules described herein. By a fragment of an FN 1 -ALK polynucleotide, an FNl-tmALK polynucleotide or truncated ALK polynucleotide is intended fragments at least about 15 nucleotides, or at least about 20 nucleotides, still more preferably at least about 30 nucleotides, or at least about 40 nucleotides in length, which are useful as diagnostic probes and primers as discussed herein. Of course, larger fragments of about 50-1500 nucleotides in length are also useful according to the present invention, as are fragments corresponding to most, if not all, of the nucleotide sequence of the cDNAs having sequences set forth in SEQ ID NOs: 4, 6, 8, 13, 16, or 19. By "a portion (or fragment) at least 20 nucleotides in length", for example, is meant fragments that include 20 or more contiguous bases from the respective nucleotide sequences from which the fragments are derived. Generation of such DNA fragments is routine to the skilled artisan, and may be accomplished, by way of example, by restriction endonuclease cleavage or shearing by sonication of DNA obtainable from the cDNA clone described herein or synthesized according to the sequence disclosed herein. Alternatively, such fragments can be directly generated synthetically.

Of course, polynucleotides hybridizing to a larger portion of the reference polynucleotide

(e.g. the FNl-ALKvariantl fusion polynucleotide having the sequence set forth in SEQ ID NO: 4, for instance, a portion 50-750 nt in length, or even to the entire length of the reference

polynucleotide, are useful as probes according to the present invention, as are polynucleotides corresponding to most, if not all, of the nucleotide sequence of the cDNAs described herein or the nucleotide sequences set forth in SEQ ID NOs: 4, 6, 8, 13, 16, or 19.

As indicated, such portions or fragments are useful as nucleotide probes for use diagnostically according to conventional DNA hybridization techniques or for use as primers for amplification of a target sequence by the polymerase chain reaction (PCR), as described, for instance, in MOLECULAR CLONING, A LABORATORY MANUAL, 2nd. edition, Sambrook, J., Fritsch, E. F. and Maniatis, T., eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.

(1989) and in Ausubel et al, supra, the entire disclosure of which is hereby incorporated herein by reference. Of course, a polynucleotide which hybridizes only to a poly A sequence (such as the 3' terminal poly(A) tract of an FNl -tmALK sequences (e.g., SEQ ID NOs: 4, 6, or 8), an FN1-ALK sequence (e.g., SEQ ID NOs: 13, 16, or 19) or to a complementary stretch of T (or U) resides, would not be included in a polynucleotide used to hybridize to a portion of a nucleic acid, since such a polynucleotide would hybridize to any nucleic acid molecule containing a poly (A) stretch or the complement thereof (e.g., practically any double-stranded cDNA clone).

As indicated, nucleic acid molecules of the present invention, which encode a mutant ALK polypeptide, may include but are not limited to those encoding the amino acid sequence of the mature polypeptide, by itself; the coding sequence for the mature polypeptide and additional sequences, such as those encoding the leader or secretory sequence, such as a pre-, or pro- or pre- pro-protein sequence; the coding sequence of the mature polypeptide, with or without the aforementioned additional coding sequences, together with additional, non-coding sequences, including for example, but not limited to introns and non-coding 5' and 3' sequences, such as the transcribed, non-translated sequences that play a role in transcription, mRNA processing, including splicing and polyadenylation signals, for example— ribosome binding and stability of mR A; an additional coding sequence which codes for additional amino acids, such as those which provide additional functionalities.

Thus, the sequence encoding the polypeptide may be fused to a marker sequence, such as a sequence encoding a peptide that facilitates purification of the fused polypeptide. In certain embodiments of this aspect of the invention, the marker amino acid sequence is a hexa-histidine peptide, such as the tag provided in a pQE vector (Qiagen, Inc.), among others, many of which are commercially available. As described in Gentz et al., Proc. Natl. Acad. Sci. USA 86: 821-824

(1989), for instance, hexa-histidine provides for convenient purification of the fusion protein. The

"HA" tag is another peptide useful for purification which corresponds to an epitope derived from the influenza hemagglutinin protein, which has been described by Wilson et al., Cell 37: 767

(1984). As discussed below, other such fusion proteins include the FN1-ALK fusion polypeptide or the FNl-tmALK itself fused to Fc at the N- or C-terminus.

Recombinant constructs may be introduced into host cells using well-known techniques such infection, transduction, transfection, transvection, electroporation and transformation. The vector may be, for example, a phage, plasmid, viral or retroviral vector. Retroviral vectors may be replication competent or replication defective. In the latter case, viral propagation generally will occur only in complementing host cells.

The polynucleotides may be joined to a vector containing a selectable marker for propagation in a host. Generally, a plasmid vector is introduced in a precipitate, such as a calcium phosphate precipitate, or in a complex with a charged lipid. If the vector is a virus, it may be packaged in vitro using an appropriate packaging cell line and then transduced into host cells. The invention may be practiced with vectors comprising cis-acting control regions to the

polynucleotide of interest. Appropriate trans-acting factors may be supplied by the host, supplied by a complementing vector or supplied by the vector itself upon introduction into the host. In certain embodiments in this regard, the vectors provide for specific expression, which may be inducible and/or cell type-specific (e.g., those inducible by environmental factors that are easy to manipulate, such as temperature and nutrient additives).

The DNA insert comprising a FN1-ALK polynucleotide, a FNl-tmALK fusion

polynucleotide, or truncated ALK polynucleotide should be operatively linked to an appropriate promoter, such as the phage lambda PL promoter, the E. coli lac, trp and tac promoters, the SV40 early and late promoters and promoters of retroviral LTRs, to name a few. Other suitable promoters are known to the skilled artisan. The expression constructs will further contain sites for transcription initiation, termination and, in the transcribed region, a ribosome binding site for translation. The coding portion of the mature transcripts expressed by the constructs may include a translation initiating at the beginning and a termination codon (UAA, UGA or UAG) appropriately positioned at the end of the polypeptide to be translated.

As indicated, the expression vectors may include at least one selectable marker. Such markers include dihydrofolate reductase or neomycin resistance for eukaryotic cell culture and tetracycline or ampicillin resistance genes for culturing in E. coli and other bacteria.

Representative examples of appropriate hosts include, but are not limited to, bacterial cells, such as E. coli, Streptomyces and Salmonella typhimurium cells; fungal cells, such as yeast cells; insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS and Bowes melanoma cells; and plant cells. Appropriate culture mediums and conditions for the above- described host cells are known in the art.

Non-limiting vectors for use in bacteria include pQE70, pQE60 and pQE-9, available from Qiagen; pBS vectors, Phagescript vectors, Bluescript vectors, pNH8A, pNH16a, pNH18A, pNH46A, available from Stratagene; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 available from Pharmacia. Non-limiting eukaryotic vectors include pWLNEO, pSV2CAT, pOG44, pXTl and pSG available from Stratagene; and pSVK3, pBPV, pMSG and pSVL available from Pharmacia. Other suitable vectors will be readily apparent to the skilled artisan.

Non-limiting bacterial promoters suitable for use in the present invention include the E. coli lacl and lacZ promoters, the T3 and T7 promoters, the gpt promoter, the lambda PR and PL promoters and the trp promoter. Suitable eukaryotic promoters include the CMV immediate early promoter, the HSV thymidine kinase promoter, the early and late SV40 promoters, the promoters of retroviral LTRs, such as those of the Rous sarcoma virus (RSV), and metallothionein promoters, such as the mouse metallothionein-I promoter.

In the yeast, Saccharomyces cerevisiae, a number of vectors containing constitutive or inducible promoters such as alpha factor, alcohol oxidase, and PGH may be used. For reviews, see Ausubel et ah, supra, and Grant et ah, Methods Enzymol. 153: 516-544 (1997).

Introduction of the construct into the host cell can be effected by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection or other methods. Such methods are described in many standard laboratory manuals, such as Davis et al, BASIC METHODS IN MOLECULAR BIOLOGY (1986). The host cell can, of course, be an embryonic stem cell used to make a transgenic animal expressing a protein with ALK kinase activity (e.g., a mutant ALK, an FNl -tmALK fusion, or a FN 1 -ALK fusion). Similarly, xenografts expressing a protein with ALK kinase activity are contemplated within the invention.

Transcription of DNA encoding a FN 1 -ALK fusion polypeptide or a FN 1 -tmALK fusion polypeptide of the present invention by higher eukaryotes may be increased by inserting an enhancer sequence into the vector. Enhancers are cis-acting elements of DNA, usually about from 10 to 300 bp that act to increase transcriptional activity of a promoter in a given host cell-type. Examples of enhancers include the SV40 enhancer, which is located on the late side of the replication origin at basepairs 100 to 270, the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.

For secretion of the translated protein into the lumen of the endoplasmic reticulum, into the periplasmic space or into the extracellular environment, appropriate secretion signals may be incorporated into the expressed polypeptide. The signals may be endogenous to the polypeptide or they may be heterologous signals.

The polypeptide may be expressed in a modified form, such as a fusion protein (e.g., a GST-fusion), and may include not only secretion signals, but also additional heterologous functional regions. For instance, a region of additional amino acids, particularly charged amino acids, may be added to the N-terminus of the polypeptide to improve stability and persistence in the host cell, during purification, or during subsequent handling and storage. Also, peptide moieties may be added to the polypeptide to facilitate purification. Such regions may be removed prior to final preparation of the polypeptide. The addition of peptide moieties to polypeptides to engender secretion or excretion, to improve stability and to facilitate purification, among others, are familiar and routine techniques in the art.

In one non- limiting example, a FN 1 -ALK fusion polypeptide or a FNl-tmALK fusion polypeptide may comprise a heterologous region from an immunoglobulin that is useful to solubilize proteins. For example, EP-A-0 464 533 (Canadian counterpart 2045869) discloses fusion proteins comprising various portions of constant region of immunoglobin molecules together with another human protein or part thereof. In many cases, the Fc part in a fusion protein is thoroughly advantageous for use in therapy and diagnosis and thus results, for example, in improved pharmacokinetic properties (EP-A 0232 262). On the other hand, for some uses it would be desirable to be able to delete the Fc part after the fusion protein has been expressed, detected and purified in the advantageous manner described. This is the case when Fc portion proves to be a hindrance to use in therapy and diagnosis, for example when the fusion protein is to be used as antigen for immunizations. In drug discovery, for example, human proteins, such as, hIL5- has been fused with Fc portions for the purpose of high-throughput screening assays to identify antagonists of hIL-5. See Bennett et al., Journal of Molecular Recognition 8: 52-58 (1995) and Johanson et al, The Journal of Biological Chemistry 270(16): 9459-9471 (1995).

FN1-ALK polypeptides can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin

chromatography. In some embodiments, high performance liquid chromatography ("HPLC") is employed for purification. Polypeptides of the present invention include naturally purified products, products of chemical synthetic procedures, and products produced by recombinant techniques from a prokaryotic or eukaryotic host, including, for example, bacterial, yeast, higher plant, insect and mammalian cells. Depending upon the host employed in a recombinant production procedure, the polypeptides of the present invention may be glycosylated or may be non-glycosylated. In addition, polypeptides disclosed herein may also include an initial modified methionine residue, in some cases as a result of host-mediated processes.

Accordingly, in one embodiment, the invention provides a method for producing a recombinant FN1-ALK fusion or a FNl-tmALK fusion polypeptide by culturing a recombinant host cell (as described above) under conditions suitable for the expression of the fusion polypeptide and recovering the polypeptide. Culture conditions suitable for the growth of host cells and the expression of recombinant polypeptides from such cells are well known to those of skill in the art. See, e.g., Ausubel et al, supra, particularly Volume 2, Chapter 16.

In further aspects, the invention provides an isolated polypeptide comprising an amino acid sequence at least 95% identical to a sequence selected from the group consisting of: (a) an amino acid sequence comprising the amino acid sequence of SEQ ID NOs: 3, 5, 7, 10, 11, 12, 14, 15, 17, 18, 20, or 21; (b) an amino acid sequence comprising amino acid residues 1-1085 of SEQ ID NO: 26 and a C-terminal portion comprising amino acid residues 1039-1392 of SEQ ID NO: 1; (c) an amino acid sequence encoding a polypeptide comprising at least six contiguous amino acids encompassing the fusion junction (residues 1999-1204 of SEQ ID NO: 3, residues 1263-1268 of SEQ ID NO: 5, or residues 1114-1119 of SEQ ID NO: 7) of an FNl-tmALK fusion polypeptide; (d) an amino acid sequence encoding a polypeptide comprising at least six contiguous amino acids encompassing the fusion junction (residues 1999-1204 of SEQ ID NO: 14, residues 1263-1268 of SEQ ID NO: 17, or residues 1 1 14-1 1 19 of SEQ ID NO: 20) of an FNl -ALK fusion polypeptide;

(e) an amino acid sequence comprising amino acid residues 1039-1392 of SEQ ID NO: 1 , wherein said polypeptide does not comprise amino acid residues 19-1038 of SEQ ID NO: 1 ; and (f) an amino acid sequence comprising amino acid sequence of SEQ ID NO: 29, wherein said polypeptide does not comprise amino acid residues 19-1038 of SEQ ID NO: 1.

As used herein, by "polypeptide" (or "amino acid sequence" or "protein") refers to a polymer formed from the linking, in a defined order, of preferably, a-amino acids, D-, L-amino acids, and combinations thereof. The link between one amino acid residue and the next is referred to as an amide bond or a peptide bond. Non-limiting examples of polypeptides include refers to an oligopeptide, peptide, polypeptide, or protein sequence, and fragments or portions thereof, and to naturally occurring or synthetic molecules. Polypeptides also include derivatized molecules such as glycoproteins and lipoproteins as well as lower molecular weight polypeptides. "Amino acid sequence" and like terms, such as "polypeptide" or "protein", are not meant to limit the indicated amino acid sequence to the complete, native amino acid sequence associated with the recited protein molecule.

It will be recognized in the art that some amino acid sequences of a polypeptide disclosed herein (e.g., FNl -ALKvariantl polypeptide) can be varied without significant effect of the structure or function of the mutant protein. If such differences in sequence are contemplated, it should be remembered that there will be critical areas on the protein which determine activity (e.g. the kinase domain of ALK). In general, it is possible to replace residues that form the tertiary structure, provided that residues performing a similar function are used. In other instances, the type of residue may be completely unimportant if the alteration occurs at a non-critical region of the protein.

Thus, in some embodiments, the invention further includes a variant of a FNl-tmALK or a FNl -ALK fusion polypeptide that shows substantial ALK kinase activity and/or that includes regions of FNl and ALK proteins. In some embodiments, a FNl-tmALK variant disclosed herein contains conservative substitutions as compared to FNl-ALKvariantl , FNl-ALKvariant3, or FN1- ALKvariant5. In some embodiments, a FN1-ALK variant disclosed herein contains conservative substitutions as compared to FNl-ALKvariant 2, FNl-ALKvariant4, or FNl-ALKvariant6. Some non-limiting conservative substitutions include the exchange, one for another, among the aliphatic amino acids Ala, Val, Leu and He; exchange of the hydroxyl residues Ser and Thr; exchange of the acidic residues Asp and Glu; exchange of the amide residues Asn and Gin; exchange of the basic residues Lys and Arg; and exchange of the aromatic residues Phe and Tyr. Further examples of conservative amino acid substitutions known to those skilled in the art are: Aromatic:

phenylalanine tryptophan tyrosine (e.g., a tryptophan residue is replaced with a phenylalanine); Hydrophobic: leucine isoleucine valine; Polar: glutamine asparagines; Basic: arginine lysine histidine; Acidic: aspartic acid glutamic acid; Small: alanine serine threonine methionine glycine. As indicated in detail above, further guidance concerning which amino acid changes are likely to be phenotypically silent (i.e., are not likely to have a significant deleterious effect on a function) can be found in Bowie et al., Science 247, supra.

In some embodiments, a variant may have "nonconservative" changes, e.g., replacement of a glycine with a tryptophan. Similar variants may also include amino acid deletions or insertions, or both. Guidance in determining which amino acid residues may be substituted, inserted, or deleted without abolishing biological or immunological activity may be found using computer programs well known in the art, for example, DNASTAR software.

The FN1-ALK fusion polypeptides, FNl-tmALK fusion polypeptides, fragments (also called portions) thereof, and variants thereof of the present invention may be provided in an isolated or purified form. A recombinantly produced version of a FN1-ALK or FNl-tmALK fusion polypeptide disclosed herein can be substantially purified by the one-step method described in Smith and Johnson, Gene 67: 31 -40 (1988).

The polypeptides of the present invention include the fusion polypeptides having the sequences set forth in SEQ ID NOs: 3, 5, 7, 10, 11, 12, 14, 15, 17, 18, 20, or 21 (whether or not including a leader sequence), an amino acid sequence encoding a polypeptide comprising at least six contiguous amino acids encompassing the fusion junction of a FN1-ALK or a FNl-tmALK fusion polypeptide, as well as polypeptides that have at least 90% similarity, more preferably at least 95% similarity, and still more preferably at least 96%>, 97%, 98% or 99% similarity to those described above.

An FN1-ALK or an FNl-tmALK fusion polypeptide of the present invention could be used as a molecular weight marker on SDS-PAGE gels or on molecular sieve gel filtration columns, for example, using methods well known to those of skill in the art.

As further described in detail below, the polypeptides of the present invention can also be used to generate fusion polypeptide specific reagents, such as polyclonal and monoclonal antibodies, which are useful in assays for detecting ALK polypeptide expression and/or ALK kinase activity as described below or as agonists and antagonists capable of enhancing or inhibiting ALK protein function/activity. Further, such polypeptides can be used in the yeast two- hybrid system to "capture" FN 1 -ALK or an FNl-tmALK fusion polypeptide-binding proteins, which are also candidate agonist and antagonist according to the present invention. The yeast two hybrid system is described in Fields and Song, Nature 340: 245-246 (1989).

In another aspect, the invention provides a peptide or polypeptide comprising an epitope- bearing portion of a polypeptide disclosed herein, such as an epitope comprising the fusion junction of a FN 1 -ALK fusion or an FNl-tmALK polypeptide variant An "epitope" refers to either an immunogenic epitope (i.e., capable of eliciting an immune response) or an antigenic epitope (i.e., the region of a protein molecule to which an antibody can specifically bind. The number of immunogenic epitopes of a protein generally is less than the number of antigenic epitopes. See, for instance, Geysen et al., Proc. Natl. Acad. Sci. USA 57:3998-4002 (1983). The production of FN 1 -ALK fusion polypeptide-specific or FNl-tmALK fusion polypeptide-specific antibodies is described in further detail below.

The antibodies that specifically bind to an epitope-bearing peptides or polypeptides are useful to detect a mimicked protein, and antibodies to different peptides may be used for tracking the fate of various regions of a protein precursor which undergoes post-translational processing. The peptides and anti-peptide antibodies may be used in a variety of qualitative or quantitative assays for the mimicked protein, for instance in competition assays since it has been shown that even short peptides (e.g., about 9 amino acids) can bind and displace the larger peptides in immunoprecipitation assays. See, for instance, Wilson et al., Cell 37: 767-778 (1984) at 777. The anti-peptide antibodies also are useful for purification of the mimicked protein, for instance, by adsorption chromatography using methods well known in the art. Immunological assay formats are described in further detail below.

Recombinant mutant ALK kinase polypeptides are also within the scope of the present invention, and may be producing using fusion polynucleotides disclosed herein, as described above. For example, in some embodiments, the invention provides a method for producing a recombinant FNl-tmALK fusion polypeptide by culturing a recombinant host cell (as described above) under conditions suitable for the expression of the fusion polypeptide and recovering the polypeptide. Culture conditions suitable for the growth of host cells and the expression of recombinant polypeptides from such cells are well known to those of skill in the art. In further aspects, the invention provides a reagent (e.g., an isolated reagent) that specifically binds to the polynucleotides or polypeptides disclosed herein.

In a further aspect, the invention provides a reagent (e.g., a binding agent) that specifically binds to a polypeptide or polynucleotide of the invention (e.g., a FNl-ALKvariantl fusion). In some embodiments, the reagent specifically binds to a portion of ALK that is present in the FNl - ALK fusions and the FNl-tmALK fusions described herein (e.g., the antibody specifically binds to the kinase domain of ALK). In some embodiments, the reagent specifically binds to a fusion junction between a FN1 portion and an ALK portion of a FNl-ALK fusion or a FNl-tmALK fusion. In some embodiments, the FNl-tmALK fusion is a FNl-ALKvariantl fusion, a FN1- ALKvariant3 fusion, or a FNl-ALKvariant5 fusion. In some embodiments, the FNl-ALK fusion is a FNl-ALKvariant2 fusion, a FNl-ALKvariant4 fusion, or a FNl-ALKvariant6 fusion. In some embodiments, where the reagent specifically binds to a polynucleotide, the reagent does not specifically bind to a polynucleotide having a nucleotide sequence consisting of only adenine (A) residues or of only thymine (T) residues. The reagent itself may be another polynucleotide (e.g., a nucleic acid probe).

In some embodiments, the isolated polynucleotide or the reagent may further comprise a detectable label (e.g., a fluorescent label or an infrared label). By "detectable label" with respect to a polypeptide, polynucleotide, or reagent (e.g., binding agent or antibody) disclosed herein means a chemical, biological, or other modification of or to the polypeptide, polynucleotide, or binding agent, including but not limited to fluorescence, infrared, mass, residue, dye, radioisotope, label, or tag modifications, etc., by which the presence of the molecule of interest may be detected. The detectable label may be attached to the polypeptide, polynucleotide, or binding agent by a covalent (e.g., peptide bond or phosphodiester bond) or non-covalent chemical bond (e.g., an ionic bond).

The invention provides binding agents, such as antibodies or AQUA peptides, or binding fractions thereof, that specifically bind to the FNl-tmALK fusion polypeptides disclosed herein (e.g., a FNl-ALKvariantl fusion, a FNl-ALKvariant3 fusion, or a FNl-ALKvariant5 fusion) or specifically bind to a FNl-ALK fusion polypeptide disclosed herein (e.g., a FNl -ALKvariant2 fusion, a FNl-ALKvariant4 fusion, or a FNl -ALKvariant6 fusion). By "specifically binding" or "specifically binds" means that a reagent or binding agent (e.g. , a nucleic acid probe, an antibody, or AQUA peptide) interacts with its target molecule (e.g., a FNl-tmALK fusion polypeptide or polynucleotide, a FNl-ALK fusion polypeptide or polynucleotide, a truncated ALK fusion polypeptide or polynucleotide, or a full-length ALK polypeptide or polynucleotide), where the interaction is dependent upon the presence of a particular structure (e.g., the antigenic determinant or epitope on the polypeptide or the nucleotide sequence of the polynucleotide); in other words, the reagent is recognizing and binding to a specific polypeptide or polynucleotide structure rather than to all polypeptides or polynucleotides in general. By "binding fragment thereof means a fragment or portion of a reagent that specifically binds the target molecule (e.g., an Fab fragment of an antibody).

A reagent that specifically binds to the target molecule may be referred to as a target- specific reagent or an anti-target reagent. For example, an antibody that specifically binds to a FNl-ALKvariantl polypeptide may be referred to as a FNl-ALKvariantl -specific antibody or an anti-FNl-ALKvariantl antibody. Likewise, an antibody that specifically binds to the full length ALK polypeptide may be referred to as an ALK-specific antibody or an anti-ALK antibody. Similarly, a nucleic acid probe that specifically binds to a FNl-ALKvariantl polynucleotide may be referred to as a FNl-ALKvariantl -specific nucleic acid probe or an anti- FNl-ALKvariantl nucleic acid probe.

In some embodiments, where the target molecule is a polypeptide, a reagent (e.g., an antibody) that specifically binds its target molecule has a binding affinity (KD) for its target molecule (e.g., a full length ALK polypeptide, a FNl-tmALK fusion polypeptide, or an FN1-ALK fusion polypeptide) of lx 10^"6 M or less. In some embodiments, a reagent that specifically binds

-7 -8 to its target molecule binds to its target molecule with a K_D of 1 xlO^" M or less, or a K_D of 1 xlO^" M or less, or a K_D of 1 x 10^"9 M or less, or a K_D of 1 x 10^"10 M or less, of a K_D of 1 x 10^"11 M or

-12

less, of a KD of 1 x 10^" M or less. In certain embodiments, a reagent that specifically binds to its target molecule binds to its target molecule with a K_D of 1 pM to 500 pM, or between 500 pM to 1 μΜ, or between 1 μΜ to 100 nM, or between 100 mM to 10 nM.

In some embodiments, where the target molecule is a polynucleotide, a reagent that specifically binds its target molecule is a reagent that hybridizes under stringent conditions to its target polynucleotide. The term "stringent conditions" with respect to nucleotide sequence or nucleotide probe hybridization conditions is the "stringency" that occurs within a range from about T_m minus 5 °C (i.e., 5 °C below the melting temperature (T_m) of the reagent or nucleic acid probe) to about 20 °C to 25 °C below T_m. Typical stringent conditions are: overnight incubation at 42 °C in a solution comprising: 50% formamide, 5 X.SSC (750 mM NaCl, 75 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5X Denhardt's solution, 10%> dextran sulfate, and 20 micrograms/ml denatured, sheared salmon sperm DNA, followed by washing the filters in 0.1 X SSC at about 65 °C. As will be understood by those of skill in the art, the stringency of hybridization may be altered in order to identify or detect identical or related polynucleotide sequences. By a "reagent (e.g., a polynucleotide or nucleotide probe) that hybridizes under stringent conditions to a target polynucleotide (e.g., a FNl-ALKvariantl fusion polynucleotide)" is intended that the reagent (e.g., the polynucleotide or nucleotide probe (e.g., DNA, RNA, or a DNA-RNA hybrid)) hybridizes along the entire length of the reference polynucleotide or hybridizes to a portion of the reference polynucleotide that is at least about 15 nucleotides (nt), or to at least about 20 nt, or to at least about 30 nt, or to about 30-70 nt of the reference

polynucleotide. These nucleotide probes are useful as diagnostic probes and primers (e.g., for PCR) as discussed herein.

Non-limiting examples of a target molecule to which a reagent specifically binds includes the full length ALK polypeptide (or polynucleotide encoding the same), the kinase domain of an ALK protein (or polynucleotide encoding the same), the transmembrane domain of ALK polypeptide (or a polynucleotide encoding the same), the FNl-ALKvariantl fusion polypeptide (or an FNl-ALKvariantl polynucleotide), the FNl-ALKvariant2 fusion polypeptide (or an FNl- ALKvariantl polynucleotide), the FNl-ALKvariant3 fusion polypeptide (or an FNl-ALKvariant3 polynucleotide), the FNl-ALKvariant4 fusion polypeptide (or an FNl-ALKvariant4

polynucleotide), the FNl-ALKvariant5 fusion polypeptide (or an FNl-ALKvariant5

polynucleotide), the FNl-ALKvariant6 fusion polypeptide (or an FNl-ALKvariant6

polynucleotide), and fragments thereof, particularly those fragments that include the junction between the FNl portion and the ALK portion of an FNl-ALK fusion or an FNl-tmALK fusion as disclosed herein.

The reagent that specifically binds to its target, including those useful in the practice of the disclosed methods, include, among others, FNl-tmALK fusion polypeptide or FNl-ALK fusion polypeptide-specific antibodies and AQUA peptides (heavy-isotope labeled peptides)

corresponding to, and suitable for detection and quantification of, FNl-tmALK fusion or FNl- ALK fusion polypeptide expression in a biological sample. Thus, a "FNl-ALK fusion polypeptide-specific reagent" is any reagent, biological or chemical, capable of specifically binding to, detecting and/or quantifying the presence/level of expressed FNl-ALK fusion polypeptide in a biological sample, and an a "FNl -tmALK fusion polypeptide-specific reagent" is any reagent, biological or chemical, capable of specifically binding to, detecting and/or quantifying the presence/level of expressed FNl-tmALK fusion polypeptide in a biological sample. The terms include, but are not limited to, the antibodies and AQUA peptide reagents discussed below, and equivalent binding agents are within the scope of the present invention.

In some embodiments, the reagent that specifically binds to a polypeptide disclosed herein (e.g., an FNl-tmALK or FN1-ALK fusion) is an antibody (e.g., a FNl-ALKvariantl fusion polypeptide-specific antibody). In some embodiments, the antibody is an isolated antibody or antibodies that specifically bind(s) to a FNl-tmALK fusion polypeptide (e.g., FNl-ALKvariantl, FNl-tmALKvariant3 or FNl-ALKvariant5) or an FN1-ALK fusion polypeptide (e.g., FNl- ALKvariantl, FNl-ALKvariant4 or FNl-ALKvariant6) but does not substantially bind either full length FNl or full-length ALK proteins. In some embodiments, the reagent that specifically binds to an FNl -ALK fusion polypeptide or an FNl-tmALK fusion polypeptide is an antibody that also specifically binds to full-length ALK polypeptide. In some embodiments, the ALK protein- specific antibody specifically binds to the transmembrane and/or kinase domain of full-length ALK polypeptide. In some embodiments, the binding agent that specifically binds to an FN1- ALK fusion polypeptide or an FNl-tmALK fusion polypeptide is an antibody is an antibody that specifically binds to full-length FNl polypeptide. In some embodiments, the FNl protein-specific antibody specifically binds to the extracellular domain of full-length FNl polypeptide.

The antibodies that specifically binds to the FNl -ALK fusion and/or FNl-tmALK fusion polypeptides may also bind to highly homologous and equivalent epitopic peptide sequences in other mammalian species, for example murine or rabbit, and vice versa. Antibodies useful in practicing the methods disclosed herein include (a) monoclonal antibodies, (b) purified polyclonal antibodies that specifically bind to the target polypeptide (e.g., the fusion junction of the fusion polypeptide, (c) antibodies as described in (a)-(b) above that specifically bind equivalent and highly homologous epitopes or phosphorylation sites in other non-human species (e.g., mouse, rat), and (d) fragments of (a)-(c) above that specifically bind to the antigen (or more preferably the epitope) bound by the exemplary antibodies disclosed herein.

The term "antibody" or "antibodies" refers to all types of immunoglobulins, including IgG, IgM, IgA, IgD, and IgE, including binding fragments thereof (i.e., fragments of an antibody that are capable of specifically binding to the antibody's target molecule, such as F_ab, and F(ab')₂ fragments), as well as recombinant, humanized, polyclonal, and monoclonal antibodies and/or binding fragments thereof. Antibodies can be derived from any species of animal, such as from a mammal. Non-limiting exemplary natural antibodies include antibodies derived from human, chicken, goats, and rodents (e.g., rats, mice, hamsters and rabbits), including transgenic rodents genetically engineered to produce human antibodies (see, e.g., Lonberg et al, W093/12227; U.S. Pat. No. 5,545,806; and Kucherlapati, et al, WO91/10741; U.S. Pat. No. 6,150,584, which are herein incorporated by reference in their entirety). Antibodies may be also be chimeric antibodies. See, e.g., M. Walker et al., Molec. Immunol. 26: 403-11 (1989); Morrision et al, Proc. Nat'l Acad. Sci. 81: 6851 (1984); Neuberger et al, Nature 312: 604 (1984)). The antibodies may be recombinant monoclonal antibodies produced according to the methods disclosed in U.S. Pat. No. 4,474,893 (Reading) or U.S. Pat. No. 4,816,567 (Cabilly et al.) The antibodies may also be chemically constructed specific antibodies made according to the method disclosed in U.S. Pat. No. 4,676,980 (Segel et al).

Natural antibodies are the antibodies produced by a host animal, however the invention contemplates also genetically altered antibodies wherein the amino acid sequence has been varied from that of a native antibody. Because of the relevance of recombinant DNA techniques to this application, one need not be confined to the sequences of amino acids found in natural antibodies; antibodies can be redesigned to obtain desired characteristics. The possible variations are many and range from the changing of just one or a few amino acids to the complete redesign of, for example, the variable or constant region. Changes in the constant region will, in general, be made in order to improve or alter characteristics, such as complement fixation, interaction with membranes and other effector functions. Changes in the variable region will be made in order to improve the antigen binding characteristics. The term "humanized antibody", as used herein, refers to antibody molecules in which amino acids have been replaced in the non-antigen binding regions in order to more closely resemble a human antibody, while still retaining the original binding ability. Other antibodies specifically contemplated are oligoclonal antibodies. As used herein, the phrase "oligoclonal antibodies" refers to a predetermined mixture of distinct monoclonal antibodies. See, e.g., PCT publication WO 95/20401; U.S. Patent Nos. 5,789,208 and 6,335,163. In one embodiment, oligoclonal antibodies consisting of a predetermined mixture of antibodies against one or more epitopes are generated in a single cell. In other embodiments, oligoclonal antibodies comprise a plurality of heavy chains capable of pairing with a common light chain to generate antibodies with multiple specificities (e.g., PCT publication WO

04/009618). Oligoclonal antibodies are particularly useful when it is desired to target multiple epitopes on a single target molecule. In view of the assays and epitopes disclosed herein, those skilled in the art can generate or select antibodies or mixtures of antibodies that are applicable for an intended purpose and desired need.

Recombinant antibodies are also included in the present invention. These recombinant antibodies have the same amino acid sequence as the natural antibodies or have altered amino acid sequences of the natural antibodies. They can be made in any expression systems including both prokaryotic and eukaryotic expression systems or using phage display methods (see, e.g., Dower et al, W091/17271 and McCafferty et al, WO92/01047; U.S. Pat. No. 5,969,108, which are herein incorporated by reference in their entirety). Antibodies can be engineered in numerous ways. They can be made as single-chain antibodies (including small modular

immunopharmaceuticals or SMIPs™), Fab and F(ab')₂ fragments, etc. Antibodies can be humanized, chimerized, deimmunized, or fully human. Numerous publications set forth the many types of antibodies and the methods of engineering such antibodies. For example, see U.S. Patent Nos. 6,355,245; 6,180,370; 5,693,762; 6,407,213; 6,548,640; 5,565,332; 5,225,539; 6,103,889; and 5,260,203. The genetically altered antibodies may be functionally equivalent to the above- mentioned natural antibodies. In certain embodiments, modified antibodies provide improved stability or/and therapeutic efficacy.

Non-limiting examples of modified antibodies include those with conservative

substitutions of amino acid residues, and one or more deletions or additions of amino acids that do not significantly deleteriously alter the antigen binding utility. Substitutions can range from changing or modifying one or more amino acid residues to complete redesign of a region as long as the therapeutic utility is maintained. Antibodies can be modified post-translationally {e.g., acetylation, and/or phosphorylation) or can be modified synthetically {e.g., the attachment of a labeling group). Antibodies with engineered or variant constant or Fc regions can be useful in modulating effector functions, such as, for example, antigen-dependent cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). Such antibodies with engineered or variant constant or Fc regions may be useful in instances where a parent singling protein is expressed in normal tissue; variant antibodies without effector function in these instances may elicit the desired therapeutic response while not damaging normal tissue. Accordingly, certain aspects and methods of the present disclosure relate to antibodies with altered effector functions that comprise one or more amino acid substitutions, insertions, and/or deletions. The term "biologically active" refers to a protein having structural, regulatory, or biochemical functions of a naturally occurring molecule. Likewise, "immunologically active" refers to the capability of the natural, recombinant, or synthetic full-length ALK protein or ALK fusion polypeptide (e.g., a FN1-ALK fusion polypeptide or an FNl-tmALK fusion polypeptide disclosed herein), or any oligopeptide thereof, to induce a specific immune response in appropriate animals or cells and to bind with specific antibodies.

Also within the invention are antibody molecules with fewer than 4 chains, including single chain antibodies, Camelid antibodies and the like and components of an antibody, including a heavy chain or a light chain. In some embodiments an immunoglobulin chain may comprise in order from 5' to 3', a variable region and a constant region. The variable region may comprise three complementarity determining regions (CDRs), with interspersed framework (FR) regions for a structure FRl, CDRl, FR2, CDR2, FR3, CDR3 and FR4. Also within the invention are heavy or light chain variable regions, framework regions and CDRs. An antibody may comprise a heavy chain constant region that comprises some or all of a CHI region, hinge, CH2 and CH3 region.

One non-limiting epitopic site of a fusion polypeptide-specific antibody is a peptide fragment consisting essentially of about 11 to 17 amino acids of a fusion polypeptide sequence, which fragment encompasses the fusion junction between the FNl portion of the molecule and the ALK portion of the molecule. It will be appreciated that antibodies that specifically binding shorter or longer peptides/epitopes encompassing the fusion junction of an FNl -ALK fusion polypeptide or an FNl-tmALK fusion polypeptide are within the scope of the present invention.

The invention is not limited to use of antibodies, but includes equivalent molecules, such as protein binding domains or nucleic acid aptamers, which bind, in a fusion-protein or truncated- protein specific manner, to essentially the same epitope to which a FNl -ALK fusion polypeptide- specific antibody, FNl-tmALK fusion polypeptide-specific antibody, a full length FNl -specific antibody, a full length ALK-specific antibody, or truncated ALK-specific antibody useful in the methods disclosed herein binds. See, e.g., Neuberger et ah, Nature 312: 604 (1984). Such equivalent non-antibody reagents may be suitably employed in the methods further described below.

Polyclonal antibodies useful in practicing the methods disclosed herein may be produced according to standard techniques by immunizing a suitable animal (e.g., rabbit, goat, etc.) with an antigen encompassing a desired fusion-protein specific epitope (e.g. the fusion junction between FNl and ALK in an FN1-ALK or FNl-tmALK fusion polypeptide), collecting immune serum from the animal, and separating the polyclonal antibodies from the immune serum, and purifying polyclonal antibodies having the desired specificity, in accordance with known procedures. The antigen may be a synthetic peptide antigen comprising the desired epitopic sequence, selected and constructed in accordance with well-known techniques. See, e.g., ANTIBODIES: A LABORATORY MANUAL, Chapter 5, p. 75-76, Harlow & Lane Eds., Cold Spring Harbor Laboratory (1988);

Czernik, Methods In Enzymology, 201: 264-283 (1991); Merrifield, J. Am. Chem. Soc. 85: 21-49 (1962)). Polyclonal antibodies produced as described herein may be screened and isolated as further described below.

Monoclonal antibodies may also be beneficially employed in the methods disclosed herein, and may be produced in hybridoma cell lines according to the well-known technique of Kohler and Milstein. Nature 265: 495-97 (1975); Kohler and Milstein, Eur. J. Immunol. 6: 511 (1976); see also, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Ausubel et al. Eds. (Wiley and Sins, New York, NY 1989 and yearly updates up to and including 2010). Monoclonal antibodies so produced are highly specific, and improve the selectivity and specificity of assay methods provided by the invention. For example, a solution containing the appropriate antigen {e.g. a synthetic peptide comprising the fusion junction of FNl-ALK or FNl-tmALK fusion polypeptide) may be injected into a mouse and, after a sufficient time (in keeping with conventional

techniques), the mouse sacrificed and spleen cells obtained. The spleen cells are then immortalized by fusing them with myeloma cells, typically in the presence of polyethylene glycol, to produce hybridoma cells. Rabbit fusion hybridomas, for example, may be produced as described in U.S Patent No. 5,675,063. The hybridoma cells are then grown in a suitable selection media, such as hypoxanthine-aminopterin-thymidine (HAT), and the supernatant screened for monoclonal antibodies having the desired specificity, as described below. The secreted antibody may be recovered from tissue culture supernatant by conventional methods such as precipitation, ion exchange or affinity chromatography, or the like.

Monoclonal Fab fragments may also be produced in Escherichia coli by recombinant techniques known to those skilled in the art. See, e.g., W. Huse, Science 246: 1275-81 (1989); Mullinax et al, Proc. Nat'lAcad. Sci. 87: 8095 (1990). If monoclonal antibodies of one isotype are desired for a particular application, particular isotypes can be prepared directly, by selecting from the initial fusion, or prepared secondarily, from a parental hybridoma secreting a monoclonal antibody of different isotype by using the sib selection technique to isolate class-switch variants (Steplewski, et al., Proc. Nat'l. Acad. Sci., 82: 8653 (1985); Spira et al., J. Immunol. Methods, 74: 307 (1984)). The antigen combining site of the monoclonal antibody can be cloned by PCR and single-chain antibodies produced as phage-displayed recombinant antibodies or soluble antibodies in E. coli (see, e.g., ANTIBODY ENGINEERING PROTOCOLS, 1995, Humana Press, Sudhir Paul editor.)

Further still, U.S. Pat. No. 5,194,392, Geysen (1990) describes a general method of detecting or determining the sequence of monomers (amino acids or other compounds) which is a topological equivalent of the epitope (i.e., a "mimotope") which is complementary to a particular paratope (antigen binding site) of an antibody of interest. More generally, this method involves detecting or determining a sequence of monomers which is a topographical equivalent of a ligand which is complementary to the ligand binding site of a particular receptor of interest. Similarly, U.S. Pat. No. 5,480,971, Houghten et al. (1996) discloses linear Ci-C-alkyl peralkylated oligopeptides and sets and libraries of such peptides, as well as methods for using such

oligopeptide sets and libraries for determining the sequence of a peralkylated oligopeptide that preferentially binds to an acceptor molecule of interest. Thus, non-peptide analogs of the epitope- bearing peptides also can be made routinely by these methods.

Antibodies useful in the methods disclosed herein, whether polyclonal or monoclonal, may be screened for epitope and fusion protein specificity according to standard techniques. See, e.g., Czernik et al, Methods in Enzymology, 201: 264-283 (1991). For example, the antibodies may be screened against a peptide library by ELISA to ensure specificity for both the desired antigen and, if desired, for reactivity only with the full-length ALK protein, a particular ALK fusion

polypeptide (e.g., an FNl-ALKvariantl polypeptide), or fragments thereof. The antibodies may also be tested by Western blotting against cell preparations containing target protein to confirm reactivity with the only the desired target and to ensure no appreciable binding to other proteins. The production, screening, and use of fusion protein-specific antibodies is known to those of skill in the art, and has been described. See, e.g., U.S. Patent Publication No. 20050214301.

Full-length ALK protein-specific and ALK fusion polypeptide-specific antibodies useful in the methods disclosed herein may exhibit some limited cross-reactivity with similar epitopes in other proteins or polypeptides, such as similar fusion polypeptides. This is not unexpected as most antibodies exhibit some degree of cross-reactivity, and anti-peptide antibodies will often cross- react with epitopes having high homology or identity to the immunizing peptide. See, e.g., Czernik, supra. Cross-reactivity with other fusion proteins is readily characterized by Western blotting alongside markers of known molecular weight. Amino acid sequences of cross-reacting proteins may be examined to identify sites highly homologous or identical to full length ALK protein sequence or the ALK fusion polypeptide (e.g., FNl-ALKvariantl polypeptide) sequence to which the antibody binds. Undesirable cross-reactivity can be removed by negative selection using antibody purification on peptide columns.

ALK-specific antibodies and ALK fusion polypeptide-specific antibodies that are useful in practicing the methods disclosed herein are ideally specific for human fusion polypeptide, but are not limited only to binding the human species, per se. The invention includes the production and use of antibodies that also bind conserved and highly homologous or identical epitopes in other mammalian species (e.g., mouse, rat, monkey). Highly homologous or identical sequences in other species can readily be identified by standard sequence comparisons, such as using BLAST, with the human ALK protein sequence (SEQ ID NO: 1), the human FNl-tmALK fusion polypeptide sequences disclosed herein (SEQ ID NOs: 3, 5, 7, 10, 11, and 12), and the human FN1-ALK fusion polypeptide sequences disclosed herein SEQ ID NOs: 14, 15, 17, 18, 20, and 21).

Antibodies employed in the methods disclosed herein may be further characterized by, and validated for, use in a particular assay format, for example FC, IHC, and/or ICC. The use of FN1- ALK fusion and/or FNl-tmALK fusion polypeptide-specific antibodies in such methods is further described herein. The antibodies described herein, used alone or in the below-described assays, may also be advantageously conjugated to fluorescent dyes (e.g. Alexa488, phycoerythrin), or labels such as quantum dots, for use in multi-parametric analyses along with other signal transduction (phospho-AKT, phospho-Erk 1/2) and/or cell marker (cytokeratin) antibodies, as further described below.

In practicing the methods disclosed herein, the expression and/or activity of an FNl -ALK or FNl-tmALK polypeptide and/or of full-length ALK in a given biological sample may also be advantageously examined using antibodies specific for (i.e., that specifically bind to) full length ALK protein or antibodies specific for ALK fusion polypeptides. For example, ALK-specific antibodies (i.e., antibodies that specifically bind full-length ALK) are commercially available (see Cell Signaling Technology, Inc., Danvers, MA, Catalog Nos. 3333 and 3791; Abeam, 2010 Catalogu No.,abl7127, ab59286, and Sigma-Aldrich, 2010 Catalog No. HPA010694, for example). In some embodiments, ALK-specific antibodies used in the methods disclosed herein specifically bind the transmembrane domain of ALK and, thus, will detect full-length ALK and the FNl-tmALK fusion polypeptide of the invention. In some embodiments, ALK-specific antibodies used in the methods disclosed herein specifically bind the kinase domain of ALK and, thus, will detect full-length ALK, the FNl -ALK fusion polypeptides described herein, and the FNl-tmALK fusion polypeptides described herein. Furthermore, ALK fusion- specific antibodies are commercially available (see Abeam, 2010 Catalog No. ab4061 (NPM-ALK), and Thermo Scientific, 2010 Catalog No. PAl -37060 (NPM-ALK), for example). Such antibodies may also be produced according to standard methods, as described above.

Detection of expression and/or activity of full-length ALK and/or FNl-ALK fusion polypeptide expression, in a biological sample (e.g. a tumor sample) can provide information on whether the fusion protein alone is driving the tumor, or whether aberrantly expressed full length ALK is also present and driving the tumor. Such information is clinically useful in assessing whether targeting the fusion protein or the full-length protein(s), or both, or is likely to be most beneficial in inhibiting progression of the tumor, and in selecting an appropriate therapeutic or combination thereof. Antibodies specific for the ALK kinase extracellular domain, which is not present in the mutant ALK disclosed herein, may be particularly useful for determining the presence/absence of the mutant ALK kinase.

It will be understood that more than one antibody may be used in the practice of the above- described methods. For example, one or more FNl-ALK fusion and/or FNl-tmALK fusion polypeptide-specific antibodies together with one or more antibodies specific for full-length ALK kinase, another kinase, receptor, or kinase substrate that is suspected of being, or potentially is, activated in a cancer in which FNl-ALK fusion or FNl-tmALK fusion polypeptide is expressed may be simultaneously employed to detect the activity of such other signaling molecules in a biological sample comprising cells from such cancer.

Those of skill in the art will appreciate that fusion polypeptides of various embodiments of the present invention and the epitope-bearing fragments thereof described above can be combined with parts of other molecules to create chimeric polypeptides. For example, an epitope-bearing fragment of an FNl-tmALK fusion or an FNl-ALK fusion polypeptide may be combined with the constant domain of immunoglobulins (IgG) to facilitate purification of the chimeric polypeptide and increase the in vivo half-life of the chimeric polypeptide (see, e.g., examples of CD4-Ig chimeric proteins in EPA 394,827; Traunecker et al., Nature 331: 84-86 (1988)). Fusion proteins that have a disulfide-linked dimeric structure (e.g., from an IgG portion may also be more efficient in binding and neutralizing other molecules than the monomeric FNl-tmALK fusion or FNl-ALK fusion polypeptide alone (see Fountoulakis et al., J Biochem 270: 3958-3964(1995)).

In some embodiments, a reagent that specifically binds to a FNl-tmALK fusion polypeptide or a FNl-ALK fusion polypeptide is a heavy-isotope labeled peptide (i.e., an AQUA peptide). Such an AQUA peptide may be suitable for the absolute quantification of an expressed FNl-tmALK fusion polypeptide or FN1-ALK fusion polypeptide in a biological sample. As used herein, the term "heavy-isotope labeled peptide" is used interchangeably with "AQUA peptide". The production and use of AQUA peptides for the absolute quantification or detection of proteins (AQUA) in complex mixtures has been described. See WO/03016861, "Absolute Quantification of Proteins and Modified Forms Thereof by Multistage Mass Spectrometry," Gygi et al. and also Gerber et al., Proc. Natl. Acad. Sci. U.S.A. 100: 6940-5 (2003) (the teachings of which are hereby incorporated herein by reference, in their entirety). The term "specifically detects" with respect to such an AQUA peptide means the peptide will only detect and quantify polypeptides and proteins that contain the AQUA peptide sequence and will not substantially detect polypeptides and proteins that do not contain the AQUA peptide sequence.

The AQUA methodology employs the introduction of a known quantity of at least one heavy-isotope labeled peptide standard (which has a unique signature detectable by LC-SRM chromatography) into a digested biological sample in order to determine, by comparison to the peptide standard, the absolute quantity of a peptide with the same sequence and protein modification in the biological sample. Briefly, the AQUA methodology has two stages: peptide internal standard selection and validation and method development; and implementation using validated peptide internal standards to detect and quantify a target protein in sample. The method is a powerful technique for detecting and quantifying a given peptide/protein within a complex biological mixture, such as a cell lysate, and may be employed, e.g., to quantify change in protein phosphorylation as a result of drug treatment, or to quantify differences in the level of a protein in different biological states.

Generally, to develop a suitable internal standard, a particular peptide (or modified peptide) within a target protein sequence is chosen based on its amino acid sequence and the particular protease to be used to digest. The peptide is then generated by solid-phase peptide

13 synthesis such that one residue is replaced with that same residue containing stable isotopes ( C, ¹⁵N). The result is a peptide that is chemically identical to its native counterpart formed by proteolysis, but is easily distinguishable by MS via a 7-Da mass shift. The newly synthesized AQUA internal standard peptide is then evaluated by LC-MS/MS. This process provides qualitative information about peptide retention by reverse-phase chromatography, ionization efficiency, and fragmentation via collision-induced dissociation. Informative and abundant fragment ions for sets of native and internal standard peptides are chosen and then specifically monitored in rapid succession as a function of chromatographic retention to form a selected reaction monitoring (LC-SRM) method based on the unique profile of the peptide standard.

The second stage of the AQUA strategy is its implementation to measure the amount of a protein or modified protein from complex mixtures. Whole cell lysates are typically fractionated by SDS-PAGE gel electrophoresis, and regions of the gel consistent with protein migration are excised. This process is followed by in-gel proteolysis in the presence of the AQUA peptides and LC-SRM analysis. {See Gerber et ah, supra.) AQUA peptides are spiked in to the complex peptide mixture obtained by digestion of the whole cell lysate with a proteolytic enzyme and subjected to immunoaffinity purification as described above. The retention time and fragmentation pattern of the native peptide formed by digestion {e.g., trypsinization) is identical to that of the

AQUA internal standard peptide determined previously; thus, LC-MS/MS analysis using an SRM experiment results in the highly specific and sensitive measurement of both internal standard and analyte directly from extremely complex peptide mixtures.

Since an absolute amount of the AQUA peptide is added {e.g., 250 fmol), the ratio of the areas under the curve can be used to determine the precise expression levels of a protein or phosphorylated form of a protein in the original cell lysate. In addition, the internal standard is present during in-gel digestion as native peptides are formed, such that peptide extraction efficiency from gel pieces, absolute losses during sample handling (including vacuum

centrifugation), and variability during introduction into the LC-MS system do not affect the determined ratio of native and AQUA peptide abundances.

An AQUA peptide standard is developed for a known sequence previously identified by the IAP-LC-MS/MS method within in a target protein. If the site is modified, one AQUA peptide incorporating the modified form of the particular residue within the site may be developed, and a second AQUA peptide incorporating the unmodified form of the residue developed. In this way, the two standards may be used to detect and quantify both the modified an unmodified forms of the site in a biological sample.

Peptide internal standards may also be generated by examining the primary amino acid sequence of a protein and determining the boundaries of peptides produced by protease cleavage. Alternatively, a protein may actually be digested with a protease and a particular peptide fragment produced can then sequenced. Suitable proteases include, but are not limited to, serine proteases {e.g. trypsin, hepsin), metallo proteases {e.g., PUMP1), chymotrypsin, cathepsin, pepsin, thermolysin, carboxypeptidases, etc. A peptide sequence within a target protein is selected according to one or more criteria to optimize the use of the peptide as an internal standard. Preferably, the size of the peptide is selected to minimize the chances that the peptide sequence will be repeated elsewhere in other non-target proteins. Thus, a peptide is preferably at least about 6 amino acids. The size of the peptide is also optimized to maximize ionization frequency. Thus, in some embodiments, the peptide is not longer than about 20 amino acids. In some embodiments, the peptide is between about 7 to 15 amino acids in length. A peptide sequence is also selected that is not likely to be chemically reactive during mass spectrometry, thus sequences comprising cysteine, tryptophan, or methionine are avoided.

A peptide sequence that does not include a modified region of the target region may be selected so that the peptide internal standard can be used to determine the quantity of all forms of the protein. Alternatively, a peptide internal standard encompassing a modified amino acid may be desirable to detect and quantify only the modified form of the target protein. Peptide standards for both modified and unmodified regions can be used together, to determine the extent of a modification in a particular sample (i.e. to determine what fraction of the total amount of protein is represented by the modified form). For example, peptide standards for both the phosphorylated and unphosphorylated form of a protein known to be phosphorylated at a particular site can be used to quantify the amount of phosphorylated form in a sample.

The peptide is labeled using one or more labeled amino acids (i.e., the label is an actual part of the peptide) or less preferably, labels may be attached after synthesis according to standard methods. Preferably, the label is a mass-altering label selected based on the following

considerations: The mass should be unique to shift fragments masses produced by MS analysis to regions of the spectrum with low background; the ion mass signature component is the portion of the labeling moiety that preferably exhibits a unique ion mass signature in MS analysis; the sum of the masses of the constituent atoms of the label is preferably uniquely different than the fragments of all the possible amino acids. As a result, the labeled amino acids and peptides are readily distinguished from unlabeled ones by the ion/mass pattern in the resulting mass spectrum.

Preferably, the ion mass signature component imparts a mass to a protein fragment that does not match the residue mass for any of the 20 natural amino acids.

The label should be robust under the fragmentation conditions of MS and not undergo unfavorable fragmentation. Labeling chemistry should be efficient under a range of conditions, particularly denaturing conditions, and the labeled tag preferably remains soluble in the MS buffer system of choice. The label preferably does not suppress the ionization efficiency of the protein and is not chemically reactive. The label may contain a mixture of two or more isotopically distinct species to generate a unique mass spectrometric pattern at each labeled fragment position.

2 13 15 17 18 34

Stable isotopes, such as H, ^1JC, ^1JN, "0, ¹⁰0, or S, are some non-limiting labels. Pairs of peptide internal standards that incorporate a different isotope label may also be prepared. Non- limiting amino acid residues into which a heavy isotope label may be incorporated include leucine, proline, valine, and phenylalanine.

Peptide internal standards are characterized according to their mass-to-charge (m/z) ratio, and preferably, also according to their retention time on a chromatographic column (e.g., an HPLC column). Internal standards that co-elute with unlabeled peptides of identical sequence are selected as optimal internal standards. The internal standard is then analyzed by fragmenting the peptide by any suitable means, for example by collision-induced dissociation (CID) using, e.g., argon or helium as a collision gas. The fragments are then analyzed, for example by multi-stage mass spectrometry (MSⁿ) to obtain a fragment ion spectrum, to obtain a peptide fragmentation signature. Preferably, peptide fragments have significant differences in m/z ratios to enable peaks corresponding to each fragment to be well separated, and a signature is that is unique for the target peptide is obtained. If a suitable fragment signature is not obtained at the first stage, additional stages of MS are performed until a unique signature is obtained.

Fragment ions in the MS/MS and MS spectra are typically highly specific for the peptide of interest, and, in conjunction with LC methods, allow a highly selective means of detecting and quantifying a target peptide/protein in a complex protein mixture, such as a cell lysate, containing many thousands or tens of thousands of proteins. Any biological sample potentially containing a target protein/peptide of interest may be assayed. Crude or partially purified cell extracts are preferably employed. Generally, the sample has at least 0.01 mg of protein, typically a

concentration of 0.1-10 mg/mL, and may be adjusted to a desired buffer concentration and pH.

A known amount of a labeled peptide internal standard, preferably about 10 femtomoles, corresponding to a target protein to be detected/quantified is then added to a biological sample, such as a cell lysate. The spiked sample is then digested with one or more protease(s) for a suitable time period to allow digestion. A separation is then performed (e.g. by HPLC, reverse- phase HPLC, capillary electrophoresis, ion exchange chromatography, etc.) to isolate the labeled internal standard and its corresponding target peptide from other peptides in the sample.

Microcapillary LC is a one non-limiting method. Each isolated peptide is then examined by monitoring of a selected reaction in the MS. This involves using the prior knowledge gained by the characterization of the peptide internal standard and then requiring the MS to continuously monitor a specific ion in the MS/MS or MSⁿ spectrum for both the peptide of interest and the internal standard. After elution, the area under the curve (AUC) for both peptide standard and target peptide peaks are calculated. The ratio of the two areas provides the absolute quantification that can be normalized for the number of cells used in the analysis and the protein's molecular weight, to provide the precise number of copies of the protein per cell. Further details of the AQUA methodology are described in Gygi et al., and Gerber et al. supra.

AQUA internal peptide standards (heavy-isotope labeled peptides) may desirably be produced, as described above, to detect any quantify any unique site {e.g., the fusion junction within a FNl -ALK fusion or a FNl-tmALK fusion polypeptide) within a mutant ALK

polypeptide. For example, an AQUA phosphopeptide may be prepared that corresponds to the fusion junction sequence of FNl-ALKvariant2 fusion polypeptide Peptide standards for may be produced for the FNl-ALKvariant2 fusion junction and such standards employed in the AQUA methodology to detect and quantify the fusion junction (i.e. the presence of FNl-ALKvariant2 fusion polypeptide) in a biological sample.

For example, an exemplary AQUA peptide comprises the amino acid sequence TTPVSP which corresponds to the three amino acids immediately flanking each side of the fusion junction in the FNl-ALKvariantl fusion polypeptide, where the amino acids encoded by the FNl gene are italicized and the amino acids encoded by the ALK gene in bold. It will be appreciated that larger AQUA peptides comprising the fusion junction sequence and, optionally, additional residues downstream (i.e., N'-terminal of the junction) or upstream (i.e., C'-terminal of the junction) of it may also be constructed. Similarly, a smaller AQUA peptide comprising less than all of the residues of such sequence (but still comprising the point of fusion junction itself) may

alternatively be constructed. Such larger or shorter AQUA peptides are within the scope of the present invention, and the selection and production of AQUA peptides may be carried out as described above (see Gygi et al., Gerber et al., supra.).

In another aspect, the invention provides a method for detecting the presence of a mutant ALK polypeptide or an FN1-ALK fusion polypeptide in a biological sample from a mammalian cancer or a suspected mammalian cancer, said method comprising the steps of: (a) obtaining a biological sample (e.g., a biological sample containing at least one polypeptide) from a mammalian cancer or suspected ovarian cancer; and (b) utilizing at least one reagent that specifically binds to a mutant ALK polypeptide or an FN 1 -ALK fusion polypeptide to determine whether said mutant ALK polypeptide is present in said biological sample, wherein specific binding of said reagent to said biological sample indicates said mutant ALK polypeptide or said FN 1 -ALK fusion polypeptide is present in said biological sample. In some embodiments, the mammalian cancer is mammalian ovarian cancer (e.g., from a human). In some embodiments, the mutant ALK polypeptide is truncated ALK polypeptide. In some embodiments, the mutant ALK polypeptide is an FNl-tmALK fusion polypeptide (e.g., an FNl -tmALK fusion polypeptide comprises an amino acid sequence of SEQ ID NO: 3, 5, 7, 10, 1 1 , or 12). In some embodiments, the FN 1 -ALK fusion polypeptide comprises an amino acid sequence of SEQ ID NO: 14, 15, 17, 18, 20, or 21.

As used throughout the specification, the term "biological sample" is used in its broadest sense, and means any biological sample suspected of containing a polypeptide with ALK kinase activity such as a mutant ALK polypeptide (including, without limitation, an FNl-tmALK fusion polypeptide or a truncated ALK), full length ALK protein (with or without the signal peptide sequence), an FN1-ALK polypeptide, an ALK fusion polypeptide (e.g., NPM-ALK or EML4- ALK), or fragments thereof. Biological samples include, without limitation, saliva, mucous, tears, blood, circulating tumor cells, serum, tissues, marrow, lymph/interstitial fluids, buccal cells, pleural effusion, fine needle aspirate, mucosal cells, cerebrospinal fluid, semen, feces, plasma, urine, a suspension of cells, or a suspension of cells and viruses or extracts thereof, and may comprise a cell, chromosomes isolated from a cell (e.g., a spread of metaphase chromosomes), genomic DNA (in solution or bound to a solid support such as for Southern analysis), RNA (in solution or bound to a solid support such as for northern analysis), cDNA (in solution or bound to a solid support). In some embodiments, a biological sample is mammalian (e.g., human) and is a biopsy sample or a blood sample including a circulating tumor cell. In some embodiments, the biological sample contains ovarian cells suspected of being cancerous.

Biological samples useful in the practice of the methods disclosed herein may be obtained from any mammal in which a cancer or suspected cancer characterized by the presence of a mutant ALK (e.g., a FNl -tmALK fusion polynucleotide or polypeptide) or a polypeptide with ALK kinase activity (or polynucleotide encoding the same) is present or might be present or developing. As used herein, the phrase "characterized by" with respect to a cancer (or suspected cancer) and indicated molecule (e.g., a polypeptide with ALK kinase activity or a mutant ALK) is meant a cancer (or suspected cancer) in which a gene translocation or mutation (e.g., causing aberrant expression of full-length ALK) and/or an expressed polypeptide with ALK kinase activity (e.g., a mutant ALK polypeptide, a ALK fusion, or a FN 1 -ALK fusion polypeptide) is present, as compared to a cancer or a normal tissue in which such translocation, aberrant expression of full- length ALK, and/or polypeptide with ALK kinase activity are not present. The presence of such translocation, aberrant expression of full-length ALK, and/or polypeptide with ALK kinase activity may drive (i.e., stimulate or be the causative agent of), in whole or in part, the growth and survival of such cancer or suspected cancer.

In one embodiment, the mammal is a human, and the human may be a candidate for an ALK-inhibiting therapeutic, for the treatment of a cancer, e.g., ovarian cancer. The human candidate may be a patient currently being treated with, or considered for treatment with, an ALK kinase inhibitor. In another embodiment, the mammal is large animal, such as a horse or cow, while in other embodiments, the mammal is a small animal, such as a dog or cat, all of which are known to develop cancers, including ovarian cancers, such as ovarian stromal cancers or ovarian clear cell carcinomas.

Any biological sample comprising cells (or extracts of cells) from a mammalian cancer is suitable for use in the methods disclosed herein. In one embodiment, the biological sample comprises cells obtained from a tumor biopsy. The biopsy may be obtained, according to standard clinical techniques, from primary tumors occurring in an organ of a mammal, or by secondary tumors that have metastasized in other tissues. In another embodiment, the biological sample comprises cells obtained from a fine needle aspirate taken from a tumor, and techniques for obtaining such aspirates are well known in the art (see Cristallini et al., Acta Cytol. 36(3): 416-22 (1992)).

In some embodiments, the biological sample comprises circulating tumor cells.

Circulating tumor cells ("CTCs") may be purified, for example, using the kits and reagents sold under the trademarks Vita- Assays™, Vita-Cap™, and CellSearch® (commercially available from Vitatex, LLC (a Johnson and Johnson corporation). Other methods for isolating CTCs are described (see, for example, PCT Publication No. WO/2002/020825, Cristofanilli et al, New Engl. J. of Med. 351 (8):781-791 (2004), and Adams et al, J. Amer. Chem. Soc. 130(27): 8633- 8641 (July 2008)). In a particular embodiment, a circulating tumor cell ("CTC") may be isolated and identified as having originated from the lung. Accordingly, the invention provides a method for isolating a CTC, and then screening the CTC one or more assay formats to identify the presence of a polypeptide with ALK kinase activity or nucleic acid molecule encoding the same (e.g., mutant ALK polypeptide or polynucleotide such as a FNl-ALKvariantl fusion polypeptide or polynucleotide or a FNl-ALKvariant2 fusion polypeptide or polynucleotide) in the CTC. Some non- limiting assay formats include Western blotting analysis, flow-cytometry (FC), immuno-histochemistry (IHC), immuno-fluorescence (IF), fluorescence in situ hybridization (FISH) and polymerase chain reaction (PCR). A CTC from a patient that is identified as comprising a polypeptide with ALK kinase activity or polynucleotide encoding the same (e.g., a FNl-ALKvariantl fusion polypeptide or polynucleotide) may indicate that the patient's originating cancer (e.g., an ovarian cancer such as an ovarian stromal cancer or an ovarian clear cell carcinoma) is likely to respond to a composition comprising at least one ALK kinase-inhibiting therapeutic.

As used herein, by "likely to respond" is meant that a cancer is more likely to show growth retardation or abrogation in response to {e.g., upon contact with or treatment by) an ALK inhibitor. In some embodiments, a cancer that is likely to respond to an ALK inhibitor is one that dies {e.g., the cancer cells apoptose) in response to the ALK inhibitor.

A biological sample may comprise cells (or cell extracts) from a cancer in which polypeptide with ALK kinase activity (e.g., a FN1-ALK fusion polypeptide or mutant ALK polypeptide) is expressed and/or activated but full-length ALK polypeptide is not. Alternatively, the sample may comprise cells from a cancer in which both a mutant ALK fusion polypeptide and a full-length ALK kinase are expressed and/or activated, or in which full-length ALK kinase is expressed and/or active, but an ALK fusion polypeptide (such as an FN 1 -ALK fusion polypeptide) is not.

Cellular extracts of the foregoing biological samples may be prepared, either crude or partially (or entirely) purified, in accordance with standard techniques, and used in the methods disclosed herein. Alternatively, biological samples comprising whole cells may be utilized in assay formats such as immunohistochemistry (IHC), flow cytometry (FC), and

immunofluorescence (IF), as further described above. Such whole-cell assays are advantageous in that they minimize manipulation of the tumor cell sample and thus reduce the risks of altering the in vivo signaling/activation state of the cells and/or introducing artifact signals. Whole cell assays are also advantageous because they characterize expression and signaling only in tumor cells, rather than a mixture of tumor and normal cells. In practicing the disclosed method for determining whether a compound inhibits progression of a tumor characterized by the presence of a polypeptide with ALK kinase activity (or polynucleotide encoding the same), biological samples comprising cells from mammalian xenografts (or bone marrow transplants) may also be advantageously employed. Non-limiting xenografts (or transplant recipients) are small mammals, such as mice, harboring human tumors (or leukemias) that express a polypeptide with ALK kinase activity (e.g., FNl-tmALK fusion polypeptide, FN 1 -ALK fusion polypeptide, full length ALK, or a truncated ALK kinase).

Xenografts harboring human tumors are well known in the art (see Kal, Cancer Treat Res. 72: 155-69 (1995)) and the production of mammalian xenografts harboring human tumors is well described (see Winograd et ah, In Vivo. 1(1): 1-13 (1987)). Similarly the generation and use of bone marrow transplant models is well described (see, e.g., Schwaller, et ah, EMBO J. 17: 5321- 333 (1998); Kelly et al, Blood 99: 310-318 (2002)).

In assessing the presence of a polypeptide with ALK kinase activity (or polynucleotide encoding the same) in a biological sample comprising cells from a mammalian cancer tumor, a control sample representing a cell in which such a polypeptide with ALK kinase activity does not occur (e.g., healthy ovarian cells) may desirably be employed for comparative purposes. Ideally, the control sample comprises cells from a subset of the particular cancer (e.g., ovarian cancer) that is representative of the subset in which the polypeptide with ALK kinase activity (or

polynucleotide encoding the same) does not occur. Comparing the level in the control sample versus the test biological sample thus identifies whether the mutant polynucleotide and/or polypeptide is/are present. Alternatively, since a polypeptide with ALK kinase activity (or polynucleotide encoding the same) may not be present in the majority of cancers, any tissue that similarly does not express polypeptide with ALK kinase activity (or polynucleotide encoding the same) may be employed as a control.

The methods described below will have valuable diagnostic utility for cancers

characterized by the presence of a polypeptide with ALK kinase activity, and treatment decisions pertaining to the same. For example, biological samples may be obtained from a subject that has not been previously diagnosed as having a cancer characterized by the presence of polypeptide with ALK kinase activity, nor has yet undergone treatment for such cancer, and the method is employed to diagnostically identify a tumor in such subject as belonging to a subset of tumors (e.g., an ovarian stromal tumor or an ovarian clear cell carcinoma) in which a polypeptide with ALK kinase activity (or polynucleotide encoding the same) is present/expressed. Alternatively, a biological sample may be obtained from a subject that has been diagnosed as having a cancer characterized by the presence of one type of kinase, such as EFGR, and has been receiving therapy, such as EGFR inhibitor therapy (e.g., Tarceva™, Iressa™) for treatment of such cancer, and the method can be employed to identify whether the subject's tumor is also characterized by the presence of polypeptide with ALK kinase activity (or polynucleotide encoding the same) such as mutant ALK or an FN 1 -ALK fusion polypeptide, and is therefore likely to fully respond to the existing therapy and/or whether alternative or additional ALK- inhibiting therapy is desirable or warranted. The methods disclosed herein may also be employed to monitor the progression or inhibition of a polypeptide with ALK kinase activity-expressing cancer following treatment of a subject with a composition comprising an ALK-inhibiting therapeutic or combination of therapeutics.

Such diagnostic assay may be carried out subsequent to or prior to preliminary evaluation or surgical surveillance procedures. The identification method may be advantageously employed as a diagnostic to identify patients having cancer, such as ovarian cancer, characterized by the presence of a polypeptide with ALK kinase activity such as an FNl-tmALK or FN 1 -ALK fusion protein, which patients would be most likely to respond to therapeutics targeted at inhibiting ALK kinase activity. The ability to select such patients would also be useful in the clinical evaluation of efficacy of future ALK-targeted therapeutics as well as in the future prescription of such drugs to patients.

The ability to selectively identify cancers in which a polypeptide with ALK kinase activity

(or polynucleotide encoding the same) such as a FNl-tmALK or a FN 1 -ALK polynucleotide and/or encoded fusion polypeptide, is/are present enables important new methods for accurately identifying such tumors for diagnostic purposes, as well as obtaining information useful in determining whether such a tumor is likely to respond to a ALK-inhibiting therapeutic composition, or likely to be partially or wholly non-responsive to an inhibitor targeting a different kinase when administered as a single agent for the treatment of the cancer.

As used herein, by "cancer" or "cancerous" is meant a cell that shows abnormal growth as compared to a normal (i.e., non-cancerous) cell of the same cell type. For example, a cancerous cell may be metastatic or non-metastatic. A cancerous cell may also show lack of contact inhibition where a normal cell of that same cell type shows contact inhibition. Any cancer cell is included in this definition including, without limitation, leukemia, lymphoma, ovarian cancer, liver cancer, renal cancer, lung cancer (e.g., non-small cell lung cancer or small cell lung cancer), prostate cancer, breast cancer, colon cancer, brain and/or nerve cancer (e.g., glioblastoma), bone cancer, and skin cancer (e.g., myeloma). As used herein, by "suspected cancer" or "tissue suspected of being cancerous" is meant a cell or tissue that has some aberrant characteristics (e.g., hyperplastic or lack of contact inhibition) as compared to normal cells or tissues of that same cell or tissue type as the suspected cancer, but where the cell or tissue is not yet confirmed by a physician or pathologist as being cancerous.

In another aspect, the invention provides a method for detecting the presence of a polypeptide with ALK kinase activity in a biological sample from a mammalian ovarian cancer or suspected mammalian ovarian cancer, said method comprising the steps of: (a) obtaining a biological sample from a mammalian ovarian cancer or suspected mammalian ovarian cancer and (b) utilizing a reagent that specifically binds said polypeptide with ALK kinase activity to determine whether said polypeptide with ALK kinase activity is present in said biological sample, wherein specific binding of said reagent to said biological sample indicates said polypeptide with ALK kinase activity is present in said biological sample. In some embodiments, the polypeptide is aberrantly expressed full-length ALK protein. In some embodiments, the polypeptide is a mutant ALK polypeptide, such as a truncated ALK polypeptide or an FNl-tmALK fusion polypeptide (e.g., comprising the amino acid sequence of SEQ ID NO: 3, 5, 7, 10, 11, or 12). In some embodiments, the polypeptide is an ALK fusion polypeptide (e.g., an NPM-ALK fusion polypeptide, an AL017-ALK fusion polypeptide, an TFG-ALK fusion polypeptide, an MSN- ALK fusion polypeptide, an TPM3-ALK fusion polypeptide, an TPM4-ALK fusion polypeptide, an ATIC-ALK fusion polypeptide, an MYH9-ALK fusion polypeptide, an CLTC-ALK fusion polypeptide, an SEC31L1-ALK fusion polypeptide, an RANBP2-ALK fusion polypeptide, an CARS-ALK fusion polypeptide, an EML4-ALK fusion polypeptide, an KIF5B-ALK fusion polypeptide, and an TFG-ALK fusion polypeptide). In some embodiments, the polypeptide is an FN1-ALK fusion polypeptide (e.g., comprising the amino acid sequence selected from the group consisting of SEQ ID NO: 14, 15, 17, 18, 20, or 21).

The various methods disclosed herein may be carried out in a variety of different assay formats known to those of skill in the art. Some non-limiting examples of methods include immunoassays and peptide and nucleotide assays.

Immunoassays.

Immunoassays useful in the practice of the methods disclosed herein may be homogenous immunoassays or heterogeneous immunoassays. In a homogeneous assay the immunological reaction usually involves a specific reagent (e.g. a FNl-ALKvariantl fusion polypeptide-specific antibody or an ALK-specific antibody), a labeled analyte, and the biological sample of interest. The signal arising from the label is modified, directly or indirectly, upon the binding of the antibody to the labeled analyte. Both the immunological reaction and detection of the extent thereof are carried out in a homogeneous solution. Immunochemical labels that may be employed include free radicals, radio-isotopes, fluorescent dyes, enzymes, bacteriophages, coenzymes, and so forth. Semi-conductor nanocrystal labels, or "quantum dots", may also be advantageously employed, and their preparation and use has been well described. See generally, K. Barovsky, Nanotech. Law & Bus. 1(2): Article 14 (2004) and patents cited therein.

In a heterogeneous assay approach, the reagents are usually the biological sample, binding reagent {e.g., an antibody), and suitable means for producing a detectable signal. Biological samples as further described below may be used. The antibody is generally immobilized on a support, such as a bead, plate or slide, and contacted with the sample suspected of containing the antigen in a liquid phase. The support is then separated from the liquid phase and either the support phase or the liquid phase is examined for a detectable signal employing means for producing such signal. The signal is related to the presence of the analyte in the biological sample. Means for producing a detectable signal include the use of radioactive labels, fluorescent labels, enzyme labels, quantum dots, and so forth. For example, if the antigen to be detected contains a second binding site, an antibody which binds to that site can be conjugated to a detectable group and added to the liquid phase reaction solution before the separation step. The presence of the detectable group on the solid support indicates the presence of the antigen in the test sample. Examples of suitable immunoassays are the radioimmunoassay, immunofluorescence methods, enzyme-linked immunoassays, and the like.

Immunoassay formats and variations thereof, which may be useful for carrying out the methods disclosed herein, are well known in the art. See generally E. Maggio, Enzyme -

Immunoassay, (1980) (CRC Press, Inc., Boca Raton, Fla.); see also, e.g., U.S. Pat. No. 4,727,022 (Skold et al, "Methods for Modulating Ligand-Receptor Interactions and their Application"); U.S. Pat. No. 4,659,678 (Forrest et al, "Immunoassay of Antigens"); U.S. Pat. No. 4,376,110 (David et al, "Immunometric Assays Using Monoclonal Antibodies"). Conditions suitable for the formation of reagent-antibody complexes are well known to those of skill in the art. See id. FN1- ALK fusion polypeptide-specific monoclonal antibodies may be used in a "two-site" or

"sandwich" assay, with a single hybridoma cell line serving as a source for both the labeled monoclonal antibody and the bound monoclonal antibody. Such assays are described in U.S. Pat. No. 4,376,110. The concentration of detectable reagent should be sufficient such that the binding of a protein with ALK kinase activity (e.g., a full-length ALK protein, a truncated ALK, an FN1- tmALK fusion polypeptide or an FN 1 -ALK fusion polypeptide) is detectable compared to background.

Antibodies useful in the practice of the methods disclosed herein may be conjugated to a solid support suitable for a diagnostic assay (e.g., beads, plates, slides or wells formed from materials such as latex or polystyrene) in accordance with known techniques, such as

precipitation. Antibodies or other binding reagents binding reagents may likewise be conjugated

35 125 131

to detectable groups such as radiolabels (e.g., S, I, I), enzyme labels (e.g., horseradish peroxidase, alkaline phosphatase), and fluorescent labels (e.g., fluorescein) in accordance with known techniques.

Cell-based assays, such flow cytometry (FC), immuno-histochemistry (IHC), or immunofluorescence (IF) are particularly desirable in practicing the methods disclosed herein, since such assay formats are clinically-suitable, allow the detection of expression of a protein with ALK kinase activity (e.g., wild-type ALK polypeptide, a mutant ALK polypeptide or an FN1- ALK fusion polypeptide) in vivo, and avoid the risk of artifact changes in activity resulting from manipulating cells obtained from, e.g. a tumor sample in order to obtain extracts. Accordingly, in some embodiments, the methods disclosed herein are implemented in a flow-cytometry (FC), immuno-histochemistry (IHC), or immunofluorescence (IF) assay format.

Flow cytometry (FC) may be employed to determine the expression of polypeptide with ALK kinase activity in a mammalian tumor before, during, and after treatment with a drug targeted at inhibiting ALK kinase activity. For example, tumor cells from a fine needle aspirate may be analyzed by flow cytometry for expression and/or activation of a polypeptide with ALK kinase activity or polynucleotide encoding the same (e.g., a mutant ALK or an FN1-ALK fusion polynucleotide or polypeptide), as well as for markers identifying cancer cell types, etc., if so desired. Flow cytometry may be carried out according to standard methods. See, e.g. Chow et al, Cytometry (Communications in Clinical Cytometry) 46: 72-78 (2001). Briefly and by way of example, the following protocol for cytometric analysis may be employed: fixation of the cells with 2% paraformaldehyde for 10 minutes at 37 °C followed by permeabilization in 90% methanol for 10 minutes on ice. Cells may then be stained with the primary antibody (e.g., a full- length ALK-specific or a FNl-ALKvariantl fusion polypeptide-specific antibody), washed and labeled with a fluorescent-labeled secondary antibody. The cells would then be analyzed on a flow cytometer (e.g. a Beckman Coulter FC500) according to the specific protocols of the instrument used. Such an analysis would identify the level of expressed full-length ALK or FN1- ALKvariantl fusion polypeptide in the tumor. Similar analysis after treatment of the tumor with an ALK-inhibiting therapeutic would reveal the responsiveness of a full-length ALK-expressing tumor or a FN1- ALKvariantl fusion polypeptide-expressing tumor to the targeted inhibitor of ALK kinase.

Immunohistochemical (IHC) staining may be also employed to determine the expression and/or activation status of polypeptide with ALK kinase activity in a mammalian cancer (e.g., an ovarian cancer or other type of cancer such as a lung, kidney, or colon cancer) before, during, and after treatment with a drug targeted at inhibiting ALK kinase activity. IHC may be carried out according to well-known techniques. See, e.g., ANTIBODIES: A LABORATORY MANUAL, Chapter 10, Harlow & Lane Eds., Cold Spring Harbor Laboratory (1988). Briefly, and by way of example, paraffin-embedded tissue (e.g. tumor tissue from a biopsy) is prepared for immunohistochemical staining by deparaffmizing tissue sections with xylene followed by ethanol; hydrating in water then PBS; unmasking antigen by heating slide in sodium citrate buffer; incubating sections in hydrogen peroxide; blocking in blocking solution; incubating slide in primary antibody (e.g., an ALK-specific or ALK fusion polypeptide-specific antibody) and secondary antibody; and finally detecting using ABC avidin/biotin method according to manufacturer's instructions.

Immunofluorescence (IF) assays may be also employed to determine the expression and/or activation status of a polypeptide with ALK kinase activity (e.g., full length ALK polypeptide or a FN 1 -ALK fusion polypeptide) in a mammalian cancer before, during, and after treatment with a drug targeted at inhibiting ALK kinase activity. IF may be carried out according to well-known techniques. See, e.g., J.M. polak and S. Van Noorden (1997) INTRODUCTION TO

iMMUNOCYTOCHEMiSTRY, 2nd Ed.; ROYAL MICROSCOPY SOCIETY MICROSCOPY HANDBOOK 37, Bio Scientific/Springer- Verlag. Briefly, and by way of example, patient samples may be fixed in paraformaldehyde followed by methanol, blocked with a blocking solution such as horse serum, incubated with a primary antibody against (i.e., that specifically binds to) a polypeptide with ALK kinase activity (e.g., an FNl-tmALK fusion polypeptide) followed by a secondary antibody labeled with a fluorescent dye such as Alexa 488 and analyzed with an epifluorescent microscope.

A variety of other protocols, including enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescent-activated cell sorting (FACS), for measuring expression and/or activity of a polypeptide with ALK kinase activity are known in the art and provide a basis for diagnosing the presence of the polypeptide with ALK kinase activity (e.g., a mutant ALK polypeptide, full-length ALK, or an ALK fusion polypeptide such as an FNl-ALKvariant4 fusion polypeptide). Normal or standard values for full-length ALK polypeptide expression are established by combining body fluids or cell extracts taken from normal mammalian subjects, preferably human, with an antibody that specifically binds to full length ALK polypeptide under conditions suitable for complex formation. The amount of standard complex formation may be quantified by various methods, but preferably by photometric means. Quantities of full length ALK polypeptide expressed in subject, control, and disease samples from biopsied tissues are compared with the standard values. Deviation between standard and subject values establishes the parameters for diagnosing disease. Of course, since the FN 1 -ALK fusion polypeptides and the mutant ALK polypeptide (including the FNl-tmALK fusion polypeptides) described herein are discovered in cancerous cells, no normal biological samples are expected to contain these polynucleotides or polypeptides.

Peptide & Nucleotide Assays.

Similarly, AQUA peptides for the detection/quantification of a polypeptide with ALK kinase activity in a biological sample comprising cells from a tumor may be prepared and used in standard AQUA assays, as described in detail above. Accordingly, in some embodiments of the methods disclosed herein, the binding reagent comprises a heavy isotope labeled phosphopeptide (AQUA peptide) corresponding to a peptide sequence comprising the fusion junction of FN 1- ALK fusion polypeptide or an FNl-tmALK fusion polypeptide, as described above.

FN 1 -ALK fusion polypeptide or FNl-tmALK fusion polypeptide-specific binding reagents useful in practicing the methods disclosed herein may also be mRNA, oligonucleotide or DNA probes that can directly hybridize to, and detect, fusion or truncated polypeptide expression transcripts in a biological sample. Such probes (also referred to a "primers" herein) are discussed in detail herein. Briefly, and by way of example, formalin- fixed, paraffin-embedded patient samples may be probed with (i.e., contacted with under conditions where the probe will hybridize to a nucleic acid molecule in the sample if that nucleic acid molecule shares sufficient sequence identity with the probe to allow hybridization) a fluorescein-labeled RNA probe followed by washes with formamide, SSC and PBS and analysis with a fluorescent microscope. Polynucleotides encoding a polypeptide with ALK kinase activity may also be used for diagnostic purposes. The polynucleotides that may be used include oligonucleotide sequences, antisense RNA and DNA molecules, and PNAs. The polynucleotides may be used to detect and quantitate gene expression in biopsied tissues in which expression of a polypeptide with ALK kinase activity (e.g., a FN1-ALK fusion polypeptide, full length ALK, or mutant ALK polypeptide (e.g., truncated ALK or an FNl-tmALK fusion) may be correlated with disease. The diagnostic assay may be used to distinguish between absence, presence, and excess expression of a polypeptide with ALK kinase activity, and to monitor regulation of levels of a polypeptide with ALK kinase activity during therapeutic intervention.

In one embodiment, hybridization with PCR probes which are capable of detecting polynucleotide sequences, including genomic sequences, encoding a polypeptide with ALK kinase activity including an FNl -ALK fusion polypeptide or a mutant ALK kinase polypeptide or closely related molecules, may be used to identify nucleic acid sequences that encode such polypeptides with ALK kinase activity. The construction and use of such probes is described herein. The specificity of the probe, whether it is made from a highly specific region, e.g., 10 unique nucleotides in the fusion junction, or a less specific region, e.g., the 3' coding region, and the stringency of the hybridization or amplification (maximal, high, intermediate, or low) will determine whether the probe identifies only naturally occurring sequences encoding mutant ALK kinase polypeptide, alleles, or related sequences.

Probes may also be used for the detection of related sequences, and should preferably contain at least 50% of the nucleotides from any of the mutant ALK polypeptide encoding sequences. The hybridization probes of the subject invention may be DNA or RNA and derived from the nucleotide sequences of SEQ ID NOs: 2, 4, 6, 8, 13, 16, or 19 most preferably encompassing the fusion junction, or from genomic sequence including promoter, enhancer elements, and introns of the naturally occurring FNl and ALK genes, as further described above. The probes may alternatively hybridize nucleotides encoding the C-terminal domain located at amino acids 1376-1620 of SEQ ID NO: 1), amino acid residues 1504- 1507 of SEQ ID NO: l making up the phosphotyrosine-binding site of the C-terminal domain of ALK, or amino acid residues 1603-1606 of SEQ ID NO: 1 representing the interaction site for the phosphotyrosine- dependent binding of the substrate phosphlipase C-γ (PLC-γ).

An FNl -ALK fusion polynucleotide, FNl-tmALK fusion polynucleotide, or truncated ALK polynucleotide (e.g., lacking sequences encoding the extracellular domain of full length ALK polypeptide), or full length ALK polynucleotide may be used in Southern or northern analysis, dot blot, or other membrane-based technologies; in PCR technologies; or in dip stick, pin, ELISA or chip assays utilizing fluids or tissues from patient biopsies to detect altered expression of a polypeptide with ALK kinase activity. Such qualitative or quantitative methods are well known in the art. In a particular aspect, the nucleotide sequences encoding a polypeptide with ALK kinase activity may be useful in assays that detect activation or induction of various cancers, including cancers of the liver, pancreas, kidneys, and testes (as well as cancers that arise in the ducts, such as the bile duct, of these tissues). Polynucleotides encoding a polypeptide with ALK kinase activity may be detectably labeled by standard methods, and added to a fluid or tissue sample from a patient under conditions suitable for the formation of hybridization complexes. After a suitable incubation period, the sample is washed and the signal is quantitated and compared with a standard value. If the amount of signal in the biopsied or extracted sample is significantly altered from that of a comparable control sample, the nucleotide sequences have hybridized with nucleotide sequences in the sample, and the presence of altered levels of nucleotide sequences encoding a polypeptide with ALK kinase activity (e.g., an FN1-ALK fusion polypeptide, FNl-tmALK polypeptide, or truncated ALK kinase polypeptide) in the sample indicates the presence of the associated disease. Such assays may also be used to evaluate the efficacy of a particular therapeutic treatment regimen in animal studies, in clinical trials, or in monitoring the treatment of an individual patient.

Another aspect of the invention provides a method for diagnosing a patient as having a cancer or a suspected cancer driven by an ALK kinase. The method includes contacting a biological sample of said cancer or a suspected cancer (where the biological sample comprising at least one nucleic acid molecule) with a probe that hybridizes under stringent conditions to a nucleic acid molecule encoding a polypeptide with ALK kinase activity such as a full length ALK polynucleotide, a mutant ALK polynucleotide, or a ALK fusion polynucleotide (e.g., a NPM-ALK fusion polynucleotide, an FN 1 -ALK fusion polynucleotide, or an EML4-ALK fusion

polynucleotide), and wherein hybridization of said probe to at least one nucleic acid molecule in said biological sample identifies said patient as having a cancer or a suspected cancer driven by a ALK kinase.

Yet another aspect of the invention provides a method for diagnosing a patient as having a cancer or a suspected cancer driven by a ALK kinase. The method includes contacting a biological sample of said cancer or suspected cancer (where said biological sample comprises at least one polypeptide) with a binding agent that specifically binds to a polypeptide with ALK kinase activity, wherein specific binding of said binding agent to at least one polypeptide in said biological sample identifies said patient as having a cancer or a suspected cancer driven by a ALK kinase.

In order to provide a basis for the diagnosis of disease (e.g., a cancer) characterized by expression of a polypeptide with ALK kinase activity (e.g., a FN1- ALKvariantl fusion polypeptide), a normal or standard profile for expression is established. This may be

accomplished by combining body fluids or cell extracts taken from normal subjects, either animal or human, with a sequence, or a fragment thereof, which encodes a polypeptide with ALK kinase activity (e.g., a FNl-ALK fusion polypeptide, a FNl-tmALK fusion polypeptide, or a truncated ALK kinase polypeptide), under conditions suitable for hybridization or amplification. Standard hybridization may be quantified by comparing the values obtained from normal subjects with those from an experiment where a known amount of a substantially purified polynucleotide is used. Standard values obtained from normal samples may be compared with values obtained from samples from patients who are symptomatic for disease. Deviation between standard and subject values is used to establish the presence of disease.

Once disease is established and a treatment protocol is initiated, hybridization assays may be repeated on a regular basis to evaluate whether the level of expression in the patient begins to approximate that which is observed in the normal patient. The results obtained from successive assays may be used to show the efficacy of treatment over a period ranging from several days to months.

Additional diagnostic uses for polynucleotides encoding a polypeptide with ALK kinase activity (e.g., mutant ALK polynucleotides and FNl-ALK polynucleotides) may involve the use of polymerase chain reaction (PCR), another assay format that is standard to those of skill in the art. See, e.g., MOLECULAR CLONING, A LABORATORY MANUAL, 2nd. edition, Sambrook, J., Fritsch, E. F. and Maniatis, T., eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989). PCR oligomers may be chemically synthesized, generated enzymatically, or produced from a recombinant source. Oligomers will preferably consist of two nucleotide sequences, one with sense orientation (5' to 3') and another with antisense (3' to 5'), employed under optimized conditions for identification of a specific gene or condition. The same two oligomers, nested sets of oligomers, or even a degenerate pool of oligomers may be employed under less stringent conditions for detection and/or quantitation of closely related DNA or RNA sequences. Methods which may also be used to quantitate the expression of a polypeptide with ALK kinase activity (e.g., an FN1-ALK fusion polypeptide, an FNl-tmALK fusion polypeptide, or a truncated ALK kinase polypeptide include radiolabeling or biotinylating nucleotides, co- amplification of a control nucleic acid, and standard curves onto which the experimental results are interpolated (Melby et al., J. Immunol. Methods, 159: 235-244 (1993); Duplaa et al. Anal. Biochem. 229-236 (1993)). The speed of quantitation of multiple samples may be accelerated by running the assay in an ELISA format where the oligomer of interest is presented in various dilutions and a spectrophotometric or colorimetric response gives rapid quantitation.

In another embodiment of the invention, the polynucleotides encoding a polypeptide with ALK kinase activity may be used to generate hybridization probes which are useful for mapping the naturally occurring genomic sequence. The sequences may be mapped to a particular chromosome or to a specific region of the chromosome using well known techniques. Such techniques include fluorescence in-situ hybridization (FISH), FACS, or artificial chromosome constructions, such as yeast artificial chromosomes, bacterial artificial chromosomes, bacterial PI constructions or single chromosome cDNA libraries, as reviewed in Price, C. M., Blood Rev. 7: 127-134 (1993), and Trask, B. J., Trends Genet. 7: 149-154 (1991).

In one embodiment, fluorescence in-situ hybridization (FISH) is employed (as described in Verma et al. HUMAN CHROMOSOMES: A MANUAL OF BASIC TECHNIQUES, Pergamon Press, New York, N.Y. (1988)) and may be correlated with other physical chromosome mapping techniques and genetic map data. The FISH technique is well known (see, e.g., US Patent Nos. 5,756,696; 5,447,841; 5,776,688; and 5,663,319). Examples of genetic map data can be found in the 1994 Genome Issue of Science (265: 198 If). Correlation between the location of the gene encoding ALK protein and/or the gene encoding FN1 protein on a physical chromosomal map and a specific disease, or predisposition to a specific disease, may help delimit the region of DNA associated with that genetic disease. The nucleotide sequences of the subject invention may be used to detect differences in gene sequences between normal, carrier, or affected individuals.

In situ hybridization of chromosomal preparations and physical mapping techniques such as linkage analysis using established chromosomal markers may be used for extending genetic maps. Often the placement of a gene on the chromosome of another mammalian species, such as mouse, may reveal associated markers even if the number or arm of a particular human chromosome is not known. New sequences can be assigned to chromosomal arms, or parts thereof, by physical mapping. This provides valuable information to investigators searching for disease genes using positional cloning or other gene discovery techniques. Once the disease or syndrome has been crudely localized by genetic linkage to a particular genomic region, for example, AT to 1 lq22-23 (Gatti et al., Nature 336: 577-580 (1988)), any sequences mapping to that area may represent associated or regulatory genes for further investigation. The nucleotide sequence of the subject invention may also be used to detect differences in the chromosomal location due to translocation, inversion, etc., among normal, carrier, or affected individuals.

It shall be understood that all of the methods (e.g., PCR and FISH) that detect

polynucleotides encoding a polypeptide with ALK kinase activity (e.g., full-length ALK, mutant ALK, or an ALK fusion polynucleotides such as FNl-ALKvariant2 or NPM-ALK), may be combined with other methods that detect polypeptides with ALK kinase activity or

polynucleotides encoding a polypeptide with ALK kinase activity. For example, detection of a FNl-tmALK polynucleotide in the genetic material of a biological sample (e.g., FN1- ALKvariantl in a circulating tumor cell) may be followed by Western blotting analysis or immuno-histochemistry (IHC) analysis of the proteins of the sample to determine if the FN1- ALKvariantl polynucleotide was actually expressed as a FN1- ALKvariantl fusion polypeptide in the biological sample. Such Western blotting or IHC analyses may be performed using an antibody that specifically binds to the polypeptide encoded by the detected FN1- ALKvariantl polynucleotide, or the analyses may be performed using antibodies that specifically bind either to full length FN1 (e.g., bind to the N-terminus of the protein) or to full length ALK (e.g., bind an epitope in the kinase domain of ALK). Such assays are known in the art (see, e.g., US Patent 7,468,252).

In another example, the CISH technology of Dako allows chromatogenic in situ hybridization with immuno-histochemistry on the same tissue section. See Elliot et al., Br J Biomed Sci 2008; 65(4): 167- 171, 2008 for a comparison of CISH and FISH.

In various embodiments of the methods of the invention, the reagent is an antibody. In some embodiments, the reagent (e.g., the antibody) specifically binds to a full length ALK polypeptide or to a full length FN1 polypeptide. In some embodiments, the reagent (e.g., the antibody) specifically binds to an FN 1 -ALK fusion polypeptide and does not specifically bind to either full-length FN1 polypeptide or full-length ALK polypeptide. In some embodiments, the method is implemented in a format selected from the group consisting of a flow cytometry assay, an immunohistochemistry (IHC) assay, an immunofluorescence (IF) assay, an Enzyme-linked immunosorbent assay (ELISA) assay, and a Western blotting analysis assay. In some embodiments, the reagent is a heavy-isotope labeled (AQUA) peptide. In some embodiments, the AQUA peptide comprises an amino acid sequence comprising a fusion junction of an FNl-ALK fusion polypeptide or of an FNl-tmALK fusion polypeptide. In some embodiments, the method is implemented using mass spectrometry analysis.

In further aspects, the invention provides a method for detecting the presence of a mutant

ALK polynucleotide or an or an FNl-ALK fusion polynucleotide in a biological sample from a mammalian cancer or suspected mammalian cancer, said method comprising the steps of: (a) obtaining a biological sample from said mammalian cancer or suspected mammalian cancer; and (b) utilizing at least one reagent that specifically binds to a mutant ALK polynucleotide or to an FNl-ALK fusion polynucleotide to determine whether said mutant ALK polynucleotide or said or said FNl-ALK fusion polynucleotide is present in said biological sample, wherein specific binding of said reagent to said biological sample indicates said mutant ALK polynucleotide is present in said biological sample. In some embodiments, the mammalian cancer is mammalian ovarian cancer (e.g., from a human). In some embodiments, the mutant ALK polynucleotide is a truncated ALK polynucleotide. In some embodiments, the mutant ALK polynucleotide is an FNl- tmALK fusion polynucleotide (e.g., a FNl-ALK fusion polynucleotide encoding a polypeptide comprising an amino acid sequence of SEQ ID NOs: 3, 5, 7, 10, 11, or 12). In some

embodiments, the FNl-tmALK fusion polynucleotide comprises a nucleotide sequence of SEQ ID NOs: 4, 6, or 8. In some embodiments, the FNl-ALK fusion polynucleotide comprises a nucleotide sequence of SEQ ID NO: 13, 16, or 19. In some embodiments, the FNl-ALK fusion polynucleotide encodes a polypeptide comprising an amino acid sequence of SEQ ID NO: 14, 15, 17, 18, 20, or 21. In some embodiments, the mammalian cancer or suspected mammalian cancer is mammalian ovarian cancer or suspected mammalian ovarian cancer.

In yet further aspects, the invention provides a method for detecting the presence of a polynucleotide encoding a polypeptide with ALK kinase activity in a biological sample from a mammalian ovarian cancer or suspected mammalian ovarian cancer, said method comprising the steps of: (a) obtaining a biological sample from a mammalian ovarian cancer or suspected mammalian ovarian cancer and (b) utilizing a reagent that specifically binds to said polynucleotide encoding said polypeptide with ALK kinase activity to determine whether said polynucleotide is present in said biological sample, wherein specific binding of said reagent to said biological sample indicates said polynucleotide encoding said polypeptide with ALK kinase activity is present in said biological sample. In various embodiments, the polypeptide is aberrantly expressed full-length ALK polypeptide (e.g., aberrantly expressed in mammalian ovarian cancer or suspected mammalian ovarian cancer). In some embodiments, the polypeptide is a mutant ALK polypeptide, such as a truncated ALK polypeptide or an FNl-tmALK fusion polypeptide (e.g., comprising an amino acid sequence of SEQ ID NO: 3, 5, 7, 10, 11, or 12). In some embodiments, the polynucleotide comprises a nucleotide sequence of SEQ ID NO: 4, 6, or 8. In some embodiments, the polypeptide is an ALK fusion polypeptide (e.g., an NPM-ALK fusion polypeptide, an AL017-ALK fusion polypeptide, an TFG-ALK fusion polypeptide, an MSN- ALK fusion polypeptide, an TPM3-ALK fusion polypeptide, an TPM4-ALK fusion polypeptide, an ATIC-ALK fusion polypeptide, an MYH9-ALK fusion polypeptide, an CLTC-ALK fusion polypeptide, an SEC31L1-ALK fusion polypeptide, an RANBP2-ALK fusion polypeptide, an CARS-ALK fusion polypeptide, an EML4-ALK fusion polypeptide, an KIF5B-ALK fusion polypeptide, and an TFG-ALK fusion polypeptide). In some embodiments, the polypeptide is an FN1-ALK fusion polypeptide (e.g., comprising the amino acid sequence selected from the group consisting of SEQ ID NO: 14, 15, 17, 18, 20, or 21).

In various embodiments, the reagent used in the methods of the invention is a nucleic acid probe.

In various embodiments, the reagent used in the methods of the invention is a reagent that specifically detects the isolated polynucleotide. Thus, in further embodiments, the invention provides a reagent that specifically detects a polynucleotide disclosed herein (e.g., a

polynucleotide encoding an FN1-ALK variantl polypeptide). It should be noted that a reagent that specifically detects the isolated polynucleotide need not necessarily specifically bind to or hybridize to the polynucleotide or a nucleotide sequence complementary thereto. For example, the reagent may comprise a primer pair, where one member of the primer pair hybridizes to a nucleotide sequence located 3' to a polynucleotide disclosed herein (e.g., a polynucleotide encoding full length ALK or encoding a FNl-ALKvariantl fusion polypeptide) and the other member hybridizes to a nucleotide sequence located 3 ' to the complementary sequence of the desired polynucleotide. Using DNA polymerase (e.g., Taq DNA polymerase or DNA polymerase 1), the two primers can be extended and the polynucleotide (e.g., encoding a FNl-ALKvariantl fusion polypeptide) is amplified (e.g., duplicated in a standard DNA polymerase methods or amplified multiple times as during a PCR reaction). The amplified polynucleotide can then be detected. In some embodiments, the isolated polynucleotide or the reagent may further comprise a detectable label (e.g., a fluorescent label or an infrared label). In some embodiments, the reagent is a polymerase chain reaction (PCR) probe or a fluorescence in situ hybridization (FISH) probe. In some embodiments, the reagent comprises a detectable label. In some embodiments, the reagent is a fluorescence in-situ hybridization (FISH) probe and said method is implemented in a FISH assay. In some embodiments, the reagent is a polymerase chain reaction (PCR) probe and said method is implemented in a PCR assay.

In various embodiments of the methods of the invention, the mammalian ovarian cancer or suspected mammalian ovarian cancer is a stromal tumor or a clear cell carcinoma. In various embodiments, mammalian ovarian cancer or suspected mammalian ovarian cancer is from a human. In various embodiments, the biological sample is a circulating tumor cell from a mammalian ovarian cancer or suspected mammalian ovarian cancer.

In various embodiments of the methods of the invention, the activity of said polypeptide is detected. In various embodiments of the methods of the invention, the expression of said polypeptide is detected. In various embodiments, the mammalian ovarian cancer or suspected mammalian ovarian cancer from which the biological sample was obtained and to which the reagent specifically binds is a mammalian ovarian cancer or suspected mammalian ovarian cancer likely to respond to an ALK-inhibiting therapeutic. One non-limiting example of an ALK- inhibiting therapeutic is crizotinib (also known as PF-02341066). Additional non- limiting examples of ALK-inhibiting therapeutics include NVT TAE-684, AP26113, CEP- 14083, CEP- 14513, CEP11988, WHI-P131 and WHI-P154.

In various embodiments, the patient from whom said biological sample is obtained, where the reagent specifically binds to the biological sample, is diagnosed as having a mammalian ovarian cancer or suspected mammalian ovarian cancer driven by mutant ALK polynucleotide or mutant ALK polypeptide, or is diagnosed as having a mammalian ovarian cancer or suspected mammalian ovarian cancer driven by aberrant expression of a polypeptide with ALK activity.

In yet another aspect, the invention provides a method for determining whether a compound inhibits the progression of a mammalian cancer characterized by the expression of a mutant ALK polynucleotide or a FN 1 -ALK polynucleotide, said method comprising the step of determining whether said compound inhibits the expression of said mutant ALK polynucleotide as a mutant ALK polypeptide or inhibits the expression of said FN 1 -ALK polynucleotide as a FN1- ALK polypeptide in said cancer. In another aspect, the invention provides a method for determining whether a compound inhibits the progression of a mammalian cancer characterized by the expression of a mutant ALK polypeptide or an FN 1 -ALK polypeptide, said method comprising the step of determining whether said compound inhibits the expression and/or activity of said mutant ALK polypeptide or said FN 1 -ALK polypeptide in said cancer. In various embodiments, the mutant ALK polypeptide is an FNl-ALKvariantl polypeptide, an FN1- ALKvariant3 polypeptide, an FNl-ALKvariant5 polypeptide, or a truncated ALK polypeptide. In various embodiments, the FN 1 -ALK polypeptide is an FNl-ALKvariant2 polypeptide, an FN1- ALKvariant4 polypeptide, or an FNl-ALKvariant6 polypeptide.

In another aspect, the invention provides a method for inhibiting the progression of a mammalian cancer or suspected mammalian cancer that expresses a mutant ALK polypeptide or an FN 1 -ALK polypeptide, said method comprising the step of inhibiting the expression and/or activity of said polypeptide in said mammalian ovarian cancer or suspected mammalian ovarian cancer by treating said mammalian cancer or suspected mammalian cancer with an effective amount of an ALK inhibitor. In some embodiments, the cancer is from a human.

In further aspects, the invention provides a method for determining whether a compound inhibits the progression of a mammalian ovarian cancer or suspected mammalian ovarian cancer characterized by the expression of a polypeptide with ALK activity, said method comprising the step of determining whether said compound inhibits the expression of said polypeptide in said cancer. In another aspect, the invention provides a method for inhibiting the progression of a mammalian cancer or suspected mammalian cancer characterized by the expression of a polypeptide with ALK activity, said method comprising the step of inhibiting the expression and/or activity of said polypeptide in said mammalian ovarian cancer or suspected mammalian ovarian cancer by treating said mammalian cancer or suspected mammalian cancer with an effective amount of an ALK inhibitor. In some embodiments, the cancer is from a human.

In various embodiments, the polypeptide with ALK activity is aberrantly expressed full length ALK polypeptide, an FNl-tm ALK polypeptide, a truncated ALK polypeptide, or an ALK fusion polypeptide (e.g., an NPM-ALK fusion polypeptide, an AL017-ALK fusion polypeptide, an TFG-ALK fusion polypeptide, an MSN-ALK fusion polypeptide, an TPM3-ALK fusion polypeptide, an TPM4-ALK fusion polypeptide, an ATIC-ALK fusion polypeptide, an MYH9- ALK fusion polypeptide, an CLTC-ALK fusion polypeptide, an SEC31L1-ALK fusion polypeptide, an RANBP2-ALK fusion polypeptide, an CARS-ALK fusion polypeptide, an EML4- ALK fusion polypeptide, an KIF5B-ALK fusion polypeptide, and an TFG-ALK fusion polypeptide).

In various embodiments, the inhibition is determined using at least one reagent selected from the group consisting of a reagent that specifically binds to a polynucleotide disclosed herein, a reagent that specifically binds to polypeptide disclosed herein, a reagent that specifically binds to a full length ALK polynucleotide, a reagent that specifically binds to a full length ALK

polypeptide, a reagent that specifically binds to a full length FN1 polynucleotide, and a reagent that specifically binds to a full length FN1 polypeptide.

In various embodiments, the expression and/or activity of said polypeptide is inhibited with a composition comprising a therapeutic selected from the group consisting of crizotinib (also known as PF-02341066), NVT TAE-684, AP261 13, CEP-14083, CEP-14513, CEP1 1988, WHI- P131 and WHI-P154.

As used herein, a "ALK inhibitor" or a "ALK-inhibiting compound" means any composition comprising one or more compounds, chemical or biological, which inhibits, either directly or indirectly, the expression and/or activity of a polypeptide with ALK kinase activity. Such inhibition may be in vitro or in vivo. "ALK inhibitor therapeutic" or "ALK-inhibiting therapeutic" means a ALK-inhibiting compound used as a therapeutic to treat a patient harboring a cancer (e.g. , a liver, testicular, kidney, or pancreatic cancer) characterized by the presence of a polypeptide with ALK kinase activity such as a mutant ALK (e.g., an FNl-tmALK fusion polypeptide) or an ALK fusion polypeptide such as one of the FN 1 -ALK fusion polypeptides disclosed herein.

In some embodiments of the invention, the ALK inhibitor is a binding agent that specifically binds to a FNl-tmALK or a FN 1 -ALK fusion polypeptide, a binding agent that specifically binds to a mutant ALK polypeptide, an siRNA targeting a FNl -tmALK fusion polynucleotide (e.g., a FNl-ALKvariantl fusion polynucleotide) or an FN1-ALK fusion polynucleotide (e.g., a FNl-ALKvariant6 fusion polynucleotide), or an siRNA targeting a mutant ALK polynucleotide.

The ALK-inhibiting compound may be, for example, a kinase inhibitor, such as a small molecule or antibody inhibitor. It may be a pan-kinase inhibitor with activity against several different kinases, or a kinase-specific inhibitor. Since ALK, ROS, LTK, InsR, and IGFIR belong to the same family of tyrosine kinases, they may share similar structure in the kinase domain. Thus, in some embodiments, an ALK inhibitor also inhibits the activity of an ALK kinase an LTK kinase, an insulin receptor, or an IGF1 receptor. ALK-inhibiting compounds are discussed in further detail below. Patient biological samples may be taken before and after treatment with the inhibitor and then analyzed, using methods described above, for the biological effect of the inhibitor on ALK kinase activity, including the phosphorylation of downstream substrate protein. Such a pharmacodynamic assay may be useful in determining the biologically active dose of the drug that may be preferable to a maximal tolerable dose. Such information would also be useful in submissions for drug approval by demonstrating the mechanism of drug action.

In accordance with the present invention, the polypeptide with ALK kinase activity (e.g., an FN 1 -ALK fusion polypeptide or an FNl-tmALK fusion polypeptide) may occur in at least one subgroup of human ovarian cancer. Accordingly, the progression of a mammalian cancer in which a polypeptide with ALK kinase activity is expressed may be inhibited, in vivo, by inhibiting the activity of ALK kinase in such cancer. ALK activity in cancers characterized by expression of a polypeptide with ALK kinase activity (e.g., an FN1-ALK fusion polypeptide or a mutant ALK polypeptide such as an FNl-tmALK fusion or a truncated ALK) may be inhibited by contacting the cancer {e.g., a tumor) with a therapeutically effective amount of an ALK-inhibiting

therapeutic. Accordingly, the invention provides, in part, a method for inhibiting the progression of polypeptide with ALK kinase activity -expressing ovarian cancer by inhibiting the expression and/or activity of ALK kinase in the ovarian cancer by contacting the cancer {e.g., a tumor) with a therapeutically effective amount of an ALK-inhibiting therapeutic. Similarly, the invention provides, in part, a method for inhibiting the progression of a mutant ALK or a FN 1 -ALK fusion- expressing cancer by inhibiting the expression and/or activity of ALK kinase in the cancer by contacting the cancer {e.g., a tumor) with a therapeutically effective amount of an ALK-inhibiting therapeutic.

As used herein, by "therapeutically effective amount" or "pharmaceutically effective amount" is mean an amount of an ALK-inhibiting therapeutic that is adequate to inhibit the cancer (or cell thereof) or suspected cancer (or cells thereof), as compared to an untreated cancer or suspected cancer, by either slowing the growth of the cancer or suspected cancer, reducing the mass of the cancer or suspected cancer, reducing the number of cells of the cancer or suspected cancer, or killing the cancer.

An ALK-inhibiting therapeutic may be any composition comprising at least one ALK inhibitor. Such compositions also include compositions comprising only a single ALK- inhibiting compound, as well as compositions comprising multiple therapeutics (including those against other RTKs), which may also include a non-specific therapeutic agent like a chemotherapeutic agent or general transcription inhibitor.

In some embodiments, an ALK-inhibiting therapeutic useful in the practice of the methods disclosed herein is a targeted, small molecule inhibitor. Small molecule targeted inhibitors are a class of molecules that typically inhibit the activity of their target enzyme by specifically, and often irreversibly, binding to the catalytic site of the enzyme, and/or binding to an ATP -binding cleft or other binding site within the enzyme that prevents the enzyme from adopting a conformation necessary for its activity. An exemplary small-molecule targeted kinase inhibitor is Pfizer, Inc.'s compound Crizotinib (also known as PF-02341066), which inhibits ALK kinase activity, and its properties have been well described. See You et al, Cancer Res 67: 4408 (2007) and U.S. Patent Pub. No. 2008/0300273. Additional small molecule kinase inhibitors that may target ALK include TAE-684 (from Novartis), AP26113 (Ariad Pharmaceuticals, Inc.), and CEP- 14083, CEP-14513, and CEP-11988 (Cephalon; see Wan et al, Blood 107: 1617-1623, 2006).

PF-02341066 has the structure:

TAE-684, a 5-chloro-2,4-diaminophenylpyrimidine, has the structure:

and has been shown to inhibit the ALK kinase. Galkin, et al., Proc. National Acad. Sci 104(1) 270- 275, 2007.

Additional small molecule inhibitors and other inhibitors (e.g. , indirect inhibitors) of ALK kinase activity may be rationally designed using X-ray crystallographic or computer modeling of ALK three dimensional structure, or may found by high throughput screening of compound libraries for inhibition of key upstream regulatory enzymes and/or necessary binding molecules, which results in inhibition of ALK kinase activity. Such approaches are well known in the art, and have been described. ALK inhibition by such therapeutics may be confirmed, for example, by examining the ability of the compound to inhibit ALK activity, but not other kinase activity, in a panel of kinases, and/or by examining the inhibition of ALK activity in a biological sample comprising cancer cells (e.g., ovarian cancer cells). Methods for identifying compounds that inhibit a cancer characterized by the expression/presence of polypeptide with ALK kinase activity, are further described below.

ALK-inhibiting therapeutics useful in the methods disclosed herein may also be targeted antibodies that specifically bind to critical catalytic or binding sites or domains required for ALK activity, and inhibit the kinase by blocking access of ligands, substrates or secondary molecules to a and/or preventing the enzyme from adopting a conformation necessary for its activity. The production, screening, and therapeutic use of humanized target-specific antibodies has been well- described. See Merluzzi et al., Adv Clin Path. 4(2): 77-85 (2000). Commercial technologies and systems, such as Morphosys, Inc.'s Human Combinatorial Antibody Library (HuCAL®), for the high-throughput generation and screening of humanized target-specific inhibiting antibodies are available. The production of various anti-receptor kinase targeted antibodies and their use to inhibit activity of the targeted receptor has been described. See, e.g. U.S. Patent Publication No.

20040202655, U.S. Patent Publication No. 20040086503, U.S. Patent Publication No.

20040033543, Standardized methods for producing, and using, receptor tyrosine kinase activity- inhibiting antibodies are known in the art. See, e.g., European Patent No. EP1423428,

Phage display approaches may also be employed to generate ALK-specific antibody inhibitors, and protocols for bacteriophage library construction and selection of recombinant antibodies are provided in the well-known reference text CURRENT PROTOCOLS IN IMMUNOLOGY, Colligan et al. (Eds.), John Wiley & Sons, Inc. (1992-2000), Chapter 17, Section 17.1. See also U.S. Patent No. 6,319,690, U.S. Patent No. 6,300,064, U.S. Patent No. 5,840,479, and U.S. Patent Publication No. 20030219839.

A library of antibody fragments displayed on the surface of bacteriophages may be produced {see, e.g. U. S. Patent 6,300,064) and screened for binding to a polypeptide with ALK kinase activity such as the FN 1 -ALK fusions and FNl-tmALK fusions. An antibody fragment that binds to a FN1-ALK fusion polypeptide (e.g., a FNl-ALKvariant2 fusion polypeptide) or an FNl-tmALK fusion polypeptide (e.g., FNl-ALKvariantl fusion polypeptide) is identified as a candidate molecule for blocking constitutive activation of that fusion polypeptide in a cell. See European Patent No. EP1423428.

ALK-binding targeted antibodies identified in screening of antibody libraries as describe above may then be further screened for their ability to block the activity of ALK, both in vitro kinase assay and in vivo in cell lines and/or tumors. ALK inhibition may be confirmed, for example, by examining the ability of such antibody therapeutic to inhibit ALK kinase activity in a panel of kinases, and/or by examining the inhibition of ALK activity in a biological sample comprising cancer cells, as described above. In some embodiments, a ALK-inhibiting compound reduces ALK kinase activity, but reduces the kinase activity of other kinases to a lesser extent (or not at all). Methods for screening such compounds for ALK kinase inhibition are further described above.

ALK-inhibiting compounds that useful in the practice of the disclosed methods may also be compounds that indirectly inhibit ALK activity by inhibiting the activity of proteins or molecules other than ALK kinase itself. Such inhibiting therapeutics may be targeted inhibitors that modulate the activity of key regulatory kinases that phosphorylate or de-phosphorylate (and hence activate or deactivate) ALK itself, or interfere with binding of ligands. As with other receptor tyrosine kinases, ALK regulates downstream signaling through a network of adaptor proteins and downstream kinases. As a result, induction of cell growth and survival by ALK activity may be inhibited by targeting these interacting or downstream proteins.

ALK kinase activity may also be indirectly inhibited by using a compound that inhibits the binding of an activating molecule necessary for full length ALK, an ALK fusion polypeptide (e.g., an FN1-ALK fusion polypeptide), or mutant ALK (e.g., a truncated ALK polypeptide or an FN1- tmALK fusion polypeptide) to adopt its active conformation. For example, the production and use of anti-PDGF antibodies has been described. See U.S. Patent Publication No. 20030219839, "Anti-PDGF Antibodies and Methods for Producing Engineered Antibodies," Bowdish et al. Inhibition of ligand (PDGF) binding to the receptor directly down-regulates the receptor activity.

ALK inhibiting compounds or therapeutics may also comprise anti-sense and/or transcription inhibiting compounds that inhibit ALK kinase activity by blocking transcription of the gene encoding ALK, an FN 1 -ALK fusion-encoding gene, or a mutant ALK-encoding gene. The inhibition of various receptor kinases, including VEGFR, EGFR, and IGFR, and FGFR, by antisense therapeutics for the treatment of cancer has been described. See, e.g., U.S. Patent Nos. 6,734,017; 6, 710,174, 6,617,162; 6,340,674; 5,783,683; 5,610,288.

Antisense oligonucleotides may be designed, constructed, and employed as therapeutic agents against target genes in accordance with known techniques. See, e.g. Cohen, J., Trends in Pharmacol. Sci. 10(11): 435-437 (1989); Marcus-Sekura, Anal. Biochem. 172: 289-295 (1988); Weintraub, H., Sci. AM. pp. 40-46 (1990); Van Der Krol et al., BioTechniques 6(10): 958-976 (1988); Skorski et al, Proc. Natl. Acad. Sci. USA (1994) 91: 4504-4508. Inhibition of human carcinoma growth in vivo using an antisense RNA inhibitor of EGFR has recently been described. See U.S. Patent Publication No. 20040047847. Similarly, an ALK-inhibiting therapeutic comprising at least one antisense oligonucleotide against a mammalian ALK gene, FN 1 -ALK fusion polynucleotide or mutant ALK polynucleotide may be prepared according to methods described above. Pharmaceutical compositions comprising ALK-inhibiting antisense compounds may be prepared and administered as further described below.

Small interfering RNA molecule (siRNA) compositions, which inhibit translation, and hence activity, of ALK through the process of RNA interference, may also be desirably employed in the methods disclosed herein. RNA interference, and the selective silencing of target protein expression by introduction of exogenous small double-stranded RNA molecules comprising sequence complimentary to mRNA encoding the target protein, has been well described. See, e.g. U.S. Patent Publication No. 20040038921, U.S. Patent Publication No. 20020086356, and U.S.

Patent Publication 20040229266.

Double-stranded RNA molecules (dsRNA) have been shown to block gene expression in a highly conserved regulatory mechanism known as RNA interference (RNAi). Briefly, the RNAse III Dicer processes dsRNA into small interfering RNAs (siRNA) of approximately 22 nucleotides, which serve as guide sequences to induce target-specific mRNA cleavage by an RNA-induced silencing complex RISC (see Hammond et al., Nature (2000) 404: 293-296). RNAi involves a catalytic-type reaction whereby new siRNAs are generated through successive cleavage of longer dsRNA. Thus, unlike antisense, RNAi degrades target RNA in a non-stoichiometric manner. When administered to a cell or organism, exogenous dsRNA has been shown to direct the sequence-specific degradation of endogenous messenger RNA (mRNA) through RNAi.

A wide variety of target-specific siRNA products, including vectors and systems for their expression and use in mammalian cells, are now commercially available. See, e.g., Promega, Inc.

(www.promega.com); Dharmacon, Inc. (www. dharmacon. com) . Detailed technical manuals on the design, construction, and use of dsRNA for RNAi are available. See, e.g., Dharmacon' s

"RNAi Technical Reference & Application Guide"; Promega's "RNAi: A Guide to Gene

Silencing." ALK-inhibiting siRNA products are also commercially available, and may be suitably employed in the methods disclosed herein. See, e.g., Dharmacon, Inc., Lafayette, CO (Cat Nos.

M-003162-03, MU-003162-03, D-003162-07 thru -10 (siGENOME™ SMARTselection and SMARTpool® siRNAs).

It has recently been established that small dsRNA less than 49 nucleotides in length, and preferably 19-25 nucleotides, comprising at least one sequence that is substantially identical to part of a target mRNA sequence, and which dsRNA optimally has at least one overhang of 1-4 nucleotides at an end, are most effective in mediating RNAi in mammals. See U.S. Patent Publication Nos. 20040038921 and 20040229266. The construction of such dsRNA, and their use in pharmaceutical preparations to silence expression of a target protein, in vivo, are described in detail in such publications.

If the sequence of the gene to be targeted in a mammal is known, 21-23 nt RNAs, for example, can be produced and tested for their ability to mediate RNAi in a mammalian cell, such as a human or other primate cell. Those 21-23 nt RNA molecules shown to mediate RNAi can be tested, if desired, in an appropriate animal model to further assess their in vivo effectiveness.

Target sites that are known, for example target sites determined to be effective target sites based on studies with other nucleic acid molecules, for example ribozymes or antisense, or those targets known to be associated with a disease or condition such as those sites containing mutations or deletions, can be used to design siRNA molecules targeting those sites as well.

Alternatively, the sequences of effective dsRNA can be rationally designed/predicted screening the target mRNA of interest for target sites, for example by using a computer folding algorithm. The target sequence can be parsed in silico into a list of all fragments or subsequences of a particular length, for example 23 nucleotide fragments, using a custom Perl script or commercial sequence analysis programs such as Oligo, MacVector, or the GCG Wisconsin Package.

Various parameters can be used to determine which sites are the most suitable target sites within the target RNA sequence. These parameters include but are not limited to secondary or tertiary RNA structure, the nucleotide base composition of the target sequence, the degree of homology between various regions of the target sequence, or the relative position of the target sequence within the RNA transcript. Based on these determinations, any number of target sites within the RNA transcript can be chosen to screen siRNA molecules for efficacy, for example by using in vitro RNA cleavage assays, cell culture, or animal models. See, e.g., U.S. Patent Publication No. 20030170891. An algorithm for identifying and selecting RNAi target sites has also recently been described. See U.S. Patent Publication No. 20040236517.

Commonly used gene transfer techniques include calcium phosphate, DEAE-dextran, electroporation and microinjection and viral methods (Graham et al. (1973) Virol. 52: 456;

McCutchan et al, (1968), J. Natl. Cancer Inst. 41: 351; Chu et al. (1987), Nucl. Acids Res. 15: 1311; Fraley et al. (1980), J. Biol. Chem. 255: 10431; Capecchi (1980), Cell 22: 479). DNA may also be introduced into cells using cationic liposomes (Feigner et al. (1987), Proc. Natl. Acad. Sci USA 84: 7413). Commercially available cationic lipid formulations include Tfx 50 (Promega) or Lipofectamin 200 (Life Technologies). Alternatively, viral vectors may be employed to deliver dsRNA to a cell and mediate RNAi. See U.S Patent Publication No. 20040023390.

Trans fection and vector/expression systems for RNAi in mammalian cells are

commercially available and have been well described. See, e.g., Dharmacon, Inc.,

DharmaFECT™ system; Promega, Inc., siSTRIKE™ U6 Hairpin system; see also Gou et al. (2003) FEBS. 548, 113-118; Sui, G. et al. A DNA vector-based RNAi technology to suppress gene expression in mammalian cells (2002) Proc. Natl. Acad. Sci. 99, 5515-5520; Yu et al. (2002) Proc. Natl. Acad. Sci. 99, 6047-6052; Paul, C. et al. (2002) Nature Biotechnology 19, 505- 508; McManus et al. (2002) RNA 8, 842-850.

siRNA interference in a mammal using prepared dsRNA molecules may then be effected by administering a pharmaceutical preparation comprising the dsRNA to the mammal. The pharmaceutical composition is administered in a dosage sufficient to inhibit expression of the target gene. dsRNA can typically be administered at a dosage of less than 5 mg dsRNA per kilogram body weight per day, and is sufficient to inhibit or completely suppress expression of the target gene. In general a suitable dose of dsRNA will be in the range of 0.01 to 2.5 milligrams per kilogram body weight of the recipient per day, preferably in the range of 0.1 to 200 micrograms per kilogram body weight per day, more preferably in the range of 0.1 to 100 micrograms per kilogram body weight per day, even more preferably in the range of 1.0 to 50 micrograms per kilogram body weight per day, and most preferably in the range of 1.0 to 25 micrograms per kilogram body weight per day. A pharmaceutical composition comprising the dsRNA is administered once daily, or in multiple sub-doses, for example, using sustained release

formulations well known in the art. The preparation and administration of such pharmaceutical compositions may be carried out accordingly to standard techniques, as further described below.

Such dsRNA may then be used to inhibit ALK expression and activity in a cancer, by preparing a pharmaceutical preparation comprising a therapeutically-effective amount of such dsRNA, as described above, and administering the preparation to a human subject having a cancer (e.g., a liver, pancreatic, kidney, or testicular cancer) expressing FN 1 -ALK fusion protein or mutant ALK polypeptide (such as, for example, aberrant expression of full length ALK), for example, via direct injection to the tumor. The similar inhibition of other receptor tyrosine kinases, such as VEGFR and EGFR using siRNA inhibitors has recently been described. See U.S. Patent Publication No. 20040209832, U.S. Patent Publication No. 20030170891, and U.S. Patent Publication No. 20040175703.

ALK-inhibiting therapeutic compositions useful in the practice of the methods disclosed herein may be administered to a mammal by any means known in the art including, but not limited to oral or peritoneal routes, including intravenous, intramuscular, intraperitoneal, subcutaneous, transdermal, airway (aerosol), rectal, vaginal and topical (including buccal and sublingual) administration.

For oral administration, an ALK-inhibiting therapeutic will generally be provided in the form of tablets or capsules, as a powder or granules, or as an aqueous solution or suspension. Tablets for oral use may include the active ingredients mixed with pharmaceutically acceptable carriers and excipients such as inert diluents, disintegrating agents, binding agents, lubricating agents, sweetening agents, flavoring agents, coloring agents and preservatives. Suitable inert diluents include sodium and calcium carbonate, sodium and calcium phosphate, and lactose, while corn starch and alginic acid are suitable disintegrating agents. Binding agents may include starch and gelatin, while the lubricating agent, if present, will generally be magnesium stearate, stearic acid or talc. If desired, the tablets may be coated with a material such as glyceryl monostearate or glyceryl distearate, to delay absorption in the gastrointestinal tract.

Capsules for oral use include hard gelatin capsules in which the active ingredient is mixed with a solid diluent, and soft gelatin capsules wherein the active ingredients is mixed with water or an oil such as peanut oil, liquid paraffin or olive oil. For intramuscular, intraperitoneal, subcutaneous and intravenous use, the pharmaceutical compositions disclosed herein will generally be provided in sterile aqueous solutions or suspensions, buffered to an appropriate pH and isotonicity. Suitable aqueous vehicles include Ringer's solution and isotonic sodium chloride. The carrier may consist exclusively of an aqueous buffer ("exclusively" means no auxiliary agents or encapsulating substances are present which might affect or mediate uptake of the ALK- inhibiting therapeutic). Such substances include, for example, micellar structures, such as liposomes or capsids, as described below. Aqueous suspensions may include suspending agents such as cellulose derivatives, sodium alginate, polyvinyl-pyrrolidone and gum tragacanth, and a wetting agent such as lecithin. Suitable preservatives for aqueous suspensions include ethyl and n- propyl p-hydroxybenzoate.

ALK-inhibiting therapeutic compositions may also include encapsulated formulations to protect the therapeutic (e.g., a dsRNA compound or an antibody that specifically binds a FN1- ALK fusion polypeptide) against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,81 1 ; PCT publication WO 91/06309; and European patent publication EP-A-43075. An encapsulated formulation may comprise a viral coat protein. The viral coat protein may be derived from or associated with a virus, such as a polyoma virus, or it may be partially or entirely artificial. For example, the coat protein may be a Virus Protein 1 and/or Virus Protein 2 of the polyoma virus, or a derivative thereof.

ALK-inhibiting compounds can also comprise a delivery vehicle, including liposomes, for administration to a subject, carriers and diluents and their salts, and/or can be present in pharmaceutically acceptable formulations. For example, methods for the delivery of nucleic acid molecules are described in Akhtar et al, 1992, Trends Cell Bio., 2, 139; DELIVERY STRATEGIES FOR ANTISENSE OLIGONUCLEOTIDE THERAPEUTICS, ed. Akbtar, 1995, Maurer et al, 1999, Mol. Membr. Biol, 16, 129-140; Hofland and Huang, 1999, Handb. Exp. Pharmacol, 137, 165-192; and Lee et al, 2000, ACS Symp. Ser., 752, 184-192. U.S. Pat. No. 6,395,713 and PCT

Publication No. WO 94/02595 further describe the general methods for delivery of nucleic acid molecules. These protocols can be utilized for the delivery of virtually any nucleic acid molecule.

ALK-inhibiting therapeutics (i.e., a ALK-inhibiting compound being administered as a therapeutic) can be administered to a mammalian tumor by a variety of methods known to those of skill in the art, including, but not restricted to, encapsulation in liposomes, by iontophoresis, or by incorporation into other vehicles, such as hydrogels, cyclodextrins, biodegradable nanocapsules, and bioadhesive microspheres, or by proteinaceous vectors (see PCT Publication No. WO

00/53722). Alternatively, the therapeutic/vehicle combination is locally delivered by direct injection or by use of an infusion pump. Direct injection of the composition, whether

subcutaneous, intramuscular, or intradermal, can take place using standard needle and syringe methodologies, or by needle-free technologies such as those described in Corny et al, 1999, Clin. Cancer Res., 5, 2330-2337 and PCT Publication No. WO 99/3 1262.

Pharmaceutically acceptable formulations of ALK-inhibitor therapeutics include salts of the above described compounds, e.g., acid addition salts, for example, salts of hydrochloric, hydrobromic, acetic acid, and benzene sulfonic acid. A pharmacological composition or formulation refers to a composition or formulation in a form suitable for administration, e.g., systemic administration, into a cell or patient, including for example a human. Suitable forms, in part, depend upon the use or the route of entry, for example oral, transdermal, or by injection. Such forms should not prevent the composition or formulation from reaching a target cell. For example, pharmacological compositions injected into the blood stream should be soluble. Other factors are known in the art, and include considerations such as toxicity and forms that prevent the composition or formulation from exerting its effect.

Administration routes that lead to systemic absorption (e.g. , systemic absorption or accumulation of drugs in the blood stream followed by distribution throughout the entire body), are desirable and include, without limitation: intravenous, subcutaneous, intraperitoneal, inhalation, oral, intrapulmonary and intramuscular. Each of these administration routes exposes the ALK-inhibiting therapeutic to an accessible diseased tissue or tumor. The rate of entry of a drug into the circulation has been shown to be a function of molecular weight or size. The use of a liposome or other drug carrier comprising the compounds of the instant invention can potentially localize the drug, for example, in certain tissue types, such as the tissues of the reticular endothelial system (RES). A liposome formulation that can facilitate the association of drug with the surface of cells, such as, lymphocytes and macrophages is also useful. This approach can provide enhanced delivery of the drug to target cells by taking advantage of the specificity of macrophage and lymphocyte immune recognition of abnormal cells, such as cancer cells.

By "pharmaceutically acceptable formulation" is meant, a composition or formulation that allows for the effective distribution of the nucleic acid molecules of the instant invention in the physical location most suitable for their desired activity. Nonlimiting examples of agents suitable for formulation with the nucleic acid molecules of the instant invention include: P-glycoprotein inhibitors (such as Pluronic P85), which can enhance entry of drugs into the CNS (Jolliet-Riant and Tillement, 1999, Fundam. Clin. Pharmacol., 13, 16-26); biodegradable polymers, such as poly (DL-lactide-coglycolide) microspheres for sustained release delivery after intracerebral implantation (Emerich et al, 1999, Cell Transplant, 8, 47-58) (Alkermes, Inc. Cambridge, Mass.); and loaded nanoparticles, such as those made of polybutylcyanoacrylate, which can deliver drugs across the blood brain barrier and can alter neuronal uptake mechanisms (Prog Neuro- psychopharmacol Biol Psychiatry, 23, 941-949, 1999). Other non-limiting examples of delivery strategies for the ALK-inhibiting compounds useful in the methods disclosed hereinn include material described in Boado et al, 1998, J. Pharm. Sci., 87, 1308-1315; Tyler et al., 1999, FEBS Lett., 421, 280-284; Pardridge et al, 1995, PNAS USA., 92, 5592-5596; Boado, 1995, Adv. Drug Delivery Rev., 15, 73-107; Aldrian-Herrada et al, 1998, Nucleic Acids Res., 26, 4910-4916; and Tyler et al, 1999, PNAS USA., 96, 7053-7058.

Therapeutic compositions comprising surface-modified liposomes containing poly

(ethylene glycol) lipids (PEG-modified, or long-circulating liposomes or stealth liposomes) may also be suitably employed in the methods disclosed herein. These formulations offer a method for increasing the accumulation of drugs in target tissues. This class of drug carriers resists opsonization and elimination by the mononuclear phagocytic system (MPS or RES), thereby enabling longer blood circulation times and enhanced tissue exposure for the encapsulated drug (Lasic et al. Chem. Rev. 1995, 95, 2601-2627; Ishiwata et al, Chem. Pharm. Bull. 1995, 43, 1005- 1011). Such liposomes have been shown to accumulate selectively in tumors, presumably by extravasation and capture in the neovascularized target tissues (Lasic et al., Science 1995, 267, 1275-1276; Oku et al., 1995, Biochim. Biophys. Acta, 1238, 86-90). The long-circulating liposomes enhance the pharmacokinetics and pharmacodynamics of DNA and RNA, particularly compared to conventional cationic liposomes which are known to accumulate in tissues of the

MPS (Liu et al, J. Biol. Chem. 1995, 42, 24864-24870; PCT Publication No. WO 96/10391; PCT Publication No. WO 96/10390; and PCT Publication No. WO 96/10392). Long-circulating liposomes are also likely to protect drugs from nuclease degradation to a greater extent compared to cationic liposomes, based on their ability to avoid accumulation in metabolically aggressive MPS tissues such as the liver and spleen.

Therapeutic compositions may include a pharmaceutically effective amount of the desired compounds in a pharmaceutically acceptable carrier or diluent. Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art, and are described, for example, in REMINGTON'S PHARMACEUTICAL SCIENCES, Mack Publishing Co. (A. R. Gennaro edit. 1985). For example, preservatives, stabilizers, dyes and flavoring agents can be provided. These include sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid. In addition, antioxidants and suspending agents can be used.

A pharmaceutically effective dose is that dose required to prevent, inhibit the occurrence, or treat (alleviate a symptom to some extent, preferably all of the symptoms) of a disease state. The pharmaceutically effective dose depends on the type of disease, the composition used, the route of administration, the type of mammal being treated, the physical characteristics of the specific mammal under consideration, concurrent medication, and other factors that those skilled in the medical arts will recognize. Generally, an amount between 0.1 mg/kg and 100 mg/kg body weight/day of active ingredients is administered dependent upon potency of the negatively charged polymer.

Dosage levels of the order of from about 0.1 mg to about 140 mg per kilogram of body weight per day are useful in the treatment of the above-indicated conditions (about 0.5 mg to about 7 g per patient per day). The amount of active ingredient that can be combined with the carrier materials to produce a single dosage form varies depending upon the host treated and the

particular mode of administration. Dosage unit forms generally contain between from about 1 mg to about 500 mg of an active ingredient. It is understood that the specific dose level for any particular patient depends upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, route of administration, and rate of excretion, drug combination and the severity of the particular disease undergoing therapy.

For administration to non-human animals, the composition can also be added to the animal feed or drinking water. It can be convenient to formulate the animal feed and drinking water compositions so that the animal takes in a therapeutically appropriate quantity of the composition along with its diet. It can also be convenient to present the composition as a premix for addition to the feed or drinking water.

An ALK-inhibiting therapeutic useful in the practice of the various methods described herein may comprise a single compound as described above, or a combination of multiple compounds, whether in the same class of inhibitor (e.g., antibody inhibitor), or in different classes (e.g., antibody inhibitors and small-molecule inhibitors). Such combination of compounds may increase the overall therapeutic effect in inhibiting the progression of a fusion protein-expressing cancer. For example, the therapeutic composition may a small molecule inhibitor, such as Crizotinib (also known as PF- 02341066) produced by Pfizer, Inc. (see U.S. Pub. No. 2008/0300273) alone, or in combination with other Crizotinib analogues targeting ALK activity and/or small molecule inhibitors of ALK, such as NVP-TAE684 produced by Novartis, Inc. The therapeutic composition may also comprise one or more non-specific chemotherapeutic agent in addition to one or more targeted inhibitors. Such combinations have recently been shown to provide a synergistic tumor killing effect in many cancers. The effectiveness of such combinations in inhibiting ALK activity and tumor growth in vivo can be assessed as described below.

The invention also provides, in part, a method for determining whether a compound inhibits the progression of a cancer (e.g., a ovarian cancer) characterized by a polypeptide with ALK kinase activity or polypeptide encoding the same by determining whether the compound inhibits the ALK kinase activity of the polypeptide in the cancer. In some embodiments, inhibition of activity of ALK is determined by examining a biological sample comprising cells from bone marrow, blood, or a tumor. In another embodiment, inhibition of activity of ALK is determined using at least one mutant ALK polynucleotide or polypeptide-specific reagent.

The tested compound may be any type of therapeutic or composition as described above. Methods for assessing the efficacy of a compound, both in vitro and in vivo, are well established and known in the art. For example, a composition may be tested for ability to inhibit ALK in vitro using a cell or cell extract in which ALK kinase is activated. A panel of compounds may be employed to test the specificity of the compound for ALK (as opposed to other targets, such as EGFR or PDGFR).

Another technique for drug screening which may be used provides for high throughput screening of compounds having suitable binding affinity to a protein of interest, as described in PCT Publication No. WO 84/03564. In this method, as applied to FN 1 -ALK fusion polypeptides ant mutant ALK polypeptides, large numbers of different small test compounds are synthesized on a solid substrate, such as plastic pins or some other surface. The test compounds are reacted with a polypeptide, or fragments thereof, and washed. Bound polypeptide {e.g., FNl-ALKvariantl, FN1- ALKvariant2, FN 1 -ALKvariant3 , FN 1 -ALKvariant4, FN 1 -ALKvariant5 , or FN 1 -ALKvariant6 fusion polypeptides or truncated ALK polypeptide) is then detected by methods well known in the art. A purified polypeptide can also be coated directly onto plates for use in the aforementioned drug screening techniques. Alternatively, non-neutralizing antibodies can be used to capture the peptide and immobilize it on a solid support.

A compound found to be an effective inhibitor of ALK activity in vitro may then be examined for its ability to inhibit the progression of a cancer expressing a polypeptide with kinase activity (such as ovarian cancer or other cancer such as a liver cancer, lung cancer, colon cancer, kidney cancer, or a pancreatic cancer), in vivo, using, for example, mammalian xenografts harboring human ovarian, liver, pancreatic, kidney, lung, or colon tumors that are express a polypeptide with kinase activity (e.g., a polypeptide resulting from the translocation of an FN1 gene and an ALK gene). In this procedure, cancer cell lines known to express a FN 1 -ALK fusion protein (e.g., a FN 1 -ALK variant2, FNl-ALKvariant4, or a FNl-ALKvariant6), a FNl-tmALK fusion protein (e.g., a FNl-ALKvariantl, FNl-tmALKvariant3, or a FNl-tmALKvariant5), or a truncated ALK protein may be placed subcutaneously in an animal (e.g., into a nude or SCID mouse, or other immune-compromised animal). The cells then grow into a tumor mass that may be visually monitored. The animal may then be treated with the drug. The effect of the drug treatment on tumor size may be externally observed. The animal is then sacrificed and the tumor removed for analysis by IHC and Western blot. Similarly, mammalian bone marrow transplants may be prepared, by standard methods, to examine drug response in hematological tumors expressing a mutant ALK kinase. In this way, the effects of the drug may be observed in a biological setting most closely resembling a patient. The drug's ability to alter signaling in the tumor cells or surrounding stromal cells may be determined by analysis with phosphorylation- specific antibodies. The drug's effectiveness in inducing cell death or inhibition of cell proliferation may also be observed by analysis with apoptosis specific markers such as cleaved caspase 3 and cleaved PARP.

Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. In some embodiments, the compounds exhibit high therapeutic indices.

The following Examples are provided only to further illustrate the invention, and are not intended to limit its scope, except as provided in the claims appended hereto. The present invention encompasses modifications and variations of the methods taught herein which would be obvious to one of ordinary skill in the art. Materials, reagents and the like to which reference is made are obtainable from commercial sources, unless otherwise noted.

EXAMPLE 1

Identification of ALK Kinase Activity in Ovarian Cancer Patients

by Global Phosphopeptide Profiling Ovarian cancer tissue samples

Ovarian tumor tissue (n=37) and normal tissue (n=l 1) were collected from surgical resections from patients when sufficient material for PhosphoScan analysis, RNA, and DNA extractions were available. The collected tumors were frozen in liquid nitrogen and stored at -80°C.

Additional patient samples were also tested. A summary of the 69 ovarian tumor patients is provided in Tables 3 and 4 below. Most of the 43 serous type tissues are high-grade serous carcinoma characterized by cancerous spreading in the pelvic area.

Table 3 Characteristics of Ovarian Tumor Patients All Patients (n=69)

Characteristics

No. %

I. Age at surgery, years

Median 47

14-40

41-55

55-65

II. Histologic type

Adenocarcinoma

Serous

Endometrioid

Mucinous

Clear Cell

Stromal tumor

Granulosa-Theca Cell Tumor

Sarcoma

Others

Secondary Tumor

Malignant Brenner Tumor

Teratoma

Squamous Carcinoma

Table 4

Primary Ovarian Tissue Information

Sample ID Age Tissue Type Diagnosis

OC22 19 Malignant Serous Carcinoma

OC29 26 Malignant Serous Carcinoma

A14 35 Malignant Serous Carcinoma

A18 35 Malignant Serous Carcinoma

OC08 38 Malignant Serous Carcinoma

A23 38 Malignant Serous Carcinoma

A27 40 Malignant Serous Carcinoma

OC09 41 Malignant Serous Carcinoma

OC34 43 Malignant Serous Carcinoma

A16 43 Malignant Serous Carcinoma

A21a 43 Malignant Serous Carcinoma

OC28 45 Malignant Serous Carcinoma A20 45 Malignant Serous Carcinoma

A21 45 Malignant Serous Carcinoma

A22 45 Malignant Serous Carcinoma

A37 46 Malignant Serous Carcinoma

OC02 47 Malignant Serous Carcinoma

OC29a 48 Malignant Serous Carcinoma

A09 48 Malignant Serous Carcinoma

OC16 49 Malignant Serous Carcinoma

OC05 50 Malignant Serous Carcinoma

A04 50 Malignant Serous Carcinoma

OC26 52 Malignant Serous Carcinoma

A07 52 Malignant Serous Carcinoma

A08 52 Malignant Serous Carcinoma

A25 52 Malignant Serous Carcinoma

A29 52 Malignant Serous Carcinoma

OC24 53 Malignant Serous Carcinoma

OC35 53 Malignant Serous Carcinoma

A30 53 Malignant Serous Carcinoma

OC13 54 Malignant Serous Carcinoma

OC07 55 Malignant Serous Carcinoma

OC31 55 Malignant Serous Carcinoma

OC10 56 Malignant Serous Carcinoma

OC04 59 Malignant Serous Carcinoma

OC27 59 Malignant Serous Carcinoma

A35 60 Malignant Serous Carcinoma

A33 61 Malignant Serous Carcinoma

A24 62 Malignant Serous Carcinoma

A31 62 Malignant Serous Carcinoma

OCOl 63 Malignant Serous Carcinoma

OC14 63 Malignant Serous Carcinoma

OC11 65 Malignant Serous Carcinoma

OC17 47 Malignant Endometrioid Carcinoma

A17 55 Malignant Endometrioid Carcinoma

OC26a 57 Malignant Endometrioid Carcinoma

A19 36 Malignant Mucinous Carcinoma

A34 40 Malignant Mucinous Carcinoma

A15 41 Malignant Mucinous Carcinoma

Al l 54 Malignant Mucinous Carcinoma

OC36 58 Malignant Mucinous Carcinoma

A10 38 Malignant Clear Cell Carcinoma

OC30 17 Benign Granulosa-Theca Cell Tumor

A13 39 Benign Granulosa-Theca Cell Tumor

OC23 42 Benign Granulosa-Theca Cell Tumor

OC18 58 Benign Granulosa-Theca Cell Tumor

OC19 41 Malignant Stromal Sarcoma

A03 52 Malignant Endometrial Stromal Sarcoma OC03 28 Malignant Secondary Tumor

OC20 40 Malignant Secondary Tumor

A06 45 Malignant Secondary Tumor

OC21 47 Malignant Secondary Tumor

A32 50 Malignant Secondary Tumor

OC06 44 Malignant Malignant Brenner Tumor

A01 14 Benign Teratoma

A39 32 Benign Teratoma

A02 54 Benign Teratoma

A28 43 Malignant Squamous Carcinoma

A36 45 Malignant Squamous Carcinoma

Phosphopeptide Immunoprecipitation.

Samples were analyzed using the PhosphoScan® the immunoaffmity phospho-tyrosine peptide profiling method commercially available from Cell Signaling Technology, Inc. (Danvers, MA). This method has been described (see Rush et al, Nat Biotechnol 23:94-101, 2005, 2005; Rikova et al, Cell 131 : 1190-203, 2007; and US Patent Nos. 7,300,753 and 7,198,896). Briefly, an average of 15 milligrams of peptides were prepared from 0.2g to 0.5 g tumor tissue was homogenized and lysed in urea lysis buffer (20mM HEPES pH 8.0, 9M urea, 1 mM sodium vanadate, 2.5 mM sodium pyrophosphate, ImM beta-glycerophosphate) at 1.25 x 10 cells/ml and sonicated. Sonicated lysates were cleared by centrifugation at 20,000 x g, and proteins were reduced and alkylated as described previously (see Rush et al., Nat. Biotechnol. 23(1): 94-101 (2005)). Samples were diluted with 20 mM HEPES pH 8.0 to a final urea concentration of 2M. Trypsin (lmg/ml in 0.001 M HC1) was added to the clarified lysate at 1 : 100 v/v. Samples were digested overnight at room temperature.

Following digestion, lysates were acidified to a final concentration of 1% TFA.

Phosphopeptides were prepared using the PhosphoScan kit commercially available from Cell Signaling Technology, Inc. (Danvers, MA). Briefly, peptide purification was carried out using Sep-Pak C₁₈ columns as described previously (see Rush et al, supra . Following purification, all elutions (10%, 15%, 20%, 25%, 30%, 35% and 40% acetonitrile in 0.1 % TFA) were combined and lyophilized. Dried peptides were resuspended in 1.4 ml MOPS buffer (50 mM MOPS/NaOH pH 7.2, 10 mM Na₂HP0₄, 50 mM NaCl) and insoluble material removed by centrifugation at

12,000 x g for 10 minutes.

The phosphotyrosine monoclonal antibody P-Tyr-100 (Cell Signaling Technology, Inc., Danvers, MA). After coupling, antibody-resin was washed twice with PBS and three times with MOPS buffer. Immobilized antibody (40 μΐ, 160 μg) was added as a 1 : 1 slurry in MOPS IP buffer to the solubilized peptide fraction, and the mixture was incubated overnight at 4°C. The immobilized antibody beads were washed three times with MOPS buffer and twice with ddH₂0. Peptides were eluted twice from beads by incubation with 50 μΐ of 0.15% TFA for 15 minutes each, and the fractions were combined.

Analysis by LC-MS/MS Mass Spectrometry.

Peptides in the IP eluate (50 μΐ) were concentrated and separated from eluted antibody using Stop and Go extraction tips (StageTips) (see Rappsilber et ah, Anal. Chem., 75(3): 663-70 (2003)). Peptides were eluted twice from the microcolumns, each with 10 μΐ of 1% TFA. The sample was loaded onto a 10 cm x 75 μιη PicoFrit capillary column (New Objective) packed with Magic C 18 AQ reversed-phase resin (Michrom Bioresources) using a Famos autosampler with an inert sample injection valve (Dionex). The column was developed with a 45-min linear gradient of acetonitrile in 0.4% acetic acid, 0.005% HFBA delivered at 280 nl/min (Ultimate, Dionex).

After concentration on reverse-phase microtips, the samples were analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS). Briefly, samples were collected with an LTQ-Orbitrap mass spectrometer, using a top-ten method, a dynamic exclusion repeat count of 1 , and a repeat duration of 30 seconds. MS and MS/MS spectra were collected in the Orbitrap and LTQ component of the mass spectrometer, respectively. SORCERER-SEQUEST (TM, v4.0.3 (c) 2008, Sage-N Research, Inc., Milpitas, CA) searches were done against the NCBI human RefPept database downloaded on Jan. 6, 2009 (containing 37,742 proteins) or Mar. 1, 2010 (containing 36,500 proteins), allowing for serine, threonine and tyrosine phosphorylation (STY+80) and methionine oxidation (M+16) as differential modifications. The PeptideProphet probability threshold was chosen to give a false positive rate of 5% for the peptide identification.

Additionally, cluster analysis was performed to assess potentially aberrant tyrosine phosphorylation of receptor tyrosine kinases (RTK) in tumor tissues, spectral counts per RTK were summed and normalized to the amount of peptide subjected to pY immuno-precipitation (15mg), elevated spectral count in each tumor sample was calculated by subtracting average spectral count in 19 normal ovarian tissues. Elevated pY spectral counts of RTKs were observed in 60 tumor samples and were used as basis for hierarchical clustering using the Pearson correlation distance metric and average linkage (MultiExperiment Viewer version 4.4).

In addition, label free quantification was performed on 33 tumor samples and 10 normal samples that were run back to back on the LTQ-Orbitrap mass spectrometer. To compare the abundance of phospho-peptides in tumor samples, the average raw intensities of MSI peaks in the normal samples were used as basal intensity and calculated the ratio of MSI peak intensity of individual tumor sample to the basal intensity. An intensity of 20,000 (noise level intensity) was used as intensities for normal samples that had no MS 1 intensity value to calculate the basal intensity.

The foregoing analyses identified many tyrosine phosphorylated proteins, the majority of which are novel (data not shown). Among the 36 patients with ovarian cancer initially tested, 31 had adenocarcinoma, 4 had stromal tumor. Three patients with adenocarcinoma, namely patients XY1-OC7, XY1-OC16 and XY1-OC26, and 2 patients with stromal tumor, namely XY1-OC19 and XY1-OC23, had tyrosine phosphorylated ALK. Moreover, the level of tyrosine

phosphorylation in Patient XY1-OC19 was the highest with multiple phosphorylation sites. ALK phosphorylation was not detected by MS analysis in the rest of the tumor or normal ovarian tissue samples.

When all 69 tumors and 19 normal tissues were analyzed, LC-MS/MS identified 2484 tyroine phosphorylation sites in 1349 proteins, with global false positive rate less than 5%.

Previous studies have demonstrated that elevated tyrosine phosphorylation of receptor tyrosine kinases (RTKs) may represent aberrant kinase activity in tumor tissues, which could drive oncogenesis (Rikova et al, supra). Using receptor tyrosine kinase (RTK) tyrosine

phosphorylation in 19 normal ovarian tissues as basal phosphorylation level, potentially aberrant tyrosine phosphorylation of RTKs in tumor tissues was assessed. Elevated tyrosine

phosphorylation in many RTKs across the 60 tumor tissues was observed including discoidin domain receptors (DDRs), ephrin receptor kinases (EphA, EphB), as well as EGFR and FGFR family kinases. Most interestingly, ALK was found as the top kinase with the highest elevated tyrosine phosphorylation among other RTKs.

ALK phosphorylation was found in 4 patients, including 3 serous carcinoma patients

OC07, OC16 and OC26 and a stromal sarcoma patient OC19. The sequences of the peptides found in these patients is shown in Table 5.

Table 5

1078 KHQELQAMQM ELQSPEyK 2

1092 TSTIMTDyN PNYCFAGK 1

TDYN PNyCFAGK;

TSTIMTDYN PNyCFAGK;

1096 LRTStlMTDYN PNyCFAG K 4

DlyRASyYRK;

DlyRASyyRK;

1278 DlyRASYyRK 3

DlyRASyYRK;

1282 DlyRASyyRK 2

DlyRASyyRK;

1283 DlyRASYyRK 2

N KPTSLWN PTyGSWFTEKPTK;

1507 N KPTSLWN PTyGSWFTEKPTKK 1 2 2 2

H FPCG N VN YGyQQQG LPLEAAT

1586 APGAGHYEDtI LK 1

pY, phospho-tyrosine. "y" in the peptide sequence represents phospho-tyrosine .

Hyper-phosphorylation of ALK in OC19 was indicated by multiple pY sites in the juxtamembrane, kinase and C-terminal regulatory domains, many of which were seen previously in other type of cancers (data not shown). Phosphorylation of Y1507, which is equivalent to Y567 in NPM-ALK found in anaplastic large cell lymphoma, was detected in all four patients (Table Z). This phospho-site was also observed in primary tumor tissues and cell lines of non-small cell lung cancer bearing EML-ALK fusions, as well as neuroblastoma cell lines bearing ALK activating mutations (data not shown). Extracted ion chromatograms, MS2 spectra of two tryptic peptides containing phosho-Y1507 indicated the detection of ALK peptides in these patients. MS2 spectra with highest normalized intensities of 1.4xl0⁴ and 2.0xl0⁴ in OC19 and OC26, respectively, were matched to NKPTSLWNPTyGSWFTEKPTK and NKPTSLWNPTyGSWFTEKPTKK, respectively.

Previous studies have shown that Y1507 (Y527 in NPM-ALK) located at the carboxyl- terminal region of ALK is a direct docking site for SH2 domain-containing transforming protein (SHC1). Recruitment and phosphorylation of SHC1 activate Ras/extracellular signal regulated kinase (ERK) pathway and promotes tumorigenesis (Fujimoto et al., Proc Natl Acad Sci USA 93: 4181-6, 1996; Chiarle et al, Nat. Rev. Cancer 8: 1 1-23, 2008). Consistent with these observations, using label free quantification analysis, hyperphosphorylation of SHC1, MAPK14 and CDK1 at specific sites was found in the three serous carcinoma patients bearing ALK phosphorylation. In addition, phosphorylation of other ALK downstream signaling molecules, including those that are involved in PI3K/AKT, Jak/Stat3 pathways were found to be up-regulated in these tumors. These results suggest that in serous carcinoma patients OC07, OC16 and OC26, ALK might contribute to activation of multiple signaling pathways that promote tumor cell survival, growth and

proliferation.

In summary, 4 out of 69 (5.8%) ovarian cancer patients were found that carried

phosphorylated ALK at sites that correlate with ALK oncogenesis, including 3 out of 43 (7.0%) serous carcinoma and one stromal sarcoma patients. This is the first time that ALK tyrosine phosphorylation is reported in ovarian cancer patients. EXAMPLE 2

Isolation & Sequencing of FN1-ALK Fusion Gene

Given the presence of highly activated form of ALK kinase in patient XY1-OC19, 5' rapid amplification of cDNA ends on the sequence encoding the kinase domain of ALK was conducted in order to determine whether a chimeric ALK transcript was present.

Rapid Amplification of Complementary DNA Ends

RNeasy Mini Kit (Qiagen) was used to extract RNA from human tumor samples. Rapid amplification of cDNA ends was performed with the use of 5' RACE system (Invitrogen) with primers ALK-GSP1 for cDNA synthesis and ALK-GSP2 and ALK-GSP3 for a nested PCR reaction, followed by cloning and sequencing the PCR products.

For the 5 'RACE system, the following primers were used:

ALK-GSP1 : 5 ' G C AGTAGTTG G G GTTGTAGTC

For the nested PCR reaction, the following primers were used.

ALK-GSP2: 5'GCGGAGCTTGCTCAGCTTGT

ALK-GSP3.1 : 5 ' TG C AG CTCCTG GTG CTTCC Sequencing of the PCR products revealed that the ALK kinase in the patient samples of

XY1-OC19, was a product of a chimeric transcript of a novel fusion gene, which encodes N- terminus of fibronectin 1 (FNl) and C-terminus of ALK. The gene fusion in XY1-OC19 was in- frame. The cDNA and protein sequences that cover the junction of FNl and ALK are shown in Figures 1A and IB. The putative FNl-ALKvariantl gene contains the first 23 exons of FNl and the last 11 exons (exonl9 to exon29) of ALK, which encodes the first (i.e., N-terminal) 1201 amino acids of FNl and the last (i.e., C'terminal) 598 amino acids of ALK. Unlike previously reported ALK fusions, in the FNl-ALKvariantl fusion, the breakpoint point in ALK occurred in intron 18, which allows exonl9, encoding the transmembrane domain, to remain in the fusion gene. The nucleic acid sequence for the coding region of FNl-ALKvariantl fusion gene is provided in SEQ ID NO: 4 and the amino acid sequence for the fusion polypeptide encoded by the FNl-ALKvariantl fusion gene is provided in SEQ ID NO: 3 (with signal peptide) and in SEQ ID NO: 10 (without signal peptide). Two schematic diagrams of this putative fusion protein FNl- ALKvariantl are shown in Figures 2 A and 2B.

Considering the common breakpoint in the intron 19 of ALK as well as other possible in frame breakpoints in FNl (i.e., after exon 24 and after exon 21), the cDNA and protein sequences of five other putative FN 1 -ALK variants are provided in Table 2 and in SEQ ID NOs: 5-8, 11-21.

EXAMPLE 3

Detection of genomic DNA breakpoint in FN1-ALK fusion gene in

patient XY1-OC19 using a PCR Assay

PCR assay

DNeasy Tissue Kit (Qiagen) was used to extract DNA from frozen ovarian tumor samples. Amplification of genomic DNA sequence between Exon 23 of FNl and Exon 19 of ALK in Patient XY1-OC19 was performed with the use of Qiagen LongRange PCR Kit with primers FNlE23f and ALKE19r.

The following primers were used for PCR assay of genomic DNA isolated from Patient XY1-OC19:

FNlE23f: 5' TGACACTGGAGTGCTCACAGTCTC

ALKE19r: 5' GAGGATCAGCGAGAGTGGCAGGTG

The PCR product was run on a 1% agarose gel. As shown in Figure 3, a single product of which the size is around lkb was detected. This PCR product was sequenced.

Sequencing of the PCR products revealed the breakpoint in the genomic DNA of XYl- OC19 to be between l-946bp of FNl-intron 23 and 1343-1356bp of ALK intron 18. The complete sequence of the fused intron (960bp) between exon 23 of FN1 and exonl9 of ALK is shown in Fig. 1C.

EXAMPLE 4

Detection of Wild Type ALK and FN1-ALK Kinase Expression in Primary Human Ovarian Tumor Tissue and Human Ovarian Cancer Cell lines Using RT-PCR Assay

The presence or absence of wild type ALK kinase and/or a FNl-tmALK fusion protein or an FN 1 -ALK fusion protein (e.g., an FNl-ALKvariantl) in several primary human ovarian tumor tissues as well as three ovarian cell lines, OVMANA, OVSAHO, and OVMIU was detected using reverse transcription and polymerase chain reaction assay (RT-PCR). These methods have been previously described. See, e.g., Cools et al, N. Engl. J. Med. 348: 1201-1214 (2003). The OVMANA, OVSAHO, and OVMIU were purchased from Japanese Collection of Research Bioresources/Health Science Research Resources Bank.

PCR Assay

To confirm that a fusion between the FN1 gene and the ALK gene had occurred, RT-PCR was performed on RNA extracted from ovarian tumor tissues XY1-OC16, XY1-OC19, XYl- OC26, XY1-OC7 and XY1-OC8, as well as three ovarian cancer cell lines, Ovamana, Ovsaho and Ovmiu. For RT-PCR, first-strand cDNA was synthesized from 2.5 ug of total RNA with the use of Superscript™ III first-strand synthesis system (Invitrogen) with oligo (dT)₂₀. Then, wild type ALK transcript was amplified with the use of primer pairs ALKE16f and ALK GSP3. The transcript from FN 1 -ALK fusion gene was amplified with the use of primer pairs FNlE21f and ALK GSP3. The annealing position of these primers are shown schematically in Figure 4A. The transcript from control gene GAPDH was amplified with the use of primer pairs GAPDH F and GAPDH R. The sequences of these primers are:

ALKE16f: GAGGATATATAGGCGGCAAT

FNlE21f: 5'TAAGCTGGGTGTACGACCAA

ALK GSP3: TGCAGCTCCTGGTGCTTCC

GAPDH F: 5'GATTCCACCCATGGCAAATTCC

GAPDH R: 5 ' CACGTTGGCAGTGGGGAC

As shown in Figure 4B, patient XY1-OC 19 contained mRNA predicted to encode the FNl-

ALKvariantl fusion polypeptide (middle gel) but not the wild type ALK mRNA (i.e., no band in the ALK exon 16-GSP3 upper gel). Consistent with the mass spec data (see Example 1), patients XY1-OC 16, XY1-OC26 and XY1-OC7 expressed full length ALK mR A, which is surprising since ALK expression (i.e., full length or fused) is not found in healthy ovary tissue. Patient XY1- OC8 does not express either form of ALK transcript. In addition, two ovarian cancer cell lines, OVMANA and OVSAHO, expressed full length ALK transcripts again, surprisingly since ALK expression has never been described before in ovarian cells. Ovarian cell line OVMIU, does not express either form of ALK transcript. These three cell lines were purchased from Japanese Collection of Research Bioresources and grown in RPMI 1640 with 10% FBS. They are derived from ovarian adenocarcinoma, with OVMANA being derived from ovarian clear cell carcinoma, a subtype of ovarian adenocarcinoma or ovarian epithelial tumor.

This assay may be used to detect the presence of a full length ALK kinase or a fusion protein disclosed herein (e.g., resulting from a translocation between the ALK gene and the FNl gene) in a human cancer sample in other biological tissue samples (e.g., tumor tissue samples may be obtained from a patient having ovarian, pancreatic, kidney, or testicular cancer). Such an analysis will identify a patient having a cancer characterized by expression of full length ALK kinase, truncated ALK kinase (FNl-tmALK fusion protein), or a ALK fusion protein (e.g., a FN1 - ALK fusion protein) who is likely to respond to treatment with a ALK inhibitor.

Example 5

Detection of full length ALK and/or fusion protein resulting from an FNl and ALK gene translocation in ovarian tumor tissue and ovarian cell lines

To detect ALK protein in ovarian tumor tissue and ovarian cell lines, Western blot analysis was done with tissue lysates and cell lysates.

Western blot analysis

Frozen ovarian tumor tissue were minced in liquid nitrogen and resuspended in lx cell lysis buffer diluted from CST's product #9803. Tissue suspension was then sonicated and cleared by centrifugation. Cell line lysates were made by harvesting cultured cells with lx cell lysis buffer with sonication and centrifugation. Two part of the cleared tissue or cell lysate was then mixed with one part of the 3xSDS loading buffer, and heated at 100°C for 3 minutes. Around 30 micrograms of protein was loaded into each lane of pre-cast 4-20% gradient SDS protein gel (Invitrogen (Carlsbad, CA); catalog no. EC60285). Protein were then separated by electrophoresis and transferred onto nitrocellulose membrane. The membrane was then blotted with anti-ALK antibody manufactured by Cell Signaling Technology (which was generated against the kinase domain of ALK) and imaged according to CST's standard western blot procedure.

Antibodies that specifically bind to ALK (clone D5F3, specifically binds to the

intracellular domain of ALK), phospho-ALK (Y1278/1282/1283), EGFR, phospho-EGFR

(Y1068) and β-Actin were obtained from Cell Signaling Technology, Inc., Danvers, MA.

As shown in Figure 5 A, full length ALK protein expression at 220kd is detected in lysates made from tumor samples of patients XY1-OC16, and XY1-OC26 (serous carcinoma), as well as the 140kd potential proteolytically cleaved form of ALK in OC26, which might be too weak to see in OC16. In patient XY1-OC19 (stromal tumor), however, multiple ALK signals of variable sizes were detected with the most prominent signal at approximately 78 kd, suggesting the presence of a novel form of ALK that is different from the EML4-ALK Variant 3 fusion protein in the lung cancer cell line H2228 (see US Patent No. 7,700,339). These results suggest that OC19 might carry a different gene translocation involving ALK.

ALK protein localization and expression by immunohistochemical (IHC) analysis with patient tissue was next performed. Similar to neuroblastoma cells and tumor tissue that over- express ALK (Osajima-Hakomori et al., Am J Pathol 167: 213-22, 2005), a diffused ALK staining was observed at the cytoplasm of serous carcinoma OC26 (Fig. 5C), but not in another serous carcinoma OC29a (Fig. 5B). In the stromal tumor OC19, strong ALK signal is present at the plasma membrane and the cytoplasm, with membrane accentuation in some cells (Fig. 5D).

As shown in Figure 6, full length ALK expression was also surprisingly observed in 2 ovarian cell lines, Ovsaho and Ovmana, with expression in Ovmana much higher than that in Ovsaho. ALK expression was not detected in 12 other ovarian cell lines, namely RMGl,

Kuramuchi, Rmug S, Caov3, Skov3, Ovmiu, Ovtoko, Ovkate, Tovl l2D, Ovise, TYK-nu, and MCAS. These cell lines were purchased from Japanese Collection of Research Bioresources or the American Type Culture Collection (ATCC; Manassas, VA) and grown as the cell provider recommended.

As a neuronal receptor kinase, ALK is not normally expressed in the ovary. Upon the discovery of expression and phosphorylation of ALK in four ovarian cancer patients, the ALK gene was next tested to determine if it had undergone a genetic alteration in these patients. As no activating mutations reported in neuroblastoma (Janoueix-Lerosey et al. Nature 455: 967-970, 2008; Chen et al, Nature 455: 971-974, 2008; George et al, Nature 455: 975-978, 2008)were detected in the cytoplasmic region of ALK in OC07, OC16, OC26 and OC19 (data not shown), other possible alterations of ALK, including gene amplification and translocation, were considered.

EXAMPLE 6

ALK Gene Amplification in Primary Ovarian Tumor Tissue and Ovarian Cancer Cell Lines

Ovarian tissue normally does not express ALK. The presence of phosphorylated ALK in several ovarian tumor tissues could be due to gene amplification. To see if there is any gene amplification on the ALK locus located on Chromosome 2, quantitative PCR assay was used to measure the gene dose of ALK relative to that of GAPDH, a control gene located on Chromosome 12.

Quantitative PCR assay

DNeasy Blood and Tissue Kit (Qiagen, Inc., Germantown, MD) was used to extract genomic DNA from frozen ovarian tumor samples and ovarian cell lines Ovamana, Ovsaho and Ovmiu. Quantitative PCR was performed using iQ™ SYBR Green Supermix from Bio-Rad (Hercules, CA; Cat #170-8880) with primers ALKgDNA qF and ALKgDNA qR for ALK gDNA and primers GAPDHgDNA qF and GAPDHgDNA qR for GAPDH gDNA. The sequences of these primers are:

ALKgDN A qF 1 : 5'ACAAGGTCCACGGATCCAGAAACA

ALKgDN A qR 1 : 5'AGTCTCCCAGTTGCAACGTTAGGT

GAPDHgDNA qF: 5' CACAGTCCATGCCATCACTGC

GAPDHgDNA qR: 5 ' ATGCCAGTGAGCTTCCCGTTC

Each qPCR reaction contains lOul of SYBR Green Master Mix, lul of 10 nM forward primer, lul of 10 nM reverse primer, 6ul of nuclease free water and 2ul of genomic DNA at 10 ng/ml. qPCR reaction was carried out with the following parameters: 95°C for 10 minutes followed by 40 cycles of 95C for 15 seconds and 65°C for 15 seconds. A dissociation run from 55 °C to 95 °C was added to the end of qPCR reaction for melting curve analysis. Non-specific amplification was not detected with the primers used in the study. PCR reactions were set up in duplicates for each sample tested. Data was exported from CFX96 Real-Time PCR Detection System and analyzed using Microsoft Excel.

Starting quantity of each gene was obtained with iQ5 RealTime PCR System manufactured by Bio-Rad (#1709780). The relative quantity of a gene is reflected by the ratio of the starting quantity of this gene against that of GAPDH. As shown in Figure 7A, the red bars represent relative quantity of ALK, whereas the blue bars represent the relative quantity of GAPDH in each tumor tissue or cell line. Standard errors are determined by duplicate data points in each sample for each gene. As shown in Figure 7A, ALK gene quantity is 3 fold as high as GAPDH in ovarian tumor samples XY1-OC16 and XY1-OC26, indicating that ALK gene amplification occurred in these tumor samples, which is consistent with the Phosphoscan®, RT-PCR as well as western blot results. ALK gene amplification also occurred in Ovmana, as indicated by an ALK versus

GAPDH ratio of 2 in this cell line. The ratio of ALK versus GAPDH in tumor samples XYl- OC19, XY1-OC7, XY1-OC18, XY1-OC23, XY1-OC30 and XYl-B22, as well as in cell lines Ovsaho and Ovmiu, is close to 1, indicating that ALK is not amplified in these samples.

To further examine whether there is copy number variation (CNV) of ALK in ovarian tumors where ALK phosphorylation was detected, real time quantitative PCR was employed with genomic DNA isolated from serous carcinoma OC07, OC16, OC26, and stromal sarcoma OC19, as well as serous carcinoma OC08, in which ALK phosphorylation is not detected. In this analysis, an Ultra Conservative Element (UCE) located on human Chromosome 7 (Chr7: 1,234,865- 1,235,026) that has been identified to have a copy number of two was used as reference DNA segment, and the AC_t was calculated by subtracting the C_t value of the UCE reactions from that of the ALK reactions in each sample. This normalizes the variation in the amount of templates between samples. The AC_t values from each individual was then used to calculate relative copy number against calibrator DNA isolated from NA10851 (Coriell Cell Repository, Camden, NJ). Calculations were performed to normalize a copy number of two to a value of 1 in the bar graph. Standard deviations were based on normalized Ct values of duplicates.

While no obvious copy number variation of ALK was detected in OC08 and OC19, 1.5 and 1.7 fold ALK copy number gains were detected in OC16 and OC26, respectively (Fig. 7B). The result is consistent with results from high-resolution single nucleotide polymorphism (SNP) array analysis, which indicated a copy number of 3 in the ALK region in Patient OC26 (data not shown). The ALK copy number gain in OC16 and OC26 may account for the aberrant expression of ALK in these serous carcinoma patients. Surprisingly, a loss of ALK gene copies was observed in serous carcinoma OC07 (Fig. 7B; OC7 sample), in which ALK phosphorylation (see Fig. 1 A and 1C, Suppl. Fig. 1) and mRNA expression (see below) were detected, suggesting that aberrant ALK expression and phosphorylation is independent of ALK copy number loss in this patient.

Example 7

Generation of Recombinant Vectors Encoding FN1-ALK and FNl-tmALK Fusion Polypeptides and Expression of the Polypeptides

The open reading frame of the FNl-tmALK fusions and the FN 1 -ALK fusions are amplified by PCR and inserted into an expression cloning vectors (e.g., pcDNA3 commercially available from Invitrogen, Carlsbad, CA). These PCR products may also be cloned into vectors that can be packaged into retroviruses (e.g., the retroviral vectors MSCV-Neo and MSCV-puro which are both commercially available from Clontech Laboratories, Inc., Mountain View, CA).

The resulting recombinant vectors (e.g., containing FNl-tmALK fusions and the FN1- ALK fusions-encoding polynucleotides) may be transfected (e.g., electroporation, DEAE-Dextran, PEI, etc..) into a host cell (e.g., 293T cells, Hela cells, 3T3 cells, COS cells, etc..) to make recombinant retrovirus.

In one example, NIH3T3 cells are purchased from American Type Culture Collection (Manassas, VA) and grown as per manufacturer's instructions.

3T3 cells may be infected with (i.e., transduced with) a recombinant retrovirus encoding a FNl-tmALK fusion or a FN 1 -ALK fusion. Two days after transduction, 0.5 mg/ml G418 will be added to the cell culture media. Two weeks after being transduced (i.e., 12 days after selection in G418), cells are lysed and Western blotting analysis performed, staining the electrophoretically resolved cell lysates with an antibody that specifically binds to a portion of the ALK protein present in the FNl-tmALK and FN1-ALK fusion polypeptides (e.g., the C-19 ALK antibody, catalog no. sc-6344 from Santa Cruz Biotechnology, Inc. or the ALK antibody, catalog no.

ab59286 from Abeam, Cambridge, MA).

Expression of all six FNl-tmALK and FN 1 -ALK fusion polypeptides is expected.

In one example, the coding sequences of wild type ALK and FNl-ALKvariant 1 were cloned from primary tumor samples OC26 and OC19, respectively, into retroviral vector MSCV- Neo. Figure 8A provides schematic diagrams of the multiple cloning site of empty vector (top), vector containing full-length ALK (middle), and vector containing FNl-ALKvariantl (bottom).

The src kinase was used as a control. The expression plasmids were transfected into 293T cells by FuGene 6 (Roche Diagnostics, Indianapolis, IN) and retrovirus was harvested 48 hours later. NIH3T3 cells were infected with recombinant retroviruses encoding MSCV-neo/ALK or MSCV- neo/FNlALKvariantl and selected in G418-containing media for 7 days.

EXAMPLE 8 EFFECT OF F I-TMALK AND FN 1 -ALK FUSION POLYPEPTIDE EXPRESSION ON 3T3 CELLS'

GROWTH IN VITRO AND IN VIVO

3T3 cells have contact inhibition, meaning that they do not form colonies in soft agar. To determine if the presence of a polypeptide with ALK kinase activity in these cells removes their contact inhibition, retrovirally transduced 3T3 cells are selected for G418 (0.5 mg/ml) for 7 days, and the cells are then cultured in soft agar in triplicate for 17 days. A retrovirus encoding full length ALK polypeptide and a retrovirus encoding truncated ALK are also used to transduce 3T3 cells. As a control, a retrovirus encoding the src kinase was also used to transducer 3T3 cells.

The 3T3 soft agar assay is well known (see, e.g., Colburn et al, Molecular and Cellular Biology 5(4): 890-893, 1985 and Platica et al., Biochemical and Biophysical Research

Communications 314 (3): 891-896, 2004).

For these studies, NIH3T3 cells were infected with recombinant retroviruses encoding MSCV-neo/ALK or MSCV-neo/FNlALKvariantl described in Example 7 and selected in G418- containing media for 7 days. The cells were then cultured in soft agar in triplicates or injected subcutaneously into nude mice (see Example 10 below). 3T3 cells transduced with one of the six FNl-tmALK and FN 1 -ALK fusion polypeptides, full length ALK, and truncated ALK are expected to lose their contact inhibition. This provides evidence that the presence of a FN 1 -ALK fusion, an FNl-tmALK fusion, or a truncated ALK is able to drive a cell into a cancerous state of growth.

The transduced cells were also lysed to tested by Western blotting with ALK-specific antibody to determine if they expressed ALK protein or FNl-ALKvariant 1 protein. As shown in Fig. 8B, as expected, both full length ALK (220 kd) and the shorter form of ALK (140 kd) due to proteolytic cleavage (Moog-Lutz et al, J. Biol. Chem 280: 26039-26048, 2005; Mazot et al, Oncogene 30: 2017-2025, 2011) were observed in 3T3/ALK cells (Fig. 8B, "ALK" lane).

Interestingly, in Fig. 8B, "FN1-ALK" lane, in addition to full length FNl-ALKvariantl proteins, a prominent ALK signal of ~78kd in 3T3/FN1-ALK cells was observed, which is reminiscent of the strong ALK signal in Patient OC19 tumor (see Fig.5 A).

The NIH3T3 cells stably expressing full length ALK or FN 1 -ALK were also used to determine whether determine whether full length ALK and FNl-ALKvariantl have transforming potential). Using immunofluorescence analysis with anti-ALK antibodies (green), keratin (red) and DRAQ5® (which stains the nucleus blue), both full length ALK and FN 1 -ALK showed intracellular reticulum/Golgi localization (Fig. 8C, upper panels), probably due to a defect of glycosylation in 3T3 cells (Mazot et al, supra). After 3T3/Neo, 3T3/ALK or 3T3/FN1-ALK cells were injected subcutaneously into nude mice, tumor growth induced by both 3T3/ALK and 3T3/FN1-ALK cells was observed, with the FNl-ALK tumors growing more aggressively than the full length ALK tumors (Fig. 8C, lower panels). The average tumor sizes 12 days after injection

3 3

were around 2000 mm and 100 mm for FNl-ALK and ALK tumors, respectively. If allowed to grow, ALK tumors could reach 2000 mm 20 days after injection (data not shown). These results indicate that both the full length ALK and the FNl-ALK fusion gene isolated from the ovarian cancer patients have transforming potential and that FNl-ALK is more oncogenic than the full length ALK.

In addition, the ability of 3T3 cells transduced with the other variants of FNl-ALK to form tumors in vivo is analyzed. Immunocompromised nude mice (which lack a thymus, available from the Jackson Laboratory, Bar Harbor, Maine) are injected with 1 x 10⁶ 3T3 cells that are transduced with retrovirus containing empty vector, or vector encoding FNl-ALKvariant 2, or variant3, or variant4, or variant5, or variant6 fusion polypeptides, full length ALK, or truncated ALK. Mice are monitored daily for tumor formation and size, and will be sacrificed when tumors reach approximately 1 cm x 1 cm.

Tumor formation is expected to be observed in all injected nude mice except for those nude mice which are injected with empty vector. EXAMPLE 9

FNl-tmALK and FNl-ALK Fusion Polypeptide Activity in Transduced BaF3 Cells

Murine BaF3 cells normally need interleukin-3 (IL-3) to survive. BaF3 cells arre obtained from DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Germany) and will be maintained at 37°C in RPMI-1640 medium (Invitrogen) with 10% fetal bovine serum (FBS) (Sigma) and 1.0 ng/ml murine IL-3 (R&D Systems).

To determine if expression of a FNl-tmALK and FNl-ALK fusion polypeptide, truncated ALK, or full length ALK can enable BaF3 cells to survive without IL-3, BaF3 cells are transduced with the retroviruses described in Example 7.

It is expected that the presence of FNl-tmALK and FNl-ALK fusion polypeptide, truncated ALK, or full length ALK will enable BaF3 cells to survive without IL-3.

Next, an in vitro kinase assay can be performed to determine if the ALK kinase portion of the FNl-tmALK and FNl-ALK fusion polypeptide, truncated ALK , or full length ALK is active. Cell lysates from the transduced BaF3 cells will be subjected to immunoprecipitation with anti- Myc-Tag antibody (which will pull down the Myc-tagged polypeptides). The pulled-down immune complex will washed 3 times with cell lysis buffer, followed by kinase buffer

(commercially available from Cell Signaling Technology, Inc.). Kinase reactions will be initiated by re-suspending the immune complex into 25 ul kinase buffer that contains 50uM ATP, 0.2 uCi/ul [gamma32p] ATP, with 1 mg/ml of Poly (EY, 4: 1) and substrate. Reactions will be stopped by spotting reaction cocktail onto p81 filter papers. Samples may then be washed and assayed for kinase activity by detection with a scintillation counter.

It is expected that all of the FN1- tmALK fusion (i.e., variant 1, variant 3, and variant 5), the FNl-ALK fusions (i.e., variant 2, variant 4, and variant 6), the truncated ALK, and the full length ALK have active ALK kinase activity and all can phosphorylate their substrates (e.g., a src- related peptide).

EXAMPLE 10

Sensitivity of FNl-tmALK and FNl-ALK Fusion Polypeptide Activity

to PF-02341066 and TAE684

To determine whether the polypeptides disclosed herein are sensitive to PF-02341066, BaF3 cells are obtained from DSMZ (Deutsche Sammlung von Mikroorganismen und

Zellkulturen GmbH, Germany) and are transduced with transduced with retrovirus encoding FN1- ALKvariantl, FNl-ALKvariant 2, FNl-ALKvariant 3, FNl-ALKvariant 4, FNl-ALKvariant5, FNl-ALKvariant6, truncated ALK, or full length ALK, and selected for IL3 independent growth in the presence of absence of PF-02341066. Karpas 299 cells (commercially available from DSMZ) express NPM-ALK, and are used as a positive control.

A MTS assay will be performed using the CellTiter 96 Aqueous One Solution Reagent, (Promega, Catalog No. G3582).

It is expected that the BaF3 cells transduced with retrovirus expressing one of the following polypeptides: FN 1 -ALKvariantl, FNl-ALKvariant 2, FNl-ALKvariant 3, FNl- ALKvariant 4, FNl-ALKvariant5, FNl-ALKvariant6, truncated ALK, or full length ALK, will stop growing in the presence of PF-02341066.

The ability of full length ALK and the FN 1 -ALKvariantl expressed in 3T3 cells to respond to TAE or PF-02341066 in vivo was next determined. For these studies, 1.5-2xl0⁶ transduced 3T3 cells expressing Src, FNl-ALK or ALK (see Examples 7 and 8) were resuspended in Matrigel (BD Biosciences) and injected subcutaneous ly into the upper or lower right flank of 6- 8 weeks old female NCR NU-F mice purchased from Taconic (Hudson, NY). Mice were randomized to 3 treatment groups (n=6 per group) once the tumors became palpable (-100 mm ) and gavaged orally with vehicle (10% l-methy-2-pyrrolidinone/90% PEG-300) alone,

lOOmg/kg/day Crizotinib or lOmg/kg/day TAE684 (NVP-TAE684) for 7-1 1 days. Crizotinib and TAE684 were purchased from ChemieTek (Indianapolis, IN). Tumors were measured every other day using calipers, and tumor volume was calculated as 0.52xwidth xlength. Mice were monitored daily for general conditions. The experiment was terminated when the mean size of the treated or control mice reached 1500 mm .

As shown in Figs. 9A-C, administration of Crizotinib (lOOmg/kg/day) drastically inhibited the growth of both ALK (Fig. 9A) and FNl-ALKvariantl (Fig. 9B) tumors, but not the 3T3/SRC tumors (Fig. 9C), where the control is shown in green squares and crizotinib-treated is shown in blue diamonds. Consistent with these observations, western blot analysis using antibodies specific for ALK and phospho-ALK revealed that Crizotinib abolished phosphorylation of both full length FN 1 -ALK variantl and the -78 kd ALK variant 24 hours after treatment (Fig. 9D). An ALK signal with lower molecular weight next to the full length FN 1 -ALK variantl was also observed, which is likely to be non-phophorylated FN1-ALK variantl (Fig. 9D). Similar tumor growth inhibition effects were observed with TAE684: when administered at lOmg/kg/day, TAE684 effectively inhibited the growth of both ALK and FN 1 -ALK, but not SRC tumor (Figs. lOA-lOC). These results suggest that ALK and FNl-ALKvariantl tumors are highly sensitive to ALK inhibitors.

EXAMPLE 11

Detection of ALK Expression in a Human Ovarian Cancer Sample Using FISH Assay

The presence of a polynucleotide encoding a polypeptide with ALK kinase activity (e.g., FNl-ALKvariantl, FNl-ALKvariant 2, FNl-ALKvariant 3, FNl-ALKvariant 4, FN1- ALKvariant5, FNl-ALKvariant6, truncated ALK, or full length ALK) in ovarian cancer (e.g., in a stromal tumor or a clear cell tumor), or in other cancers such as pancreatic cancer, kidney cancer, lung cancer, or colon cancer is detected using a fluorescence in situ hybridization (FISH) assay. Such FISH assays are well known in the art (see, e.g., Verma et al. Human Chromosomes: A Manual of Basic Techniques, Pergamon Press, New York, N.Y. (1988); Colleoni et al, American Journal of Pathology. 156:781-789, 2000).

I l l To do this, paraffin-embedded human tumor samples are examined. Some tissues that are examined include liver, pancreas, ovarian, colon, long, and kidney cancers.

For analyzing rearrangements involving the ALK gene, a dual color break-apart probe will be obtained from Vysis (Vysis, Downers Grove, III, USA) and used according to the

manufacturer's instructions.

The ALK rearrangement probe contains two differently labeled probes on opposite sides of the breakpoint of the ALK gene (at nucleotide 3171) in the wild type sequence (see, e.g., SEQ ID NO: 2). When hybridized, the native ALK region will appear as an orange/green fusion signal, while rearrangement at this locus (as occurs in the translocations between the FNl gene and the ALK gene) will result in separate orange and green signals. The truncated ALK may also be due to a rearrangement at the ALK gene locus.

The FISH analysis will likely reveal a low incidence of ALK gene translocations (e.g., with the FNl gene) in the sample population having ovarian cancer and other cancers. However, it is predicted that a subset of the studied cancers will contain a ALK gene translocation. These cancers containing the ALK gene translocation (e.g., with the FNl gene) are identified as those cancers likely to respond to an ALK inhibitor. In other words, cells of the cancer, upon treatment (or contact) with a ALK inhibitor are predicted to show growth retardation, growth abrogation (i.e., stop growing) or actually die (e.g., by apoptosis) as compared to untreated cancer cells (i.e., cells not contacted with the ALK inhibitor).

EXAMPLE 12

Detection of ALK Expression in a Large Patient Cohort

To test possible aberrant ALK expression in a larger patient cohort, immunohistochemical analysis was performed on ovarian tissue microarrays (TMA). For these studies, formalin- fixed and paraffin-embedded (FFPE) ovarian tumor tissue microarray (TMA) slides were purchased from Folio Biosciences (Powell, Ohio) and Biochain Institute, Inc. (Hayward, CA). 4 μιη ovarian tissue sections or FFPE TMA slides were deparaffmized and rehydrated through xylene and graded ethanol, respectively. Antigen retrieval was performed in a Decloaking Chamber (Biocare

Medical, Concord, CA) using 1.0 mM EDTA, pH 8.0. Slides were then quenched in 3% H₂0₂ for

10 minutes, washed in deionized H₂0 and blocked with Tris buffered saline /0.5% Tween-20

(TBST)/5% goat serum in a humidified chamber for 60 minutes. Sections were then exposed to

ALK (D5F3) XP® Rabbit mAb (from Cell Signaling Technology, Inc.) overnight at 4°C. Detection was performed with SignalStain® Boost IHC Detection Reagent (Cell Signaling Technology, Inc.) for 30 minutes. All slides were exposed to NovaRed (Vector Laboratories, Inc., Burlingame, CA) for 1 minute before they were rinsed, dehydrated, cleared and cover-slipped.

Similar to what was observed in the Chinese cohort (69 tumors) using PhosphoScan® immunoaffinity profiling method, while no ALK staining was detected in normal tissues, granulosa-theca cell tumors or clear cell carcinomas, ALK staining was detected in 14 out of 353 (4%) serous carcinoma and 3 out of 37 endometrioid carcinoma specimens, with half of the specimens having strong ALK signal (Figs. 11 A-l ID). The results are summarized in Table 6, which lists the total specimen numbers of each subtype and numbers of ALK weak (+) or strong (++ or +++) specimens.

Table 6

Ovarian Tissue Type Total ALK + (%) ALK ++/ +++ (%)

Serous Carcinoma 353 7 (2.0%) 7 (2.0%)

Endometrioid Carcinoma 37 2 (5.4%) 1 (2.2%)

Mucinous Carcinoma 88 7 (8.0%) 0

Clear Cell Carcinoma 14 0 0

Granulosa-theca Cell Tumor 19 0 0

Normal 37 0 0

Mostly weak ALK staining in some mucinous carcinoma specimens was also observed (see Table 6, images not shown). Similar to that in Patient OC26, most of the ALK staining observed in serous carcinomas in TMA is diffusedly localized in the cytoplasm (Figs. 1 lA-1 ID), suggesting that ALK might be activated and signaling in these tumors, as what has been observed in neuroblastomas where wild type ALK is overexpressed (Osajima-Hakomori et al., Am. J. Pathol. 167: 213-222, 2005). Considering the notion that a significant number of high-grade serous carcinoma has been misclassified as endometrioid carcinoma (Vaghan et al., supra;

Kelemen and Kobel, Lancet Oncol. 12: 1071-1080, 2011), these data suggest that strong ALK expression resides mainly in a portion (~2%) of serous carcinoma.

While the invention has been described with particular reference to the illustrated embodiments, it will be understood that numerous modifications thereto will appear to those skilled in the art. Accordingly, the above description, the following claims, and accompanying drawings should be taken as illustrative of the invention and not in a limiting sense.

What is claimed is:

Claims

Claims

1. An isolated polynucleotide comprising a nucleotide sequence at least 95% identical to a sequence selected from the group consisting of:

(a) a nucleotide sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 3, 5, 7, 10, 1 1 , 12, 14, 15, 17, 18, 20, or 21 ;

(b) a nucleotide sequence comprising the nucleotide sequence of SEQ ID NO: 4, 6, 8; 13, 16, or 19;

(c) a nucleotide sequence encoding polypeptide comprising an N-terminal portion comprising amino acid residues 1-1085 of SEQ ID NO: 26 or amino acid residues 1 -1 1 16 of SEQ ID NO: 27 and a C-terminal portion comprising amino acid residues 1039-1392 of SEQ ID NO: 1 ;

(d) a nucleotide sequence encoding a polypeptide comprising an N-terminal portion comprising amino acid residues 1-1085 of SEQ ID NO: 26 or amino acid residues 1 -1 1 16 of SEQ ID NO: 27 and a C-terminal portion comprising the amino acid sequence set forth in SEQ ID NO: 29;

(e) a nucleotide sequence comprising (i) at least six contiguous nucleotides encompassing the fusion junction (nucleotides 3601-3606 of SEQ ID NO: 4, nucleotides 3793-3798 of SEQ ID NO: 6, or nucleotides 3346-3351 of SEQ ID NO: 8) or encoding at least six contiguous polypeptides encompassing the fusion junction (residues 1999-1204 of SEQ ID NO: 3, residues 1263-1268 of SEQ ID NO: 5, or residues 1 1 14-1 1 19 of SEQ ID NO: 7) of an FNl-tmALK fusion polypeptide;

(f) a nucleotide sequence comprising (i) at least six contiguous nucleotides encompassing the fusion junction (nucleotides 3601-3606 of SEQ ID NO: 13, nucleotides 3793-3798 of SEQ ID

NO: 16, nucleotides 3346-3351 of SEQ ID NO: 19) or encoding at least six contiguous

polypeptides encompassing the fusion junction (residues 1999-1204 of SEQ ID NO: 14, residues 1263-1268 of SEQ ID NO: 17, or residues 1 1 14-1 1 19 of SEQ ID NO: 20) of an FNl-ALK fusion polypeptide;

(g) a nucleotide sequence encoding a polypeptide comprising amino acid residues 1039-1392 of SEQ ID NO: 1 , wherein said polypeptide does not comprise amino acid residues 19-1038 of SEQ ID NO: 1 ; and

(h) a nucleotide sequence encoding a polypeptide comprising amino acid sequence of SEQ ID NO: 29, wherein said polypeptide does not comprise amino acid residues 19-1038 of SEQ ID NO: 1 ; and

(i) a nucleotide sequence complementary to any of the nucleotide sequences of (a)-(h).

2. An isolated reagent that specifically binds to a polynucleotide of claim 1, wherein said reagent does not specifically bind to a polynucleotide having a nucleotide sequence consisting of only A residues or of only T residues.

3. The isolated polynucleotide of claim 1, wherein said polynucleotide further comprises a detectable label.

4. A method for producing a recombinant vector comprising inserting an isolated nucleic acid molecule of claim 1 into a vector.

5. A recombinant vector produced by the method of claim 4.

6. A method for making a recombinant host cell comprising introducing the recombinant vector of claim 5 into a host cell.

7. A recombinant host cell produced by the method of claim 6.

8. A method for producing a recombinant polypeptide, said method comprising culturing the recombinant host cell of claim 7 under conditions suitable for the expression of said fusion polypeptide and recovering said polypeptide.

9. A recombinant polypeptide produced by the method of claim 8.

10. The reagent of claim 2, wherein said reagent is a polymerase chain reaction (PCR) probe or a fluorescence in situ hybridization (FISH) probe.

11. An isolated polypeptide comprising an amino acid sequence at least 95% identical to a sequence selected from the group consisting of:

(a) an amino acid sequence comprising the amino acid sequence of SEQ ID NOs: 3, 5, 7, 10, 11, 12, 14, 15, 17, 18, 20, or 21;

(b) an amino acid sequence comprising amino acid residues 1-1085 of SEQ ID NO: 26 and a C- terminal portion comprising amino acid residues 1039-1392 of SEQ ID NO: 1; (c) an amino acid sequence encoding a polypeptide comprising at least six contiguous amino acids encompassing the fusion junction (residues 1999-1204 of SEQ ID NO: 3, residues 1263-1268 of SEQ ID NO: 5, or residues 1114-1119 of SEQ ID NO: 7) of an FNl-tmALK fusion polypeptide;

(d) an amino acid sequence encoding a polypeptide comprising at least six contiguous amino acids encompassing the fusion junction (residues 1999-1204 of SEQ ID NO: 14, residues 1263-1268 of

SEQ ID NO: 17, or residues 1114-1119 of SEQ ID NO: 20) of an FN1-ALK fusion polypeptide;

(e) an amino acid sequence comprising amino acid residues 1039-1392 of SEQ ID NO: 1, wherein said polypeptide does not comprise amino acid residues 19-1038 of SEQ ID NO: 1; and

(f) an amino acid sequence comprising amino acid sequence of SEQ ID NO: 29, wherein said polypeptide does not comprise amino acid residues 19-1038 of SEQ ID NO: 1.

12. An isolated reagent that specifically binds to a polypeptide of claim 11, but does not specifically bind to either full-length FN1 protein or full-length ALK protein.

13. The isolated reagent of claim 12, wherein said reagent is an antibody or a heavy-isotope labeled (AQUA) peptide.

14. The isolated reagent of claim 2 or 12, wherein said reagent comprises a detectable label.

15. A method for detecting the presence of a mutant ALK polypeptide or a FN 1 -ALK fusion polypeptide in a biological sample from a mammalian subject having cancer or a mammalian subject suspected of having cancer, said method comprising the steps of:

(a) obtaining a biological sample from a mammalian subject having cancer or mammalian subject suspected of having cancer; and

(b) utilizing at least one reagent that specifically binds to a mutant ALK polypeptide or a FN1- ALK fusion polypeptide to determine whether said mutant ALK polypeptide or said FN 1 -ALK fusion polypeptide is present in said biological sample, wherein specific binding of said reagent to said biological sample indicates said mutant ALK polypeptide or said FN 1 -ALK fusion polypeptide is present in said biological sample.

16. The method of claim 15, wherein said mutant ALK polypeptide is truncated ALK

polypeptide.

17. The method of claim 15, wherein said mutant ALK polypeptide is an FNl-tmALK fusion polypeptide.

18. The method of claim 17, wherein said FNl-tmALK fusion polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 3, 5, 7, 10, 11, and 12.

19. A method for detecting the presence of a polypeptide with ALK kinase activity in a biological sample from a mammalian subject having ovarian cancer or a mammalian subject suspected of having ovarian cancer, said method comprising the steps of:

(a) obtaining a biological sample from a mammalian subject having ovarian cancer or a mammalian subject suspected of having ovarian cancer and

(b) utilizing a reagent that specifically binds said polypeptide with ALK kinase activity to determine whether said polypeptide with ALK kinase activity is present in said biological sample, wherein specific binding of said reagent to said biological sample indicates said polypeptide with ALK kinase activity is present in said biological sample.

20. The method of claim 19, wherein said polypeptide is aberrantly expressed full-length ALK protein.

21. The method of claim 19, wherein said polypeptide is an FNl-tmALK fusion polypeptide or a truncated ALK polypeptide.

22. The method of claim 21, wherein said FNl-tmALK fusion polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 3, 5, 7, 10, 11, and 12.

23. The method of claim 19, wherein said polypeptide is an ALK fusion polypeptide.

24. The method of claim 23, wherein said polypeptide is selected from the group consisting of an NPM-ALK fusion polypeptide, an AL017-ALK fusion polypeptide, an TFG-ALK fusion polypeptide, an MSN-ALK fusion polypeptide, an TPM3-ALK fusion polypeptide, an TPM4- ALK fusion polypeptide, an ATIC-ALK fusion polypeptide, an MYH9-ALK fusion polypeptide, an CLTC-ALK fusion polypeptide, an SEC31L1-ALK fusion polypeptide, an RANBP2-ALK fusion polypeptide, an CARS-ALK fusion polypeptide, an EML4-ALK fusion polypeptide, an KIF5B-ALK fusion polypeptide, and an TFG-ALK fusion polypeptide, and an FNl-ALK fusion polypeptide.

25. The method of claim 24, wherein said FNl-ALK fusion polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 14, 15, 17, 18, 20, and 21.

26. The method of claim 15 or 19, wherein the reagent is an antibody.

27. The method of claim 26, wherein the antibody specifically binds to a full length ALK polypeptide.

28. The method of claim 26, wherein the antibody specifically binds to an FNl-ALK fusion polypeptide or an FNl-tnALK fusion polypeptide and does not specifically bind to either full- length FN1 polypeptide or full-length ALK polypeptide.

29. The method of claim 26, wherein the antibody specifically binds to a full-length FN1 polypeptide.

30. The method of claim 26, wherein said method is implemented in a format selected from the group consisting of a flow cytometry assay, an immunohistochemistry (IHC) assay, an immunofluorescence (IF) assay, an Enzyme-linked immunosorbent assay (ELISA) assay, and a Western blotting analysis assay.

31. The method of claim 15 or 19, wherein said reagent is a heavy-isotope labeled (AQUA) peptide.

32. The method of claim 31, wherein said heavy-isotope labeled (AQUA) peptide comprises an amino acid sequence comprising a fusion junction of an FNl-ALK fusion polypeptide.

33. The method of claim 31, wherein said method is implemented using mass spectrometry analysis.

34. A method for detecting the presence of a mutant ALK polynucleotide or a FN1-ALK fusion polynucleotide in a biological sample from a mammalian subject having cancer or a mammalian subject suspected of having cancer, said method comprising the steps of:

(a) obtaining a biological sample from said mammalian subject having cancer or mammalian subject suspected of having cancer; and

(b) utilizing at least one reagent that specifically binds to a mutant ALK polynucleotide or a FN1- ALK fusion polynucleotide to determine whether said mutant ALK polynucleotide or said FN1-

ALK fusion polynucleotide is present in said biological sample, wherein specific binding of said reagent to said biological sample indicates said mutant ALK polynucleotide or said FN 1 -ALK fusion polynucleotide is present in said biological sample.

35. The method of claim 34, wherein said mutant ALK polynucleotide is a truncated ALK polynucleotide.

36. The method of claim 34, wherein said mutant ALK polynucleotide is an FNl-tmALK fusion polynucleotide.

37. The method of claim 36, wherein said FNl-tmALK fusion polynucleotide encodes a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 3, 5, 7, 10, 11, and 12.

38. The method of claim 36, wherein said FNl-tmALK fusion polynucleotide comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: 4, 6, and 8.

39. A method for detecting the presence of a polynucleotide encoding a polypeptide with ALK kinase activity in a biological sample from a mammalian subject having ovarian cancer or a mammalian subject suspected of having ovarian cancer, said method comprising the steps of: (a) obtaining a biological sample from a mammalian subject having ovarian cancer or a mammalian subject suspected of having ovarian cancer and (b) utilizing a reagent that specifically binds to said polynucleotide encoding said polypeptide with ALK kinase activity to determine whether said polynucleotide is present in said biological sample, wherein specific binding of said reagent to said biological sample indicates said polynucleotide encoding said polypeptide with ALK kinase activity is present in said biological sample.

40. The method of claim 39, wherein said polypeptide is aberrantly expressed full-length ALK polypeptide.

41. The method of claim 39, wherein said polypeptide is an FNl-tmALK fusion polypeptide or a truncated ALK polypeptide.

42. The method of claim 41, wherein said polynucleotide comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: 4, 6, and 8.

43. The method of claim 39, wherein said polypeptide is an ALK fusion polypeptide.

44. The method of claim 43, wherein said polypeptide is selected from the group consisting of an NPM-ALK fusion polypeptide, an AL017-ALK fusion polypeptide, an TFG-ALK fusion polypeptide, an MSN-ALK fusion polypeptide, an TPM3-ALK fusion polypeptide, an TPM4- ALK fusion polypeptide, an ATIC-ALK fusion polypeptide, an MYH9-ALK fusion polypeptide, an CLTC-ALK fusion polypeptide, an SEC31L1-ALK fusion polypeptide, an RANBP2-ALK fusion polypeptide, an CARS-ALK fusion polypeptide, an EML4-ALK fusion polypeptide, an KIF5B-ALK fusion polypeptide, and an TFG-ALK fusion polypeptide, and an FN 1 -ALK fusion polypeptide.

45. The method of claim 34 or 39, wherein said reagent is a nucleic acid probe.

46. The method of claim 45, wherein said nucleic acid probe is a fluorescence in-situ

hybridization (FISH) probe and said method is implemented in a FISH assay.

47. The method of claim 45, wherein said nucleic acid probe is a polymerase chain reaction (PCR) probe and said method is implemented in a PCR assay.

48. The method of claim 19, or 39, wherein said mammalian ovarian cancer or suspected mammalian ovarian cancer is a stromal tumor or a clear cell carcinoma.

49. The method of claim 15, 19, 34, or 39, wherein said biological sample is a circulating tumor cell from said cancer or suspected cancer.

50. The method of claim 15, 19, 34, or 39, wherein said cancer or suspected cancer is from a human.

51. The method of claim 15, 19, 34, or 39, wherein said reagent is detectably labeled.

52. The method of claim 15 or 19, wherein the activity of said polypeptide is detected.

53. The method of claim 15, 19, 34, or 39, wherein said mammalian ovarian cancer or suspected mammalian ovarian cancer is likely to respond to an ALK-inhibiting therapeutic.

54. The method of claim 53, wherein said therapeutic is selected from the group consisting of PF- 02341066, NVT TAE-684, and AP26113.

55. The method of claim 15 or 34, wherein a patient from whom said biological sample is obtained is diagnosed as having a mammalian cancer or suspected mammalian cancer driven by mutant ALK polynucleotide or mutant ALK polypeptide.

56. The method of claim 19 or 39, wherein a patient from whom said biological sample is obtained is diagnosed as having a mammalian ovarian cancer or suspected mammalian ovarian cancer driven by aberrant expression of a full length ALK polypeptide.

57. A method for determining whether a compound inhibits the progression of a mammalian cancer characterized by the expression of a mutant ALK polypeptide or an FN1-ALK fusion polypeptide, said method comprising the step of determining whether said compound inhibits the expression and/or activity of said mutant ALK polypeptide or said FNl-ALK fusion polypeptide in said cancer.

58. A method for inhibiting the progression of a mammalian cancer or suspected mammalian cancer that expresses a mutant ALK polypeptide or FNl-ALK fusion polypeptide, said method comprising the step of inhibiting the expression and/or activity of said mutant ALK polypeptide or said FNl-ALK fusion polypeptide in said mammalian cancer or suspected mammalian cancer.

59. The method of claim 57 or 58, wherein said mutant ALK polypeptide is selected from the group consisting of an FNl-ALKvariantl polypeptide, an FNl-ALKvariant3 polypeptide, an FN1- ALKvariant5 polypeptide, and a truncated ALK polypeptide.

60. The method of claim 57 or 58, wherein said mammalian cancer is a mammalian ovarian cancer.

61. A method for determining whether a compound inhibits the progression of a mammalian ovarian cancer characterized by the expression of a polypeptide with ALK activity, said method comprising the step of determining whether said compound inhibits the expression of said polypeptide in said cancer.

62. A method for inhibiting the progression of a mammalian ovarian cancer or suspected mammalian ovarian cancer that expresses a polypeptide with ALK kinase activity, said method comprising the step of inhibiting the expression and/or activity of said polypeptide in said mammalian ovarian cancer or suspected mammalian ovarian cancer.

63. The method of claim 57 or 61, wherein inhibition is determined using at least one reagent selected from the group consisting of a reagent that specifically binds to a polynucleotide of claim 1, a reagent that specifically binds to polypeptide of claim 9, a reagent that specifically binds to a full length ALK polynucleotide, a reagent that specifically binds to a full length ALK polypeptide, a reagent that specifically binds to a full length FN1 polynucleotide, and a reagent that specifically binds to a full length FN1 polypeptide.

64. The method of claim 61 or 62, wherein said polypeptide is a full length ALK polypeptide aberrantly expressed in said mammalian ovarian cancer.

65. The method of claim 61 or 62, wherein said polypeptide is an FNl-tmALK fusion polypeptide.

66. The method of claim 61 or 62, wherein said polypeptide is an ALK fusion polypeptide.

67. The method of claim 66, wherein said polypeptide is selected from the group consisting of an NPM-ALK fusion polypeptide, an ALO 17-ALK fusion polypeptide, an TFG-ALK fusion polypeptide, an MSN-ALK fusion polypeptide, an TPM3-ALK fusion polypeptide, an TPM4- ALK fusion polypeptide, an ATIC-ALK fusion polypeptide, an MYH9-ALK fusion polypeptide, an CLTC-ALK fusion polypeptide, an SEC31L1-ALK fusion polypeptide, an RANBP2-ALK fusion polypeptide, an CARS-ALK fusion polypeptide, an EML4-ALK fusion polypeptide, an KIF5B-ALK fusion polypeptide, and an TFG-ALK fusion polypeptide, and an FN 1 -ALK fusion polypeptide.

68. The method of claim 57, 58, 61, or 62, wherein said mammalian cancer or suspected mammalian cancer is from a human.

69. The method of claim 58 or 62, wherein expression and/or activity of said polypeptide is inhibited with a composition comprising a therapeutic selected from the group consisting of PF- 02341066, NVT TAE-684, and AP26113.

70. The method of claim 58 or 62, wherein the expression and/or activity of said polypeptide is inhibited with a composition comprising a therapeutic selected from the group consisting of CEP- 14083, CEP-14513, CEP11988, WHI-P131 and WHI-P154.

71. An isolated reagent that specifically detects a polynucleotide of claim 1.

72. The isolated reagent of claim 71, wherein the reagent does not specifically bind to or hybridize to the polynucleotide.

73. The isolated reagent of claim 71, wherein the reagent comprises a primer pair, wherein each member of the primer pair hybridizes to nucleotide sequences adjacent to the polynucleotide or complement thereof and wherein the primer pair can amplify a nucleic acid molecule comprising the polynucleotide.

74. The isolated reagent of claim 71, wherein said reagent comprises a detectable label.