WO2005107803A2 - Methods of determining the prognosis and treatment of subjects with lung cancer - Google Patents

Abstract

The instant invention provides methods of selecting subjects that will benefit from treatment with a Pinl modulator by determining the level and phosphorylation state of Pinl present in a biological sample from the subject. The invention also provides methods of determining the prognosis of a subject having a cell proliferative disorder, e.g., colon cancer, by determining the level of pPinl in a biological sample. The invention further provides methods of treating subjects with a Pinl associated disorder, and methods of monitoring the efficacy of treatment.

Description

Methods of Determining the Prognosis and Treatment of Subjects with Lung Cancer

Related Applications This application claims the benefit of U.S. Patent Application Serial No.

60/569,149 filed May 6, 2004, the contents of which is hereby incorporated by reference.

Background of the invention The GLOBOCAN registry, which provides estimates of cancer incidence and mortality worldwide, shows that in the year 200 there were more than 10 million new cases of cancer, over 6 million cancer deaths and 22.4 million people living with cancer (Ferlay et al., GLOBOCAN 2000: Cancer Incidence, Mortality and Prevalence World Wide, Version 1.0. IARC CancerBase No. 5. Lyon, IARC Press, 2001; Parkin et al, Eur. J. Cancer 2001;37(suppl 8):S4-S66). This represents and increase of approximately 23% in cancer incidence and mortality since 1990. The most common cancers in terms of new cases were lung, breast, colorectal, stomach and liver, and it is estimated that, in 2020, there will be 15 million new cases and 10 million deaths.

Worldwide, lung cancer is the leading cause of cancer death, with an estimated 1.1 million lung-cancer-related deaths in the year 2000 alone, this disease kills more people every year than breast and prostate cancer combined (Ferlay, et al, supra). Moreover, for patients with advanced or metastatic non-small cell lung carcinoma, median survival has improved only about 2 months over the past three decades (Breathanch, et al. J. Clin. Oncol. 2001;19:1734-153). The epidermal growth factor receptor (EGFR) is one of a family of receptors that use tyrosine kinase as the signal transduction trigger (Sedlacek, et al. , Drugs 2000;59:435-476). EGFR is abnormally expressed in many human solid tumors, including non-small cell lung cancer (NSCLC), prostate, breast, colorectal, head and neck, ovarian, gastric and pancreatic cancers (Salomon, et al, Crit. Rev. Oncol. Hematol. 1995; 19: 183-232). From precancerous lesions to malignant tumors, elevated EGFR tyrosine kinase activity has been detected at all stages of tumor progression (Kelloff et al. Cancer Epidemiol Biomarker Prev 1996;5:657-666; Kurei et al, Clin. Cancer Res. 1996;2:1787-1793). The EGFR pathway contributes to a number of processes involved in tumor survival and growth including cell proliferation, inhibition of apoptosis, angiogenesis, and metastasis.

Current anticancer treatments include surgery, radiotherapy, chemotherapy, and endocrine therapy. Typically, the first-line treatment for patients with EGFR related solid tumors (e.g., NSCLC) consists of platinum-based combination chemotherapy (e.g., carboplatin plus paclitaxel, cisplatin plus docetaxel, or cisplatin plus gemcitobine or vinorelbine). However, these regimens have limited efficacy, with a number of combination regimens showing 2-year survival rates of less than 15% (Kelly et al, J. Clin. Oncol. 2001; 19-3210-3218). There are also several small-molecule EGFR-TK inhibitors that have been approved or are in clinical development, such as STI571 (imatinib mesylate, Gleevec™; Novartis Pharmaceuticals Corporation, East Hanover, NJ); gefitinib (Iressa®; Astra- Zeneca Pharmacruticals LP; Wilmington, DE), and OSI-774 (erlotinib, Tarceva ™; OSI Pharmaceuticals; Melville, NE/Genetech; South San Francisco, CA); CI-1033 (Pfizer Inc.; New York, NY) and GW572016 (GlaxoSmithKline; Research Triangle Park, NC). All of these molecules act by competing for occupation of the ATP-binding site on the TK domain of the receptor (Nlahovic et al, The Oncologist;2003;8:531-538). While many of these EGFR-TK inhibitors have been shown to have minimal adverse side effects, tumor responses, for example using genfitinib, have been observed in only 10-19 percent of patients with chemotherapy-refractory advanced cancer (Kris, et al, JAMA 2003;290:2149-58; Fukuoka et al, J. Clin. Oncol. 2003:21:2237-46). Moreover, recent studies have indicated that the majority of lung cancer patients that are responsive to genfitinib, have somatic mutations EGFR (Lynch et al, Ν.Engl.J. Med; April 30, 2004;350(21); Paez et al, www. scienceexpress. or g /29 April 2004/page /10.1126/science.1099314). Given the limitations of the current available treatment options have significant toxicity with limited efficacy, there is still a need for further development of innovative strategies to combat disease, provide maintenance therapy, and enhance the quality of life.

The peptidyl-prolyl cis-trans isomerases (PPIases), or rotamases, are a family of ubiquitous enzymes that catalyze the cis/trans isomerization of the peptide bond on the n-terminal side of proline residues in proteins (Hunter, Cell 92:141-142, 1998). PPIases are divided into three classes, cyclophilins (Cyps), FK-506 binding proteins (FKBPs) and the Pinl/parvulin class. Pinl is a highly conserved protein that catalyzes the isomerization of only phosphorylated Ser/Thr-Pro bonds (Rananathan, R. et al (1997) Cell 89:875-86; Yaffe, et al. 1997, Science 278:1957-1960; Shen, et al. 1998,Genes Dev. 12:706-720; Lu, et al. 1999, Science 283:1325-1328; Crenshaw, et al. 1998, Embo J. 17:1315-1327; Lu, et al. 1999, Nature 399:784-788; Zhou, et al. 1999, Cell Mol. Life Sci. 56:788-806). In addition, Pinl contains an N-terminal WW domain, which functions as a phosphorylated Ser/Thr-Pro binding module (Sudol, M. (1996) Prog. Biophys. Mol Biol 65:113-32). This phosphorylation-dependent interaction targets Pinl to a subset of phosphorylated substrates, including Cdc25, Wee 1, Mytl, Tau-Rad4, and the C-terminal domain of RNA polymerase II large domain (Crenshaw, D.G., et al (1998) Embo. J 17: 1315-27; Shen, M. (1998) Genes Dev. 12:706-20; Wells, NJ. (1999) J Cell. Sci. 112: 3861-71).

It is becoming increasingly apparent that Pinl is involved in the onset and progression of many types of cell proliferative and neurodegenerative disorders (see, for example, Lu, K.P. (2003) Cancer Cell 4:175-80; Wulf, G. et al. (2003) Breast Cancer Res. 5:76-82; Ryo, A. et al. (2003) J. Cell Sci. 116: 773-83; Wulf, G.M. et al. (2001) EMBO J. 20:3459-72; Liou et al. (2003) Nature 424:556-61; Lu, P.J. et al. (1999) Nature 399:784-8). Pinl activity has also been shown to restore the conformation and function of phosphorylated tau by promoting its dephosphorylation, indicating that Pinl is involved in neurodegeneration (Liou, Y.C. et al. (2003) Nature 424:556-61). Accordingly, the present invention is based on the use of for modulators of Pinl , and methods of selecting and treating subjects with disorders characterized by the misexpression of Pinl and/or EGFR, e.g., cancer.

Summary of the Invention It has been determined that the level and location of Pinl is of great importance to the progression of disease. Further, it has been shown that the phosphorylation state of Pinl is important in determining the ability of Pinl to bind substrate. The phosphorylation state and levels of Pinl are important factors in determining which subjects will be likely to benefit from treatment with a Pinl modulator and for determining the prognosis of subjects with a cell proliferative disorders including cancer (see USSN 60/556,008, the entire contents of which are herein incorporated by reference). The present invention is based, at least in part, on the discovery that there is a correlation between EGFR and pPinl expression and the survival of patients having lung cancer. Accordingly, the ability of a clinician to obtain information regarding the expression of EGFR and Pinl would be advantageous in the determination of which subjects are likely to benefit from treatment with a Pinl modulator and/or an EGFR modulator, and in determining the prognosis of a subject with a cell proliferative disorder, e.g., cancer.

Accordingly, the instant invention provides methods to determine which subjects in a population would benefit from treatment with a Pinl modulator and/or an EGFR modulator. The instant invention also provides methods for determining the prognosis of a subject with a cell proliferative disorder. The invention further provides methods to monitor the efficacy of treatment of individuals having a cell proliferative disorder with a Pinl modulator and/or an EGFR modulator. Further, kits are provided to perform the methods of the invention. In one aspect, the invention provides a method for treating a subject having a cell proliferative disorder that has been preselected based on increased levels of EGFR by administering a Pinl modulator and/or an EGFR modulator to the subject such that treatment of the subject occurs.

In another aspect, the invention provides a method for treating a subject having a cell proliferative disorder that has been preselected based on decreased levels of phosphorylated Pinl and increased levels of EGFR by administering a Pinl modulator and/or an EGFR modulator to the subject such that the treatment of the subject occurs.

In a further aspect, the invention provides a method of treating a subject having a cell proliferative disorder that has been preselected based on the increase levels of unphosphorylated Pinl and EGFR, comprising administering a Pinl modulator and/or an EGFR modulator to the subject such that the treatment of the subject occurs.

In another aspect, the invention provides a method of determining the prognosis of a subject having a cell proliferative disorder by obtaining a first biological sample from the subject and determining the level of pPinl in the sample, then obtaining a second biological sample from the subject at a time after collection of the first biological sample and determining the level of pPinl in the sample, wherein an increase in the level of pPinl is indicative of good prognosis. In certain embodiments, the cell proliferative disorder is an EGFR related disorder. In one aspect, the invention provides a method of determining the prognosis of a subject having a cell proliferative disorder by obtaining a first biological sample from the subject and determining the level of pPinl in the sample, then obtaining a second biological sample from the subject at a time after collection of the first biological sample and determining the level of pPinl in the sample, wherein a decrease in the level of pPinl is indicative of poor prognosis. In certain embodiments, the cell proliferative disorder is an EGFR disorder.

In another aspect, the invention provides a method of monitoring the efficacy of treatment of a subject by determining the levels of phosphorylated and/or unphosphorylated Pinl after the administration of a Pinl modulator and/or EGFR modulator to the subject; wherein a change in the level of phosphorylated Pinl and/or unphosphorylated Pinl and/or EGFR is indicative of the efficacy of treatment. In certain embodiments, the level of phosporylated Pinl is increased. In other embodiments the level of unphosphorylated Pinl is decreased. In other embodiments, the level of EGFR is decreased. In one embodiment, the level of EGFR is decreased and the level of phosphorylated Pinl is increased.

Embodiments of the present invention include methods in which the cell proliferative disorder is an EGFR related cancer. In certain embodiments, the cell proliferative disorder is a solid tumor including, but not limited to, lung, prostate, breast, colorectal, head and neck, ovarian, gastric and pancreatic cancer. In one embodiment, the cell proliferative disorder is lung cancer. In related embodiments, the lung cancer is adenocarcinoma, non-small cell or squamous cell lung cancer. In other embodiments, the tumor is non-responsive to traditional cancer therapies, e.g., surgery, radiotherapy, chemotherapy and endocrine therapy. In certain embodiments, the levels of Pinl are determined by measuring the level of phosphorylated and/or unphosphorylated Pinl and/or EGFR using immunohistochemistry. In specific embodiments, the immunohistochemistry is performed using antibodies specific for phosphorylated, unphosphorylated Pinl, or EGFR. In a certain embodiments, the pPinl levels are determined using an antibody specific for pPinl . In other embodiments, the levels of pPinl and EGFR are determined using antibodies specific for pPinl and EGFR, respectively. In other embodiments, the levels of pPinl and/or EGFR are determined using fluorescence in situ hybridization (FISH). In certain embodiments, the subject is classified into one of the following groups based on the level, location and phosphorylation state of Pinl, e.g., nPinlStr, nPinlMod, nPinlweak, nPinlNeg. In other embodiments, the subject is classified based on the level or amplification of EGFR as described herein. In certain embodiments, the subject is classified based on the level of EGFR. In other embodiments, the methods include optionally, and one or more other anti-cancer treatments including, but not limited to, surgery, radiotherapy, chemotherapy, and endocrine therapy. In further embodiments, the other chemotherapy is an EGFR inhibitor such as, but not limited to, STI571 (imatinib mesylate, Gleevec™; Novartis Pharmaceuticals Corporation, East Hanover, NJ); gefitinib (Iressa®; Astra-Zeneca Pharmacruticals LP; Wilmington, DE), and OSI-774 (erlotinib, Tarceva ™; OSI Pharmaceuticals; Melville, NE/Genetech; South San Francisco, CA); CI-1033 (Pfizer Inc.; New York, NY) and GW572016 (GlaxoSmithKline; Research Triangle Park, NC). In other embodiments, the subject is further classified based on the presence of a somatic mutation in EGFR. In one embodiment, the subject is treated with a Pinl modulator when the subject is classified as not having a somatic mutation, e.g., in the tyrosine kinase domain, and is non-responsive to small molecule EGFR inhibitors (e.g., gefitinib).

In a further aspect, the invention provides a kit for selecting a subject that would benefit from treatment with a Pinl modulator and/or an EGFR modulator including a phosphorylated Pinl specific antibody and/or an unphosphorylated Pinl specific antibody and/or an EGFR specific antibody, and directions for use.

In one aspect, the invention provides a kit for determining the prognosis of a subject with a cell proliferative disorder comprising an antibody specific for pPinl and instructions for use. In specific embodiments the antibody enclosed in the kit is a monoclonal or a polyclonal antibody. In a related embodiment, the kit can contain a second antibody specific for specific for EGFR.

Brief Description of the Drawings Figure 1 depicts the polypeptide sequence of Pinl (SEQ ID NO:l). The serine residue identified in Lu et al. ((2002) J Biol Chem. 277: 2381-4) is underlined and in bold. Figure 2 is a bar graph that depicts the fraction of samples with cytoplasmic and nuclear pPINl staining.

Figure 3 depicts a survival curve based on subject's expression of pPinl. The graph depicts the fraction of subjects surviving as a function of time (in months) and negative, weak, moderate or strong pPINl expression.

Figure 4 depicts a survival curve based on the two group classification of pPinl expression.

Figure 5 depicts a survival curve that shows the influence of cytoplasmic pPINl expression. Figures 6A and 6B is a bar graph that depicts the fraction of samples with nuclear and cytoplasmic Pinl staining as a function of pPinl staining, respectively.

Figure 7 A and 7B depict the fraction of samples that are positive for EGFR (IHC) or the fraction of altered EGFR samples (FISH) as a function of pPinl staining, respectively. Figure 8 and 9 depict survival curves of subjects based on the expression of pPinl/EGFR co-expression and pPinl/EGFR co-alterations. The graphs depict the fraction of subjects surviving as a function of time (in months).

Detailed Description of the Invention The term "biological sample" includes solid and body fluid samples. The biological samples of the present invention may include cells, protein or membrane extracts of cells, blood or biological fluids such as ascites fluid or brain fluid (e.g., cerebrospinal fluid). Examples of solid biological samples include samples taken from the lung, prostate, breast, colorectal, head and neck, ovaries, gastric and pancreas. Examples of "body fluid samples" include samples taken from the blood, serum, cerebrospinal fluid, semen, prostate fluid, seminal fluid, urine, saliva, sputum, mucus, bone marrow, lymph, and tears. Samples for use in the methods of the invention can be obtained by standard methods including venous puncture and surgical biopsy. In certain embodiments^ the biological sample is a tissue or organ sample obtained by surgery or by needle biopsy.

The term "Pinl status" indicates a classification of a subject into one of a defined series of groups based on the presence and level of phosphorylated and unphosphorylated Pinl. Exemplary classifications of Pinl status defined herein in the Examples. However, a skilled artisan could further define the categories presented herein to better define Pinl status. For example, Pinl status can be represented as ratio of phosphorylated Pinl to unphosphorylated Pinl. The presence and level of Pinl can be determined using art recognized techniques, e.g., immunohistochemistry using Pinl specific antibodies. Subjects can be treated for Pinl -associated states based on their Pinl status.

The term "nuclear Pinl" is intended to include Pinl polypeptide that is localized to the nucleus of a cell. In certain embodiments, nuclear Pinl is predominantly phosphorylated. The term "cytoplasmic Pinl" is intended to include Pinl polypeptide that is localized to the cytoplasm of a cell. In certain embodiments, cytoplasmic Pinl is predominantly unphosphorylated.

The term "phosphorylation state" is intended to denote that the Pinl polypeptide can exist in either a phosphorylated or unphosphorylated state. The phosphorylation state denotes whether the Pinl in a biological sample is phosphorylated or unphosphorylated, or the relative ratios of phosphorylated to unphosphorylated Pinl in a sample. For example, Lu et al. ((2002) J Biol Chem. 277: 2381-4) demonstrated the importance of the phosphorylation of serine 16 on the ability of Pinl to bind phosphorylated substrate. The term "Pinl -associated state" or "Pinl associated disorder" includes disorders and states (e.g., a disease state) that are associated with the abnormal activity of Pinl. This abnormal activity can be as a result of the misexpression or misregulation of the production, degradation, or regulation of Pinl, e.g., the phosphorylation dephosphorylation of Pinl . Without being bound by theory, Pinl associated disorders that are related to higher than necessary levels of Pinl can be caused by (1) an increase in the level of transcription or translation, or a decrease in the level of degradation, of Pinl such that an abnormally high amount of Pinl polypeptide is present in a cell, or (2) the amount Pinl that is present in the unphosphorylated, i.e., active form, is abnormally high due to either an increase in the dephosphorylation of Pinl or a decrease in the phosphorylation of Pinl. Pinl disorders are often associated with abnormal cell growth, abnormal cell proliferation, or misexpression of Pinl (e.g., Pinl protein or nucleic acid). Pinl -associated states include states resulting from an elevation in the expression of cyclin Dl and/or Pinl . Pinl -associated states also include states resulting from an elevation in the phosphorylation level of c-Jun, particularly phosphorylation of c-Jun on Ser -Pro and/or from an elevation in the level of c-Jun amino terminal kinases (JNKs) present in a cell. Pinl -associated states include neoplasia, cancer, undesirable cell growth, and/or tumor growth. Pinl -associated states include states caused by DNA damage, an oncogenic protein (i.e. Ha-Ras), loss of or reduced expression of a tumor suppressor (i.e. Brcal), and/or growth factors. Pinl -associated state is also intended to include diseases or disorders caused by, or associated with, deregulation of genes and/or gene products involved in a biological pathway that includes Pinl and/or cyclin Dl (e.g. beta-catenin, APC or WNT). Beta-catenin, APC and WNT have been linked to cancer development as demonstrated in Biocliim Biophys Acta. (2003) 1653: 1-24 and Eur J Surg Oncol. (2003) 29: 107-117. Pinl associated states further include disorders and states associated with regulation or activity of Pinl in the brain, e.g., Alzheimer's disease, wherein the phosphorylation state of tau is influenced by the activity of Pinl. The term "EGFR related disorder" as used herein, includes cell proliferative disosrders or states associated with the abnormal activity of EGFR. This abnormal activity can be as a result of the misexpression or misregulation of the production, degradation, or regulation of EGFR. In certain embodiments, misregulation of EGFR is associated with a mutation in the EGFR gene that results in increased EGFR tyrosine kinase activity, e.g., a somatic mutation such as described by Lynch et al, supra, and Paez, et al, supra.

The term "misexpression" includes a non-wild type pattern of gene expression. Expression as used herein includes transcriptional, post transcriptional, e.g., mRNA stability, translational, and post translational stages. Misexpression includes: expression at non-wild type levels, i.e., over or under expression; a pattern of expression that differs from wild type in terms of the time or stage at which the gene is expressed, e.g., increased or decreased expression (as compared with wild type) at a predetermined developmental period or stage; a pattern of expression that differs from wild type in terms of decreased expression (as compared with wild type) in a predetermined cell type or tissue type; a pattern of expression that differs from wild type in terms of the splicing size, amino acid sequence, post-transitional modification, or biological activity of the expressed polypeptide; a pattern of expression that differs from wild type in terms of the effect of an environmental stimulus or extracellular stimulus on expression of the gene, e.g., a pattern of increased or decreased expression (as compared with wild type) in the presence of an increase or decrease in the strength of the stimulus. Misexpression includes any expression from a transgenic nucleic acid. Misexpression includes the lack or non-expression of a gene or transgene, e.g., that can be caused by a deletion of all or part of the gene or its control sequences. The term "carcinoma" includes malignancies of epithelial or endocrine tissues, including respiratory system carcinomas, gastrointestinal system carcinomas, genitourinary system carcinomas, testicular carcinomas, breast carcinomas, prostate carcinomas, endocrine system carcinomas, melanomas, choriocarcinoma, and carcinomas of the cervix, lung, head and neck, colon, and ovary. The term "carcinoma" also includes carcinosarcomas, which include malignant tumors composed of carcinomatous and sarcomatous tissues. The term "adenocarcinoma" includes carcinomas derived from glandular tissue or a tumor in which the tumor cells form recognizable glandular structures.

For example, the therapeutic methods of the present invention can be applied to cancerous cells of mesenchymal origin, such as those producing sarcomas (e.g., fibrosarcoma, myxosarcoma, liosarcoma, chondrosarcoma, osteogenic sarcoma or chordosarcoma, angiosarcoma, endotheliosardcoma, lympangiosarcoma, synoviosarcoma or mesothelisosarcoma); leukemias and lymphomas such as granulocytic leukemia, monocytic leukemia, lymphocytic leukemia, malignant lymphoma, plasmocytoma, reticulum cell sarcoma, or Hodgkin's disease; sarcomas such as leiomysarcoma or rhabdomysarcoma, tumors of epithelial origin such as squamous cell carcinoma, basal cell carcinoma, sweat gland carcinoma, sebaceous gland carcinoma, adenocarcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, undifferentiated carcinoma, bronchogenic carcinoma, melanoma, renal cell carcinoma, hepatoma-liver cell carcinoma, bile duct carcinoma, cholangiocarcinoma, papillary carcinoma, transitional cell carcinoma, chorioaencinoma, semonoma, or embryonal carcinoma; and tumors of the nervous system including gioma, menigoma, medulloblastoma, schwannoma or epidymoma. Additional cell types amenable to treatment according to the methods described herein include those giving rise to mammary carcinomas, gastrointestinal carcinoma, such as colonic carcinomas, bladder carcinoma, prostate carcinoma, and squamous cell carcinoma of the neck and head region. Examples of cancers amenable to treatment according to the methods described herein include colorectal cancers, e.g., colon cancer. The language "inhibiting undesirable cell growth" is intended to include the inhibition of undesirable or inappropriate cell growth. The inhibition is intended to include inhibition of cell proliferation, including rapid proliferation. For example, undesirable cell growth can result in benign masses or malignant tumors. Examples of benign conditions which result from inappropriate cell growth or angiogenesis are diabetic retinopathy, retrolental fϊbrioplasia, neovascular glaucoma, psoriasis, angiofibromas, rheumatoid arthritis, hemangiomas, Karposi's sarcoma, and other conditions or dysfunctions characterized by dysregulated endothelial cell division.

The language "inhibiting tumor growth" or "inhibiting neoplasia" includes the prevention of the growth of a tumor in a subject or a reduction in the growth of a preexisting tumor in a subject, or can be the inhibition of the metastasis of a tumor from one site to another. In particular, the language "tumor" is intended to encompass both in vitro, e.g., in cell culture, and in vivo tumors that form in any organ or body part of the subject. The tumors preferably are tumors sensitive to the Pinl -modulating compounds of the present invention. .

The term "cancer" includes malignancies characterized by deregulated or uncontrolled cell growth, for instance carcinomas, sarcomas, leukemias, and lymphomas. The terms "cancer" and "tumor" may be used interchangeably herein. The term "cancer" includes primary malignant tumors, e.g., those whose cells have not migrated to sites in the subject's body other than the site of the original tumor, and secondary malignant tumors, e.g., those arising from metastasis, the migration of tumor cells to secondary sites that are different from the site of the original tumor.

Pinl modulating agents may be used to treat, inhibit, and/or prevent undesirable cell growth, neoplasia, neurodegenerative diseases, and/or cancer in a subject. Pinl modulating compounds may be used to inhibit Pinl activity in a subject. In one embodiment, Pinl modulating compounds may be used to inhibit cyclin Dl expression in a subject. In a further embodiment, Pinl modulating compounds of the invention can be used to treat subjects having Alzheimer's disease.

In certain embodiments Pinl modulators have a Kj for Pinl of less than 0.2mM, less than 0. ImM, less than 750 μM, less than 500 μM, less than 250 μM, less than 100 μM, less than 50 μM, less than 500 nM, less than 250nM, less than 50 nM, less than 10 nM, less than 5 nM, or less than 2 nM. The language "Pinl modulating compound" refers to compounds that modulate, e.g., inhibit, promote, or otherwise alter, the activity of Pinl. Pinl modulating compounds include both Pinl agonists and antagonists. In certain embodiments, the Pinl modulating compounds include compounds that interact with the peptidyl prolyl isomrease domain (PPI) and/or the WW domain of Pinl . In certain embodiments, the Pinl modulating compound is substantially specific to Pinl. The phrase "substantially specific for Pinl" is intended to include inhibitors of the invention that have a Kj or K_d that is at least 2, 3, 4, 5, 10, 15, or 20 times less than the Kj or K_d for other peptidyl prolyl isomerases, e.g., hCyP-A, hCyP-B, hCyP-C, NKCA, hFKBP-12, hFKBP-13, and hFKBP-25.

Examples of Pinl modulating compounds include compounds described in U.S. Provisional Application No. 60/537,171, filed January 16, 2004, entitled "Pinl- Modulating Compounds and Methods of Use Thereof; U.S. Application No. 10/361,206 filed March 3, 2003, entitled "Pinl -Modulating Compounds and Methods of Use Thereof; U.S. Application Serial No. 10/379,410, filed March 3, 2003, entitled "Pinl -Modulating Compounds and Methods of Use Thereof; U.S. Application Serial No: 10/379,404, filed March 3, 2003, entitled "Pinl -Modulating Compounds and Methods of Use Thereof; U.S. Application Serial No. 10/379,115, filed on March 3, 2003; entitled "Methods for Designing Specific Inhibitors for Pinl Proline Isomerase and Pinl-Related Molecules"; U.S. Application No. 10/379,161, filed March 3, 2003, entitled "Methods of Treating Pinl Associated Disorders"; U.S. Provisional Application No. 60/463271, entitled "PHOTOCHEMOTHERAPEUTIC COMPOUNDS FOR USE IN TREATMENT OF PIN1 -ASSOCIATED STATES", filed April 16, 2003; and U.S. Application No. 10/379,408, entitled "Pinl -Modulating Compounds and Methods of Use Thereof, filed March 3, 2003. The entire contents of each of the aforementioned applications are hereby expressly incorporated herein by reference in their entireties. In certain embodiments, the Pinl inhibiting compounds include compounds that interact with the PPI and/or the WW domain of Pinl.

The term "preselected" is intended to mean that a subject has been identified based on their level and/or phosphorylation state of Pinl to be likely to benefit from treatment with a Pinl modulator. In certain embodiments, a subject is preselected based on the levels of unphosphorylated Pinl, phosphorylated Pinl, or the relative amounts of phosphorylated and unphosphorylated Pinl . The term "prognosis" is intended to mean the probable course and outcome of a disease, e.g., a Pinl associated disease. In certain embodiments, the term is intended to mean the likelihood that a subject will live longer than the average length of time that a population of subjects with a similar disease will live. In related embodiments, a subject's prognosis is indicative of the aggressiveness and course of treatment that a subject will receive.

Polvpeptides

This invention relates to methods of diagnosis, treatment and prognosis of subjects with a cell proliferative disorder such as cancer, e.g., an EGFR related disorder.. Pinl is a highly conserved protein (SEQ ID NO:l) that catalyzes the isomerization of only phosphorylated Ser/Thr-Pro bonds (Rananathan, R. et al. (1997) Cell 89:875-86; Yaffe, et al. 1997, Science 278:1957-1960; Shen, et al. 1998,Genes Dev. 12:706-720; Lu, et al. 1999, Science 283:1325-1328; Crenshaw, et al. 1998, Embo J. 17:1315-1327; Lu, et al. 1999, Nature 399:784-788; Zhou, et al. 1999, Cell Mol. Life Sci. 56:788-806). In addition, Pinl contains an N-terminal WW domain, which functions as a phosphorylated Ser/Thr-Pro binding module (Sudol, M. (1996) Prog. Biophys. Mol. Biol. 65:113-32). This phosphorylation-dependent interaction targets Pinl to a subset of phosphorylated substrates, including Cdc25, Wee 1, Mytl, Tau-Rad4, and the C- terminal domain of RNA polymerase II large domain (Crenshaw, D.G., et al. (1998) Embo. J. 17:1315-27; Shen, M. (1998) Genes Dev. 12:706-20; Wells, NJ. (1999) J. Cell. Sci. 112: 3861-71).

The specificity of Pinl activity is essential for cell growth; depletion or mutations of Pinl cause growth arrest, affect cell cycle checkpoints and induce premature mitotic entry, mitotic arrest and apoptosis in human tumor cells, yeast or Xenopus extracts (Lu, et al. 1996, Nature 380:544-547; Winkler, et al. 200, Science 287:1644-1647; Hani, et al. 1999. J. Biol. Chem. 274:108-116). In addition, Pinl is dramatically misexpressed in human cancer samples and the total level or concentration of Pinl is correlated with the aggressiveness of tumors. Moreover, inhibition of Pinl by various approaches, including Pinl antisense polynucleotides or genetic depletion, kills human and yeast dividing cells by inducing premature mitotic entry and apoptosis.

Thus, Pinl -catalyzed prolyl isomerization regulates the conformation and function of these phosphoprotein substrates and facilitates dephosphorylation because of the conformational specificity of some phosphatases. Thus, Pinl -dependent peptide bond isomerization is an important post-phosphorylation regulatory mechanism, allowing cells to turn phosphoprotein function on or off with high efficiency and specificity during temporally regulated events, including the cell cycle (Lu et al., supra). The invention further provides a method of determining the prognosis of a subject with an EGFR related cell proliferative disorder, e.g., lung cancer, comprising determining the levels of pPinl and EGFR in a biological sample. The sequence of the EGFR polypeptide can be found at, for example, SwissProt Accession Number P00533.

Antibodies

The invention provides a method of detecting the presence and amount of unphosphorylated Pinl and/or phosphorylated Pinl in a biological sample. The invention also provides a method of determining the amount of phosphorylated Pinl relative to the amount of unphosphorylated Pinl in a sample. Accordingly, the methods of the invention may use antibodies that recognize phosphorylated Pinl and unphosphorylated Pinl, antibodies that are specific for phosphorylated Pinl, and antibodies that are specific for unphosphorylated Pinl . Antibodies that are specific for Pinl are described in US patent 6,596,848, the entire contents of which are incorporated herein by reference. Phosphorylation specific Pinl antibodies are commercially available from, for example, Cell Signaling Technology (Beverly, MA).

The invention also provides methods of detecting levels of Pinl in combination with EGFR. Levels of EGFR can be determined using antibodies as described in the Examples set forth below, or using nucleic acid probes, specific for the EGFR coding sequence uisng methods standard to one of skill in the art. The phrase "antibodies specific for phosphorylated Pinl" is intended to include antibodies that preferentially bind to an antigen of Pinl that contains a phosphorylated residue. Antibodies that are specific for phosphorylated Pinl bind to Pinl with at least twice the affinity that they bind to a nonspecific antigen (e.g., BSA or casein). Further, an antibody that is specific for phosphorylated Pinl has more affinity for phosphorylated Pinl than it does unphosphorylated Pinl . In certain embodiments, the antibody specific for phosphorylated Pinl binds with at least 2, 3, 4, 5, 10, 20, 50, 100, 500, or 1000 times the affinity to phosphorylated Pinl as it does unphosphorylated Pinl . In at least one embodiment, the antibody specific for phosphorylated Pinl recognizes a Pinl molecule that is phosphorylated on serine 16 of SEQ ID NO: 1.

The phrase "antibodies specific for unphosphorylated Pinl" is intended to include antibodies that preferentially bind a Pinl polypeptide that is not phosphorylated. Antibodies that are specific for unphosphorylated Pinl bind to Pinl with at least twice the affinity that they bind to a nonspecific antigen (e.g., BSA or casein). Further, an antibody that is specific for unphosphorylated Pinl has more affinity for unphosphorylated Pinl than it does phosphorylated Pinl. In certain embodiments, the antibody specific for unphosphorylated Pinl binds with at least 2, 3, 4, 5, 10, 20, 50, 100, 500, or 1000 times the affinity to unphosphorylated Pinl as it does phosphorylated Pinl. In at least one embodiment, the antibody specific for unphosphorylated Pinl recognizes an epitope of Pinl that comprising a residue that is capable of being phosphorylated, e.g., serine 16 of SEQ ID NO:l.

Polyclonal antibodies for use in the present invention can be produced by immunizing animals, usually a mammal, by multiple subcutaneous or intraperitoneal injections of an immunogen (antigen) and an adjuvant as appropriate. As an illustrative embodiment, animals are typically immunized against a protein, peptide or derivative by combining about 1 μg to 1 mg of protein capable of eliciting an immune response, along with an enhancing carrier preparation, such as Freund's complete adjuvant, or an aggregating agent such as alum, and injecting the composition intradermally at multiple sites. Animals are later boosted with at least one subsequent administration of a lower amount, as 1/5 to 1/10 the original amount of immunogen in Freund's complete adjuvant (or other suitable adjuvant) by subcutaneous injection at multiple sites. Animals are subsequently bled, serum assayed to determine the specific antibody titer, and the animals are again boosted and assayed until the titer of antibody no longer increases (i.e., plateaus).

Such populations of antibody molecules are referred to as "polyclonal" because the population comprises a large set of antibodies each of which is specific for one of the many differing epitopes found in the immunogen, and each of which is characterized by a specific affinity for that epitope. An epitope is the smallest determinant of antigenicity, which for a protein, comprises a peptide of six to eight residues in length (Berzofsky, J. and I. Berkower, (1993) in Paul, W., Ed., Fundamental Immunology-,

Raven Press, N.Y., p.246). Affinities range from low, e.g. 10"6 M, to high, e.g., 10~11 M. The polyclonal antibody fraction collected from mammalian serum is isolated by well known techniques, e.g. by chromatography with an affinity matrix that selectively binds immunoglobulin molecules such as protein A, to obtain the IgG fraction. To enhance the purity and specificity of the antibody, the specific antibodies may be further purified by immunoaffinity chromatography using solid phase-affixed immunogen. The antibody is contacted with the solid phase-affixed immunogen for a period of time sufficient for the immunogen to immunoreact with the antibody molecules to form a solid phase-affixed immunocomplex. Bound antibodies are eluted from the solid phase by standard techniques, such as by use of buffers of decreasing pH or increasing ionic strength, the eluted fractions are assayed, and those containing the specific antibodies are combined.

"Monoclonal antibody" or "monoclonal antibody composition" as used herein refers to a preparation of antibody molecules of single molecular composition. A monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope. Monoclonal antibodies can be prepared using a technique which provides for the production of antibody molecules by continuous growth of cells in culture. These include but are not limited to the hybridoma technique originally described by Kohler and Milstein (1975, Nature 256:495-497; see also Brown et al. 1981 J Immunol 127:539-46; Brown et al., 1980, JBiol Chem 255:4980-83; Yeh et al., 1976, PNAS 76:2927-31 ; and Yeh et al, 1982, Int. J Cancer 29:269-75) and the more recent human B cell hybridoma technique (Kozbor et al., 1983, Immunol Today 4:72), EBN-hybridoma technique (Cole et al., 1985, Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96), and trioma techniques. The technology for producing hybridomas is well known (see generally Current Protocols in Immunology, Coligan et al. ed., John Wiley & Sons, New York, 1994). Hybridoma cells producing a monoclonal antibody of the invention are detected by screening the hybridoma culture supematants for antibodies that bind the polypeptide of interest, e.g., using a standard ELISA assay.

A monoclonal antibody can be produced by the following steps. In all procedures, an animal is immunized with an antigen such as a protein (or peptide thereof) as described above for preparation of a polyclonal antibody. The immunization is typically accomplished by administering the immunogen to an immunologically competent mammal in an immunologically effective amount, i.e., an amount sufficient to produce an immune response. Preferably, the mammal is a rodent such as a rabbit, rat or mouse. The mammal is then maintained on a booster schedule for a time period sufficient for the mammal to generate high affinity antibody molecules as described. A suspension of antibody-producing cells is removed from each immunized mammal secreting the desired antibody. After a sufficient time to generate high affinity antibodies, the animal (e.g., mouse) is sacrificed and antibody-producing lymphocytes are obtained from one or more of the lymph nodes, spleens and peripheral blood. Spleen cells are preferred, and can be mechanically separated into individual cells in a physiological medium using methods well known to one of skill in the art. The antibody-producing cells are immortalized by fusion to cells of a mouse myeloma line. Mouse lymphocytes give a high percentage of stable fusions with mouse homologous myelomas, however rat, rabbit and frog somatic cells can also be used. Spleen cells of the desired antibody-producing animals are immortalized by fusing with myeloma cells, generally in the presence of a fusing agent such as polyethylene glycol. Any of a number of myeloma cell lines suitable as a fusion partner are used with to standard techniques, for example, the P3-NSl/l-Ag4-l, P3-x63-Ag8.653 or Sp2/O-Agl4 myeloma lines, available from the American Type Culture Collection (ATCC), Rockville, MD.

The fusion-product cells, which include the desired hybridomas, are cultured in selective medium such as HAT medium, designed to eliminate unfused parental myeloma or lymphocyte or spleen cells. Hybridoma cells are selected and are grown under limiting dilution conditions to obtain isolated clones. The supematants of each clonal hybridoma is screened for production of antibody of desired specificity and affinity, e.g., by immunoassay techniques to determine the desired antigen such as that used for immunization. Monoclonal antibody is isolated from cultures of producing cells by conventional methods, such as ammonium sulfate precipitation, ion exchange chromatography, and affinity chromatography (Zola et al, Monoclonal Hybridoma Antibodies: Techniques And Applications, Hurell (ed.), pp. 51-52, CRC Press, 1982). Hybridomas produced according to these methods can be propagated in culture in vitro or in vivo (in ascites fluid) using techniques well known to those with skill in the art. Alternative to preparing monoclonal antibody-secreting hybridomas, a monoclonal antibody directed against a polypeptide of the invention can be identified and isolated by screening a recombinant combinatorial immunoglobulin library (e.g., an antibody phage display library) with the polypeptide of interest. Kits for generating and screening phage display libraries are commercially available (e.g., the Pharmacia Recombinant Phage Antibody System, Catalog No. 27-9400-01; and the Stratagene SurfZAP Phage Display Kit, Catalog No. 240612). Additionally, examples of methods and reagents particularly amenable for use in generating and screening an antibody display library can be found in, for example, U.S. Patent No. 5,223,409; PCT Publication No. WO 92/18619; PCT Publication No. WO 91/17271; PCT Publication No. WO 92/20791; PCT Publication No. WO 92/15679; PCT Publication No. WO 93/01288; PCT Publication No. WO 92/01047; PCT Publication No. WO 92/09690; PCT Publication No. WO 90/02809; Fuchs et al (1991) Bio/Technology 9:1370-1372; Hay et al. (1992) Hum. Antibod. Hybridomas 3:81-85; Huse et al. (1989) Science 246:1275- 1281; Griffiths et al. (1993) EMBO J. 12:725-734.

Additionally, recombinant antibodies, such as chimeric and humanized monoclonal antibodies, comprising both human and non-human portions, which can be made using standard recombinant DNA techniques, are within the scope of the invention. Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art, for example using methods described in PCT Publication No. WO 87/02671; European Patent Application 184,187; European Patent Application 171,496; European Patent Application 173,494; PCT Publication No. WO 86/01533; U.S. Patent No. 4,816,567; European Patent Application 125,023; Better et al (1988) Science 240:1041-1043; Liu et al (1987) Proc. Natl Acad. Sci. USA 84:3439-3443; Liu et al. (1987) J Immunol 139:3521- 3526; Sun et al (1987) Proc. Natl Acad. Sci. USA 84:214-218; Nishimura et α . (1987) Cancer Res. 47:999-1005; Wood et al (1985) Nature 314:446-449; and Shaw et al. (1988) J Natl Cancer Inst. 80:1553-1559); Morrison (1985) cience 229:1202-1207; i et al (1986)

Bio/Techniques 4:214; U.S. Patent 5,225,539; Jones et al (1986) Nature 321:552-525; Nerhoeyan et al. (1988) Science 239:1534; and Beidler et al (1988) J. Immunol. 141 :4053-4060.

"Labeled antibody" as used herein includes antibodies that are labeled by a detectable means and includes enzymatically, radioactively, fluorescently, chemiluminescently, and/or bioluminescently labeled antibodies.

One of the ways in which an antibody can be detectably labeled is by linking the same to an enzyme. This enzyme, in turn, when later exposed to its substrate, will react with the substrate in such a manner as to produce a chemical moiety which can be detected, for example, by spectrophotometric, fluorometric or by visual means. Enzymes which can be used to detectably label the Pinl -specific or a EGFR-specific antibody include, but are not limited to, malate dehydrogenase, staphylococcal nuclease, delta-N- steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-NI- phosphate dehydrogenase, glucoamylase and acetylcholinesterase.

Detection may be accomplished using any of a variety of immunoassays. For example, by radioactively labeling an antibody, it is possible to detect the antibody through the use of radioimmune assays. A description of a radioimmune assay (RIA) may be found in Laboratory Techniques and Biochemistry in Molecular Biology, by Work, T. S., et al., North Holland Publishing Company, NY (1978), with particular reference to the chapter entitled "An Introduction to Radioimmune Assay and Related Techniques" by Chard, T.

The radioactive isotope can be detected by such means as the use of a gamma counter or a scintillation counter or by audioradiography. Isotopes which are particularly useful for the purpose of the present invention are: H, I, S, C, and preferably I. It is also possible to label an antibody with a fluorescent compound. When the fluorescently labeled antibody is exposed to light of the proper wave length, its presence can then be detected due to fluorescence. Among the most commonly used fluorescent labeling compounds are fluorescein isothiocyanate, rhodamine, phycoerytherin, phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine.

An antibody can also be detectably labeled using fluorescence emitting metals such as Eu, or others of the lanthanide series. These metals can be attached to the antibody using such metal chelating groups as diethylenetriaminepentaacetic acid (DTP A) or ethylenediaminetetraacetic acid (EDTA).

An antibody also can be detectably labeled by coupling it to a chemiluminescent compound. The presence of the chemiluminescent-tagged antibody is then determined by detecting the presence of luminescence that arises during the course of a chemical reaction. Examples of particularly useful chemiluminescent labeling compounds are luminol, luciferin, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester. Likewise, a bioluminescent compound may be used to label an antibody of the present invention. Bioluminescence is a type of chemiluminescence found in biological systems in which a catalytic protein increases the efficiency of the chemiluminescent reaction. The presence of a bioluminescent protein is determined by detecting the presence of luminescence. Important bioluminescent compounds for purposes of labeling are luciferin, luciferase and aequorin.

Methods of the Invention Selection Methods In one embodiment, the invention provides methods for evaluating subjects for the level and phosphorylation state of Pinl . In related embodiments, the invention provides methods for evaluating subjects for the level on Pinl and EGFR. These results can be used to preselect a subject for treatment with a Pinl modulator and/or and EGFR modulator. The amount of phosphorylated and/or unphosphorylated Pinl and/or EGFR in a biological sample may be determined by a radioimmunoassay, an immunoradiometric assay, enzyme immunoassay, and /or by immunohistochemistry using antibodies specific for phosphorylated Pinl or unphosphorylated Pinl, or EGFR, respectively. "Radioimmunoassay" is a technique for detecting and measuring the concentration of an antigen using a labeled (i.e. radioactively labeled) form of the antigen. Examples of radioactive labels for antigens include ³H, ¹⁴C, and ¹²⁵I. The concentration of phosphorylated and/or unphosphorylated Pinl and/or EGFR in a sample (i.e. biological sample) is measured by having the antigen in the sample compete with a labeled (i.e. radioactively) antigen for binding to an antibody to the antigen. To ensure competitive binding between the labeled antigen and the unlabeled antigen, the labeled antigen is present in a concentration sufficient to saturate the binding sites of the antibody. The higher the concentration of antigen in the sample, the lower the concentration of labeled antigen that will bind to the antibody.

In a radioimmunoassay, to determine the concentration of labeled antigen bound to antibody, the antigen-antibody complex must be separated from the free antigen. One method for separating the antigen-antibody complex from the free antigen is by precipitating the antigen-antibody complex with an anti-isotype antiserum. Another method for separating the antigen-antibody complex from the free antigen is by precipitating the antigen-antibody complex with formalin-killed S. aureus. Yet another method for separating the antigen-antibody complex from the free antigen is by performing a "solid-phase radioimmunoassay" where the antibody is linked (i.e. covalently) to Sepharose beads, polystyrene wells, polyvinylchloride wells, or microtiter wells. By comparing the concentration of labeled antigen bound to antibody to a standard curve based on samples having a known concentration of antigen, the concentration of antigen in the biological sample can be determined.

An "Immunoradiometric assay" (IRMA) is an immunoassay in which the antibody reagent is radioactively labeled. An IRMA requires the production of a multivalent antigen conjugate, by techniques such as conjugation to a protein e.g., rabbit serum albumin (RSA). The multivalent antigen conjugate must have at least 2 antigen residues per molecule and the antigen residues must be of sufficient distance apart to allow binding by at least two antibodies to the antigen. For example, in an IRMA the multivalent antigen conjugate can be attached to a solid surface such as a plastic sphere. Unlabeled "sample" antigen and antibody to antigen which is radioactively labeled are added to a test tube containing the multivalent antigen conjugate coated sphere. The antigen in the sample competes with the multivalent antigen conjugate for antigen antibody binding sites. After an appropriate incubation period, the unbound reactants are removed by washing and the amount of radioactivity on the solid phase is determined. The amount of bound radioactive antibody is inversely proportional to the concentration of antigen in the sample. ,

The most common enzyme immunoassay is the "Enzyme-Linked Immunosorbent Assay (ELISA)." The "Enzyme-Linked Immunosorbent Assay (ELISA)" is a technique for detecting and measuring the concentration of an antigen using a labeled (i.e. enzyme linked) form of the antibody.

In a "sandwich ELISA", an antibody (i.e. to phosphorylated and unphosphorylated Pinl) is linked to a solid phase (i.e. a microtiter plate) and exposed to a biological sample containing, phosphorylated and/or unphosphorylated Pinl . The solid phase is then washed to remove unbound antigen. A labeled (i.e. enzyme linked) is then bound to the bound-antigen (if present) forming an antibody-antigen-antibody sandwich. Examples of enzymes that can be linked to the antibody are alkaline phosphatase, horseradish peroxidase, luciferase, urease, and β-galactosidase. The enzyme linked antibody reacts with a substrate to generate a colored reaction product that can be assayed for.

In a "competitive ELISA", antibody is incubated with a sample containing phosphorylated and unphosphorylated Pinl . The antigen-antibody mixture is then contacted with an antigen-coated solid phase (i.e. a microtiter plate). The more antigen present in the sample, the less free antibody that will be available to bind to the solid phase. A labeled (i.e. enzyme linked) secondary antibody is then added to the solid phase to determine the amount of primary antibody bound to the solid phase.

In an "immunohistochemistry assay" a section of tissue for is tested for specific proteins by exposing the tissue to antibodies that are specific for the type of Pinl protein that is being assayed (e.g., phosphorylated and unphosphorylated Pinl. The antibodies are then visualized by any of a number of methods to determine the presence and amount of the protein present. Examples of methods used to visualize antibodies are, for example, through enzymes linked to the antibodies (e.g., luciferase, alkaline phosphatase, horseradish peroxidase, or β-galactosidase), or chemical methods (e.g.,

DAB/Substrate chromagen). Examples of immunohistochemsitry assays are provided in the Examples.

Once the levels of phosphorylated and/or unphosphorylated Pinl and/or EGFR in a biological sample are determined the subject can be classified based on the level of phosphorylated and/or unphosphorylated Pinl and/or EGFR. Exemplary classifications provided in the Examples. The data presented in the Examples indicates that the majority of phosphorylated Pinl localizes to the nucleus while the majority of unphosphorylated Pinl localizes to the cytoplasm.

In one embodiment, subjects with high levels of unphosphorylated Pinl localized in the cytoplasm or low levels of pPin in the nucleus are preselected for treatment with a Pinl modulator. In another embodiment, subjects with high levels of EGFR and low levels of pPinl are selected for treatment with a Pinl modulator and an EGFR modulator.

Methods of Prognosis

The instant invention provides method of determining the prognosis of a subject with a Pinl associated disorder or an EGFR related cell proliferative disorder, such as cancer. In certain embodiments, the cancer is a solid tumor such as, but not limited to lung, prostate, breast, colorectal, head and neck, ovarian, gastric and pancreatic cancer. In certain embodiments the cancer is lung cancer such as non-small cell carcinoma, adenocarcinoma and squamous cell carcinoma. The instant invention provides for the determination of the prognosis of a subject by evaluating the levels of pPinl and/or EGFR in the subject at one or more points in time.

In one embodiment, the level of pPinl and/or EGFR in a subject can be compared to the statistical mean level in a population of subjects with similar diseases and the prognosis of the subject can be determined based on the level of pPinl and/or EGFR relative to the statistical mean. For example, if the level of pPinl in a subject is lower than the mean, the prognosis of the individual is considered poor, e.g., the subject will likely not survive for as long as the mean length of survival of the population. If the level of pPinl in a subject is higher than the mean , the prognosis of the individual is considered good, e.g., the subject will survive for as long as the mean length of survival of the population, or longer. The term "statistical mean" is used herein in a manner consistent with the well- understood definitions in the art of statistics. The statistical mean can be determined by quantitating the level of pPinl and/or EGFR in a statistically significant number of subjects and determining the mean value of that population.

In another embodiment, the prognosis of an individual can be determined by evaluating the level of pPinl and/or EGFR in biological samples isolated at different time points. If the levels of pPinl decrease from a first sample to a second sample, the prognosis of the subject is poor. If the levels of pPinl in a sample stay the same, or increase, the prognosis is good. If the levels of EGFR increase from the first sample to a second sample, the prognosis of the subject is poor. The levels of pPinl and/or EGFR can be determined and compared with a survival curve generated with data from a statistically significant number of subjects having a similar disease. The comparison of the pPinl and/or EGFR levels in a subject to the survival curve will determine the prognosis of a subject, i.e., the chance the subject has to survive for a given amount of time. In one embodiment, the levels of pPinl in a biological sample can be determined and used in combination with the levels of EGFR to determine the prognosis^' of a subject. Exemplary methods to predict the prognosis of a subject in are provided in the Examples. Survival curves are provided for subjects based on the determination of pPinl levels and/or EGFR levels or alterations. One of ordinary skill in the art would understand that these markers are exemplary and this analysis could be performed with any other known cancer marker. The use of pPinl and one or more additional makers allows for a more accurate determination of a subject's prognosis.

The prognosis of a subject as determined by the methods disclosed herein, can aide in the determination of what course of treatment to provide a subject. Further, the prognosis can indicate the aggressiveness of treatment that is required.

Methods of Treatment

The term "subject" is intended to include living organisms, e.g., prokaryotes and eukaryotes. Examples of subjects include mammals, e.g., humans, dogs, cows, horses, pigs, sheep, goats, cats, mice, rabbits, rats, and transgenic non-human animals. Most preferably the subject is a human.

The term "subject that would benefit from treatment with a Pinl modulator" is intended to include subjects having a Pinl associated disorder wherein treatment of said subject with a Pinl modulator would alleviate, reduce or eliminate one or more symptoms of the Pinl disorder and/or an EGFR related cell proliferative disorder, e.g., cancer..

The language "effective amount" of the compound is that amount necessary or sufficient to treat or prevent or alleviate at least one symptom of a cell proliferative morphological and/or somatic symptom of the cell proliferative disorder. In an example, an effective amount of a Pinl modulator and/or EGFR modulator is the amount sufficient to inhibit undesirable cell growth in a subject. In another example, an effective amount of the modulator compound(s) is the amount sufficient to reduce the size of a pre-existing benign cell mass or malignant tumor in a subject. The effective amount can vary depending on such factors as the size and weight of the subject, the type of illness, or the particular Pinl compound, EGFR compound or combination thereof. For example, the choice of the Pinl modulator and/or EGFR modulator can affect what constitutes an "effective amount". One of ordinary skill in the art would be able to study the aforementioned factors and make the determination regarding the effective amount of the Pinl modulating compound and/or EGFR modulating compound without undue experimentation.

The regimen of administration can affect what constitutes an effective amount of the compound(s). A Pinl modulator compound and/or EGFR modulator can be administered to the subject either prior to or after the onset of a Pinl associated state, EGFR related disorder, e.g., cell proliferative disorder. Further, several divided dosages, as well as staggered dosages, can be administered daily or sequentially, or the dose can be continuously infused, or can be a bolus injection. Further, the dosages of the modulators can be proportionally increased or decreased as indicated by the exigencies of the therapeutic or prophylactic situation.

The term "treated," "treating" or "treatment" includes the diminishment or alleviation of at least one symptom associated or caused by the state, disorder or disease being treated. For example, treatment can be diminishment of one or several symptoms of a disorder or complete eradication of a disorder. While it is possible for a compounds to be administered alone, it is preferable to administer the compound as a pharmaceutical composition. The language "pharmaceutical composition" includes preparations suitable for administration to mammals, e.g., humans. When the modulators are administered as pharmaceuticals to mammals, e.g., humans, they can be given per se or as a pharmaceutical composition containing, for example, 0.1 to 99.5% (more preferably, 0.5 to 90%) of active ingredient in combination with a pharmaceutically acceptable carrier.

The phrase "pharmaceutically acceptable carrier" is art recognized and includes a pharmaceutically acceptable material, composition or vehicle, suitable for administering compounds of the present invention to mammals. The carriers include liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting the subject agent from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which can serve as pharmaceutically acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as com starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, com oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffer solutions; and other non-toxic compatible substances employed in pharmaceutical formulations.

Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.

Examples of pharmaceutically acceptable antioxidants include: water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, α-tocopherol, and the like; and metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

Formulations of the present invention include those suitable for oral, nasal, topical, transdermal, buccal, sublingual, rectal, vaginal and/or parenteral administration. The formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound which produces a therapeutic effect. Generally, out of one hundred per cent, this amount will range from about 1 percent to about ninety-nine percent of active ingredient, preferably from about 5 percent to about 70 percent, most preferably from about 10 per cent to about 30 per cent.

Methods of preparing these formulations or compositions include the step of bringing into association a compound of the present invention with the carrier and, optionally, one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association a compound of the present invention with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product. In solid dosage forms for oral administration (capsules, tablets, pills, dragees, powders, granules and the like), the active ingredient is mixed with one or more pharmaceutically acceptable carriers, such as sodium citrate or dicalcium phosphate, and/or any of the following: fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and/or silicic acid; binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and/or acacia; humectants, such as glycerol; disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; solution retarding agents, such as paraffin; absorption accelerators, such as quaternary ammonium compounds; wetting agents, such as, for example, cetyl alcohol and glycerol monostearate; absorbents, such as kaolin and bentonite clay; lubricants, such a talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof; and coloring agents. In the case of capsules, tablets and pills, the pharmaceutical compositions may also comprise buffering agents. Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like.

A tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared using binder (for example, gelatin or hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (for example, sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), surface-active or dispersing agent. Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. The tablets, and other solid dosage forms of the pharmaceutical compositions, such as dragees, capsules, pills and granules, may optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical-formulating art. They may also be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, • hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile, other polymer matrices, liposomes and/or microspheres. They may be sterilized by, for example, filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved in sterile water, or some other sterile injectable medium immediately before use. These compositions may also optionally contain opacifying agents and may be of a composition that they release the active ingredient(s) only, or preferentially, in a certain portion of the gastrointestinal tract, optionally, in a delayed manner. Examples of embedding compositions which can be used include polymeric substances and waxes. The active ingredient can also be in micro-encapsulated form, if appropriate, with one or more of the above-described excipients.

Liquid dosage forms for oral administration of the compounds of the invention include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active ingredient, the liquid dosage forms may contain inert diluent commonly used in the art, such as, for example, water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3- butylene glycol, oils (in particular, cottonseed, groundnut, co , germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.

Besides inert dilutents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents. Suspensions, in addition to the active compounds, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar- agar and tragacanth, and mixtures thereof.

Formulations of pharmaceutical compositions for use in the methods of the invention for rectal or vaginal administration may be presented as a suppository, which may be prepared by mixing one or more compounds of the invention with one or more suitable nonirritating excipients or carriers comprising, for example, cocoa butter, polyethylene glycol, a suppository wax or a salicylate, and which is solid at room temperature, but liquid at body temperature and, therefore, will melt in the rectum or vaginal cavity and release the active compound.

Dosage forms for the topical or transdermal administration of a compound include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants. The active compound may be mixed under sterile conditions with a pharmaceutically acceptable carrier, and with any preservatives, buffers, or propellants which may be required.

The ointments, pastes, creams and gels may contain, in addition to an active compound, excipients, such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.

Transdermal patches have the added advantage of providing controlled delivery of a compound to the body. Such dosage forms can be made by dissolving or dispersing the compound in the proper medium. Absorption enhancers can also be used to increase the flux of the compound across the skin. The rate of such flux can be controlled by either providing a rate controlling membrane or dispersing the active compound in a polymer matrix or gel.

Pharmaceutical compositions suitable for parenteral administration comprise one or more compounds in combination with one or more pharmaceutically acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents. Examples of suitable aqueous and nonaqueous carriers which may be employed in the pharmaceutical compositions for use in the methods of the invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms may be. ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.

Injectable depot forms are made by forming microencapsule matrices of the subject compounds in biodegradable polymers such as polylactide-polyglycolide. Depending on the ratio of drug to polymer, and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and ρoly(anhydrides). Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissue. For the methods of treatment of the instant invention, formulations may be given orally, parenterally, topically, or rectally. They are of course given by forms suitable for each administration route. For example, they are administered in tablets or capsule form, by injection, inhalation, eye lotion, ointment, suppository, etc. administration by injection, infusion or inhalation; topical by lotion or ointment; and rectal by suppositories. Oral administration is preferred.

The phrases "parenteral administration" and "administered parenterally" as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal and intrastemal injection and infusion.

The phrases "systemic administration," "administered systemically," "peripheral administration" and "administered peripherally" as used herein mean the administration of a compound, drug or other material other than directly into the central nervous system, such that it enters the patient's system and, thus, is subject to metabolism and other like processes, for example, subcutaneous administration.

These compounds may be administered to humans and other animals for therapy by any suitable route of administration, including orally, nasally, as by, for example, a spray, rectally, intravaginally, parenterally, intracisternally and topically, as by powders, ointments or drops, including buccally and sublingually.

Actual dosage levels of the active ingredients in the pharmaceutical compositions of this invention may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.

The selected dosage level will depend upon a variety of factors including the activity of the particular compound of the present invention employed, or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compound employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts. A physician or veterinarian having ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician or veterinarian could start doses of the compounds of the invention employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.

In general, a suitable daily dose of a compound of the invention will be that amount of the compound which is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend upon the factors described above. Generally, intravenous and subcutaneous doses of the compounds of this invention for a patient, when used for the indicated analgesic effects, will range from about 0.0001 to about 100 mg per kilogram of body weight per day, more preferably from about 0.01 to about 50 mg per kg per day, and still more preferably from about 1.0 to about 100 mg per kg per day. An effective amount is that amount treats a Pinl associated or EGFR related disorder, e.g., a cell proliferative disorder. If desired, the effective daily dose of the active compound may be administered as two, three, four, five, six or more sub- doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms.

Exemplification The invention is further illustrated by the following examples which should not be construed as limiting. Examples

Materials

A lung cancer tissue microarray containing 1516 lung tissue samples was analyzed. The breakdown of histopathological features is presented in the results section.

Methods

Immunostaining protocol

Immunohistochemistry assay was used to detect expression of PIN1 in tissue microarrays (TMAs). Phosphorylated Pinl was detected using a phosphor-PINl specific antibody (Cell Signaling Cat#3721), total PIN1 was detected using a PIN1 polyclonal antibody (Oncogene Research Cat#PC270). Freshly cut tissue microarray sections were used to avoid the possibility of reduced assay sensitivity due to age-dependent oxidation of stored slides.

Protocol for phosphorylated PIN1 detection

The following steps were taken to analyze the level of phosphorylated Pinl in lung tissue samples.

SLIDE PREPARATION -Deparaffinize TMAs overnight in Xylene -descending ethanol series -rinse 5 minutes in PBS buffer

PRETREATMENT -Water bath 30 minutes at 99°C in (DAKO Target Retrieval Solution Puffer Code No.: SI 699)

-Let slides cool -wash 5 minutes in PBS buffer

PEROXIDASE-BLOCKING

-incubate 30 min in 0,3% H2O2/Methanol

-wash 2x5 min in PBS buffer

-incubate 30 min in normal serum (Normal goat Serum, Vector S-1000)

PRIMARY ANTIBODY and Visualization

-dilution 1 :100 (Pintex,PIN-l phospho Ab #3721)

-incubate 2h at 37°C in moist chamber

-wash 2x5 min in PBS buffer (1 :10)

-place slides in LINK reagent (Bioninylated Anti-rabbit Vector BA-1000) -wash 2x5 min in TRIS-PBS buffer

-incubate with ABC reagent (Vectastain ABC-Kit, Elite standard, Vector PK 6100) -wash 2x5 min in TRIS-PBS buffer

CHROMOGEN

-cover slides for 6 min with DAB-Chromogen(Liquid DAB DAKO) -wash slides in PBS-Puffer thoroughly

-counterstain for 20 sec with Hamatoxylin(Harris Hamatoxylin HTX 31000, Medite GmbH)

-rinse with water -differentiate in HCL-Ethanol -hemalaun "blue" for 5 min in water -ascending ethanol series -xylene -cover

Protocol for unphosphorylated Pinl detection

The following steps were taken to analyze the level of unphosphorylated Pinl in lung tissue samples.

SLIDE PREPARATION -Deparaffinize TMAs overnight in Xylene -descending ethanol series -rinse 5 minutes in PBS buffer

PRETREATMENT -Microwave 30 minutes at 90°C in citrate buffer pH 6 (ProTaqstura Code No.: 400300692) -wash 5 minutes in PBS buffer

PEROXIDASE-BLOCKING -incubate 30 min in 0,3%H2O2/Methanol -wash 2x5 min in PBS buffer -incubate 30 min in normal serum(Normal horse Serum, Vector S-2000)

PRIMARY ANTIBODY AND VISUALIZATION -dilution 1:600 (Pintex,monoclonal,m- 156)

-incubate 2h at 37°C in moist chamber

-wash 2x5 min in PBS buffer (1:10)

-place slides 30 min in LINK reagent (Biotinylated Anti-mouse Vector BA-2000)

-wash 2x5 min in TRIS-PBS buffer -incubate 30 min with ABC reagent (Vectastain ABC-Kit, Elite standard, Vector PK

6100)

-wash 2x5 min in TRIS-PBS buffer

CHROMOGEN -cover slides for 6 min with DAB-Chromogen(Liquid DAB DAKO Code No.: K 3467) -wash slides in PBS-Puffer thoroughly

-counterstain for 20 sec with Hamatoxylin(Harris Hamatoxylin HTX 31000, Medite GmbH) -rinse with water -differentiate in HCL-Ethanol -hemalaun for 5 min in water -ascending ethanol series -xylene -cover

Analysis of the immunostaining

For consistency, a single pathologist evaluated all immunostainings. For phosphorylated Pinl staining intensity was estimated by visual inspection in a four step scale (0, 1, 2, 3). In addition to staining intensity, the percentage of positive cells was estimated. Cytoplasmic staining was observed in rare cases (49/1146 analyzable samples) and noted if present.

The criteria for pPP l classification was as follows: Negative: pPINl intensity (int) = 0

Weak: pPP l int = 1 or pPINl int = 2 and pPPNl pet <30%

Moderate: pPr l int = 2 and pPINl pet >30% but <= 70% or pPINl int = 3 and pPPNl pet <30%

Strong: pPINl int = 2 and pPINl pet >70% or pPINl int = 3 and pPINl pet >30%

For unphosporylated PIN1 (uPIN), nuclear and cytoplasmic staining was evaluated separately. In both cases, staining intensity was estimated by visual inspection in a four step scale (0, 1, 2, 3). In addition to staining intensity, the percentage of positive cells was estimated for nuclear staining. This was not done for cytoplasmatic staining as all cells showed a similar cytoplasmatic staining intensity in most cases. Based on both score components (percentage and intensity), the nuclear PINl stainings were classified as "negative", "weak", "moderate" or "strong". The cut-off criteria for classification was as follows:

Negative:

PINl int = 0

Weak:

Pinl int = 1 or Pinl in = 2 and pet < 10% of cells

Moderate:

2+ intensity in >10% but < 70% of cells or 3+ intensity in < 30% of cells Strong:

2+ intensity in >70% of cells or 3+ intensity in >30% of cells

For analysis purposes, negative subjects were considered uPinl negative, and subjects with weak, moderate and strong uPinl levels were considered uPinl positive. EGFR IHC data were analyzed according the following criteria: EGFR Thresh-holding Criteria Negative No staining Weak 1 + intensity in > 50% of cells or

2+ intensity in < 20% of cells Moderate 1+ intensity in > 50% cells or

2+ intensity in > 20% but < 70% of cells or

3+ intensity in < 20% of cells Strong 2+ intensity in > 70% cells or

3+ intensity in > 20% cells

For analysis of the co-expression of pPIN and EGFR, a two-group classification was used for both nuclear and cytoplasmic staining in order to reduce the complexity of the dataset and to better reflect the biological properties of the analyzed tissues. These criteria for classification are as follows:

pPINl Thresh-holding Criteria

Nuclear Negative pPINl IHC result - negative

Nuclear Positive pPINl IHC result - weak, moderate or strong

Cytoplasmic negative pPINl cytoplasmic IHC result - 0

Cytoplasmic positive pPINl cytoplasmic IHC 1+, 2+ or 3+

EGFR Thresh-holding Criteria

EGFR High Weak, moderate or strong staining EGFR Low No staining Fluorescence in situ hybridization (FISH) analysis scoring

EGFR FISH data were defined as follows: EGFR normal Ratio gene/centromer < 1.0 or < 4 FISH signals

EGFR gain Ratio gene/centromer > 1.0 but < 3.0 or

>5 but < 10 EGFR gene signals EGFR amplification Ratio gene/centromer > 3.0 or > 10 EGFR signals

In analyses involving multiple molecular markers the following two-group classification was used: EGFR normal normal

EGFR altered gain or amplification

Statisitics

Chi-square test was applied to evaluate associations between pPINl expression and tumor phenotype co-expression of other markers. Survival analysis were calculated according to Kaplan-Meier. Log-rank p-value are given for survival difference. For multivariate analysis, Cox' proportional hazards model was utilized.

Example 1: Analysis of Phosphorylated Pinl levels in Lung Tissue

Out of 1516 tissue samples, 1146 provided interpretable results when immunostained for pPINl. For the other 370 tissue samples, analysis failed due to missing tissue spots in the microarray sample or due to too few tumor cells in the tissue spot.

Intracellular localization ofpPINl immunostaining

Nuclear pPINl expression was found in 353/391 (90.3%) of pPFNl positive samples. Cytoplasmic expression was found in only 49 tissue samples, all in samples with no or weak nuclear pPINl staining (Figure 2). Because of the low frequency of cytoplasmic pPINl staining, the following analyses were generally limited to samples with nuclear pPINl expression, with analyses of tissues with cytoplasmic pPPNl positive staining added where appropriate. Association ofpPINl alterations with tumor phenotype

The association between nuclear pPINl staining and lung cancer phenotype is summarized in Table 1.

Table 1 : Nuclear pPINl expression and tumor phenotype in lung cancers. pPINl IHC Result n n NEG WEAK MOD STR p-value

(on TMA) (analyzed)

Histology Adenocarcinoma 364 286 69.6% 19.6% 6.3% 4.6%

Adenosquamous ca. 13 10 40.o% 50.0% 10.0% 0/0%

Bronchioloalveolar ca. 81 53 62.3% 22.6% 11.3% 3.8%

Large cell ca. 258 197 72.6% 22.8% 2.0% 2.5%

Neuroendocrine ca. 63 55 47.3% 41.8% 5.5% 5.5%

Squamous cell ca. 730 541 71.2% 22.6% 3.1% 3.1%

Stage pTl 274 192 56.3% 28.1% 8.3% 7.3% O.0001 pT2 854 652 71.3% 21.8% 3.5% 3.4% pT3 251 199 72.9% 22.1% 4.0% 1.0% pT4 83 79.5% 19.3% 0.0% 1.2%

Social Stage pNO 769 566 66.6% 24.0% 5.1% 4.2% 0.0364 pNl 332 247 69.2% 23.15 4.5% 3.2% pN2 338 277 77.3% 18.1% 2.5% 2.2% pN3 39 31 67.7% 32.3% 0.0% 0.0%

Nuclear PINl staining was found in approximaltely 30% of adenocarcinomas, large cell carcinomas and squamous cell carcinomas, which represent the largest subgroup of lung cancers tissues. A decrease in nuclear staining intensity was significantly associated with tumor stage (pO.OOOl) and nodal stage (p=0.0364).

A significant link between pPINl expression and patient survival was not found using the predefined thresh-holding criteria (Figure 3), or if the dataset was reduced to pPINl negative versus positive (combining weak, moderate and strong expression, (Figure 4).

Kaplan-Meier analysis of the 49 samples with cytoplasmic pPINl expression did not reveal any statistically significant findings (Figure 5).

Co-expression of PINl (unphosphorylated) andpPINl (phosphorylated)

PPN1 (unphosphorylated) expression data of lung tissues were analyzed and compared to nuclear pPINl immunostaining. Nuclear pPINl immunostaining was strongly associated with both cytoplasmic (pO.OOOl) and nuclear (pO.OOOl) PINl positive staining (Figure 6). In contrast, most of the 47 samples with data on both pPPNl and uPrNl were negative for nuclear and cytoplasmic PINl (27/47).

Example 2: Analysis of pPinl Co-expression of pPinl and EGFR pPinl levels were evaluated as described above. Immunohistochemistry and

FISH analyses were performed according to standard assays were also used to detect EGFR expression and amplification in the lung tissue microarrays (EGFR antibody, PharmDX, DALO Cat #1492).

For co-expression, a trend towards reduced EGFR expression in tumors with increased pPINl expression was observed. For survival analysis, data were grouped according to the simplified criteria described above. A survival analysis including the four groups of pPINl/EGFR co-expression pattern indicates shows that positive pPINl expression provides additional prognostic information in EGFR expressing tumors.(Figures 8 and 9) These analyses indicate that a high level of phosphorylated (inactive) PINl in tumor cell nuclei is associated with low pT stage and with an absence of nodal metastases in lung cancer. These data further indicate that a high level of pPINl is associated with a significantly improved prognosis in the subset of tumors with high EGFR expression. A continuous decrease in pPINl with increasing EGFR levels was also observed. These date indicate that better prognostic marker than EGFR in subjects with lung cancer, although the combination of both markers is beneficial in further defining the prognosis of a subject.

Equivalents Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

What is claimed is:

1. A method for treating a subject having an EGFR related cell proliferative disorder by administering an effective amount of a Pinl modulator such that treatment of the subject occurs.

2. The method of claim 1, further comprising administering an EGFR modulator.

3. A method of treating a subj ect having a EGFR related cell proliferative disorder that has been selected based on increased levels of EGFR and decreased levels of pPINl by administering an effective amount of a Pinl modulator such that treatment of the subject occurs.

4. The method of claim 3, further comprising administering an EGFR modulator.

5. A method of treating a subject having a EGFR related cell proliferative disorder that has been preselected based on increased levels of unphosphorylated Pinl and increased levels of EGFR by administering an effective amoung of a Pinl modulator such that treatment of the subject occurs.

6. The method of claim 6, further comprising administering an EGFR modulator.

7. The method of any of claims 1 to 6, wherein the EGFR related cell proliferative disorder is a solid tumor.

8. The method of claim 7, wherein the tumor is selected from the group comprising lung, prostate, breast, colorectal, head and neck, ovarian, gastric and pancreatic tumor.

9. The method of claim 8, wherein the tumor is lung cancer.

10. The method of claim 9, wherein the lung cancer is selected from the group comprising non-small lung carcinoma, adenocarcinoma and squamous cell carcinoma.

11. The method of any one of claims 1 to 10, wherein the EGFR related cell proliferative disorder is characterized by a somatic mutation in EGFR.

12. The method of claim 11, wherein the subject is treated with one or more EGFR tyrosine kinase inhibitors.

13. The method of any of claims 1-12, further comprising treating the subject with one or more additional cancer therapies,

14. The method of claim 13, wherein the one or more additional cancer therapies are surgery, radiotherapy, chemotherapy or endocrine therapy.

15. The method of claim 14, wherein the subject is treated with radiotherapy.

16. The method of any of claims 1-12, wherein the subject is non-responsive to surgery, radiotherapy, chemotherapy or endocrine therapy.

17. A method for determining the prognosis of a subject having an EGFR related cell proliferative disorder comprising the steps of: determining the level of pPinl in a biological sample from the subject; wherein an increased level of pPinl in the sample compared to the statistical mean of a population having the EGFR cell proliferative disorder is indicative of a good prognosis.

18. A method for determining the prognosis of a subject having an EGFR related cell proliferative disorder comprising the steps of: determining the level of pPinl in a biological sample from the subject; wherein an decreased level of pPinl in the sample compared to the statistical mean of a population having the EGFR cell proliferative disorder is indicative of a poor prognosis.

19. The method of claim 17 or 18, wherein the pPINl level is determined using an antibody specific for pPinl .

20. The method of claim 19, wherein the level of pPinl is determined by immunohistochemistry.

21. The method of claim 20, wherein the level of pPinl is determined by FISH.

22. A method of determining the prognosis of a subject having an EGFR related cell proliferative disorder comprising: obtaining a first biological sample from said subject and determining the level of pPinl in said sample; obtaining a second biological sample from said subject at a time after collection of said first biological sample and determining the level of pPinl in said sample; wherein an increase in the level of pPinl is indicative of good prognosis.

23. A method of determining the prognosis of a subj ect having an EGFR related cell proliferative disorder comprising: obtaining a first biological sample from said subject and determining the level of pPinl in said sample; obtaining a second biological sample from said subject at a time after collection of said first biological sample and determining the level of pPinl in said sample; wherein a decrease in the level of pPinl is indicative of poor prognosis.

24. The method of any of claim 22 or 23, wherein the EGFR related cell proliferative disorder is a solid tumor.

25. The method of claim 24, wherein the tumor is selected from the group comprising lung, prostate, breast, colorectal, head and neck, ovarian, gastric and pancreatic tumor.

26. The method of claim 25, wherein the tumor is lung cancer.

27. The method of claim 26, wherein the lung cancer is non-small lung carcinoma, adenocarcinoma or squamous cell carcinoma.

28. A kit for determining the prognosis of a subject with an EGFR related cell proliferative disorder comprising an antibody specific for pPinl and instructions for use.

29. The kit of claim 28, wherein the antibody is a monoclonal antibody.

30. The kit of claim 28, wherein said antibody is a polyclonal antibody.

31. The kit of claim 28, further comprising a second antibody specific for a second cancer marker.

32. The kit of claim 31, wherein said antibody is specific for EGFR.

33. A method for determining the course of treatment for a subject having an

EGFR related cell proliferative disorder comprising determining the level of pPinl in a biological sample from the subject, wherein the lower the level of pPinl the more aggressive the treatment of the subject with an anticancer agent.

34. The method of claim 33, wherein the EGFR cell proliferative is a solid tumor.

35. The method of claim 34, wherein the tumor is selected from the group comprising lung, prostate, breast, colorectal, head and neck, ovarian, gastric and pancreatic tumor.

36. The method of claim 35, wherein the tumor is lung cancer.

37. The method of claim 36, wherein the lung cancer is selected from the group comprising non-small lung carcinoma, adenocarcinoma and squamous cell carcinoma.

38. The method of any of claim 33-37, wherein the anticancer agent is a Pinl inhibitor.

39. The method of claim 38, further comprising administering one or more additional cancer therapies.

40. The method of claim 39, wherein the wherein the one or more additional cancer therapies are surgery, radiotherapy, chemotherapy, endocrine therapy and an EGFR inhibitor.

41. The method of claim 40, wherein the subject is treated with radiotherapy.