WO2009117004A1

WO2009117004A1 - Reagents and methods for use in head and neck cancer diagnosis, classification and therapy

Info

Publication number: WO2009117004A1
Application number: PCT/US2008/057840
Authority: WO
Inventors: Brian Z. Ring; Douglas T. Ross; Robert S. Seitz
Original assignee: Applied Genomics Inc.
Priority date: 2008-03-21
Filing date: 2008-03-21
Publication date: 2009-09-24

Abstract

Methods and reagents for classifying head and neck cancers and for identifying new head and neck cancer classes and subclasses. Methods for correlating class or subclass with therapeutic regimen or outcome, for identifying appropriate (new or known) therapies for particular classes or subclasses, and for predicting outcomes based on class or subclass.

Description

REAGENTS AND METHODS FOR USE IN HEAD AND NECK CANCER DIAGNOSIS,

CLASSIFICATION AND THERAPY

Background of the Invention

[0001] A major challenge of cancer treatment is the selection of chemotherapies that maximize efficacy and minimize toxicity for a given patient. A related challenge lies in the attempt to provide accurate diagnostic, prognostic and predictive information. At present, tumors are generally classified under the tumor-node-metastasis (TNM) system. This system, which uses the size of the tumor, the presence or absence of tumor in regional lymph nodes, and the presence or absence of distant metastases, to assign a stage to the tumor is described in the AJCC Cancer Staging Manual, Lippincott, 5th ed., pp. 171-180 (1997). The assigned stage is used as a basis for selection of appropriate therapy and for prognostic purposes. In addition to the TNM parameters, morphologic appearance is used to further classify tumors into tumor types and thereby aid in selection of appropriate therapy. However, this approach has serious limitations. Tumors with similar histopathologic appearance can exhibit significant variability in terms of clinical course and response to therapy. For example, some tumors are rapidly progressive while others are not. Some tumors respond readily to hormonal therapy or chemotherapy while others are resistant.

[0002] Assays for cell surface markers, e.g., using immunohistochemistry, have provided means for dividing certain tumor types into subclasses. Though useful, these analyses only in part predict the clinical behavior of tumors. There is phenotypic diversity present in cancers that current diagnostic tools fail to detect. As a consequence, there is still much controversy over how to stratify patients amongst potential treatments in order to optimize outcome (e.g., for breast cancer see "NIH Consensus Development Conference Statement: Adjuvant Therapy for Breast Cancer, November 1-3, 2000", J. Nat. Cancer Inst. Monographs, 30:5-15, 2001 and Di Leo et al, Int. J. Clin. Oncol. 7:245-253, 2002).

[0003] There clearly exists a need for improved methods and reagents for classifying cancers including head and neck cancers. Once these methods and reagents are available, clinical studies can be performed that will allow the identification of classes or subclasses of patients having different prognosis and/or responses to therapy. Such prognostic tools will allow more rationally based choices governing the aggressiveness of therapeutic interventions; such predictive tools

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4313517vl will also be useful for directing patients into appropriate treatment protocols.

Summary of the Invention

[0004] The inventors have identified markers and panels of markers that are differentially expressed in cancer samples from head and neck cancer patients. The inventors have also observed that expression of certain markers and panels of markers correlate with prognosis (e.g., likelihood of death from head and neck cancer and/or likelihood of recurrence). In one aspect, the present invention provides methods of using these markers and panels of markers to classify patients with head and neck cancer. In one aspect, the present invention provides methods of using these markers and panels of markers to predict the prognosis of patients with head and neck cancer.

[0005] Expression of the markers that are described herein can be detected using any known method. Thus, while the inventive methods have been exemplified by detecting marker expression using antibodies, marker expression may be detected using other polypeptide interaction partners or primers that hybridize with polynucleotide markers (e.g., mRNA).

Brief Description of the Appendix

[0006] This patent application refers to material comprising a table and data presented as Appendix A. Specifically, Appendix A is a table that lists a variety of markers that could be used in a classification or prognostic panel in conjunction with other markers that are described herein. The table includes the antibody ID, parent gene name, Entrez Gene ID, known aliases for the parent gene, peptides that were used in preparing antibodies and exemplary antibody titer for staining. Using the parent gene name, Entrez Gene ID and/or known aliases for the parent gene, a skilled person can readily obtain the nucleotide (and corresponding amino acid) sequences for each and every one of the parent genes that are listed in Appendix A from a public database (e.g., GenBank, Swiss-Prot or any future derivative of these). The nucleotide and corresponding amino acid sequences for each and every one of the parent genes that are listed in Appendix A are hereby incorporated by reference from these public databases. Antibodies with AGI IDs that begin with S5 or S6 were obtained from commercial sources as indicated. [0007] Appendix B is a table that lists exemplary antibodies whose binding patterns have been shown by the inventors to correlate with tumor prognosis in head and neck cancer patients.

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4313517vl The results are grouped into two prognostic categories (likelihood of death from head and neck cancer or "DOD" and likelihood of recurrence). For each antibody, the upper and lower results correspond to separate analyses that were performed using the following scoring systems: method 1 (0 = negative; 1 = weak staining; 2 = strong staining) and method 2 (0 = negative; 1 = weak or strong staining), respectively. This table was prepared using samples from the Stanford cohort as described in Example 3.

Brief Description of the Drawing

[0008] Figure 1 shows Kaplan-Meier curves that were generated using a cohort of head and neck cancer patients (Stanford cohort) after prognostic classification based on staining with the panel of antibodies set forth in Table 1. Patients from the cohort were placed into the following outcome groups based on their respective scores using the overall panel prediction function in

Table 1 : Good < -0.15 < Moderate < 0.64 < Bad (likelihood of survival) and/or Good < 0.64 <

Bad (likelihood of recurrence). Kaplan-Meier outcome curves were then calculated for patients within each prognostic group. Figure IA shows the survival curves that were obtained for patients in the "Good", "Moderate" and "Bad" survival groups. Figure IB shows the recurrence curves that were obtained for patients in the "Good" and "Bad" recurrence groups.

[0009] Figure 2 shows Kaplan-Meier curves that were generated using a cohort of head and neck cancer patients (Stanford cohort) after prognostic classification based on staining with the panel of antibodies set forth in Table 2 (upper) and Table 3 (lower).

[0010] Figure 3 shows Kaplan-Meier curves that were generated using a cohort of head and neck cancer patients (Stanford cohort) after prognostic classification based on staining with the panel of antibodies set forth in Table 4 (upper) and Table 5 (lower).

[0011] Figure 4 shows Kaplan-Meier curves that were generated using a cohort of head and neck cancer patients (Stanford cohort) after prognostic classification based on staining with the panel of antibodies set forth in Table 6 (upper) and Table 7 (lower).

[0012] Figure 5 shows Kaplan-Meier curves that were generated using a cohort of head and neck cancer patients (Stanford cohort) after prognostic classification based on staining with the panel of antibodies set forth in Table 8 (upper) and Table 9 (lower).

[0013] Figure 6 shows Kaplan-Meier curves that were generated using a cohort of head and neck cancer patients (Stanford cohort) after prognostic classification based on staining with the

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4313517vl panel of antibodies set forth in Table 10 (survival, any recurrence and distant recurrence, i.e., more than 5 years).

Definitions

[0014] Binds - When an interaction partner "binds" a marker they are linked by direct non-covalent interactions.

[0015] Cancer markers - "Cancer markers" or "markers" are molecular entities that are detectable in cancer samples. Generally, markers may be polypeptides (e.g., marker protein) or polynucleotides (e.g., marker mRNA) that are indicative of the expression of a gene (e.g., marker gene) and present within the cancer sample, e.g., within the cytoplasm or membranes of cancerous cells and/or secreted from such cells.

[0016] Cancer sample - As used herein, the term "cancer sample" or "sample" is taken broadly to include cell or tissue samples removed from a cancer patient (e.g., from a tumor, from the bloodstream, etc.), cells derived from a tumor that may be located elsewhere in the body (e.g., cells in the bloodstream or at a site of metastasis), or any material derived from such a sample. Derived material may include, for example, polynucleotides or polypeptides extracted from the sample, cell progeny, etc. In one embodiment, a cancer sample may be a tumor sample. [0017] Correlation - "Correlation" refers to the degree to which one variable can be predicted from another variable, e.g., the degree to which a patient's likely prognosis can be predicted from the expression of a marker in a cancer sample. A variety of statistical methods may be used to measure correlation between two variables, e.g., without limitation the student t- test, the Fisher exact test, the Pearson correlation coefficient, the Spearman correlation coefficient, the Chi squared test, etc. Results are traditionally given as a measured correlation coefficient with a p-value that provides a measure of the likelihood that the correlation arose by chance. A correlation with a p-value that is less than 0.1 is generally considered to be statistically significant. In various embodiments, correlations may have p-values that are less than 0.01, especially less than 0.001.

[0018] Hybridized - When a primer and a marker are physically "hybridized" with one another as described herein, they are non-covalently linked by base pair interactions. [0019] Interaction partner - An "interaction partner" is an entity that binds a polypeptide marker. For example and without limitation, an interaction partner may be an antibody or a

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4313517vl fragment thereof that binds a marker. In general, an interaction partner is said to "bind specifically" with a marker if it binds at a detectable level with the marker and does not bind detectably with unrelated molecular entities (e.g., other markers) under similar conditions. Specific association between a marker and an interaction partner will typically be dependent upon the presence of a particular structural feature of the target marker such as an antigenic determinant or epitope recognized by the interaction partner. In general, it is to be understood that specificity need not be absolute. For example, it is well known in the art that antibodies frequently cross-react with other epitopes in addition to the target epitope. Such cross-reactivity may be acceptable depending upon the application for which the interaction partner is to be used. Thus the degree of specificity of an interaction partner will depend on the context in which it is being used. In general, an interaction partner exhibits specificity for a particular marker if it favors binding with that partner above binding with other potential partners, e.g., other markers. One of ordinary skill in the art will be able to select interaction partners having a sufficient degree of specificity to perform appropriately in any given application (e.g., for detection of a target marker, for therapeutic purposes, etc.). It is also to be understood that specificity may be evaluated in the context of additional factors such as the affinity of the interaction partner for the target marker versus the affinity of the interaction partner for other potential partners, e.g., other markers. If an interaction partner exhibits a high affinity for a target marker and low affinity for non-target molecules, the interaction partner will likely be an acceptable reagent for diagnostic purposes even if it lacks specificity.

[0020] Primer - A "primer" is an polynucleotide entity that physically hybridizes with a polynucleotide marker. As is well known in the art, a primer may be directly detectable (e.g., via a detectable label) and/or indirectly detectable (e.g., by interaction with a secondary primer which is detectable). In general, a primer is said to "hybridize specifically" with a marker if it hybridizes at a detectable level with the marker and does not hybridize detectably with unrelated molecular entities (e.g., other markers) under similar conditions. Specific hybridization between a marker and a primer will typically be dependent upon the presence of a particular nucleotide sequence of the target marker which is complementary to the nucleotide sequence of the primer. In general, it is to be understood that specificity need not be absolute. The degree of specificity of a primer will depend on the context in which it is being used. In general, a primer exhibits specificity for a particular marker if it favors hybridization with that partner above hybridization

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4313517vl with other potential partners, e.g., other markers. One of ordinary skill in the art will be able to select primers having a sufficient degree of specificity to perform appropriately in any given application. It is also to be understood that specificity may be evaluated in the context of additional factors such as the affinity of the primer for the target marker versus the affinity of the primer for other potential partners, e.g., other markers. If a primer exhibits a high affinity for a target marker and low affinity for non-target molecules, the primer will likely be an acceptable reagent for diagnostic purposes even if it lacks specificity.

Detailed Description of Certain Embodiments of the Invention

[0021] As noted above, the present invention provides techniques and reagents for the classification and subclassification, of patients with head and neck cancer. Such classification (or subclassification) has many beneficial applications. For example, a particular class or subclass of patients may correlate with prognosis and/or susceptibility to a particular therapeutic regimen. As such, the classification or subclassification may be used as the basis for a prognostic or predictive kit and may also be used as the basis for identifying previously unappreciated therapies. Therapies that are effective against only a particular class or subclass of patient may have been lost in studies whose data were not stratified by subclass; the present invention allows such data to be re-stratified, and allows additional studies to be performed, so that class- or subclass-specific therapies may be identified and/or implemented. Alternatively or additionally, the present invention allows identification and/or implementation of therapies that are targeted to genes identified as class- or subclass-specific.

Patient Classification

[0022] In general, according to the present invention, head and neck cancer patients are classified or subclassified on the basis of individual markers and panels of markers whose expression is correlated with a particular class or subclass. As discussed in more detail below, expression of markers described herein can be detected using any known method. [0023] In one aspect, the present invention provides systems of identifying suitable classification markers and marker panels. In general, these systems involve identifying patterns of marker expression across a set of cancer samples. For example, marker panels that identify a particular class or subclass of head and neck cancer patient may be defined based on expression

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4313517vl levels of individual markers in a collection of cancer samples from a cohort of head and neck cancer patients. Individual classification markers will be expressed in only a subset of the cancer samples. Similarly, panels of markers may be identified using these methods by searching for combined expression patterns that are specific to a subset of the cancer samples [0024] The inventive process of identifying useful classification markers and panels of markers as described herein may itself result in the identification of new patient classes or subclasses. That is, through the process of analyzing marker expression, investigators will often discover new patient classes or subclasses based on the patterns of marker expression. Thus, the processes of (a) defining classification markers for given cancer classes or subclasses; and (b) identifying new cancer classes or subclasses may well be experimentally interrelated. In general, the greater the number of cancer samples tested, the greater the likelihood that new classes or subclasses will be defined.

[0025] Often, when identifying markers that can act as a classification (or subclassification) markers, it will be desirable to obtain the largest set of cancer samples possible, and also to collect the largest amount of information possible about the individual samples. For example, the origin of the sample, the gender of the patient, the age of the patient, the location and staging of the tumor (e.g., according to the TNM system), any microscopic or submicroscopic characteristics of the tumor that may have been determined, may be recorded. Those of ordinary skill in the art will appreciate that the more information that is known about a cancer sample, the more aspects of that sample are available for correlation with marker expression. [0026] In certain embodiments, expression of one of the markers described herein (e.g., in Appendix B) in a cancer sample (or any panel of markers) may be used to classify a head and neck cancer patient. For example, for purposes of illustration and without limitation, a patient may be classified as FABP5+ or FABP5- depending on whether expression of the FABP5 marker is detected in the cancer sample. In one embodiment, this information may be used to stratify the patient within a clinical trial (e.g., FABP5+ patients in the trial receive treatment A while FABP5- patients in the trial receive treatment B). Alternatively, the classification may be used to select a treatment for a patient outside the context of a clinical trial and/or to predict the patient's likely prognosis.

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4313517vl Assessing Patient Prognosis

[0027] The present invention provides specific markers and methods for assessing the likely prognosis of a patient having head and neck cancer. In general, the methods involve determining a level of expression of a panel of one or more markers in a cancer sample from a patient with head and neck cancer. Any one of the markers listed in Appendix B (or any combination thereof) may be used for this purpose. For example, in various embodiments any one of CYP4Z1, MMP7, TRIM29, IRX3, S100A8, SLPI, CSTA, CDH3, FABP4, XPRl, NCSTN, CDHF7, FABP5, LTB, ABCG2, TLE3, TERF2IP, SLC7A11, ITGB4, SLC7A5, NDRGl, HTF9C, AKRlCl, CASP7, MMPl, CDHl, CEACAM5, TP53, LOX, CAIX (optionally taking into account the location of the stain, i.e., membrane or cytoplasm), EFNAl and CDKN2A may be used. The patient's likely prognosis is then assessed based on the determined level of expression for the at least one marker in the panel. In general, expression of markers in Appendix B with a hazard ratio (HR) or more than 1.0 is correlated with a higher likelihood of an unfavorable prognosis while expression of markers in Appendix B with a hazard ratio (HR) of less than 1.0 is correlated with a lower likelihood of an unfavorable prognosis. As set forth in Appendix B, across a population of patients with head and neck cancer, a higher level of expression of a marker selected from the group consisting of MMP7, TRIM29, IRX3, S100A8, SLPI, CDH3, FABP4, XPRl, NCSTN, FABP5, LTB, ABCG2, SLC7A11, ITGB4, SLC7A5, NDRGl, CASP7, MMPl, TP53, LOX, CAIX and EFNAl is indicative of a higher likelihood of an unfavorable prognosis. In contrast, a higher level of expression of a marker selected from the group consisting of CYP4Z1, CDHF7, TERF2IP, CEAC AM5, and CDKN2A is indicative of a lower likelihood of an unfavorable prognosis. Other correlations will be apparent to a person of ordinary skill in the art from the data presented in Appendix B.

[0028] In one embodiment, the unfavorable prognosis is recurrence or death from head and neck cancer. In another embodiment, the unfavorable prognosis is recurrence. In yet another embodiment, the unfavorable prognosis is death from head and neck cancer. In certain embodiments, these outcomes are associated with specific temporal aspects, e.g., likelihood of recurrence within 5 years, etc.

[0029] These methods may further comprise a step of comparing the level of expression of the panel in the cancer sample with the level of expression of the panel in a negative control sample. Alternatively or additionally the level of expression of the panel in the cancer sample

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4313517vl may be compared with the level of expression of the panel in a positive control sample. While the use of negative and/or positive control samples will facilitate the identification of different levels of marker expression it will be appreciated that a trained pathologist may also be able to assess marker expression in cancer samples without the aid of negative and positive samples (e.g., when antibody based detection of marker expression is used and the diagnostic test calls for the classification of cancer samples into samples with negative and positive stains). [0030] In general, it will be appreciated that the prognostic methods may be practiced using a panel that includes just one of the markers from Appendix B. For example, in various embodiments, the prognostic methods may be practiced using a panel that includes just one of the following markers: FABP5, NDRGl, CDKN2A, CEACAM5, NCSTN, XPRl, ABCG2 or TRIM29. As demonstrated in the Examples, in certain embodiments, it may prove advantageous to use panels that include 2, 3, 4, 5, 6, 7 or all 8 of these markers, optionally with yet other markers from Appendix B. All combinations are encompassed by the present invention. Indeed, the prognostic power of these multimarker panels can be more significant than when certain markers are used alone. In various embodiments, the prognostic methods may also be practiced using a panel that includes just one of the following markers: FABP5, NDRGl , CDKN2A, NCSTN, ABCG2, SLC7A11, SLC7A5, LOX, CAIX or TRIM29. In certain embodiments, it may prove advantageous to use panels that include 2, 3, 4, 5, 6, 7, 8, 9 or all 10 of these markers, optionally with yet other markers from Appendix B. All combinations are encompassed by the present invention. It will be appreciated that yet other markers (e.g., any of those listed in Appendix A) may also be added to an inventive panel.

[0031] Without limitation and solely for purposes of illustration, the inventors have identified several exemplary panels that include both FABP5 and NDRGl . Thus in certain embodiments, an inventive panel may comprise FABP5 and/or NDRGl . In certain embodiments, CDKN2A can be added to the panel optionally with CEACAM5 and/or NCSTN. In certain embodiments, FABP5 and NDRGl can be combined with XPRl and NCSTN. In certain embodiments, FABP5 and NDRGl can be combined with ABCG2 and CEAC AM5. In certain embodiments, FABP5 and NDRGl can be combined with CSTA and optionally with TRIM29 and/or ABCG2. In certain embodiments, FABP5 and NDRGl can be combined with CSTA and optionally with TRIM29 and/or CDKN2A. In these and other embodiments described herein involving a panel with two, three, four, five or more markers, a higher level of

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4313517vl expression of CSTA across a population of patients with head and neck cancer is indicative of a lower likelihood of an unfavorable prognosis.

[0032] In one aspect, the methods may be practiced using a panel that comprises at least two markers selected from the group consisting of FABP5, NDRGl, CDKN2A, CEAC AM5, NCSTN, XPRl, ABCG2, CSTA, TRIM29, SLC7A5 and TLE3. Thus, in certain embodiments, a panel may include FABP5 in combination with NDRGl, CDKN2A, CEAC AM5, NCSTN, XPRl, ABCG2, CSTA, TRIM29, SLC7A5 or TLE3. In certain embodiments, a panel may include NDRGl in combination with CDKN2A, CEACAM5, NCSTN, XPRl, ABCG2, CSTA, TRIM29, SLC7A5 or TLE3. In certain embodiments, a panel may include CDKN2A in combination with CEACAM5, NCSTN, XPRl, ABCG2, CSTA, TRIM29, SLC7A5 or TLE3, etc. It is to be understood that these combinations are presented without limitation and that any panel including two markers selected from the group consisting of FABP5, NDRGl, CDKN2A, CEACAM5, NCSTN, XPRl, ABCG2, CSTA, TRIM29, SLC7A5 and TLE3 may be used in the present methods. In these and other embodiments described herein involving a panel with two, three, four, five or more markers, a higher level of expression of TLE3 across a population of patients with head and neck cancer is indicative of a higher likelihood of an unfavorable prognosis.

[0033] In another aspect, the methods may be practiced using a panel that comprises at least three markers selected from the group consisting of FABP5, NDRGl, CDKN2A, CEACAM5, NCSTN, XPRl, ABCG2, CSTA, TRIM29, SLC7A5 and TLE3. For example, the methods may be practiced using a panel that comprises FABP5 and NDRGl and at least one of CDKN2A, CEACAM5, NCSTN, XPRl, ABCG2, CSTA, TRIM29, SLC7A5 and TLE3. In certain embodiments, NDRGl and CDKN2A may be combined with at least one of CEACAM5, NCSTN, XPRl, ABCG2, CSTA, TRIM29, SLC7A5 and TLE3, etc.

[0034] In another aspect, the methods may be practiced using a panel that comprises at least four markers selected from the group consisting of FABP5, NDRGl, CDKN2A, CEAC AM5, NCSTN, XPRl, ABCG2, CSTA, TRIM29, SLC7A5 and TLE3. For example, the methods may be practiced using a panel that comprises FABP5, NDRGl, CDKN2A and at least one of CEACAM5, NCSTN, XPRl, ABCG2, CSTA, TRIM29, SLC7A5 and TLE3. In certain embodiments, NDRGl, CDKN2A and CEAC AM5 may be combined with at least one of NCSTN, XPRl, ABCG2, CSTA, TRIM29, SLC7A5 and TLE3, etc.

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4313517vl [0035] In another aspect, the methods may be practiced using a panel that comprises at least five markers selected from the group consisting of FABP5, NDRGl, CDKN2A, CEACAM5, NCSTN, XPRl, ABCG2, CSTA, TRIM29, SLC7A5 and TLE3. For example, the methods may be practiced using a panel that comprises FABP5, NDRGl, CDKN2A, CEACAM5 and at least one of NCSTN, XPRl, ABCG2, CSTA, TRIM29, SLC7A5 and TLE3. In certain embodiments, NDRGl, CDKN2A, CEACAM5 and NCSTN may be combined with at least one of XPRl, ABCG2, CSTA, TRIM29, SLC7A5 and TLE3, etc. In one embodiment a panel may comprise at least CEAC AM5, NCSTN, ABCG2, TRIM29 and TLE3. In one embodiment a panel may comprise at least CEAC AM5, NCSTN, ABCG2, TRIM 29 and SLC7A5.

Marker Expression

[0036] As mentioned above, expression of any of the markers described herein can be determined using any known method. In one embodiment, expression may be determined by detecting polypeptide markers using interaction partners (e.g., antibodies). In another embodiment, expression may be determined by detecting polynucleotide markers using primers.

Detecting Polypeptide Markers

[0037] Polypeptide markers may be detected using any interaction partner that binds a polypeptide marker (which could be a full length protein or an antigenic fragment thereof). Thus, any entity that binds detectably to the polypeptide marker may be utilized as an interaction partner in accordance with the present invention, so long as it binds the marker with an appropriate combination of affinity and specificity.

[0038] In various embodiments, interaction partners are antibodies, or fragments (e.g., F(ab) fragments, F(ab')₂ fragments, Fv fragments, or sFv fragments, etc.; see, for example, Inbar et al., Proc. Nat. Acad. Sci. USA 69:2659, 1972; Hochman et al., Biochem. 15:2706, 1976; and Ehrlich et al., Biochem. 19:4091, 1980; Huston et al., Proc. Nat. Acad. Sci. USA 85:5879, 1998; U.S. Pat. Nos. 5,091,513 and 5,132,405 to Huston et al.; and U.S. Pat. No. 4,946,778 to Ladner et al., each of which is incorporated herein by reference). In certain embodiments, interaction partners may be selected from libraries of mutant antibodies (or fragments thereof). For example, collections of antibodies that each include different point mutations may be screened for their association with a marker of interest. Yet further, chimeric antibodies may be used as interaction partners,

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4313517vl e.g., "humanized" or "veneered" antibodies as described in greater detail below. [0039] When antibodies are used as interaction partners, these may be prepared by any of a variety of techniques known to those of ordinary skill in the art (e.g., see Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988, see also the Examples). For example, antibodies can be produced by cell culture techniques, including the generation of monoclonal antibodies, or via transfection of antibody genes into suitable bacterial or mammalian cell hosts, in order to allow for the production of recombinant antibodies. In one technique, an "immunogen" comprising an antigenic portion of a marker of interest (or the marker itself) is initially injected into any of a wide variety of mammals (e.g., mice, rats, rabbits, sheep or goats). In this step, a marker (or an antigenic portion thereof) may serve as the immunogen without modification. Alternatively, particularly for relatively short markers, a superior immune response may be elicited if the marker is joined to a carrier protein, such as bovine serum albumin or keyhole limpet hemocyanin (KLH). The immunogen is injected into the animal host, preferably according to a predetermined schedule incorporating one or more booster immunizations and the animals are bled periodically. Polyclonal antibodies specific for the marker may then be purified from such antisera by, for example, affinity chromatography using the marker (or an antigenic portion thereof) coupled to a suitable solid support. An exemplary method is described in the Examples.

[0040] If desired for diagnostic or therapeutic purposes, monoclonal antibodies specific for a marker may be prepared, for example, using the technique of Kohler and Milstein, Eur. J. Immunol. 6:511, 1976 and improvements thereto. Briefly, these methods involve the preparation of immortal cell lines capable of producing antibodies having the desired specificity (i.e., reactivity with the marker of interest). Such cell lines may be produced, for example, from spleen cells obtained from an animal immunized as described above. The spleen cells are then immortalized by, for example, fusion with a myeloma cell fusion partner, preferably one that is syngeneic with the immunized animal. A variety of fusion techniques may be employed. For example, the spleen cells and myeloma cells may be combined with a nonionic detergent for a few minutes and then plated at low density on a selective medium that supports the growth of hybrid cells, but not myeloma cells. For example, the HAT (hypoxanthine, aminopterin, thymidine) selection technique may be used. After a sufficient time, usually about 1 to 2 weeks, colonies of hybrids are observed. Single colonies are selected and their culture supernatants

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4313517vl tested for binding activity against the marker. Hybridomas having high reactivity and specificity are typically selected.

[0041] Monoclonal antibodies may be isolated from the supernatants of growing hybridoma colonies. In addition, various techniques may be employed to enhance the yield, such as injection of the hybridoma cell line into the peritoneal cavity of a suitable vertebrate host, such as a mouse. Monoclonal antibodies may then be harvested from the ascites fluid or the blood. Contaminants may be removed from the antibodies by conventional techniques, such as chromatography, gel filtration, precipitation and extraction. The polypeptide marker of interest may be used in the purification process in, for example, an affinity chromatography step. [0042] It is to be understood that the present invention is not limited to using antibodies or antibody fragments as interaction partners. In particular, the present invention also encompasses the use of synthetic interaction partners that mimic the functions of antibodies. Several approaches to designing and/or identifying antibody mimics have been proposed and demonstrated (e.g., see the reviews by Hsieh- Wilson et al., Ace. Chem. Res. 29:164, 2000 and Peczuh and Hamilton, Chem. Rev. 100:2479, 2000). For example, small molecules that bind protein surfaces in a fashion similar to that of natural proteins have been identified by screening synthetic libraries of small molecules or natural product isolates (e.g., see Gallop et al., J. Med. Chem. 37:1233, 1994; Gordon et al., J. Med. Chem. 37:1385, 1994; DeWitt et al., Proc. Natl. Acad. Sci. U.S.A. 90:6909, 1993; Bunin et al., Proc. Natl. Acad. Sci. U.S.A. 91 :4708, 1994; Virgilio and Ellman, J. Am. Chem. Soc. 116:11580, 1994; Wang et al., J. Med. Chem. 38:2995, 1995; and Kick and Ellman, J. Med. Chem. 38:1427, 1995). Similarly, combinatorial approaches have been successfully applied to screen libraries of peptides and proteins for their ability to bind a range of proteins (e.g., see Cull et al., Proc. Natl. Acad. Sci. U.S.A. 89:1865, 1992; Mattheakis et al., Proc. Natl. Acad. Sci. U.S.A. 91 :9022, 1994; Scott and Smith, Science 249:386, 1990; Devlin et al., Science 249:404, 1990; Corey et al., Gene 128:129, 1993; Bray et al., Tetrahedron Lett. 31 :5811, 1990; Fodor et al., Science 251 :767, 1991; Houghten et al., Nature 354:84, 1991; Lam et al., Nature 354:82, 1991; Blake and Litzi-Davis, Bioconjugate Chem. 3:510, 1992; Needels et al., Proc. Natl. Acad. Sci. U.S.A. 90:10700, 1993; and Ohlmeyer et al., Proc. Natl. Acad. Sci. U.S.A. 90:10922, 1993). Similar approaches have also been used to study carbohydrate-protein interactions (e.g., see Oldenburg et al., Proc. Natl. Acad. Sci. U.S.A. 89:5393, 1992) and polynucleotide-protein interactions (e.g., see Ellington and Szostak, Nature

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4313517vl 346:818, 1990 and Tuerk and Gold, Science 249:505, 1990). These approaches have also been extended to study interactions between proteins and unnatural biopolymers such as oligocarbamates, oligoureas, oligosulfones, etc. (e.g., see Zuckermann et al, J. Am. Chem. Soc. 114:10646, 1992; Simon et al., Proc. Natl. Acad. Sci. U.S.A. 89:9367, 1992; Zuckermann et al., J. Med. Chem. 37:2678, 1994; Burgess et al., Angew. Chem., Int. Ed. Engl. 34:907, 1995; and Cho et al., Science 261 :1303, 1993). Yet further, alternative protein scaffolds that are loosely based around the basic fold of antibody molecules have been suggested and may be used in the preparation of inventive interaction partners (e.g., see Ku and Schultz Proc. Natl. Acad. Sci. U.S.A. 92:6552, 1995). Antibody mimics comprising a scaffold of a small molecule such as 3- aminomethylbenzoic acid and a substituent consisting of a single peptide loop have also been constructed. The peptide loop performs the binding function in these mimics (e.g., see Smythe et al., J. Am. Chem. Soc. 116:2725, 1994). A synthetic antibody mimic comprising multiple peptide loops built around a calixarene unit has also been described (e.g., see U.S. Pat. No. 5,770,380 to Hamilton et al.).

[0043] Any available strategy or system may be utilized to detect association between an interaction partner and its marker. In certain embodiments, association can be detected by adding a detectable label to the interaction partner. In other embodiments, association can be detected by using a labeled secondary interaction partner that binds specifically with the primary interaction partner, e.g., as is well known in the art of antigen/antibody detection. The detectable label may be directly detectable or indirectly detectable, e.g., through combined action with one or more additional members of a signal producing system. Examples of directly detectable labels include radioactive, paramagnetic, fluorescent, light scattering, absorptive and colorimetric labels. Examples of indirectly detectable include chemiluminescent labels, e.g., enzymes that are capable of converting a substrate to a chromogenic product such as alkaline phosphatase, horseradish peroxidase and the like.

[0044] Once a labeled interaction partner has bound its marker, the complex may be visualized or detected in a variety of ways, with the particular manner of detection being chosen based on the particular detectable label, where representative detection means include, e.g., scintillation counting, autoradiography, measurement of paramagnetism, fluorescence measurement, light absorption measurement, measurement of light scattering and the like. [0045] In general, association between an interaction partner and its marker may be assayed

Attorney Docket No.: 2003923-0022 Customer No.: 24280

4313517vl by contacting the interaction partner with a cancer sample that includes the marker. Depending upon the nature of the sample, appropriate methods include, but are not limited to, immunohistochemistry (IHC), radioimmunoassay, ELISA, immunoblotting and fluorescence activates cell sorting (FACS). In the case where the polypeptide marker is to be detected in a tissue sample, e.g., a biopsy sample, IHC is a particularly appropriate detection method. Techniques for obtaining tissue and cell samples and performing IHC and FACS are well known in the art.

[0046] Where large numbers of samples are to be handled (e.g., when simultaneously analyzing several samples from the same patient or samples from different patients), it may be desirable to utilize arrayed and/or automated formats. In certain embodiments, tissue arrays as described in the Examples may be used. Tissue arrays may be constructed according to a variety of techniques. According to one procedure, a commercially-available mechanical device (e.g., the manual tissue arrayer MTAl from Beecher Instruments of Sun Prairie, WI) is used to remove an 0.6-micron-diameter, full thickness "core" from a paraffin block (the donor block) prepared from each patient, and to insert the core into a separate paraffin block (the recipient block) in a designated location on a grid. In various embodiments, cores from as many as about 400 patients (or multiple cores from the same patient) can be inserted into a single recipient block; preferably, core-to-core spacing is approximately 1 mm. The resulting tissue array may be processed into thin sections for staining with interaction partners according to standard methods applicable to paraffin embedded material.

[0047] Whatever the format, and whatever the detection strategy, identification of a discriminating titer can simplify binding studies to assess the desirability of using an interaction partner. In such studies, the interaction partner is contacted with a plurality of different samples that preferably have at least one common trait (e.g., tissue of origin), and often have multiple common traits (e.g., tissue of origin, stage, microscopic characteristics, etc.). In some cases, it will be desirable to select a group of samples with at least one common trait and at least one different trait, so that a titer is determined that distinguishes the different trait. In other cases, it will be desirable to select a group of samples with no detectable different traits, so that a titer is determined that distinguishes among previously indistinguishable samples. Those of ordinary skill in the art will understand, however, that the present invention often will allow both of these goals to be accomplished even in studies of sample collections with varying degrees of similarity

Attorney Docket No.: 2003923-0022 Customer No.: 24280

4313517vl and difference.

[0048] As discussed above and in the Examples, the inventors have applied these techniques to samples from head and neck cancer patients. The invention also encompasses the recognition that markers that are secreted from the cells in which they are produced may be present in serum, enabling their detection through a blood test rather than requiring a biopsy specimen. An interaction partner that binds to such markers represents an embodiment of the invention. [0049] In general, the results of such an assay can be presented in any of a variety of formats. The results can be presented in a qualitative fashion. For example, the test report may indicate only whether or not the marker was detected, perhaps also with an indication of the limits of detection or with a qualitative assessment (e.g., weak vs. strong). Additionally the test report may indicate the subcellular location of binding, e.g., nuclear, cytoplasmic or membrane and/or the relative levels of binding in these different subcellular locations. The results may be presented in a semi-quantitative fashion. For example, various ranges may be defined and the ranges may be assigned a score (e.g., 0 to 5) that provides a certain degree of quantitative information. Such a score may reflect various factors, e.g., the number of cells in which the marker is detected, the intensity of the signal (which may indicate the level of expression of the marker), etc. The results may be presented in a quantitative fashion, e.g., as a percentage of cells in which the marker is detected, as a concentration, etc. As will be appreciated by one of ordinary skill in the art, the type of output provided by a test will vary depending upon the technical limitations of the test and the biological significance associated with detection of the marker. For example, in certain circumstances a purely qualitative output (e.g., whether or not the marker is detected at a certain detection level) provides significant information. In other cases a more quantitative output (e.g., a ratio of the level of expression of the marker in two samples) may be used.

Detecting Polynucleotide Markers

[0050] Although in many cases detection of polypeptide markers using interaction partners such as antibodies may represent the most convenient means of determining whether a marker of interest is expressed in a particular sample, the inventive methods also encompass the use of primers for the detection of polynucleotide markers. A variety of methods for detecting the presence of a particular polynucleotide marker are known in the art and may be used in the

Attorney Docket No.: 2003923-0022 Customer No.: 24280

4313517vl inventive methods. In general, these methods rely on hybridization between one or more primers and the polynucleotide marker.

[0051] Any available strategy or system may be utilized to detect hybridization between primers and a polynucleotide marker (which could be an mRNA, a cDNA produced by RT-PCR from mRNA, RNA produced from such cDNA, etc.). In certain embodiments, hybridization can be detected by using a primer with a detectable label. In other embodiments, hybridization can be detected by using a labeled secondary primer that hybridizes specifically with the primary primer (e.g., a region of the primary primer that does not hybridize with the marker). In yet other embodiments it may be advantageous to amplify the marker within the cancer sample by PCR using a set of primers designed to amplify a region of the polynucleotide marker. The resulting product can then be detected, e.g., using a labeled secondary primer that hybridizes with the amplified product. Those skilled in the art will appreciate variations on these embodiments.

[0052] Considerations for primer design are well known in the art and are described, for example, in Newton, et al. (eds.) PCR: Essential data Series, John Wiley & Sons; PCR Primer: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1995; White, et al.. (eds.) PCR Protocols: Current methods and Applications, Methods in Molecular Biology, The Humana Press, Totowa, NJ, 1993. In addition, a variety of computer programs known in the art may be used to select appropriate primers.

[0053] In general, a detectable label may be directly detectable or indirectly detectable, e.g., through combined action with one or more additional members of a signal producing system. Examples of directly detectable labels include radioactive, paramagnetic, fluorescent, light scattering, absorptive and colorimetric labels. Examples of indirectly detectable include chemiluminescent labels, e.g., enzymes that are capable of converting a substrate to a chromogenic product such as alkaline phosphatase, horseradish peroxidase and the like. [0054] Once a labeled primer has hybridized with the marker, the complex may be visualized or detected in a variety of ways, with the particular manner of detection being chosen based on the particular detectable label, where representative detection means include, e.g., scintillation counting, autoradiography, measurement of paramagnetism, fluorescence measurement, light absorption measurement, measurement of light scattering and the like. [0055] In general, hybridization between a primer and the marker may be assayed by

Attorney Docket No.: 2003923-0022 Customer No.: 24280

4313517vl contacting the primer with a cancer sample that includes the marker. Depending upon the nature of the cancer sample, appropriate methods include, but are not limited to, microarray analysis, in situ hybridization, Northern blot, and various nucleic acid amplification techniques such as PCR, RT-PCR, quantitative PCR, the ligase chain reaction, etc.

Kits

[0056] Interaction partners or primers for detecting expression of any one of the aforementioned panels may be prepared and packaged together in kits for use in classifying, diagnosing, or otherwise characterizing head and neck cancer samples.

[0057] In addition to interaction partners and primers for the markers in the inventive panels, kits for use in accordance with the present invention may include, one or more reference samples

(e.g., positive and negative controls), instructions for processing samples, performing the test, instructions for interpreting the results, buffers and/or other reagents necessary for performing the test. In certain embodiments the kit can comprise a panel of antibodies.

Identification of Novel Therapies

[0058] The prognostic power of the markers in Appendix B is useful according to the present invention not only to classify head and neck cancer patients with respect to their likely prognosis, but also to identify potential new therapies or therapeutic agents that could be useful in the treatment of head and neck cancer.

[0059] Indeed, each of the markers represents an attractive candidate for identification of new therapeutic agents (e.g., via screens to detect compounds or entities that bind or hybridize to the marker, preferably with at least a specified affinity and/or specificity, and/or via screens to detect compounds or entities that modulate (i.e., increase or decrease) expression, localization, modification, or activity of the marker. Thus, in one embodiment the present invention provides methods comprising steps of contacting a test compound with a cell expressing any one of the markers in Appendix B (e.g., individual engineered cells or in the context of a tissue, etc.); and determining whether the test compound modulates the expression, localization, modification, or activity of the marker. In many instances, interaction partners or primers (e.g., antisense or RNAi primers) themselves may prove to be useful therapeutics. [0060] Thus the present invention provides interaction partners and primers that are

Attorney Docket No.: 2003923-0022 Customer No.: 24280

4313517vl themselves useful therapeutic agents. For example, binding by an antibody raised against a marker to cancerous cells might inhibit growth of those cells. Alternatively or additionally, interaction partners defined or prepared according to the present invention could be used to deliver a therapeutic agent to a cancer cell. In particular, interaction partners (e.g., an antibody raised against a marker from Appendix B) may be coupled to one or more therapeutic agents. Suitable agents in this regard include radionuclides and drugs. Exemplary radionuclides include ⁹⁰Y, ¹²³I, ¹²⁵I, ¹³¹I, ¹⁸⁶Re, ¹⁸⁸Re, ²¹¹At and ²¹²Bi. Exemplary drugs include chlorambucil, ifosphamide, meclorethamine, cyclophosphamide, carboplatin, cisplatin, procarbazine, decarbazine, carmustine, cytarabine, hydroxyurea, mercaptopurine, methotrexate, paclitaxel, docetaxel, thioguanine, 5-fluorouracil, actinomycin D, bleomycin, daunorubicin, doxorubicin, etoposide, vinblastine, vincristine, L-asparginase, adrenocorticosteroids, canciclovir triphosphate, adenine arabinonucleoside triphosphate, 5-aziridinyl-4-hydroxylamino-2- nitrobenzamide, acrolein, phosphoramide mustard, 6-methylpurine, etoposide, benzoic acid mustard, cyanide and nitrogen mustard.

[0061] According to such embodiments, the therapeutic agent may be coupled with an interaction partner by direct or indirect covalent or non-covalent interactions. A direct interaction between a therapeutic agent and an interaction partner is possible when each possesses a substituent capable of reacting with the other. For example, a nucleophilic group, such as an amino or sulfhydryl group, on one may be capable of reacting with a carbonyl- containing group, such as an anhydride or an acid halide, or with an alkyl group containing a good leaving group (e.g., a halide) on the other. Indirect interactions might involve a linker group that is itself non-covalently bound to both the therapeutic agent and the interaction partner. A linker group can function as a spacer to distance an interaction partner from an agent in order to avoid interference with association capabilities. A linker group can also serve to increase the chemical reactivity of a substituent on an agent or an interaction partner and thus increase the coupling efficiency. An increase in chemical reactivity may also facilitate the use of agents, or functional groups on agents, which otherwise would not be possible. [0062] It will be evident to those skilled in the art that a variety of bifunctional or polyfunctional reagents, both homo- and hetero-functional (such as those described in the catalog of the Pierce Chemical Co., Rockford, 111.), may be employed as the linker group. Coupling may be effected, for example, through amino groups, carboxyl groups, sulfydryl

Attorney Docket No.: 2003923-0022 Customer No.: 24280

4313517vl groups or oxidized carbohydrate residues. There are numerous references describing such methodology, e.g., U.S. Pat. No. 4,671,958, to Rodwell et al. It will further be appreciated that a therapeutic agent and an interaction partner may be coupled via non-covalent interactions, e.g., ligand/receptor type interactions. Any ligand/receptor pair with a sufficient stability and specificity to operate in the context of the invention may be employed to couple a therapeutic agent and an interaction partner. To give but an example, a therapeutic agent may be covalently linked with biotin and an interaction partner with avidin. The strong non-covalent binding of biotin to avidin would then allow for coupling of the therapeutic agent and the interaction partner. Typical ligand/receptor pairs include protein/co-factor and enzyme/substrate pairs. Besides the commonly used biotin/avidin pair, these include without limitation, biotin/streptavidin, digoxigenin/anti-digoxigenin, FK506/FK506-binding protein (FKBP), rapamycin/FKBP, cyclophilin/cyclosporin and glutathione/glutathione transferase pairs. Other suitable ligand/receptor pairs would be recognized by those skilled in the art, e.g., monoclonal antibodies paired with a epitope tag such as, without limitation, glutathione-S-transferase (GST), c-myc, FLAG® and maltose binding protein (MBP) and further those described in Kessler pp. 105-152 of Advances in Mutagenesis " Ed. by Kessler, Springer- Verlag, 1990; "Affinity Chromatography: Methods and Protocols (Methods in Molecular Biology)" Ed. by Pascal Baillon, Humana Press, 2000; and "Immobilized Affinity Ligand Techniques" by Hermanson et al., Academic Press, 1992.

[0063] Where a therapeutic agent is more potent when free from the interaction partner, it may be desirable to use a linker group which is cleavable during or upon internalization into a cell. A number of different cleavable linker groups have been described. The mechanisms for the intracellular release of an agent from these linker groups include cleavage by reduction of a disulfide bond (e.g., U.S. Pat. No. 4,489,710 to Spitler), by irradiation of a photolabile bond (e.g., U.S. Pat. No. 4,625,014 to Senter et al.), by hydrolysis of derivatized amino acid side chains (e.g., U.S. Pat. No. 4,638,045 to Kohn et al.), by serum complement-mediated hydrolysis (e.g., U.S. Pat. No. 4,671,958 to Rodwell et al.) and by acid-catalyzed hydrolysis (e.g., U.S. Pat. No. 4,569,789 to Blattler et al.).

[0064] In certain embodiments, it may be desirable to couple more than one therapeutic agent to an interaction partner. In one embodiment, multiple molecules of an agent are coupled to one interaction partner molecule. In another embodiment, more than one type of therapeutic

Attorney Docket No.: 2003923-0022 Customer No.: 24280

4313517vl agent may be coupled to one interaction partner molecule. Regardless of the particular embodiment, preparations with more than one agent may be prepared in a variety of ways. For example, more than one agent may be coupled directly to an interaction partner molecule, or linkers that provide multiple sites for attachment can be used.

[0065] Alternatively, a carrier can be used. A carrier may bear the agents in a variety of ways, including covalent bonding either directly or via a linker group. Suitable carriers include proteins such as albumins (e.g., U.S. Pat. No. 4,507,234 to Kato et al.), peptides, and polysaccharides such as aminodextran (e.g., U.S. Pat. No. 4,699,784 to Shih et al.). A carrier may also bear an agent by non-covalent bonding or by encapsulation, such as within a liposome vesicle (e.g., U.S. Pat. Nos. 4,429,008 to Martin et al. and 4,873,088 to Mayhew et al.). Carriers specific for radionuclide agents include radiohalogenated small molecules and chelating compounds. For example, U.S. Pat. No. 4,735,792 to Srivastava discloses representative radiohalogenated small molecules and their synthesis. A radionuclide chelate may be formed from chelating compounds that include those containing nitrogen and sulfur atoms as the donor atoms for binding the metal, or metal oxide, radionuclide. For example, U.S. Pat. No. 4,673,562 to Davison et al. discloses representative chelating compounds and their synthesis. [0066] When interaction partners are themselves therapeutics, it will be understood that, in many cases, any interaction partner that binds the same marker may be so used. [0067] In one embodiment of the invention, the therapeutic agents (whether interaction partners or otherwise) are antibodies, e.g., an antibody against a marker from Appendix B. As is well known in the art, when using an antibody or fragment thereof for therapeutic purposes it may prove advantageous to use a "humanized" or "veneered" version of an antibody of interest to reduce any potential immunogenic reaction. In general, "humanized" or "veneered" antibody molecules and fragments thereof minimize unwanted immunological responses toward antihuman antibody molecules which can limit the duration and effectiveness of therapeutic applications of those moieties in human recipients.

[0068] A number of "humanized" antibody molecules comprising an antigen binding portion derived from a non-human immunoglobulin have been described in the art, including chimeric antibodies having rodent variable regions and their associated complementarity-determining regions (CDRs) fused to human constant domains (e.g., see Winter et al., Nature 349:293, 1991; Lobuglio et al., Proc. Nat. Acad. ScL USA 86:4220, 1989; Shaw et al., J. Immunol. 138:4534,

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4313517vl 1987; and Brown et al, Cancer Res. 47:3577, 1987), rodent CDRs grafted into a human supporting framework region (FR) prior to fusion with an appropriate human antibody constant domain (e.g., see Riechmann et al., Nature 332:323, 1988; Verhoeyen et al., Science 239:1534, 1988; and Jones et al. Nature 321 :522, 1986) and rodent CDRs supported by recombinantly veneered rodent FRs (e.g., see European Patent Publication No. 519,596, published Dec. 23, 1992). It is to be understood that the invention also encompasses "fully human" antibodies produced using the XenoMouse™ technology (AbGenix Corp., Fremont, CA) according to the techniques described in U.S. Patent No. 6,075,181.

[0069] Yet further, so-called "veneered" antibodies may be used that include "veneered FRs". The process of veneering involves selectively replacing FR residues from, e.g., a murine heavy or light chain variable region, with human FR residues in order to provide a xenogeneic molecule comprising an antigen binding portion which retains substantially all of the native FR protein folding structure. Veneering techniques are based on the understanding that the antigen binding characteristics of an antigen binding portion are determined primarily by the structure and relative disposition of the heavy and light chain CDR sets within the antigen-association surface (e.g., see Davies et al., Ann. Rev. Biochem. 59:439, 1990). Thus, antigen association specificity can be preserved in a humanized antibody only wherein the CDR structures, their interaction with each other and their interaction with the rest of the variable region domains are carefully maintained. By using veneering techniques, exterior (e.g., solvent-accessible) FR residues which are readily encountered by the immune system are selectively replaced with human residues to provide a hybrid molecule that comprises either a weakly immunogenic, or substantially non-immunogenic veneered surface.

[0070] Preferably, interaction partners suitable for use as therapeutics (or therapeutic agent carriers) exhibit high specificity for the target marker and low background binding to other markers. In certain embodiments, monoclonal antibodies may be used for therapeutic purposes. [0071] It is to be understood that these inventive therapeutics may be used to treat patients with head and neck cancer. These methods will typically involve the administration of a therapeutically effective amount of an inventive therapeutic.

Pharmaceutical Compositions

[0072] As mentioned above, in certain aspects, the present invention provides new therapies

Attorney Docket No.: 2003923-0022 Customer No.: 24280

4313517vl and methods for identifying these. In certain embodiments, an interaction partner or primer may be a useful therapeutic agent. Alternatively or additionally, interaction partners or primers defined or prepared according to the present invention bind to markers that serve as targets for therapeutic agents. Also, inventive interaction partners or primers may be used to deliver a therapeutic agent to a cancer cell. For example, interaction partners or primers provided in accordance with the present invention may be coupled to one or more therapeutic agents. [0073] The invention includes pharmaceutical compositions comprising these inventive therapeutic agents. In general, a pharmaceutical composition will include a therapeutic agent in addition to one or more inactive agents such as a sterile, biocompatible carrier including, but not limited to, sterile water, saline, buffered saline, or dextrose solution. The pharmaceutical compositions may be administered either alone or in combination with other therapeutic agents including other chemotherapeutic agents, hormones, vaccines and/or radiation therapy. By "in combination with", here and elsewhere in the specification, it is not intended to imply that the agents must be administered at the same time or formulated for delivery together, although these methods of delivery are within the scope of the invention. In general, each agent will be administered at a dose and on a time schedule determined for that agent. Additionally, the invention encompasses the delivery of the inventive pharmaceutical compositions in combination with agents that may improve their bioavailability, reduce or modify their metabolism, inhibit their excretion, or modify their distribution within the body. Although the pharmaceutical compositions of the present invention can be used for treatment of any subject (e.g., any animal) in need thereof, they are most preferably used in the treatment of humans. [0074] The pharmaceutical compositions of this invention can be administered to humans and other animals by a variety of routes including oral, intravenous, intramuscular, intra-arterial, subcutaneous, intraventricular, transdermal, rectal, intravaginal, intraperitoneal, topical (as by powders, ointments, or drops), bucal, or as an oral or nasal spray or aerosol. In general the most appropriate route of administration will depend upon a variety of factors including the nature of the agent (e.g., its stability in the environment of the gastrointestinal tract), the condition of the patient (e.g., whether the patient is able to tolerate oral administration), etc. At present the intravenous route is most commonly used to deliver therapeutic antibodies. However, the invention encompasses the delivery of the inventive pharmaceutical composition by any appropriate route taking into consideration likely advances in the sciences of drug delivery.

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4313517vl [0075] General considerations in the formulation and manufacture of pharmaceutical agents may be found, for example, in Remington 's Pharmaceutical Sciences, 19^th ed., Mack Publishing Co., Easton, PA, 1995.

Exemplification

Example 1 : Raising Antibodies

[0076] This example describes a method that was employed to generate the majority of the antibodies that were used in these Examples. Similar methods may be used to generate an antibody that binds to any polypeptide of interest (e.g., to polypeptides that are or are derived from other tumor markers). In some cases, antibodies may be obtained from commercial sources (e.g., Chemicon, Dako, Oncogene Research Products, NeoMarkers, etc.) or other publicly available sources (e.g., Imperial Cancer Research Technology, etc.).

Materials and Solutions

• Anisole (Cat. No. A4405, Sigma, St. Louis, MO)

• 2,2'-azino-di-(3-ethyl-benzthiazoline-sulfonic acid) (ABTS) (Cat. No. A6499, Molecular Probes, Eugene, OR)

• Activated maleimide Keyhole Limpet Hemocyanin (Cat. No. 77106, Pierce, Rockford, IL)

• Keyhole Limpet Hemocyanin (Cat. No. 77600, Pierce, Rockford, IL)

• Phosphoric Acid (H₃PO₄) (Cat. No. P6560, Sigma)

• Glacial Acetic Acid (Cat No. BPl 185-500, Fisher)

• EDC (EDAC) (Cat No. 341006, Calbiochem)

• 25% Glutaraldehyde (Cat No. G-5882, Sigma)

• Glycine (Cat No. G-8898, Sigma)

• Biotin (Cat. No. B2643, Sigma)

• Boric acid (Cat. No. B0252, Sigma)

• Sepharose 4B (Cat. No. 17-0120-01, LKB/Pharmacia, Uppsala, Sweden)

• Bovine Serum Albumin (LP) (Cat. No. 100 350, Boehringer Mannheim, Indianapolis, IN)

• Cyanogen bromide (Cat. No. C6388, Sigma)

Attorney Docket No.: 2003923-0022 Customer No.: 24280

4313517vl • Dialysis tubing Spectra/Por Membrane MWCO: 6-8,000 (Cat. No. 132 665, Spectrum Industries, Laguna Hills, CA)

• Dimethyl formamide (DMF) (Cat. No. 22705-6, Aldrich, Milwaukee, WI)

• DIC (Cat. No. BP 592-500, Fisher)

• Ethanedithiol (Cat. No. 39,802-0, Aldrich)

• Ether (Cat. No. TX 1275-3, EM Sciences)

• Ethylenediaminetetraacetatic acid (EDTA) (Cat. No. BP 120-1, Fisher, Springfield, NJ)

• l-ethyl-3-(3'dimethylaminopropyl)-carbodiimide, HCL (EDC) (Cat. no. 341-006, Calbiochem, San Diego, CA)

• Freund's Adjuvant, complete (Cat. No. M-0638-50B, Lee Laboratories, Grayson, GA)

• Freund's Adjuvant, incomplete (Cat. No. M-0639-50B, Lee Laboratories)

• Fritted chromatography columns (Column part No. 12131011; Frit Part No. 12131029, Varian Sample Preparation Products, Harbor City, CA)

• Gelatin from Bovine Skin (Cat. No. G9382, Sigma)

• Goat anti-rabbit IgG, biotinylated (Cat. No. A 0418, Sigma)

• HOBt (Cat. No. 01-62-0008, Calbiochem)

• Horseradish peroxidase (HRP) (Cat. No. 814 393, Boehringer Mannheim)

• HRP-Streptavidin (Cat. No. S 5512, Sigma)

• Hydrochloric Acid (Cat. No. 71445-500, Fisher)

• Hydrogen Peroxide 30% w/w (Cat. No. H1009, Sigma)

• Methanol (Cat. No. A412-20, Fisher)

• Microtiter plates, 96 well (Cat. No. 2595, Corning-Costar, Pleasanton, CA)

• N-α-Fmoc protected amino acids from Calbiochem. See '97-'98 Catalog pp. 1-45.

• N-α-Fmoc protected amino acids attached to Wang Resin from Calbiochem. See '97-'98 Catalog pp. 161-164.

• NMP (Cat. No. CAS 872-50-4, Burdick and Jackson, Muskegon, MI)

• Peptide (Synthesized by Research Genetics. Details given below)

• Piperidine (Cat. No. 80640, Fluka, available through Sigma)

• Sodium Bicarbonate (Cat. No. BP328-1 , Fisher)

• Sodium Borate (Cat. No. B9876, Sigma)

Attorney Docket No.: 2003923-0022 Customer No.: 24280

4313517vl • Sodium Carbonate (Cat. No. BP357-1 , Fisher)

• Sodium Chloride (Cat. No. BP 358-10, Fisher)

• Sodium Hydroxide (Cat. No. SS 255-1 , Fisher)

• Streptavidin (Cat. No. 1 520, Boehringer Mannheim)

• Thioanisole (Cat. No. T-2765, Sigma)

• Trifluoro acetic acid (Cat. No. TX 1275-3, EM Sciences)

• Tween-20 (Cat. No. BP 337-500, Fisher)

• Wetbox (Rectangular Servin' Saver™ Part No. 3862, Rubbermaid, Wooster, OH)

• BBS - Borate Buffered Saline with EDTA dissolved in distilled water (pH 8.2 to 8.4 with HCl or NaOH), 25 mM Sodium borate (Borax), 100 mM Boric Acid, 75 mM NaCl and 5 mM EDTA.

• 0.1 N HCl in saline as follows: concentrated HCl (8.3 ml/0.917 liter distilled water) and 0.154 M NaCl

• Glycine (pH 2.0 and pH 3.0) dissolved in distilled water and adjusted to the desired pH, 0.1 M glycine and 0.154 M NaCl.

• 5X Borate IX Sodium Chloride dissolved in distilled water, 0.11 M NaCl, 60 mM Sodium Borate and 250 mM Boric Acid.

• Substrate Buffer in distilled water adjusted to pH 4.0 with sodium hydroxide, 50 to 100 mM Citric Acid.

• AA solution: HOBt is dissolved in NMP (8.8 grams HOBt to 1 liter NMP). Fmoc-N-a- amino at a concentration at 0.53 M.

• DIC solution: 1 part DIC to 3 parts NMP.

• Deprotecting solution: 1 part Piperidine to 3 parts DMF.

• Reagent R: 2 parts anisole, 3 parts ethanedithiol, 5 parts thioanisole and 90 parts trifluoroacetic acid.

Equipment

• MRX Plate Reader (Dynatech, Chantilly, VA)

• Hamilton Eclipse (Hamilton Instruments, Reno, NV)

• Beckman TJ-6 Centrifuge (Model No. TJ-6, Beckman Instruments, Fullerton, CA)

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4313517vl • Chart Recorder (Recorder 1 Part No. 18- 1001 -40, Pharmacia LKB Biotechnology)

• UV Monitor (Uvicord SII Part No. 18-1004-50, Pharmacia LKB Biotechnology)

• Amicon Stirred Cell Concentrator (Model 8400, Amicon, Beverly, MA)

• 30 kD MW cut-off filter (Cat. No. YM-30 Membranes Cat. No. 13742, Amicon)

• Multi-channel Automated Pipettor (Cat. No. 4880, Corning Costar, Cambridge, MA)

• pH Meter Corning 240 (Corning Science Products, Corning Glassworks, Corning, NY)

• ACT396 peptide synthesizer (Advanced ChemTech, Louisville, KY)

• Vacuum dryer (Box from Labconco, Kansas City, MO and Pump from Alcatel, Laurel, MD).

• Lyophilizer (Unitop 600sl in tandem with Freezemobile 12, both from Virtis, Gardiner, NY)

Peptide Selection

[0077] Peptides against which antibodies would be raised were selected from within the polypeptide sequence of interest using a program that uses the Hopp/Woods method (described in Hopp and Woods, MoI. Immunol. 20:483, 1983 and Hopp and Woods, Proc. Nat. Acad. Sci. U.S.A. 78:3824, 1981). The program uses a scanning window that identifies peptide sequences of 15-20 amino acids containing several putative antigenic epitopes as predicted by low solvent accessibility. This is in contrast to most implementations of the Hopp/Woods method, which identify single short (~ 6 amino acids) presumptive antigenic epitopes. Occasionally the predicted solvent accessibility was further assessed by PHD prediction of loop structures (described in Rost and Sander, Proteins 20:216, 1994). Preferred peptide sequences display minimal similarity with additional known human proteins. Similarity was determined by performing BLASTP alignments, using a wordsize of 2 (described in Altschul et al., J. MoI. Biol. 215:403, 1990). All alignments given an EXPECT value less than 1000 were examined and alignments with similarities of greater than 60% or more than four residues in an exact contiguous non-gapped alignment forced those peptides to be rejected. When it was desired to target regions of proteins exposed outside the cell membrane, extracellular regions of the protein of interest were determined from the literature or as defined by predicted transmembrane domains using a hidden Markov model (described in Rrogh et al., J. MoI. Biol. 305:567, 2001). When the peptide sequence was in an extracellular domain, peptides were rejected if they

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4313517vl contained N-linked glycosylation sites. As shown in Appendix A, one to three peptide sequences were selected for each polypeptide marker using this procedure.

Peptide Synthesis

[0078] The sequence of the desired peptide was provided to the peptide synthesizer. The C- terminal residue was determined and the appropriate Wang Resin was attached to the reaction vessel. The peptides were synthesized C-terminus to N-terminus by adding one amino acid at a time using a synthesis cycle. Which amino acid is added was controlled by the peptide synthesizer, which looks to the sequence of the peptide that was entered into its database. The synthesis steps were performed as follows:

Step 1 - Resin Swelling: Added 2 ml DMF, incubated 30 minutes, drained DMF. Step 2 - Synthesis cycle (repeated over the length of the peptide)

2a - Deprotection: 1 ml deprotecting solution was added to the reaction vessel and incubated for 20 minutes.

2b - Wash Cycle

2c - Coupling: 750 ml of amino acid solution (changed as the sequence listed in the peptide synthesizer dictated) and 250 ml of DIC solution were added to the reaction vessel. The reaction vessel was incubated for thirty minutes and washed once. The coupling step was repeated once.

2d - Wash Cycle Step 3 - Final Deprotection: Steps 2a and 2b were performed one last time.

[0079] Resins were deswelled in methanol (rinsed twice in 5 ml methanol, incubated 5 minutes in 5 ml methanol, rinsed in 5 ml methanol) and then vacuum dried. [0080] Peptide was removed from the resin by incubating 2 hours in reagent R and then precipitated into ether. Peptide was washed in ether and then vacuum dried. Peptide was resolubilized in diH₂0, frozen and lyophilized overnight.

Conjugation of Peptide with Keyhole Limpet Hemocyanin

[0081] Peptide (6 mg) was conjugated with Keyhole Limpet Hemocyanin (KLH). When the

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4313517vl selected peptide included at least one cysteine, three aliquots (2 mg) were dissolved in PBS (2 ml) and coupled to KLH via glutaraldehyde, EDC or maleimide activated KLH (2 mg) in 2 ml of

PBS for a total volume of 4 ml. When the peptide lacked cysteine, two aliquots (3 mg) were coupled via glutaraldehyde and EDC methods.

[0082] Maleimide coupling is accomplished by mixing 2 mg of peptide with 2 mg of maleimide-activated KLH dissolved in PBS (4 ml) and incubating 4 hr.

[0083] EDC coupling is accomplished by mixing 2 mg of peptide, 2 mg unmodified KLH, and 20 mg of EDC in 4 ml PBS (lowered to pH 5 by the addition of phosphoric acid), and incubating for 4 hours. The reaction is stopped by the slow addition of 1.33 ml acetic acid (pH

4.2). When using EDC to couple 3 mg of peptide, the amounts listed above are increased by a factor of 1.5.

[0084] Glutaraldehyde coupling occurs when 2 mg of peptide are mixed with 2 mg of KLH in 0.9 ml of PBS. 0.9 ml of 0.2% glutaraldehyde in PBS is added and mixed for one hour. 0.46 ml of 1 M glycine in PBS is added and mixed for one hour. When using glutaraldehyde to couple 3 mg of peptide, the above amounts are increased by a factor of 1.5.

[0085] The conjugated aliquots were subsequently repooled, mixed for two hours, dialyzed in 1 liter PBS and lyophilized.

Immunization of Rabbits

[0086] Two New Zealand White Rabbits were injected with 250 μg (total) KLH conjugated peptide in an equal volume of complete Freund's adjuvant and saline in a total volume of 1 ml. 100 μg KLH conjugated peptide in an equal volume of incomplete Freund's Adjuvant and saline were then injected into three to four subcutaneous dorsal sites for a total volume of 1 ml two, six, eight and twelve weeks after the first immunization. The immunization schedule was as follows:

Day 0 Pre-immune bleed, primary immunization

Day 15 1st boost

Day 27 1st bleed

Day 44 2nd boost

Day 57 2nd bleed and 3rd boost

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4313517vl Day 69 3rd bleed

Day 84 4th boost

Day 98 4th bleed

Collection of Rabbit Serum

[0087] The rabbits were bled (30 to 50 ml) from the auricular artery. The blood was allowed to clot at room temperature for 15 minutes and the serum was separated from the clot using an IEC DPR-6000 centrifuge at 500Og. Cell-free serum was decanted gently into a clean test tube and stored at -2O⁰C for affinity purification.

Determination of Antibody Titer

[0088] All solutions with the exception of wash solution were added by the Hamilton Eclipse, a liquid handling dispenser. The antibody titer was determined in the rabbits using an ELISA assay with peptide on the solid phase. Flexible high binding ELISA plates were passively coated with peptide diluted in BBS (100 μl, 1 μg/well) and the plate was incubated at 4⁰C in a wetbox overnight (air-tight container with moistened cotton balls). The plates were emptied and then washed three times with BBS containing 0.1% Tween-20 (BBS-TW) by repeated filling and emptying using a semi-automated plate washer. The plates were blocked by completely filling each well with BBS-TW containing 1% BSA and 0.1% gelatin (BBS-TW-BG) and incubating for 2 hours at room temperature. The plates were emptied and sera of both pre- and post-immune serum were added to wells. The first well contained sera at 1 :50 in BBS. The sera were then serially titrated eleven more times across the plate at a ratio of 1 : 1 for a final (twelfth) dilution of 1 :204,800. The plates were incubated overnight at 4⁰C. The plates were emptied and washed three times as described.

[0089] Biotinylated goat anti-rabbit IgG (100 μl) was added to each microtiter plate test well and incubated for four hours at room temperature. The plates were emptied and washed three times. Horseradish peroxidase-conjugated Streptavidin (100 μl diluted 1 : 10,000 in BBS-TW- BG) was added to each well and incubated for two hours at room temperature. The plates were emptied and washed three times. The ABTS was prepared fresh from stock by combining 10 ml of citrate buffer (0.1 M at pH 4.0), 0.2 ml of the stock solution (15 mg/ml in water) and 10 μl of

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4313517vl 30% hydrogen peroxide. The ABTS solution (100 μl) was added to each well and incubated at room temperature. The plates were read at 414 nm, 20 minutes following the addition of substrate.

Preparation of Peptide Affinity Purification Column:

[0090] The affinity column was prepared by conjugating 5 mg of peptide to 10 ml of cyanogen bromide-activated Sepharose 4B and 5 mg of peptide to hydrazine-Sepharose 4B. Briefly, 100 μl of DMF was added to peptide (5 mg) and the mixture was vortexed until the contents were completely wetted. Water was then added (900 μl) and the contents were vortexed until the peptide dissolved. Half of the dissolved peptide (500 μl) was added to separate tubes containing 10 ml of cyanogen-bromide activated Sepharose 4B in 0.1 ml of borate buffered saline at pH 8.4 (BBS) and 10 ml of hydrazine-Sepharose 4B in 0.1 M carbonate buffer adjusted to pH 4.5 using excess EDC in citrate buffer pH 6.0. The conjugation reactions were allowed to proceed overnight at room temperature. The conjugated Sepharose was pooled and loaded onto fritted columns, washed with 10 ml of BBS, blocked with 10 ml of 1 M glycine and washed with 10 ml 0.1 M glycine adjusted to pH 2.5 with HCl and re-neutralized in BBS. The column was washed with enough volume for the optical density at 280 nm to reach baseline.

Affinity Purification of Antibodies

[0091] The peptide affinity column was attached to a UV monitor and chart recorder. The titered rabbit antiserum was thawed and pooled. The serum was diluted with one volume of BBS and allowed to flow through the columns at 10 ml per minute. The non-peptide immunoglobulins and other proteins were washed from the column with excess BBS until the optical density at 280 nm reached baseline. The columns were disconnected and the affinity purified column was eluted using a stepwise pH gradient from pH 7.0 to 1.0. The elution was monitored at 280 nm and fractions containing antibody (pH 3.0 to 1.0) were collected directly into excess 0.5 M BBS. Excess buffer (0.5 M BBS) in the collection tubes served to neutralize the antibodies collected in the acidic fractions of the pH gradient.

[0092] The entire procedure was repeated with "depleted" serum to ensure maximal recovery of antibodies. The eluted material was concentrated using a stirred cell apparatus and a

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4313517vl membrane with a molecular weight cut-off of 30 kD. The concentration of the final preparation was determined using an optical density reading at 280 nm. The concentration was determined using the following formula: mg/ml = OD₂₈₀/IA

[0093] It will be appreciated that in certain embodiments, additional steps may be used to purify antibodies of the invention. In particular, it may prove advantageous to repurify antibodies, e.g., against one of the peptides that was used in generating the antibodies. It is to be understood that the present invention encompasses antibodies that have been prepared with such additional purification or repurification steps. It will also be appreciated that the purification process may affect the binding between samples and the inventive antibodies.

Example 2: Preparing and Staining Tissue Arrays

[0094] This example describes a method that was employed to prepare the tissue arrays that were used in the Examples. This example also describes how the antibody staining was performed.

[0095] Tissue arrays were prepared by inserting full-thickness cores from a large number of paraffin blocks (donor blocks) that contain fragments of tissue derived from many different patients and/or different tissues or fragments of tissues from a single patient, into a virgin paraffin block (recipient block) in a grid pattern at designated locations in a grid. A standard slide of the paraffin embedded tissue (donor block) was then made which contained a thin section of the specimen amenable to H&E staining. A trained pathologist, or the equivalent versed in evaluating tumor and normal tissue, designated the region of interest for sampling on the tissue array (e.g., a tumor area as opposed to stroma). A commercially available tissue arrayer from Beecher Instruments was then used to remove a core from the donor block which was then inserted into the recipient block at a designated location. The process was repeated until all donor blocks had been inserted into the recipient block. The recipient block was then thin-sectioned to yield 50-300 slides containing cores from all cases inserted into the block. [0096] The selected antibodies were then used to perform immunohistochemical staining using the DAKO Envision+, Peroxidase IHC kit (DAKO Corp., Carpenteria, CA) with DAB substrate according to the manufacturer's instructions.

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4313517vl Example 3 : Correlating Individual Antibody Binding with Clinical Prognostic Data in Head and Neck Cancer

[0097] This example describes the identification of individual prognostic markers for head and neck cancer patients. The prognostic markers that were identified are summarized in Appendix B.

[0098] Tissue microarrays from a head and neck cancer cohort (Stanford cohort) were used to investigate potential IHC markers to help stratify head and neck cancer patients into different prognostic categories. Clinical information for the patients within the cohort included recurrence and survival data (both five years after diagnosis).

[0099] Tissue samples for the patients in the Stanford cohort were stained with antibodies selected from the list in Appendix A. The stained samples were then scored in a semiquantitative fashion, with 0 = negative, 1 = weak staining, and 2 = strong staining (scoring method 1). In a separate analysis, these scoring results were processed as follows: 0 = negative, 1 = weak or strong staining (scoring method 2).

[00100] The expected relationship between the staining of patient samples with each antibody and clinical outcome was measured using the Kaplan-Meier estimate of expected recurrence (e.g., see Kaplan and Meier, J. Am. Stat. Assn. 53:457-81, 1958) and with Cox regression analysis (e.g., see Cox, J. Royal Statist. Soc. B 34:187-220, 1972). Cox regression was used to determine whether individual staining results were predictive of a decreased or increased risk of (a) death due to head and neck cancer (DOD or "dead of disease") and/or (b) recurrence (e.g., see Cox and Oakes, "Analysis of Survival Data", Chapman & Hall, 1984). This produced the results that are listed for each antibody in Appendix B. Two sets of results are provided for each antibody. In each case, the upper result corresponds to analysis using scoring method 1 (i.e., 0 = negative; 1 = weak staining; 2 = strong staining) while the lower result corresponds to the same analysis using scoring method 2 (i.e., 0 = negative; 1 = weak or strong staining). Antibodies that produce a p-value of less than 0.10 or even less than 0.01 are particularly strong prognosticators. As demonstrated herein, antibodies with higher p-values can also be used as individual prognosticators and/or may provide greater prognostic information when combined with other antibodies in a panel. The "hazard ratio" (HR) listed in Appendix B for each antibody reflects the predicted increase in risk of the clinical outcome (death due to head and neck cancer or recurrence) for each increase in the staining score. Scores greater than 1.0 indicate that staining

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4313517vl predicts an increased risk compared to an average individual, scores less than 1.0 indicate that staining predicts a decreased risk. In one embodiment, an antibody with a hazard ratio of greater than 1.1 or less than 0.9 in Appendix B may be used for analyzing the prognosis of a head and neck cancer patient. In one embodiment, an antibody with a hazard ratio of greater than 1.2 or less than 0.8 in Appendix B may be used for analyzing the prognosis of a head and neck cancer patient. In one embodiment, an antibody with a hazard ratio of greater than 1.3 or less than 0.7 in Appendix B may be used for analyzing the prognosis of a head and neck cancer patient. [00101] Appendix B also includes individual prognostic data for the following markers: LOX (NCBI Locus Link ID 4015, lysyl oxidase also called MGC 105112), CAIX annotated for membrane vs. cytoplasmic staining (NCBI Locus Link ID 768, carbonic anhydrase IX also called CA9 or MN), EFNAl (NCBI Locus Link ID 1942, ephrin-Al also called B61, EFLl, ECKLG, EPLGl, LERKl or TNFAIP4), and CDKN2A (NCBI Locus Link ID 1029, cyclin- dependent kinase inhibitor 2A also called ARF, MLM, pi 4, pi 6, pi 9, CMM2, INK4, MTSl, TP16, CDK4I, CDKN2, INK4a, pl4ARF, pl6INK4 or pl6INK4a).

[00102] Appendix B also provides statistical data (obtained by a shrunken centroid analysis, e.g., see Tibshirani et al., PNAS 99:6567-6572, 2002) showing how some of the antibodies were able to predict whether lung tumors in a separate lung cancer cohort would have been diagnosed as adenocarcinoma or squamous cell carcinoma. T scores greater than 0 indicate that staining predicts an increased likelihood of a squamous cell carcinoma, T scores less than 0 indicate that staining predicts an increased likelihood of an adenocarcinoma.

Example 4: Correlating Antibody Panel Binding with Clinical Prognostic Data in Head and

Neck Cancer

[00103] It will be appreciated that the antibodies identified in Appendix B can be used alone or in combinations to predict clinical outcome (e.g., in combinations of 2, 3, 4, 5, 6, 7, 8, 9, 10 or more antibodies). It will also be appreciated that while a given antibody may not predict a clinical outcome when used alone, the same antibody may contribute to the prediction when used in combination with other antibodies.

[00104] These prognostic panels could be constructed using any method. Without limitation these include simple empirically derived rules, Cox multivariate proportional hazard models

(e.g., see Cox and Oakes, "Analysis of Survival Data", Chapman & Hall, 1984), regression trees

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4313517vl (e.g., see Segal and Bloch, Stat. Med. 8:539-50, 1989), and/or neural networks (e.g., see Ravdin et al., Breast Cancer Res. Treat. 21 :47-53, 1992). In certain embodiments a prognostic panel might include between 2-10 antibodies, for example 3-9, 4-8 or 5-7 antibodies. It will be appreciated that these ranges are exemplary and non-limiting.

[00105] To minimize the chance of identifying spurious associations, only those antibodies from Appendix B that showed statistical significance (p-value of less than 0.10) in either analysis (death from head and neck cancer or recurrence) were initially used in creating exemplary prognostic panels. Using Cox proportional hazard analysis (as described below) candidate panels were derived for prediction of clinical outcome (death from head and neck cancer or recurrence). The following describes panels that identified patients with significantly increased risks of a particular clinical outcome. It will be appreciated that these exemplary panels are non-limiting and that other panels could be readily identified based on the teachings herein.

[00106] Cox multivariate proportional hazard analysis treats the component antibodies of a panel as additive risk factors. Exemplary panels were created by initially using all applicable antibodies with a p-value of less than 0.10 in the univariate analysis, and then iteratively removing antibodies from the panel. If the removal of an antibody increased or did not affect the significance and prognostic ability of the panel as a whole, it was excluded, otherwise it was retained. In this manner exemplary panels with minimal numbers of antibodies were created. Other panels were obtained by analyzing the impact of adding one or more of some of the antibodies with a p-value of more than 0.10 in the univariate analysis of Appendix B. An exemplary panel is presented in Table 1.

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4313517vl Table 1

Death due to Cancer 1.90E-08 Hazard 3.43

P value¹

Recurrence 1.10E-07 ratio² 2.25

AGI ID Marker Terms³ sO411 FABP5 -0.26, 0.58 s0404 NDRGl -0.20, 0.66 sO147 CSTA 0.38, -0.41 s0059 TRIM29 -0.37, 0.38

- CDKN2A 0.34, -0.39

P value of overall panel

Hazard ratio of overall panel

Contribution of given antibody to exemplary overall panel prediction function depending on IHC score (e.g., staining scores of 0 [negative stain] or 1 [positive stain, weak or strong] for s0404 (NDRGl) result in its term in the overall panel prediction function equaling -0.20 or 0.66 respectively).

[00107] In general, a lower score based on the overall panel prediction function of Table 1 predicts a decreased likelihood of a poor clinical outcome (e.g., death due to head and neck cancer and/or recurrence). In one embodiment the results from the overall panel prediction function of Table 1 were used to classify patients into prognostic groups as follows: Good < - 0.15 < Moderate < 0.64 < Bad (likelihood of survival) and/or Good < 0.64 < Bad (likelihood of recurrence). It will be appreciated that these cut-offs and the overall panel prediction function (and any other functions and cut-offs that are described herein, e.g., see Tables 2-11) are exemplary and that other cut-offs and/or functions could be used with these panels. In particular, it will be appreciated that the lines between "good", "moderate" and "bad" prognosis (or between "good" and "bad" prognosis) are not absolute. It will also be appreciated that the terms for each antibody in a panel (and therefore the overall panel prediction function) may be adjusted to yield variations on the panel of Table 1 (or other panels described herein). [00108] The prognostic value of the exemplary panel of Table 1 was assessed by generating Kaplan-Meier outcome curves for head and neck cancer patients in the Stanford cohort. Patients from the cohort were placed into the following outcome groups based on their respective scores using the overall panel prediction function: Good < -0.15 < Moderate < 0.64 < Bad (likelihood of survival) and/or Good < 0.64 < Bad (likelihood of recurrence). Kaplan-Meier outcome curves were then calculated for patients within each prognostic group. Figure IA shows the survival

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4313517vl curves that were obtained for patients in the "Good", "Moderate" and "Bad" survival groups. Figure IB shows the recurrence curves that were obtained for patients in the "Good" and "Bad" recurrence groups.

[00109] Tables 2 to 9 summarize other exemplary prognostic panels that were identified by the inventors.

Table 2

Death due to Cancer 4.50E-07 Hazard 3.60

P value¹

Recurrence 3.80E-06 ratio² 3.06

AGI ID Marker Terms³ sO411 FABP5 -0.31, 0.76 s0404 NDRGl -0.21, 0.63

- CDKN2A 0.54, -0.55 sO235 CEACAM5 0.08, -0.38

' ' See Table 1

Table 3

Death due to Cancer 2.80E-06 Hazard 4.06

P value¹

Recurrence 2.10E-05 ratio² 3.29

AGI ID Marker Terms³ sO411 FABP5 -0.31, 0.76 s0404 NDRGl -0.21, 0.63

- CDKN2A 0.50, -0.48 s0260 NCSTN -0.21, 0.20

' ' See Table 1

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4313517vl Table 4

Death due to Cancer 2.10E-06 Hazard 3 .29

P value¹

Recurrence 1.50E-05 ratio² 2 .84

AGI ID Marker Terms³ sO411 FABP5 -0.20, 0.18 s0404 NDRGl -0.15, 0.51 sO586 ABCG2 -0.41, 0.31 sO235 CEACAM5 0.13, -0.66

' ' See Table 1

Table 5

Death due to Cancer 1.80E-05 Hazard 2.40 r value

Recurrence 2.30E-04 ratio² 2.08

AGI ID Marker Terms³ sO411 FABP5 -0.27, 0.68 s0404 NDRGl -0.16, 0.48 sO238 XPRl -0.08, 0.40 s0260 NCSTN -0.18, 0.16

' ' See Table 1

Table 6

Death due to Cancer 1.20E-06 Hazard 3.26

P value¹

Recurrence 1.10E-04 ratio² 2.38

AGI ID Marker Terms³ sO411 FABP5 -0.34, 0.78 s0404 NDRGl -0.23, 0.72 sO147 CSTA 0.28, -0.32

- CDKN2A 0.46, -0.50

' ' See Table 1

Attorney Docket No.: 2003923-0022 Customer No.: 24280 Table 7

Death due to Cancer 8 .60E-09 Hazard 3 .85

P value¹

Recurrence 1 .80E-07 ratio² 3 .41

AGI ID Marker Terms³ sO411 FABP5 -0.30, 0.72 s0404 NDRGl -0.18, 0.62 sO147 CSTA 0.44, -0.52 s0059 TRIM29 -0.52, 0.56

' ' See Table 1

Table 8

Death due to Cancer 2 .10E-08 Hazard 3 .85 r value

Recurrence 3 .20E-06 ratio² 2 .96

AGI ID Marker Terms³ sO411 FABP5 -0.30, 0.73 s0404 NDRGl -0.15, 0.50 sO147 CSTA 0.44, -0.54 sO586 ABCG2 -0.79, 0.61

' ' See Table 1

Table 9

Death due to Cancer 1 .50E-09 Hazard 4 .31

P value¹

Recurrence 1 .70E-06 ratio² 3 .10

AGI ID Marker Terms³ sO411 FABP5 -0.26, 0.62 s0404 NDRGl -0.14, 0.52 sO147 CSTA 0.45, -0.53 s0059 TRIM29 -0.27, 0.28 sO586 ABCG2 -0.59, 0.45

' ' See Table 1

[00110] The prognostic value of the exemplary panels of Tables 2-9 was also assessed by generating Kaplan-Meier outcome curves for head and neck cancer patients in the Stanford cohort. A single set of cut-offs was used for each panel to classify the patients in the cohort into "Good", "Moderate" and "Bad" prognosis groups based on staining patterns. Kaplan-Meier

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4313517vl outcome curves (survival or recurrence) were then calculated for patients within each prognostic group. Figure 2 (upper) shows the curves that were obtained using the panel of Table 2 (Good < 0.0 < Moderate < 0.71 < Bad). Figure 2 (lower) shows the curves that were obtained using the panel of Table 3 (Good < -0.8 < Moderate <0.18 < Bad). Figure 3 (upper) shows the curves that were obtained using the panel of Table 4 (Good < -0.6 < Moderate < 0. 1 < Bad). Figure 3 (lower) shows the curves that were obtained using the panel of Table 5 (Good < 0.0 < Moderate < 0.3 < Bad). Figure 4 (upper) shows the curves that were obtained using the panel of Table 6 (Good < 0.0 < Moderate < 0.33 < Bad). Figure 4 (lower) shows the curves that were obtained using the panel of Table 7 (Good < 0.0 < Moderate < 0.58 < Bad). Figure 5 (upper) shows the curves that were obtained using the panel of Table 8 (Good < 0.0 < Moderate < 0.67 < Bad). Figure 5 (lower) shows the curves that were obtained using the panel of Table 9 (Good < 0.0 < Moderate < 0.79 < Bad).

[00111] It is noteworthy that such a variety of panels could be constructed using different markers from Appendix B. These results highlight the fact that the panels in Tables 1-9 are exemplary panels and that other panels using these and/or additional markers from Appendix B could be readily constructed based on the teachings herein. It will also be appreciated that other panels may be prepared by omitting any 1, 2, 3 or 4 markers from any one of the 4-5 member panels described herein.

[00112] As shown in Appendix B, some of the antibodies were able to predict whether lung tumors in a separate lung cancer cohort would have been diagnosed as adenocarcinoma or squamous cell carcinoma. The results in Appendix B show that several of these antibodies correspond to antibodies that were prognostic in the head and neck cancer cohort. This unexpected link led us to apply a previously constructed lung histology antibody panel (i.e., a panel that was generated based on its ability to differentiate adenocarcinoma or squamous cell carcinoma lung cancers) to the Stanford head and neck cancer cohort. The panel is summarized in Table 10 below:

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4313517vl Table 10

Death due to Cancer 7.10E-04 Hazard 5.31

P value¹ Recurrence 8.10E-03 ratio² 3.01

AGI ID Marker Terms³ s0059 TRIM29 -1.26, 1.81 sO235 CEACAM5 0.79, -0.98 sO586 ABCG2 -0.51, 0.84 sO643 TLE3 -0.62, 0.41 s0260 NCSTN -0.35, 0.47

1,2,3

See Table 1

[00113] The prognostic value of the exemplary panel of Table 10 was assessed by generating Kaplan-Meier outcome curves for head and neck cancer patients in the Stanford cohort. Patients from the cohort were placed into the following outcome groups based on their respective scores using the overall panel prediction function: Good < 0 < Bad. Kaplan-Meier outcome curves (survival, any recurrence and distant recurrence, i.e., more than 5 years) were then calculated for patients within each prognostic group. As shown in Figure 6, this lung histology model strongly predicts outcome in the Stanford head and neck cancer cohort, with the "squamous-like" classification being associated with a poor prognosis. Table 11 below summarizes a slight variation on the panel of Table 10:

Table 11

Death due to Cancer 1.60E-03 Hazard 2.65

P value¹

Recurrence 3.00E-03 ratio² 2.29

AGI ID Marker Terms³ s0059 TRIM29 -1.59, 2.69 sO235 CEACAM5 1.19, -1.43 sO586 ABCG2 -1.16, 2.03 sO721 SLC7A5 -1.04, 1.21 s0260 NCSTN -1.26, 1.81

' ' See Table 1

[00115] Without limitation, in one embodiment, the panel of Table 11 can be used with the

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4313517vl following cut-offs: Good < -2.2 < Moderate <2.0 < Bad.

Other Embodiments

[00116] Other embodiments of the invention will be apparent to those skilled in the art from a consideration of the specification or practice of the invention disclosed herein. It is intended that the specification and examples be considered as exemplary only, with the true scope of the invention being indicated by the following claims.

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4313517vl

Claims

ClaimsWe claim:

1. A method of assessing the likely prognosis of a patient having head and neck cancer, the method comprising steps of: determining a level of expression of a panel of one or more markers in a cancer sample from a patient with head and neck cancer, wherein the panel comprises at least one marker selected from the group consisting of CYP4Z1, MMP7, TRIM29, IRX3, S100A8, SLPI, CDH3, FABP4, XPRl, NCSTN, CDHF7, FABP5, LTB, ABCG2, TERF2IP, SLC7A11, ITGB4, SLC7A5, NDRGl, CASP7, MMPl, CEACAM5, TP53, LOX, CAIX, EFNAl and CDKN2A; and assessing the patient's likely prognosis based on the determined level of expression, wherein across a population of patients with head and neck cancer, a higher level of expression of a marker selected from the group consisting of MMP7, TRIM29, IRX3, S100A8, SLPI, CDH3, FABP4, XPRl, NCSTN, FABP5, LTB, ABCG2, SLC7A11, ITGB4, SLC7A5, NDRGl, CASP7, MMPl, TP53, LOX, CAIX and EFNAl is indicative of a higher likelihood of an unfavorable prognosis and a higher level of expression of a marker selected from the group consisting of CYP4Z1, CDHF7, TERF2IP, CEACAM5, and CDKN2A is indicative of a lower likelihood of an unfavorable prognosis.

2. The method of claim 1, wherein the panel comprises at least one marker selected from the group consisting of FABP5, NDRGl, CDKN2A, CEACAM5, NCSTN, XPRl, ABCG2, SLC7A5 and TRIM29.

3. The method of claim 1, wherein the panel comprises at least one marker selected from the group consisting of FABP5, NDRGl, CDKN2A, NCSTN, ABCG2, SLC7A11, SLC7A5, LOX, CAIX or TRIM29.

4. The method of claim 1 , wherein the unfavorable prognosis is recurrence or death from head and neck cancer.

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5. The method of claim 1 , wherein the unfavorable prognosis is recurrence.

6. The method of claim 1 , wherein the unfavorable prognosis is death from head and neck cancer.

7. The method of claim 1, wherein the step of assessing comprises comparing the level of expression of the panel in the cancer sample with the level of expression of the panel in a negative control sample.

8. The method of claim 1, wherein the step of assessing comprises steps of comparing the level of expression of the panel in the cancer sample with a level of expression of the panel in a positive control sample.

9. The method of claim 1 , wherein the at least one marker in the panel is a polypeptide marker and the step of determining comprises contacting the cancer sample with an interaction partner that binds the polypeptide marker.

10. The method of claim 9, wherein the interaction partner is an antibody.

11. The method of claim 1 , wherein the at least one marker in the panel is a polynucleotide marker and the step of determining comprises contacting the cancer sample with one or more primers that hybridize with the polynucleotide marker.

12. The method of claim 1 further comprising: deciding whether to administer a treatment based upon the patient's likely prognosis.

13. The method of claim 1 , wherein the panel comprises FABP5.

14. The method of claim 1 , wherein the panel comprises NDRGl .

15. The method of claim 1 , wherein the panel comprises CDKN2A.

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16. The method of claim 1 , wherein the panel comprises CEAC AM5.

17. The method of claim 1 , wherein the panel comprises NCSTN.

18. The method of claim 1 , wherein the panel comprises XPRl .

19. The method of claim 1 , wherein the panel comprises ABCG2.

20. The method of claim 1, wherein the panel comprises TRIM29.

21. The method of claim 1 , wherein the panel comprises SLC7A5.

22. The method of claim 1 , wherein the panel comprises SLC7A11.

23. The method of claim 1 , wherein the panel comprises LOX.

24. The method of claim 1 , wherein the panel comprises CAIX.

25. The method of claim 1 , wherein the panel comprises F ABP5 and NDRG 1.

26. The method of claim 25, wherein the panel further comprises CDKN2A.

27. The method of claim 26, wherein the panel further comprises CEAC AM5.

28. The method of claim 26, wherein the panel further comprises NCSTN.

29. The method of claim 25, wherein the panel further comprises XPRl and NCSTN.

30. The method of claim 25, wherein the panel further comprises ABCG2 and CEACAM5.

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31. The method of claim 25, wherein the panel further comprises CSTA and wherein across a population of patients with head and neck cancer, a higher level of expression of CSTA is indicative of a lower likelihood of an unfavorable prognosis.

32. The method of claim 31 , wherein the panel further comprises TRIM29.

33. The method of claim 31 , wherein the panel further comprises ABCG2.

34. The method of claim 33, wherein the panel further comprises TRIM29.

35. The method of claim 31 , wherein the panel further comprises CDKN2A.

36. The method of claim 35, wherein the panel further comprises TRIM29.

37. The method of claim 1, wherein the panel comprises at least two markers selected from the group consisting of FABP5, NDRGl, CDKN2A, CEACAM5, NCSTN, XPRl, ABCG2, CSTA, TRIM29, SLC7A5 and TLE3 and wherein across a population of patients with head and neck cancer, a higher level of expression of TLE3 is indicative of a higher likelihood of an unfavorable prognosis while a higher level of expression of CSTA is indicative of a lower likelihood of an unfavorable prognosis.

38. The method of claim 1, wherein the panel comprises at least three markers selected from the group consisting of FABP5, NDRGl, CDKN2A, CEACAM5, NCSTN, XPRl, ABCG2, CSTA, TRIM29, SLC7A5 and TLE3 and wherein across a population of patients with head and neck cancer, a higher level of expression of TLE3 is indicative of a higher likelihood of an unfavorable prognosis while a higher level of expression of CSTA is indicative of a lower likelihood of an unfavorable prognosis.

39. The method of claim 1, wherein the panel comprises at least four markers selected from the group consisting of FABP5, NDRGl, CDKN2A, CEACAM5, NCSTN, XPRl, ABCG2, CSTA, TRIM29, SLC7A5 and TLE3 and wherein across a population of patients with head and

Attorney Docket No.: 2003923-0022 Customer No.: 24280

4313517vl neck cancer, a higher level of expression of TLE3 is indicative of a higher likelihood of an unfavorable prognosis while a higher level of expression of CSTA is indicative of a lower likelihood of an unfavorable prognosis.

40. The method of claim 1, wherein the panel comprises at least five markers selected from the group consisting of FABP5, NDRGl, CDKN2A, CEACAM5, NCSTN, XPRl, ABCG2, CSTA, TRIM29, SLC7A5 and TLE3 and wherein across a population of patients with head and neck cancer, a higher level of expression of TLE3 is indicative of a higher likelihood of an unfavorable prognosis while a higher level of expression of CSTA is indicative of a lower likelihood of an unfavorable prognosis.

41. The method of claim 40, wherein the panel comprises CEAC AM5, NCSTN, ABCG2, TRIM29 and TLE3.

42. The method of claim 40, wherein the panel comprises CEACAM5, NCSTN, ABCG2, TRIM29 and SLC7A5.

43. A method of classifying a patient having head and neck cancer, the method comprising steps of: determining a level of expression of a panel of one or more markers in a cancer sample from a patient with head and neck cancer, wherein the panel comprises at least one marker selected from the group consisting of CYP4Z1, MMP7, TRIM29, IRX3, S100A8, SLPI, CDH3, FABP4, XPRl, NCSTN, CDHF7, FABP5, LTB, ABCG2, TERF2IP, SLC7A11, ITGB4, SLC7A5, NDRGl, CASP7, MMPl, CEACAM5, TP53, LOX, CAIX, EFNAl and CDKN2A; and classifying the patient into a class or subclass based on the determined level of expression of the panel.

44. The method of claim 43 further comprising stratifying the patient for a clinical trial based on the class or subclass.

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45. The method of claim 43 further comprising selecting a treatment for the patient based on the class or subclass.

46. The method of claim 43 further comprising predicting the patient's likely prognosis based on the class or subclass.

47. A kit comprising interaction partners or primers for FABP5 and NDRGl .

48. The kit of claim 47 further comprising an interaction partner or primer for CDKN2A.

49. The kit of claim 48 further comprising an interaction partner or primer for CEACAM5.

50. The kit of claim 48 further comprising an interaction partner or primer for NCSTN.

51. The kit of claim 47 further comprising interaction partners or primers for XPRl and

NCSTN.

52. The kit of claim 47 further comprising interaction partners or primers for ABCG2 and CEAC AM5.

53. The kit of claim 47 further comprising an interaction partner or a primer for CSTA.

54. The kit of claim 53 further comprising an interaction partner or primer for TRIM29.

55. The kit of claim 53 further comprising an interaction partner or primer for ABCG2.

56. The kit of claim 55 further comprising an interaction partner or primer for TRIM29.

57. The kit of claim 53 further comprising an interaction partner or primer for CDKN2A.

58. The kit of claim 57 further comprising an interaction partner or primer for TRIM29.

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59. The kit of claim 47 further comprising a positive and/or negative control sample.

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