WO2009117004A1 - Réactifs et procédés d'utilisation dans le diagnostic d'un cancer de la tête et du cou, classification et thérapie - Google Patents

Réactifs et procédés d'utilisation dans le diagnostic d'un cancer de la tête et du cou, classification et thérapie Download PDF

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Publication number
WO2009117004A1
WO2009117004A1 PCT/US2008/057840 US2008057840W WO2009117004A1 WO 2009117004 A1 WO2009117004 A1 WO 2009117004A1 US 2008057840 W US2008057840 W US 2008057840W WO 2009117004 A1 WO2009117004 A1 WO 2009117004A1
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panel
expression
trim29
marker
abcg2
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PCT/US2008/057840
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English (en)
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Brian Z. Ring
Douglas T. Ross
Robert S. Seitz
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Applied Genomics Inc.
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Priority to PCT/US2008/057840 priority Critical patent/WO2009117004A1/fr
Publication of WO2009117004A1 publication Critical patent/WO2009117004A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/118—Prognosis of disease development
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/56—Staging of a disease; Further complications associated with the disease

Definitions

  • 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.
  • tumors are generally classified under the tumor-node-metastasis (TNM) system.
  • TAM tumor-node-metastasis
  • 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.
  • morphologic appearance is used to further classify tumors into tumor types and thereby aid in selection of appropriate therapy.
  • 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.
  • 4313517vl will also be useful for directing patients into appropriate treatment protocols.
  • 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).
  • prognosis e.g., likelihood of death from head and neck cancer and/or likelihood of recurrence.
  • the present invention provides methods of using these markers and panels of markers to classify patients with head and neck cancer.
  • the present invention provides methods of using these markers and panels of markers to predict the prognosis of patients with head and neck cancer.
  • marker expression can be detected using any known method.
  • 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).
  • 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.
  • Appendix A 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.
  • 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
  • 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.
  • 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).
  • 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).
  • 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).
  • 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).
  • 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
  • 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.
  • 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.
  • cancer 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.
  • a cancer sample may be a tumor sample.
  • 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.
  • correlations may have p-values that are less than 0.01, especially less than 0.001.
  • 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.
  • Interaction partner An "interaction partner" is an entity that binds a polypeptide marker.
  • an interaction partner may be an antibody or a
  • 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.
  • 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.
  • 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.
  • 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.
  • Primer - A "primer” is an polynucleotide entity that physically hybridizes with a polynucleotide marker.
  • 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).
  • 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
  • the present invention provides techniques and reagents for the classification and subclassification, of patients with head and neck cancer.
  • classification or subclassification
  • Such classification has many beneficial applications.
  • a particular class or subclass of patients may correlate with prognosis and/or susceptibility to a particular therapeutic regimen.
  • 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.
  • 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.
  • expression of markers described herein can be detected using any known method.
  • 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
  • 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.
  • expression of one of the markers described herein (e.g., in Appendix B) in a cancer sample may be used to classify a head and neck cancer patient.
  • a patient may be classified as FABP5+ or FABP5- depending on whether expression of the FABP5 marker is detected in the cancer sample.
  • 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).
  • 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.
  • the present invention provides specific markers and methods for assessing the likely prognosis of a patient having head and neck cancer.
  • 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.
  • the patient's likely prognosis is then assessed based on the determined level of expression for the at least one marker in the panel.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the level of expression of the panel in the cancer sample 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.
  • 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.
  • 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.
  • a panel that includes just one of the following markers: FABP5, NDRGl, CDKN2A, CEACAM5, NCSTN, XPRl, ABCG2 or TRIM29.
  • 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.
  • the prognostic power of these multimarker panels can be more significant than when certain markers are used alone.
  • 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.
  • a panel that includes just one of the following markers: FABP5, NDRGl , CDKN2A, NCSTN, ABCG2, SLC7A11, SLC7A5, LOX, CAIX or TRIM29.
  • 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.
  • an inventive panel may comprise FABP5 and/or NDRGl .
  • CDKN2A can be added to the panel optionally with CEACAM5 and/or NCSTN.
  • FABP5 and NDRGl can be combined with XPRl and NCSTN.
  • FABP5 and NDRGl can be combined with ABCG2 and CEAC AM5.
  • FABP5 and NDRGl can be combined with CSTA and optionally with TRIM29 and/or ABCG2.
  • FABP5 and NDRGl can be combined with CSTA and optionally with TRIM29 and/or CDKN2A.
  • TRIM29 and/or CDKN2A can be combined with CSTA and optionally with TRIM29 and/or CDKN2A.
  • 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.
  • a panel may include FABP5 in combination with NDRGl, CDKN2A, CEAC AM5, NCSTN, XPRl, ABCG2, CSTA, TRIM29, SLC7A5 or TLE3.
  • a panel may include NDRGl in combination with CDKN2A, CEACAM5, NCSTN, XPRl, ABCG2, CSTA, TRIM29, SLC7A5 or TLE3.
  • a panel may include CDKN2A in combination with CEACAM5, NCSTN, XPRl, ABCG2, CSTA, TRIM29, SLC7A5 or TLE3, etc.
  • 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.
  • 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.
  • 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.
  • 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.
  • NDRGl and CDKN2A may be combined with at least one of CEACAM5, NCSTN, XPRl, ABCG2, CSTA, TRIM29, SLC7A5 and TLE3, etc.
  • 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.
  • 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.
  • NDRGl, CDKN2A and CEAC AM5 may be combined with at least one of NCSTN, XPRl, ABCG2, CSTA, TRIM29, SLC7A5 and TLE3, etc.
  • 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.
  • 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.
  • NDRGl, CDKN2A, CEACAM5 and NCSTN may be combined with at least one of XPRl, ABCG2, CSTA, TRIM29, SLC7A5 and TLE3, etc.
  • a panel may comprise at least CEAC AM5, NCSTN, ABCG2, TRIM29 and TLE3.
  • a panel may comprise at least CEAC AM5, NCSTN, ABCG2, TRIM 29 and SLC7A5.
  • expression of any of the markers described herein can be determined using any known method.
  • expression may be determined by detecting polypeptide markers using interaction partners (e.g., antibodies).
  • expression may be determined by detecting polynucleotide markers using primers.
  • 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).
  • 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.
  • 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.
  • 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,
  • antibodies 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.
  • 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).
  • a marker or an antigenic portion thereof
  • 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).
  • KLH keyhole limpet hemocyanin
  • 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.
  • 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.
  • 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.
  • a nonionic detergent 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
  • Hybridomas having high reactivity and specificity are typically selected.
  • Monoclonal antibodies may be isolated from the supernatants of growing hybridoma colonies.
  • 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.
  • the present invention is not limited to using antibodies or antibody fragments as interaction partners.
  • the present invention also encompasses the use of synthetic interaction partners that mimic the functions of antibodies.
  • 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).
  • 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.
  • association can be detected by adding a detectable label to the interaction partner.
  • 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.
  • 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.
  • detection means include, e.g., scintillation counting, autoradiography, measurement of paramagnetism, fluorescence measurement, light absorption measurement, measurement of light scattering and the like.
  • association between an interaction partner and its marker may be assayed
  • IHC immunohistochemistry
  • ELISA ELISA
  • FACS fluorescence activates cell sorting
  • 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.
  • a commercially-available mechanical device e.g., the manual tissue arrayer MTAl from Beecher Instruments of Sun Prairie, WI
  • cores from as many as about 400 patients 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.
  • identification of a discriminating titer can simplify binding studies to assess the desirability of using an interaction partner.
  • 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.).
  • tissue of origin e.g., tissue of origin
  • multiple common traits e.g., tissue of origin, stage, microscopic characteristics, etc.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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
  • 4313517vl inventive methods In general, these methods rely on hybridization between one or more primers and the polynucleotide marker.
  • 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.).
  • hybridization can be detected by using a primer with a detectable label.
  • 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).
  • 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.
  • directly detectable labels include radioactive, paramagnetic, fluorescent, light scattering, absorptive and colorimetric labels.
  • 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.
  • 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.
  • detection means include, e.g., scintillation counting, autoradiography, measurement of paramagnetism, fluorescence measurement, light absorption measurement, measurement of light scattering and the like.
  • hybridization between a primer and the marker may be assayed by
  • 4313517vl contacting the primer with a cancer sample that includes the marker.
  • 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.
  • 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.
  • kits for use in accordance with the present invention may include, one or more reference samples
  • the kit can comprise a panel of antibodies.
  • 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.
  • 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.
  • 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.
  • interaction partners or primers e.g., antisense or RNAi primers themselves may prove to be useful therapeutics.
  • the present invention provides interaction partners and primers that are
  • interaction partners defined or prepared according to the present invention could be used to deliver a therapeutic agent to a cancer cell.
  • interaction partners e.g., an antibody raised against a marker from Appendix B
  • Suitable agents in this regard include radionuclides and drugs. Exemplary radionuclides include 90 Y, 123 I, 125 I, 131 I, 186 Re, 188 Re, 211 At and 212 Bi.
  • 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.
  • 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.
  • a nucleophilic group such as an amino or sulfhydryl group
  • a carbonyl- containing group such as an anhydride or an acid halide
  • an alkyl group containing a good leaving group e.g., a halide
  • 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.
  • 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
  • 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.
  • 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.
  • biotin/avidin pair include without limitation, biotin/streptavidin, digoxigenin/anti-digoxigenin, FK506/FK506-binding protein (FKBP), rapamycin/FKBP, cyclophilin/cyclosporin and glutathione/glutathione transferase pairs.
  • 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.
  • GST glutathione-S-transferase
  • MBP maltose binding protein
  • 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.
  • multiple molecules of an agent are coupled to one interaction partner molecule.
  • more than one type of therapeutic agents are coupled to one interaction partner molecule.
  • 4313517vl agent may be coupled to one interaction partner molecule.
  • 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.
  • 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.
  • Carriers specific for radionuclide agents include radiohalogenated small molecules and chelating compounds.
  • Carriers specific for radionuclide agents include radiohalogenated small molecules and chelating compounds.
  • 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.
  • U.S. Pat. No. 4,673,562 to Davison et al. discloses representative chelating compounds and their synthesis.
  • the therapeutic agents are antibodies, e.g., an antibody against a marker from Appendix B.
  • 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.
  • "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.
  • 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,
  • CDRs complementarity-determining regions
  • 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).
  • 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.
  • 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.
  • interaction partners suitable for use as therapeutics exhibit high specificity for the target marker and low background binding to other markers.
  • monoclonal antibodies may be used for therapeutic purposes.
  • 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.
  • the present invention provides new therapies
  • an interaction partner or primer may be a useful therapeutic agent.
  • interaction partners or primers defined or prepared according to the present invention bind to markers that serve as targets for therapeutic agents.
  • inventive interaction partners or primers may be used to deliver a therapeutic agent to a cancer cell.
  • interaction partners or primers provided in accordance with the present invention may be coupled to one or more therapeutic agents.
  • the invention includes pharmaceutical compositions comprising these inventive therapeutic agents.
  • 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.
  • compositions may be administered either alone or in combination with other therapeutic agents including other chemotherapeutic agents, hormones, vaccines and/or radiation therapy.
  • therapeutic agents including other chemotherapeutic agents, hormones, vaccines and/or radiation therapy.
  • 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.
  • each agent will be administered at a dose and on a time schedule determined for that agent.
  • 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.
  • 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.
  • 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.
  • 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.
  • the intravenous route is most commonly used to deliver therapeutic antibodies.
  • 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.
  • 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.).
  • commercial sources e.g., Chemicon, Dako, Oncogene Research Products, NeoMarkers, etc.
  • other publicly available sources e.g., Imperial Cancer Research Technology, etc.
  • Glacial Acetic Acid Cat No. BPl 185-500, Fisher
  • Sepharose 4B (Cat. No. 17-0120-01, LKB/Pharmacia, Uppsala, Sweden)
  • DMF Dimethyl formamide
  • Ethylenediaminetetraacetatic acid (Cat. No. BP 120-1, Fisher, Springfield, NJ)
  • HCL l-ethyl-3-(3'dimethylaminopropyl)-carbodiimide, HCL (EDC) (Cat. no. 341-006, Calbiochem, San Diego, CA)
  • Fritted chromatography columns Cold part No. 12131011; Frit Part No. 12131029, Varian Sample Preparation Products, Harbor City, CA
  • HRP Horseradish peroxidase
  • NMP (Cat. No. CAS 872-50-4, Burdick and Jackson, Muskegon, MI)
  • HOBt is dissolved in NMP (8.8 grams HOBt to 1 liter NMP). Fmoc-N-a- amino at a concentration at 0.53 M.
  • Reagent R 2 parts anisole, 3 parts ethanedithiol, 5 parts thioanisole and 90 parts trifluoroacetic acid.
  • Vacuum dryer Box from Labconco, Kansas City, MO and Pump from Alcatel, Laurel, MD).
  • 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.
  • 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).
  • a hidden Markov model described in Rrogh et al., J. MoI. Biol. 305:567, 2001.
  • 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.
  • 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.
  • Steps 2a and 2b were performed one last time.
  • 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.
  • 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 diH 2 0, frozen and lyophilized overnight.
  • Peptide (6 mg) was conjugated with Keyhole Limpet Hemocyanin (KLH).
  • KLH Keyhole Limpet Hemocyanin
  • 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
  • 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.
  • 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
  • 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.
  • 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 -2O 0 C for affinity purification.
  • 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 4 0 C.
  • the plates were emptied and washed three times as described.
  • Biotinylated goat anti-rabbit IgG 100 ⁇ l 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 ⁇ l 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 ⁇ l of
  • 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 ⁇ l of DMF was added to peptide (5 mg) and the mixture was vortexed until the contents were completely wetted. Water was then added (900 ⁇ l) and the contents were vortexed until the peptide dissolved.
  • 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.
  • 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.
  • Excess buffer (0.5 M BBS) in the collection tubes served to neutralize the antibodies collected in the acidic fractions of the pH gradient.
  • additional steps may be used to purify antibodies of the invention.
  • it may prove advantageous to repurify antibodies, e.g., against one of the peptides that was used in generating the antibodies.
  • the present invention encompasses antibodies that have been prepared with such additional purification or repurification steps.
  • the purification process may affect the binding between samples and the inventive antibodies.
  • 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.
  • 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.
  • donor block paraffin block
  • 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 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.
  • Tissue microarrays from a head and neck cancer 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).
  • 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
  • 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.
  • 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.
  • LOX NCBI Locus Link ID 4015, lysyl oxidase also called MGC 105112
  • CAIX annotated for membrane vs.
  • 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
  • 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).
  • 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.
  • 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.
  • prognostic panels could be constructed using any method. Without limitation these include simple empirically derived rules, Cox multivariate proportional hazard models
  • 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.
  • 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.
  • 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).
  • 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).
  • cut-offs and the overall panel prediction function are exemplary and that other cut-offs and/or functions could be used with these panels.
  • lines between "good”, “moderate” and “bad” prognosis (or between "good” and “bad” prognosis) are not absolute.
  • the terms for each antibody in a panel may be adjusted to yield variations on the panel of Table 1 (or other panels described herein).
  • 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
  • Figure IB shows the recurrence curves that were obtained for patients in the "Good” and “Bad” recurrence groups.
  • Tables 2 to 9 summarize other exemplary prognostic panels that were identified by the inventors.
  • 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
  • Figure 2 shows the curves that were obtained using the panel of Table 2 (Good ⁇ 0.0 ⁇ Moderate ⁇ 0.71 ⁇ Bad).
  • Figure 2 shows the curves that were obtained using the panel of Table 3 (Good ⁇ -0.8 ⁇ Moderate ⁇ 0.18 ⁇ Bad).
  • Figure 3 shows the curves that were obtained using the panel of Table 4 (Good ⁇ -0.6 ⁇ Moderate ⁇ 0. 1 ⁇ Bad).
  • Figure 3 shows the curves that were obtained using the panel of Table 5 (Good ⁇ 0.0 ⁇ Moderate ⁇ 0.3 ⁇ Bad).
  • Figure 4 shows the curves that were obtained using the panel of Table 6 (Good ⁇ 0.0 ⁇ Moderate ⁇ 0.33 ⁇ Bad).
  • Figure 4 shows the curves that were obtained using the panel of Table 7 (Good ⁇ 0.0 ⁇ Moderate ⁇ 0.58 ⁇ Bad).
  • Figure 5 shows the curves that were obtained using the panel of Table 8 (Good ⁇ 0.0 ⁇ Moderate ⁇ 0.67 ⁇ Bad).
  • Figure 5 shows the curves that were obtained using the panel of Table 9 (Good ⁇ 0.0 ⁇ Moderate ⁇ 0.79 ⁇ Bad).
  • 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:
  • the panel of Table 11 can be used with the

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Abstract

L’invention concerne des procédés et des réactifs permettant de classifier les cancers de la tête et du cou et d’identifier les nouvelles classes et sous-classes des cancers de la tête et du cou. L’invention concerne des procédés permettant de mettre en corrélation des classes et sous-classes avec un régime ou un résultat thérapeutique, permettant d'identifier les thérapies (nouvelles ou connues) appropriées pour des classes ou des sous-classes particulières, et de prédire les résultats en fonction de la classe ou de la sous-classe.
PCT/US2008/057840 2008-03-21 2008-03-21 Réactifs et procédés d'utilisation dans le diagnostic d'un cancer de la tête et du cou, classification et thérapie WO2009117004A1 (fr)

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Cited By (2)

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WO2013067198A1 (fr) * 2011-11-01 2013-05-10 H. Lee Moffitt Cancer Center And Research Institute, Inc. Signature génique pour prédiction de l'activité nf-kappab
CN113797219A (zh) * 2021-09-13 2021-12-17 温州医科大学附属第一医院 Xpr1抑制剂在制备抑制甲状腺癌细胞迁移和/或增殖的产品的应用

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013067198A1 (fr) * 2011-11-01 2013-05-10 H. Lee Moffitt Cancer Center And Research Institute, Inc. Signature génique pour prédiction de l'activité nf-kappab
US9115388B2 (en) 2011-11-01 2015-08-25 H. Lee Moffitt Cancer Center And Research Institute, Inc. Gene signature for the prediction of NF-kappaB activity
CN113797219A (zh) * 2021-09-13 2021-12-17 温州医科大学附属第一医院 Xpr1抑制剂在制备抑制甲状腺癌细胞迁移和/或增殖的产品的应用
CN113797219B (zh) * 2021-09-13 2023-03-31 温州医科大学附属第一医院 Xpr1抑制剂在制备抑制甲状腺癌细胞迁移和/或增殖的产品的应用

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