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Predictors of patient response to treatment with egf receptor inhibitors

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Predictors of patient response to treatment with egf receptor inhibitors


The present invention provides methods and compositions to facilitate determining whether an EGFR-expressing cancer in an individual is an EGFR inhibitor-responsive cancer, as well as methods for determining the likelihood that a patient having an EGFR-expressing cancer will exhibit a beneficial response to an EGFR inhibitor therapy. The methods generally involve determining a normalized expression level of a gene product that correlates with EGFR inhibitor responsiveness.
Related Terms: Gene Product

Browse recent Genomic Health, Inc. patents - Redwood City, CA, US
Inventors: Joffre B. Baker, Drew Watson, Tara Maddala, Steven Shak, David J. Mauro, Shirin K. Ford
USPTO Applicaton #: #20120270228 - Class: 435 612 (USPTO) - 10/25/12 - Class 435 


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The Patent Description & Claims data below is from USPTO Patent Application 20120270228, Predictors of patient response to treatment with egf receptor inhibitors.

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CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority benefit of U.S. Provisional Application Ser. No. 61/127,816 filed on May 14, 2008, the entire disclosure of which is incorporated herein by reference in its entirety.

TECHNICAL FIELD

The present disclosure provides genes and gene sets, the expression levels of which are useful for predicting response of cancer patients to an epidermal growth factor receptor (EGFR) inhibitor therapy.

INTRODUCTION

Epidermal growth factor receptor (EGFR), also known as ERBB and HER1, is a gene that encodes a receptor protein found on the surface of some cells and to which an epidermal growth factor binds, causing the cell to divide. EGFR is a member of the HER family of receptors, and the dimerization of EGFR and v-erb-b2 erythroblastic leukemia viral oncogene homolog 2 (ERBB2), also known as HER2, is an important stimulus for breast cancer growth in ERBB2-positive breast cancers. Expression levels of EGFR are amplified in a subset of breast cancers and the resulting overexpression of the receptor contributes to breast cancer etiology.

Because cancer is characterized by rapidly proliferating cells, most cancer drugs attack rapidly dividing cells indiscriminately and, as a result, often exhibited a high degree of toxicity. In most instances, little is known regarding the mechanisms of action of these cytotoxic drugs. Targeted EGFR inhibitors, e.g., have demonstrated activity against a number of cancer types. Anti-EGFR monoclonal antibodies have shown antitumor activity in advanced colorectal cancer, in squamous cell carcinomas of the head and neck, non-small-cell lung cancer (NSCLC) and renal cell carcinomas (Baselga J and Arteaga C L (2005) J Clin Oncol 2445-2459. Clinical trials are ongoing in early stage cancer and in other tumor types and are expected to show activity in cancer types where EGFR is expressed and where EGFR ligands promote tumor growth and progression.

Given the importance in selection of therapy, there has been a push to develop drugs in concert with companion diagnostic tests capable of identifying responsive patients. Such diagnostics would address the situations where some patients who might benefit from treatment do not receive the drug, and other patients who are unlikely to benefit are unnecessarily exposed to toxic side effects, incur unnecessary expense, and/or experience a delay in being treated with alternative drugs that might prove more effective.

SUMMARY

The present disclosure provides methods and compositions to facilitate determining whether an EGFR-expressing cancer in an individual is an EGFR inhibitor-responsive cancer, as well as methods for determining the likelihood that a patient having an EGFR-expressing cancer will exhibit a beneficial response to an EGFR inhibitor therapy. The methods generally involve determining a normalized expression level of a gene product that correlates with EGFR inhibitor responsiveness.

The disclosure provides methods for predicting the likelihood that a human patient with an EGFR -expressing cancer will exhibit a beneficial response to an EGFR inhibitor cancer therapy based on expression levels of one or more response indicator genes in a biological sample obtained from a tumor in the patient. Specifically, the method entails measuring an expression level of at least one response indicator gene, or its expression product. The response indicator gene is one or more selected from a group consisting of ATP5E, TITF1, CLTC, BRCA1, AREG, PTP4A3, EREG, VAV3, SATB2, CEACAM6, EGFR, CHN2, FGFR3, C13orf18, QPRT, AMACR1, CKMT2, ID1, SORBS1, SLC26A3, ErbB3, DUSP6, VDAC2, ANXA2P2, SERPINB1, NT5E, GPC3, DUSP4, PHLDA1, K-ras, DR5, VIL2, LAMC2, SFN, ANXA1, EPHA2, P14ARF, CA9, KRT17, p14ARF, Maspin, PLAUR, LAMAS, and GCNT3. The expression level is normalized, and the normalized expression level is used to determine or predict likelihood of beneficial response, wherein increased normalized expression levels of one or more of the following are measured: ATP5E, TITF1, CLTC, BRCA1, AREG, PTP4A3, EREG, VAV3, SATB2, CEACAM6, EGFR, CHN2, FGFR3, C13orf18, QPRT, AMACR1, CKMT2, ID1, SORBS1, SLC26A3, and ErbB3 are positively correlated with the likelihood that the patient will exhibit a beneficial response to the EGFR inhibitor cancer therapy; and increased normalized expression levels of DUSP6, VDAC2, ANXA2P2, SERPINB1, NT5E, GPC3, DUSP4, PHLDA1, K-ras, DR5, VIL2, LAMC2, SFN, ANXA1, EPHA2, P14ARF, CA9, KRT17, p14ARF, Maspin, PLAUR, LAMA3, and GCNT3 are negatively correlated with the likelihood that the patient will exhibit a beneficial response to the EGFR inhibitor cancer therapy. A report is generated based on the determined likelihood of response.

The disclosure provides methods for predicting a likelihood that a human patient with a KRAS-negative, EGFR-expressing cancer will exhibit a beneficial response to an EGFR inhibitor cancer therapy based on expression levels of one or more response indicator genes in a biological sample obtained from a tumor in the patient. Specifically, the method entails measuring an expression level of at least one response indicator gene, or its expression product, selected from a group consisting of EGF, ADAM17, PTP4A3, ADAM15, QPRT, SATB2, RASSF1, VAV3, CEACAM6, EREG, AREG, TITF1, SORBS1, C13orf18, CKMT2, BTC, ATP5E, B.Catenin, CCNE1, EGFR, Bclx, BRCA1, CDC25B, CHN2, ID1, SLC26A3, VDAC2, SERPINB1, PHLDA1, ANXA2P2, KRT17, EPHA2, DUSP4, CGA, CA9, Maspin, NEDD8, DUSP6, GPC3, NT5E, VIL2, and P14ARF. The expression level is normalized, and the normalized expression level is used to determine or predict likelihood of beneficial response, wherein increased normalized expression levels of one or more of the following are measured: EGF, ADAM17, PTP4A3, ADAM15, QPRT, SATB2, RASSF1, VAV3, CEACAM6, EREG, AREG, TITF1, SORBS1, C13orf18, CKMT2, BTC, ATP5E, B.Catenin, CCNE1, EGFR, Bclx, BRCA1, CDC25B, CHN2, ID1, and SLC26A3 are positively correlated with the likelihood that the patient will exhibit a beneficial response to the EGFR inhibitor cancer therapy; and increased normalized expression levels of VDAC2, SERPINB1, PHLDA1, ANXA2P2, KRT17, EPHA2, DUSP4, CGA, CA9, Maspin, NEDD8, DUSP6, GPC3, NT5E, VIL2, and P14ARF are negatively correlated with the likelihood that the patient will exhibit a beneficial response to the EGFR inhibitor cancer therapy.

The methods of the present disclosure contemplate using a normalized expression level to determine or predict likelihood of beneficial response, based on normalized expression level(s) for single response indicator genes and/or multi-gene sets. Exemplary multi-gene sets are disclosed. The expression values are normalized relative to an expression level of one or more reference genes. The disclosure provides for measurement of normalized expression level(s) of at least one response indicator gene product. For all aspects of the present disclosure, the methods may further include determining the expression levels of at least two of said genes, or their expression products. It is further contemplated that the methods of the present disclosure may further include determining the expression levels of at least three of said genes, or their expression products. It is contemplated that the methods of the present disclosure may further include determining the expression levels of at least four of said genes, or their expression products. It is contemplated that the methods of the present disclosure may further include determining the expression levels of at least five of said genes, or their expression products. It is contemplated that the methods of the present disclosure may further include determining the expression levels of at least six of said genes, or their expression products. It is contemplated that the methods of the present disclosure may further include determining the expression levels of at least seven of said genes, or their expression products. It is contemplated that the methods of the present disclosure may further include determining the expression levels of at least eight of said genes, or their expression products. It is contemplated that the methods of the present disclosure may further include determining the expression levels of at least nine of said genes, or their expression products. The methods may involve determination of the expression levels of at least ten (10) or at least fifteen (15) of the genes listed above or their products.

A normalized expression level(s), generated as discussed above, is used to determine or predict likelihood of beneficial response, The normalized expression level(s) is indicative of the likelihood that the patient will exhibit a beneficial response to an EGFR inhibitor therapy, such as an EGFR-specific antibody or small molecule. A likelihood score (e.g., a score predicting likelihood of beneficial response to EGFR inhibitor treatment) can be calculated based on the normalized expression level(s). A score may be calculated using weighted values based on a normalized expression level of a response indicator gene and its contribution to response to EGFR inhibitor cancer therapy.

In addition, the disclosure provides arrays for carrying out the methods disclose herein, or for analyzing whether a mathematical combination of the normalized expression levels of any combination of the response indicator genes is more indicative of a likelihood that a patient will respond to treatment with an EGFR inhibitor. The arrays may include, for example, probes that hybridize to a nucleic acid sequence in a response indicator genes or an activating KRAS mutation.

Determining the expression level of one or more genes may be accomplished by, for example, a method of gene expression profiling. The method of gene expression profiling may be, for example, a PCR-based method. The expression level of said genes can be determined, for example, by RT-PCR (reverse transcriptase PCR), quantitative RT-PCR (qRT-PCR), or other PCR-based methods, immunohistochemistry, proteomics techniques, an array-based method, or any other methods known in the art or their combination. In one aspect the RNA transcripts are fragmented.

Detection of an RNA transcript of a response indicator gene may be accomplished by assaying for an exon-based sequence or an intron-based sequence, the expression of which correlates with the expression of a corresponding exon sequence.

In an exemplary embodiment, the assay for the measurement of response indicator genes, or their gene products, and/or activating KRAS mutations is provided in the form of a kit or kits.

The expression levels of the genes may be normalized relative to the expression levels of one or more reference genes, or their expression products.

The tumor sample may be, for example, a tissue sample containing cancer cells, or portion(s) of cancer cells, where the tissue can be fixed, paraffin-embedded or fresh or frozen tissue. For example, the tissue may be from a biopsy (fine needle, core or other types of biopsy) or obtained by fine needle aspiration, or by obtaining body fluid containing a cancer cell, e.g. urine, blood, etc.

For all aspects of the methods of the present disclosure, it is contemplated that for every increment of an increase in the level of one or more genes or their expression products, the patient is identified to show an incremental increase in clinical outcome.

The determination of expression levels may occur more than one time in the practice of the methods disclosed herein.

The methods may further include the step of creating a report based on the determined or predicted likelihood of beneficial response. In another aspect the present disclosure provides reports for a patient containing a summary of the expression levels of the one or more response indicator genes, or their expression products, in a tumor sample obtained from said patient. In one aspect the report is in electronic form.

In one embodiment, the EGFR inhibitor is an antibody specific for EGFR. In another, the EGFR inhibitor is a small molecule, for example an EGFR-selective tyrosine kinase inhibitor.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the sequences of probes and primers used to assay each gene identified in the studies.

FIG. 2 shows the sequences of amplicons expected from the use of the probes and primers shown in FIG. 1.

FIGS. 3A-10D provide sets of graphs showing probability curves. Each graph contains three lines—a center dark line and two lighter lines above and below the dark centerline. Each graph has a center dark line representing the model-predicted relationship between normalized gene expression on the x-axis and likelihood of beneficial response to an EGFR inhibitor on the y-axis (predicted probability curve). The lighter grey lines above and below the black line represent the 95% Confidence Interval for the predicted probability, i.e. at a particular expression value for the gene, there is a 95% probability that the upper and lower grey lines include the that the actual probability of response. Confidence Intervals for the probability curves were calculated using the normal approximation method. The name of the gene analyzed is indicated below each graph by the text preceding the period; for example, “AREG.2” refers to AREG. Each of FIGS. 3A-10D are described in more detail below.

FIGS. 3A-3D show probability curves that correspond to the data reported in Table 1A for analysis of ORR in all patients (K-ras negative and K-ras positive); Odds Ratio>1.

FIGS. 4A-4F show probability curves that correspond to the data reported in Table 1B for analysis of ORR in all patients (K-ras negative and K-ras positive); Odds Ratio<1.

FIGS. 5A-5E show probability curves that correspond to the data reported in Table 2A for analysis of DC in all patients (K-ras negative and K-ras positive); Odds Ratio>1.

FIGS. 6A-6F shows probability curves that correspond to the data reported in Table 2B for analysis of DC in all patients (K-ras negative and K-ras positive); Odds Ratio<1.

FIGS. 7A-7C show probability curves that correspond to the data reported in Table 6A for analysis of ORR in K-ras negative patients; Odds Ratio>1.

FIGS. 8A-8C show probability curves that correspond to the data reported in Table 6B for analysis of ORR in K-ras negative patients; Odds Ratio<1.

FIGS. 9A-9E show probability curves that correspond to the data reported in Table 7A for analysis of DC in K-ras negative patients; Odds Ratio>1.

FIGS. 10A-10D show probability curves that correspond to the data reported in Table 7B for analysis of DC in K-ras negative patients; Odds Ratio<1.

Each of Tables 1A, 1B, 2A, 2B, 6A, 6B, 7A and 7B provides an Odds Ratio for each response indicator and gene listed in the table. The Odds Ratio reported for a gene is related to the overall slope of the probability curve shown in the corresponding Figure and is a measure of the change in the model-predicted probability of response for every unit change in normalized gene expression. As shown, genes with stronger odds ratios (further from 1) in the tables are depicted with steeper slopes in the figures.

DETAILED DESCRIPTION

Definitions

Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Singleton et al., Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley & Sons (New York, N.Y. 1994), and March, Advanced Organic Chemistry Reactions, Mechanisms and Structure 4th ed., John Wiley & Sons (New York, N.Y. 1992), provide one skilled in the art with a general guide to many of the terms used in the present application.

One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. Indeed, the present invention is in no way limited to the methods and materials described. For purposes of the present invention, the following terms are defined below.

The terms “k-ras” and “KRAS” as used herein (including in tables and figures) are used interchangeably and refer to the KRAS gene identified as of the date of this filing in the NCBI Entrez Gene database as Accession No. NM—004985.3 (Entrez Gene database, NCBI), and/or its expression products.

As used herein, the term “KRAS status”, refers to whether a patient's cancer is negative for an activating KRAS mutation (KRAS-negative) or positive for an activating KRAS mutation (KRAS-positive).

As used herein, the term “activating KRAS mutation” refers to a mutation in a k-ras gene that results in constitutive activation of a protein encoded by k-ras, i.e. the k-ras protein activates molecules downstream in its signaling pathway in the absence of receptor bound ligand. As an example, the k-ras protein might activate downstream signaling in the absence of EGF, amphiregulin, or epiregulin binding to EGFR.

As used herein, the term “EGFR-expressing cancer” refers to a cancer tumor with cells that express a cell surface epidermal growth factor receptor (EGFR) polypeptide.

As used herein, the term “epidermal growth factor receptor” (“EGFR”) refers to a gene that encodes a membrane polypeptide that binds, and is thereby activated by, epidermal growth factor (EGF). EGFR is also known in the literature as ERBB, ERBB 1 and HER1. An exemplary EGFR is the human epidermal growth factor receptor (see Ullrich et al. (1984) Nature 309:418-425; Genbank accession number NP—005219.2). Binding of an EGF ligand activates the EGFR (e.g. resulting in activation of intracellular mitogenic signaling, autophosphorylation of EGFR). One of skill in the art will appreciate that other ligands, in addition to EGF, can bind to and activate the EGFR. Examples of such ligands include, but are not limited to, amphiregulin, epiregulin, TGF-α, betacellulin, and heparin-binding EGF (HB-EGF) (Strawn and Shawver (1998) Exp. Opin. Invest. Drugs 7(4)553-573, and “The Protein Kinase Facts Book: Protein Tyrosine Kinases” (1995) Hardie, et al. (eds.), Academic Press, NY, N.Y.). See also, Oda et al. ((2005) Molec. Systems Biol. 1:2005.0010; and Moulder et al. ((2001) Cancer Res. 61:8887.

As used herein, an “EGFR gene” refers to a nucleic acid that encodes an EGFR gene product, e.g., an EGFR mRNA, an EGFR polypeptide, and the like.

As used herein, “EGFR inhibitor” refers to any agent capable of directly or indirectly inhibiting activation of an EGFR. EGFR inhibitors include agents that bind to an EGFR and inhibit its activation. EGFR inhibitors include antibodies that bind to an EGFR and inhibit activation of the EGFR; as well as small molecule tyrosine kinase inhibitors that inhibit activation of an EGFR. Antibodies to EGFR include IgG; IgM; IgA; antibody fragments that retain EGFR binding capability, e.g., Fv, Fab, F(ab)2, single-chain antibodies, and the like; chimeric antibodies; etc. Small molecule tyrosine kinase inhibitors of EGFR include EGFR-selective tyrosine kinase inhibitors. Small molecule tyrosine kinase inhibitors of EGFR can have a molecular weight in a range of from about 50 Da to about 10,000 Da.

The term “tumor,” as used herein, refers to any neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues.

The terms “cancer” and “cancerous” refer to or describe the physiological condition in mammals that is typically characterized in part by unregulated cell growth. Examples of cancer include, but are not limited to, colorectal cancer, breast cancer, ovarian cancer, lung cancer, prostate cancer, hepatocellular cancer, gastric cancer, pancreatic cancer, cervical cancer, liver cancer, bladder cancer, cancer of the urinary tract, thyroid cancer, renal cancer, carcinoma, melanoma, brain cancer, non-small cell lung cancer, squamous cell cancer of the head and neck, endometrial cancer, multiple myeloma, rectal cancer, and esophageal cancer. In an exemplary embodiment, the cancer is colorectal cancer.

As used herein, the term “pathology” of cancer includes all phenomena that compromise the well-being of the patient. This includes, without limitation, abnormal or uncontrollable cell growth, metastasis, interference with the normal functioning of neighboring cells, release of cytokines or other secretory products at abnormal levels, suppression or aggravation of inflammatory or immunological response, neoplasia, premalignancy, malignancy, invasion of surrounding or distant tissues or organs, such as lymph nodes, etc.

The terms “subject,” “individual,” and “patient” are used interchangeably herein to refer to a mammal being assessed for treatment and/or being treated. In an embodiment, the mammal is a human. The terms “subject,” “individual,” and “patient” thus encompass individuals having cancer (e.g., colorectal cancer or other cancer referenced herein), including those who have undergone or are candidates for resection (surgery) to remove cancerous tissue (e.g., cancerous colorectal tissue or other cancer referenced herein).

As used herein, the terms “treatment,” “treating,” and the like, refer to administering an agent, or carrying out a procedure (e.g., radiation, a surgical procedure, etc.), for the purposes of obtaining a effect. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of effecting a partial or complete cure for a disease and/or symptoms of the disease. “Treatment,” as used herein, covers any treatment of a disease in a mammal, particularly in a human, and includes: (a) preventing the disease or a symptom of a disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it (e.g., including diseases that may be associated with or caused by a primary disease; (b) inhibiting the disease, i.e., arresting its development; and (c) relieving the disease, i.e., causing regression of the disease.

As used herein in the context of patient response to an EGFR inhibitor treatment, the terms “beneficial response,” “beneficial patient response,” and “clinically beneficial response,” “clinical benefit,” and the like, are used interchangeably and refer to favorable patient response to a drug as opposed to unfavorable responses, i.e. adverse events. In individual patients, beneficial response can be expressed in terms of a number of clinical parameters, including loss of detectable tumor (complete response, CR), decrease in tumor size and/or cancer cell number (partial response, PR), tumor growth arrest (stable disease, SD), enhancement of anti-tumor immune response, possibly resulting in regression or rejection of the tumor; relief, to some extent, of one or more symptoms associated with the tumor; increase in the length of survival following treatment; and/or decreased mortality at a given point of time following treatment. Continued increase in tumor size and/or cancer cell number and/or tumor metastasis is indicative of lack of beneficial response to treatment.

In a population the clinical benefit of a drug, i.e. its efficacy can be evaluated on the basis of one or more endpoints. For example, analysis of overall response rate (ORR) classifies as responders those patients who experience CR or PR after treatment with drug. Analysis of disease control (DC) classifies as responders those patients who experience CR, PR or SD after treatment with drug.

As is used herein, the term “progression free survival” refers to the time interval from treatment of the patient until the progression of cancer or death of the patient, whichever occurs first.

As used herein, the term “responder” refers to a patient who has an EGFR-expressing cancer, and who exhibits a beneficial clinical response following treatment with an EGFR inhibitor.

As used herein, the term “non-responder” refers to a patient who has an EGFR-expressing cancer, and who has not shown a beneficial response following treatment with an EGFR inhibitor.

As used herein, the term “correlates,” or “correlates with,” and like terms, refers to a statistical association between instances of two events, where events include numbers, data sets, and the like. For example, when the events involve numbers, a positive correlation (also referred to herein as a “direct correlation”) means that as one increases, the other increases as well. A negative correlation (also referred to herein as an “inverse correlation”) means that as one increases, the other decreases. The correlation need not necessarily be a linear correlation and need not apply across the entire range of the variables.

The term “tumor sample” as used herein means a sample comprising tumor material obtained from a cancerous patient. The term encompasses clinical samples, for example tissue obtained by surgical resection and tissue obtained by biopsy, such as for example a core biopsy or a fine needle biopsy. The term also encompasses samples comprising tumor cells obtained from sites other than the primary tumor, e.g., circulating tumor cells. The term encompasses cells that are the progeny of the patient\'s tumor cells, e.g. cell culture samples derived from primary tumor cells or circulating tumor cells. The term encompasses samples that may comprise protein or nucleic acid material shed from tumor cells in vivo, e.g. bone marrow, blood, plasma, serum, and the like. The term also encompasses samples that have been enriched for tumor cells or otherwise manipulated after their procurement and samples comprising polynucleotides and/or polypeptides that are obtained from a patient\'s tumor material.

The terms “gene product” and “expression product” are used interchangeably herein in reference to a molecule produced using a gene\'s information. For example, a gene product or expression product would include RNA transcription products (transcripts) of the gene, including mRNA and the polypeptide translation products of such RNA transcripts, whether such product is modified post-translationally or not (e.g., unspliced RNA, a splice variant mRNA, RNA fragment, etc.). In addition, a gene product or expression product includes the polypeptide translation products of such RNA, whether such product is modified post-translationally or not (e.g., a splice variant polypeptide, etc.)

As used herein, the term “normalized expression level” refers to an expression level of a response indicator gene relative to the level of an expression product of a reference gene(s).

As used herein, the term “response indicator gene” refers to a gene, the expression of which correlates positively or negatively with beneficial patient response to EGFR inhibitor treatment. The expression of a response indicator gene may be measured by determining the expression level of an expression product of the response indicator gene.

The term “increased expression” or “increased normalized expression” with regard to a gene or an RNA transcript (or other expression product, e.g., protein) is used to refer to the level of the transcript (or fragmented RNA) determined by normalization to the level of one or more reference mRNA(s), which might be all measured transcripts in the specimen, a single reference mRNA, or a particular reference set of mRNAs. A gene exhibits “increased expression” in a subpopulation of subjects when the normalized expression level of an RNA transcript (or its gene product) is higher in one clinically relevant subpopulation of patients (e.g., patients who are responsive to an EGFR inhibitor) than in a related subpopulation (e.g., patients who are not responsive to said EGF inhibitor). In the context of an analysis of a normalized expression level of a gene in tissue obtained from an individual subject, a gene is exhibits “increased expression” when the normalized expression level of the gene trends toward or more closely approximates the normalized expression level characteristic of such a clinically relevant subpopulation of patients. Thus, for example, when the gene analyzed is a gene that shows increased expression in responsive subjects as compared to non-responsive subjects, then if the expression level of the gene in the patient sample trends toward a level of expression characteristic of a responsive subject, then the gene expression level supports a determination that the individual patient is likely to be a responder. Similarly, where the gene analyzed is a gene that is increased in expression in non-responsive patients as compared to responsive patients, then if the expression level of the gene in the patient sample trends toward a level of expression characteristic of a non-responsive subject, then the gene expression level supports a determination that the individual patient will be non-responsive. Thus normalized expression of a given gene as disclosed herein can be described as being positively correlated with an increased likelihood of positive clinical response to chemotherapy or as being positively correlated with a decreased likelihood of a positive clinical response to chemotherapy.

As used herein, the terms “label” and “detectable label” refer to a molecule capable of being detected, where such molecules include, but are not limited to, radioactive isotopes, fluorescers (fluorophores), chemiluminescers, chromophores, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, chromophores, dyes, metal ions, metal sols, ligands (e.g., biotin, avidin, strepavidin or haptens), intercalating dyes and the like. The term “fluorescer” or “fluorophore” refers to a substance or a portion thereof which is capable of exhibiting fluorescence in a detectable range.

As used herein, the term “target nucleic acid region” or “target nucleic acid” refers to a nucleic acid with a “target sequence” to be detected (e.g., in a method involving nucleic acid hybridization and/or amplification). The target nucleic acid may be either single-stranded or double-stranded and may or may not include other sequences besides the target sequence (e.g., the target nucleic acid may or may not include nucleic acid sequences upstream or 5′ flanking sequence, and may or may not include downstream or 3′ flanking sequence. Where detection is by amplification, these other sequences in addition to the target sequence may or may not be amplified with the target sequence.

The term “primer” or “oligonucleotide primer” as used herein, refers to an oligonucleotide which acts to initiate synthesis of a complementary nucleic acid strand when placed under conditions in which synthesis of a primer extension product is induced, e.g., in the presence of nucleotides and a polymerization-inducing agent such as a DNA or RNA polymerase and at suitable temperature, pH, metal ion concentration, and salt concentration. Primers are generally of a length compatible with their use in synthesis of primer extension products, and can be in the range of between about 8 nucleotides and about 100 nucleotides (nt) in length, such as about 10 nt to about 75 nt, about 15 nt to about 60 nt, about 15 nt to about 40 nt, about 18 nt to about 30 nt, about 20 nt to about 40 nt, about 21 nt to about 50 nt, about 22 nt to about 45 nt, about 25 nt to about 40 nt, and so on, e.g., in the range of between about 18 nt and about 40 nt, between about 20 nt and about 35 nt, between about 21 and about 30 nt in length, inclusive, and any length between the stated ranges. Primers can be in the range of between about 10-50 nucleotides long, such as about 15-45, about 18-40, about 20-30, about 21-25 nt and so on, and any length between the stated ranges. In some embodiments, the primers are not more than about 10, 12, 15, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, or 70 nucleotides in length. In this context, the term “about” may be construed to mean 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 more nucleotides either 5′ or 3′ from either termini or from both termini

Primers are in many embodiments single-stranded for maximum efficiency in amplification, but may alternatively be double-stranded. If double-stranded, the primer is in many embodiments first treated to separate its strands before being used to prepare extension products. This denaturation step is typically effected by heat, but may alternatively be carried out using alkali, followed by neutralization. Thus, a “primer” is complementary to a template, and complexes by hydrogen bonding or hybridization with the template to give a primer/template complex for initiation of synthesis by a polymerase, which is extended by the covalent addition of bases at its 3′ end.

A “primer pair” as used herein refers to first and second primers having nucleic acid sequence suitable for nucleic acid-based amplification of a target nucleic acid. Such primer pairs generally include a first primer having a sequence that is the same or similar to that of a first portion of a target nucleic acid, and a second primer having a sequence that is complementary to a second portion of a target nucleic acid to provide for amplification of the target nucleic acid or a fragment thereof. Reference to “first” and “second” primers herein is arbitrary, unless specifically indicated otherwise. For example, the first primer can be designed as a “forward primer” (which initiates nucleic acid synthesis from a 5′ end of the target nucleic acid) or as a “reverse primer” (which initiates nucleic acid synthesis from a 5′ end of the extension product produced from synthesis initiated from the forward primer) Likewise, the second primer can be designed as a forward primer or a reverse primer.

As used herein, the term “probe” or “oligonucleotide probe”, used interchangeable herein, refers to a structure comprised of a polynucleotide, as defined above, which contains a nucleic acid sequence complementary to a nucleic acid sequence present in the target nucleic acid analyte (e.g., a nucleic acid amplification product). The polynucleotide regions of probes may be composed of DNA, and/or RNA, and/or synthetic nucleotide analogs. Probes are generally of a length compatible with their use in specific detection of all or a portion of a target sequence of a target nucleic acid, and are in many embodiments in the range of between about 8 nt and about 100 nt in length, such as about 8 to about 75 nt, about 10 to about 74 nt, about 12 to about 72 nt, about 15 to about 60 nt, about 15 to about 40 nt, about 18 to about 30 nt, about 20 to about 40 nt, about 21 to about 50 nt, about 22 to about 45 nt, about 25 to about 40 nt in length, and so on, e.g., in the range of between about 18-40 nt, about 20-35 nt, or about 21-30 nt in length, and any length between the stated ranges. In some embodiments, a probe is in the range of between about 10-50 nucleotides long, such as about 15-45, about 18-40, about 20-30, about 21-28, about 22-25 and so on, and any length between the stated ranges. In some embodiments, the primers are not more than about 10, 12, 15, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, or 70 nucleotides in length. In this context, the term “about” may be construed to mean 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 more nucleotides either 5′ or 3′ from either termini or from both termini.

Where a nucleic acid is said to hybridize to a recited nucleic acid sequence, hybridization is under stringent conditions. An example of stringent hybridization conditions is hybridization at 50° C. or higher and 0.1×SSC (15 mM sodium chloride/1.5 mM sodium citrate). Another example of stringent hybridization conditions is overnight incubation at 42° C. in a solution: 50% formamide, 5×SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH7.6), 5×Denhardt\'s solution, 10% dextran sulfate, and 20 μg/ml denatured, sheared salmon sperm DNA, followed by washing the filters in 0.1×SSC at about 65° C. Stringent hybridization conditions are hybridization conditions that are at least as stringent as the above representative conditions, where conditions are considered to be at least as stringent if they are at least about 80% as stringent, e.g., at least about 90% as stringent as the above specific stringent conditions.

The term “computer-based system”, as used herein refers to the hardware, software, and data storage system used to analyze information. The minimum hardware of a patient computer-based system comprises a central processing unit (CPU), input device, output device, and data storage device. A skilled artisan can readily appreciate that many of the currently available computer-based system are suitable for use in the present invention and may be programmed to perform the specific measurement and/or calculation functions of the methods as disclosed herein.

To “record” data, programming or other information on a computer readable medium refers to a process for storing information, using any such methods as known in the art. Any convenient data storage structure may be chosen, based on the method and/or device used to access the stored information. A variety of data processor programs and formats can be used for storage, e.g. word processing text file, database format, etc.

A “processor” or “computing system” or “computing device” references any hardware and/or software combination that will perform the functions required of it. For example, any processor herein may be a programmable digital microprocessor such as available in the form of an electronic controller, mainframe, server or personal computer (desktop or portable). Where the processor is programmable, suitable programming can be communicated from a remote location to the processor, or previously saved in a computer program product (such as a portable or fixed computer readable storage medium, whether magnetic, optical or solid state device based). For example, a magnetic medium or optical disk may carry the programming, and can be read by a suitable reader communicating with each processor at its corresponding station.

Before the present invention and specific exemplary embodiments of the invention are described, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either both of those included limits are also included in the invention.

As used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a reference gene” includes a plurality of such genes and reference to “the EGFR inhibitor” includes reference to one or more EGFR inhibitors, and so forth. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements, or use of a “negative” limitation.

The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.

GENERAL DESCRIPTION

The practice of the methods and compositions of the present disclosure will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, and biochemistry, which are within the skill of the art. Such techniques are explained fully in the literature, such as, “Molecular Cloning: A Laboratory Manual”, 2nd edition (Sambrook et al., 1989); “Oligonucleotide Synthesis” (M. J. Gait, ed., 1984); “Animal Cell Culture” (R. I. Freshney, ed., 1987); “Methods in Enzymology” (Academic Press, Inc.); “Handbook of Experimental Immunology”, 4th edition (D. M. Weir & C. C. Blackwell, eds., Blackwell Science Inc., 1987); “Gene Transfer Vectors for Mammalian Cells” (J. M. Miller & M. P. Calos, eds., 1987); “Current Protocols in Molecular Biology” (F. M. Ausubel et al., eds., 1987); and “PCR: The Polymerase Chain Reaction”, (Mullis et al., eds., 1994).

Based on evidence of differential expression of a gene (e.g., as detected by assaying for an RNA transcript) in cancer cells that positively respond to an EGFR-inhibitor and non-responsive cancer cells, the present disclosure provides response indicator genes and gene modules. These response indicator genes and/or gene modules and associated information provided by the present disclosure allow physicians to make more intelligent treatment decisions, and to customize the treatment of EGFR-expressing cancer to the needs of individual patients, thereby maximizing the benefit of treatment and minimizing the exposure of patients to unnecessary treatments, which do not provide any significant benefits and often carry serious risks due to toxic side-effects.

The response indicator genes and/or gene modules and associated information provided by the present disclosure have utility in the development of therapies to treat EGFR-expressing cancer and screening patients for inclusion in clinical trials that test the efficacy of EGFR inhibitors. Said genes and/or gene modules and association information may also be used to design or produce a reagent that modulates the level or activity of the gene\'s transcript (i.e., RNA transcript) or its expression product. Said reagents may include but are not limited to an antisense RNA, a small inhibitory RNA, a ribozyme, a monoclonal or polyclonal antibody.

In various embodiments of the methods of the present disclosure, various technological approaches are available for determination of expression levels of the disclosed genes, including, without limitation, RT-PCR, microarrays, serial analysis of gene expression (SAGE) and nucleic acid sequencing, which will be discussed in detail below. In particular embodiments, the expression level of each gene may be determined in relation to various features of the expression products of the gene including exons, introns, protein epitopes and protein activity

EGFR Inhibitor Treatment

The present disclosure provides methods to predict the likelihood that a patient having an EGFR-expressing cancer will exhibit a beneficial response to an EGFR inhibitor therapy. Patients subject to such an assessment include: 1) patients who have an EGFR-expressing cancer and who have not yet undergone any treatment for the cancer; 2) patients who have an EGFR-expressing cancer and who have undergone complete or partial resection of the cancer, e.g., who have undergone surgical removal of cancerous tissues to the extent clinically possible; and 3) patients who have an EGFR-expressing cancer and who have been treated with a treatment regimen other than an EGFR inhibitor treatment regimen. Patients who are subject to a likelihood assessment as disclosed herein also include those whose cancer is KRAS-negative (e.g., patients who have a (KRAS−) tumor).

It will be appreciated that the same patient sample, and even the same assay, may be used for both determining whether a cancer is an EGFR-expressing cancer and assessing the likelihood that the patient having an EGFR -expressing cancer will exhibit a beneficial response to an EGFR inhibitor cancer therapy. For example, the assay(s) used to determine whether the cancer is an EGFR-expressing cancer may be carried out at the same time as the assay(s) used to assess the likelihood that the patient having an EGFR -expressing cancer will exhibit a beneficial response to an EGFR inhibitor. Alternatively, the result of the assay(s) used to determine whether the cancer is an EGFR-expressing cancer may guide the decision as to whether and how to apply an additional assay(s) used to assess the likelihood that the patient having an EGFR -expressing cancer will exhibit a beneficial response to an EGFR inhibitor.

EGFR-expressing cancers include cancers comprising cells that express an EGFR on their cell surface. Such cancers include, but are not limited to, breast cancer, lung cancer, colorectal cancer, renal cancer prostate cancer, brain cancer, liver cancer, pancreatic cancer, and head and neck cancer.

A patient who is being assessed using the method disclosed in the present disclosure is one who may be considered for treatment with an EGFR inhibitor. EGFR inhibitors include, e.g., antibodies that bind to and inhibit EGFR, EGFR-selective tyrosine kinase inhibitors, and the like. EGFR inhibitors include, but are not limited to, cetuximab (Erbitux®) and panitumumab (Vectibix®), both monoclonal antibodies that block EGFR and EGFR-dependent cell growth; gefitinib (Iressa®; N-(3-chloro-4-fluoro-phenyl)-7-methoxy-6-(3-morpholin-4-ylpropoxy)quinazolin-4-amine); OSI774 (erlotinib, Tarceva®; N-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy) quinazolin-4-amine); and alpha-cyano-beta-methyl-N-Rtrifluoromethoxylphenyl1-propenamide (LFM-A12), all small molecule tyrosine kinase inhibitors; and the like. Erbitux is a registered trademark of Bristol-Myers Squibb Co. Vectibix is a registered trademark of Amgen, Inc. Iressa is a registered trademark of AstraZeneca. Tarceva is a registered trademark of Genentech, Inc.

EGFR inhibitors also include EGFR tyrosine kinase inhibitors such as quinazolines, such as PD 153035 (Fry et al. (1994) Science 265:1093; and Traxler et al. (1997) J. Pharm. Belg. 52:1997), 4-(3-chloroanilino) quinazoline, or CP-358,774; pyrrolopyrimidines, such as CGP 59326, CGP 60261 and CGP 62706 (Traxler et al. (1997) J. Pharm. Belg. 52:1997); pyrazolopyrimidines (Strawn and Shawver (April 1998) Exp. Opin. Invest. Drugs 7:553-573); 4-(phenylamino)-7H-pyrrolo[2,3-d] pyrimidines (Traxler et al., (1996) J. Med. Chem 39:2285-2292); curcumin (diferuloyl methane) (Laxminarayana, et al., (1995), Carcinogen 16:1741-1745); 4,5-bis (4-fluoroanilino)phthalimide (Buchdunger et al. (1995) Clin. Cancer Res. 1:813-821; Dinney et al. (1997) Clin. Cancer Res. 3:161-168); tyrphostins containing nitrothiophene moieties (Brunton et al. (1996) Anti Cancer Drug Design 11:265-295); the protein kinase inhibitor ZD-1839 (AstraZeneca) (U.S. Pat. No. 5,770,599; Strawn and Shawver (April 1998) Exp. Opin. Invest. Drugs 7:553-573; and Woodburn et al. (1997) Abstract #4251, Proc. Am. Assoc. Cancer Res. 38:633); CP-358774 (Pfizer, Inc.) (Moyer et al. (1997) Cancer Res. 57:4838); PD-0183805 (Warner-Lambert); inhibitors as described in International patent application WO99/09016 (American Cyanamid); WO98/43960 (American Cyanamid); WO97/38983 (Warner Lambert); WO99/06378 (Warner Lambert); WO99/06396 (Warner Lambert); WO96/30347 (Pfizer, Inc.); WO96/33978 (AstraZeneca); WO96/33977 (AstraZeneca); and WO96/33980 (AstraZeneca).

Methods to Predict Likelihood of Response to EGFR Inhibitor Treatment


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stats Patent Info
Application #
US 20120270228 A1
Publish Date
10/25/2012
Document #
File Date
12/22/2014
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