WO2013130465A2 - Gene expression markers for prediction of efficacy of platinum-based chemotherapy drugs - Google Patents

Gene expression markers for prediction of efficacy of platinum-based chemotherapy drugs Download PDF

Info

Publication number
WO2013130465A2
WO2013130465A2 PCT/US2013/027789 US2013027789W WO2013130465A2 WO 2013130465 A2 WO2013130465 A2 WO 2013130465A2 US 2013027789 W US2013027789 W US 2013027789W WO 2013130465 A2 WO2013130465 A2 WO 2013130465A2
Authority
WO
WIPO (PCT)
Prior art keywords
genes
gene
expression level
expression
treatment
Prior art date
Application number
PCT/US2013/027789
Other languages
French (fr)
Other versions
WO2013130465A3 (en
Inventor
Steve Shak
Mark Lee
Michael Crager
Margarita Lopatin
Joffre B. Baker
Kim Clark-Langone
Robert J. Pelham
Original Assignee
Genomic Health, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Genomic Health, Inc. filed Critical Genomic Health, Inc.
Publication of WO2013130465A2 publication Critical patent/WO2013130465A2/en
Publication of WO2013130465A3 publication Critical patent/WO2013130465A3/en

Links

Classifications

    • 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
    • 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
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • the present invention relates to genes, the expression levels of which are useful for predicting response of cancer cells and cancer patients to a platinum-based chemotherapy drug.
  • Oxaiipiatin is a third-generation platinum-based ehemotherapeutic agent that has significant activity in colorectal cancer (CRC).
  • CRC colorectal cancer
  • Oxa!iplatin induces cytotoxicity through the formation of platinum-DNA adducts, which in turn, activate multiple signaling pathways (Kei!and, L,, "The Resurgence of Platinum-Based Cancer Chemotherapy,” Nat. Rev. Cancer, 2007, 7(8): p, 573-84).
  • the present invention provides response indicator genes for platinum-based chemotherapy drugs. These genes are provided in Tables 1 and 2, The present invention also provides gene subsets of "the response indicator genes based on their known function. These gene subsets include, but are not limited Co, invasion/migration, proliferation, DNA damage repair, apoptosis, cell cycle control, MAPK signaling, FGF Signaling, PI3K/AKT, antioxidant defense, Wnt signaling, thyroxine signaling, ion transport, oxidative stress response, stress induced, G-protein signaling, heat shock, pyridine metabolism, ABC transporters, glutathione metabolism, methionine metabolism, TGF beta, immune inflammation, amino acid metabolism, and folic acid metabolism gene subsets.
  • Tables 1 and 2 provide gene subsets in which each gene may be grouped.
  • the present invention also provides methods of identifying gene cliques, i.e. genes that co-express with a response indicator gene and exhibit correlation of expression with the response indicator gene, and thus may be substituted for that response indicator gene in an assay.
  • Table 3 provides a list of the top 5 genes that co-express with the genes listed in Tables 1 and 2.
  • increased expression level of one or more response indicator genes selected from Table 1 is negatively correlated with the efficacy of a platinum- based chemotherapy drug.
  • increased expression level of one or more response indicator genes selected from Table 2 is positively correlated with the efficacy of a platinum-based chemotherapy drug.
  • increased expression level of one or more genes selected from RFC1 , MTA1 , PTMA, HNRNPD, ANAPC2, SPINT2, ZNF143, SP1, EHMT1, and MUTYH is negatively correlated with the efficacy of a platinum-based chemotherapy drug in reducing the likelihood of colon cancer recurrence
  • increased expression level of one or more genes selected from TXNRD1, MAPRE1, ABCC4, SIX 1 A3, CDK2, and AVEN is positively correlated with the efficacy of a platinum-based chemotherapy drug in reducing the likelihood of colon cancer recurrence.
  • the present invention further provides methods and compositions for predicting the efficacy of a treatment comprising a platinum-based chemotherapy drug based on the expression level of one or more response indicator genes in a tumor sample obtained from the patient.
  • the method comprises assaying or measuring an expression level of one or more response indicator gene products.
  • the response indicator gene is selected from any one of the genes listed in Tables 1 and 2.
  • the expression level of the response indicator gene is normalized.
  • the expression level or the normalized expression level is used to predict the efficacy of a platinum-based chemotherapy drug, wherein increased expression level or increased normalized expression level of one or more response indicator genes selected from Table I is negatively correlated with the efficacy of a platinum -based chemotherapy drug, and increased expression level or increased normalized expression level of one or more response indicator genes selected from Table 2 is positively correlated with the efficacy of a platinum-based chemotherapy drug.
  • increased expression level of one or more genes selected from RFC 1 , MTA1 , PTMA, HNRNPD, ANAPC2, SP1NT2, Z F143, SPL EHMTI, and MUTYH is negatively correlated with the efficacy of a platinum-based chemotherapy drag
  • increased expression level of one or more genes selected from TXNRD1 , MAPREJ , ABCC4, SLC 1A3, CDK2, and AVEN is positively correlated with the efficacy of a platinum-based chemotherapy drug.
  • a report is generated based on the predicted efficacy of a platinum-based chemotherapy drug.
  • the methods of the present invention contemplate determining the expression level of at least one response indicator gene or its gene product.
  • the methods may further include determining the expression levels of at least two response indicator 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 response indicator 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 response indicator 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 response indicator genes, or their expression products.
  • the methods of the present disclosure may further include determining the expression levels of at least six response indicator 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 response indicator 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 response indicator 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 response indicator genes, or their expression products. The methods may involve determination of the expression levels of at least ten (10), at least eleven (1 1), at least twelve (12). at least thirteen (13), at least fourteen (14), at least fifteen (15), or at least sixteen (16) of the response indicator genes, or their expression products.
  • the expression level, or normalized expression level, of the response indicator gene, or its expression product is used to predict the efficacy of a platinum-based chemotherapy drug.
  • a likelihood score e.g., a score predicting the likelihood of cancer recurrence with and without adjuvant treatment with a platinum-based chemotherapy drug
  • a score may be calculated using weighted values based on the expression level or normalized expression level of a response indicator gene and its contribution to response to a platinum-based chemotherapy drug.
  • the expression product of the response indicator gene to be assayed or measured is an RNA transcript.
  • the RNA transcripts are fragmented.
  • 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 T- PCR (reverse transcriptase PGR), quantitative RT-PCR (qRT-PCR), or other PCR-based methods. inimunohistochemistry, proteomics techniques, an array-based method, polynucleotide sequencing, or any other methods known in the art or their combination.
  • 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, fresh, or frozen tissue.
  • the tissue may be obtained from a surgical resection or by body fluid containing a cancer cell, e.g. urine, blood, etc.
  • the tumor sample is obtained from a patient with colorectal cancer.
  • the patient has stage II (Dukes B) or stage III (Dukes C) colorectal cancer.
  • the platinum-based chemotherapy drug is se lected from cisplatin. carboplatin, and oxaiiplatin.
  • the platinum-based chemotherapy drug is oxaiiplatin.
  • Oxaiiplatin may be provided alone, or in combination, with one or more additional anti-cancer agents, in a specific embodiment, oxaiiplatin is provided in combination with fluorouracil (5-FU) and leucovorin.
  • FIGS. 1A-1B show the within and between patient standard deviations for 16 genes for predicting a benefit from the addition of oxaiiplatin to 5FU treatment (FIG, I A) and stromal genes (FIG. IB).
  • FIG. 2 shows a regression to the mean corrected predictiveness curve for the 9-gene mode! for predicting a benefit from the addition of oxaiiplatin to treatment with 5FU ⁇ hased
  • FIGS. 3A-3C show survival curves for the tertiles of the score from the 9-gene model.
  • FIG. 3 A shows the regression to the mean corrected estimates of survival curves for recurrence- free interval, stage 3 patients only for the first tertile.
  • FIG 3B shows the regression to the mean corrected estimates of survival curves for recurrence-free interval, stage 3 patients only for the second tertile.
  • FIG 3C shows the regression to the mean corrected estimates of survival curves for recurrence-free interval, stage 3 patients only for the third tertile.
  • amplicon refers to a piece ofDNA that has been synthesized using an amplification technique, such as the polymerase chain reaction (PGR) and ligase chain reaction.
  • PGR polymerase chain reaction
  • ligase chain reaction ligase chain reaction
  • anti-eaneer agent refers to any molecule, compound, chemical or composition that has an anti-cancer effect, such as a "positive response” as defined below.
  • Anti-cancer agents include, without limitation, chemotherapeutic agents, radtotherapeutic agents, cytokines, anti-angiogemc agents, apoptosis-inducing agents or anti-cancer immunotoxins, such as antibodies, Examples of anti-cancer agents include, without limitation, methotrexate, taxol, mercaptopurine, thioguanine.
  • anti-cancer agents are known in the art.
  • the anti-cancer agent is 5-FU and Ieucovorin,
  • test or "assaying” as used herein refer to performing a quantitative or qualitative analysis of a component in a sample.
  • the terms include laboratory or clinical observations, and/or measuring the level of the component in the sample.
  • cancer refers to or describe the physiological condition that is typically characterized by unregulated cell growth.
  • cancer in the present application include cancer of the gastrointestinal tract, such as invasive colorectal cancer or Stage ⁇ (Dukes B) or Stage Til (Dukes C) colorectal cancer,
  • co-expressed refers to a statistical correlation between the expression level of one gene and the expression level of another gene. Pairwise co-expression may be calculated by various methods known in the art, e.g., by calculating Pearson correlation coefficients or Spearman correlation coefficient. Co-expressed gene cliques may also be identified using a graph theory. An analysis of co-expression may be calculated using normalized expression data.
  • colon cancer and “colorectal cancer” are used interchangeably herein and refer in the broadest sense to (1 ) all stages and a!! forms of cancer arising from epithelial ceils of the large intestine and/or rectum and/or (2) ail stages and all forms of cancer affecting the lining of the large intestine and/or rectum.
  • the colon and rectum are treated as one organ,
  • correlates refers to a statistical association between instances of two events, where events may include numbers, data sets, and the like.
  • 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 present invention provides genes and gene subsets, the expression levels of which are correlated with a particular outcome measure, such as between the expression level of a gene and the efficacy of treating a patient with a drug.
  • the increased expression level of a gene product may be positively correlated with efficacy of a chemotherapy drug..
  • Such a positive correlation may be demonstrated statistically in various ways, e.g, by a gene expression- treatment interaction hazard ratio for cancer recurrence ⁇ 1.
  • the increased expression level of a gene product may be negatively correlated with efficacy of a chemotherapy drug.
  • chemotherapy may have decreased efficacy.
  • Such a negative correlation indicates that chemotherapy will likely be less efficacious for the patient, and this may be demonstrated statistically in various ways, e.g., a gene expression-treatment interaction hazard ratio for cancer recurrence > 1.
  • Standardization refers to a process to effectively put all the genes on a comparable scale. This is performed because some genes will exhibit more variation (a broader range of expression) than others. Standardization is performed by dividing each expression value by its standard deviation across ail samples for that gene. Hazard ratios are then interpreted as the relative risk of recurrence per 1 standard deviation increase in gene expression.
  • Ct refers to threshold cycle, the cycle number in quantitative polymerase chain reaction (qPCR) at which the fluorescence generated within a reaction well exceeds the defined threshold, i.e. the point during the reaction at which a sufficient number of amplicons have accumulated to meet the defined threshold.
  • qPCR quantitative polymerase chain reaction
  • expression level refers to qualitative or quantitative
  • Expression level may be determined for the UNA expression level of a gene or for the polypeptide expression level of a gene.
  • the term "normalized" expression level as used herein refers to an expression level of a response indicator gene relative to the level of an expression product of a reference gene(s), which might be all measured expression products in the sample, a single reference expression product, or a particular set of expression products.
  • a gene exhibits an "increased expression level" when the expression level of an expression product is higher in a first sample, such as in a clinically relevant subpopulation of patients (e.g., patients who are responsive to a platinum-based chemotherapy drug), than in a second sample, such as in a related subpopulation (e.g., patients who are not responsive to the platinum-based chemotherapy drug).
  • a gene in the context of an analysis of an expression level of a gene in tissue obtained from an individual subject, a gene exhibits "increased expression,” or “increased normalized expression,” when the expression level or normalized expression level of the gene in the subject trends toward, or more closely approximates, the expression level or normalized expression level characteristic of a clinically relevant subpopulation of patients.
  • the gene analyzed is a gene that shows increased expression in responsive subjects as compared to non-responsive subjects
  • “increased expression” or “Increased normalized” expression level of a given gene can be described as being positively correlated with efficacy of a platinum-based chemotherapy drug. If the expression level of the 'gene in the individual subject trends toward a level of expression characteristic of a responsive subject, then the gene expression level supports a determination that the individual subject ss more likely to be a responder. If the expression level of the gene in the individual subject trends toward a level of expression characteristic of a non-responsive subject then the gene expression level supports a determination that the individual subject is more likely to be a inon-responder,
  • expression product or “gene product” are used herein to refer to the RNA transcription products (transcripts) of a gene, including rnRNA, and the polypeptide translation products of such RNA transcripts.
  • An expression product may be, for example, an unspliced RNA, an rnRNA, a splice variant mRNA, a microRNA, a fragmented RNA, a polypeptide, a post- translationally modified polypeptide, a splice variant polypeptide, etc.
  • long-term survival is used herein to refer to survival for a particular time period.
  • the time period of long-term survival is for at least 3 years. In another embodiment, the time period of long-term survival is for at least 5 years.
  • measuring refers to performing a physical act of determining the dimension, quantity, or capacity of a component in a sample.
  • microarray refers to an ordered arrangement of hybrid izable array elements, e.g., oligonucleotide or polynucleotide probes, on a substrate.
  • polynucleotide generally refers to any polyribonucleotide or
  • polydeoxribonucieotide which may be unmodified RNA or DNA or modified RNA or DNA
  • polynucleotides as used herein include, without limitation, single- and double- stranded DNA, DNA including single- and double-stranded regions, single- and double-stranded RNA, and RNA including single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or. more typically, double-stranded or include single- and double-stranded regions.
  • polynucleotide refers to triple- stranded regions comprising RNA or DNA or both RNA and DNA.
  • the strands in such regions may be from the same molecule or from different molecules.
  • the regions may include all of one or more of the molecules, but more typically involve only a region of some of the moiecuies,
  • One of the moiecuies of a triple-helical region often is an oligonucleotide.
  • the term "polynucleotide” also includes DNAs (including cDNAs) and RNAs and those that contain one or more modified bases, Thus, DNAs or RNAs with backbones modified for stability or for other reasons, are "polynucleotides" as that term is used herein.
  • DNAs or RNAs comprising unusual bases, such as inosine, or modified bases, such as tritiated bases are included within the term "polynucleotides" as used herein, in general, the term “polynucleotide” embraces all chemically, enzymatieally and/or metabolically modified forms of unmodified polynucleotides, as well as the chemical forms of DNA and RNA characteristic of viruses and cells, including simple and complex ceils.
  • oligonucleotide refers to a relatively short polynucleotide, including, without limitation, single-stranded deoxyribonucleotides, single- or double-stranded ribonucleotides, RNA/DNA hybrids and double-stranded DNAs, Oligonucleotides, such as single-stranded DNA probe oligonucleotides, are often synthesized by chemical methods, for example using automated oligonucleotide synthesizers that are commercially available. However, oligonucleotides can be made by a variety of other methods, including in vitro recombinant DNA-mediated techniques and by expression of DNAs in cells and organisms.
  • primer refers to an oligonucleotide that 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.
  • 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.
  • nt nucleotides
  • 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, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19 or 20 more nucleotides either 5' or 3' from either termini or from both termini.
  • amplification but may alternatively be 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 affected by heat, but may alternatively be carried out using alkali, followed by neutralization.
  • 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 covended 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.
  • 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).
  • the second primer can be designed as a forward primer or a reverse primer.
  • probe or "oligonucleotide probe”, used interchangeably herein, refers to a structure comprised of a polynucleotide, as defined above, that contains a nucleic acid sequence complementary to a nucleic acid sequence present in the target nucleic acid anaiyte (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 ail 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 1 5 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
  • pathology of cancer includes all phenomena that comprise 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.
  • platinum-based chemotherapy drug refers to a molecule or a composition comprising a molecule containing a coordination complex comprising the chemical element platinum and useful as a chemotherapy drug.
  • Platinum-based chemotherapy drugs generally act by inhibiting DNA synthesis and have some alkylating activity. Examples of platinum-based chemotherapy drugs include cispiatin, carbopiatin, and oxaiipiatin. Platinum- based chemotherapy drugs encompass those that are currently being used as part of a
  • the platinum-based chemotherapy drug may be administered as a monotherapy, or in combination with other anti-cancer agents, or as prodrugs, or together with local therapies such as surgery and radiation, or as adjuvant or neoadjuvant chemotherapy, or as part of a multimodal approach to the treatment of neoplastic disease.
  • oxaiipiatin may be administered alone, or in combination with fluorouracii (5-FU) and/or leucovorin for the treatment of colorectal cancer.
  • efficacy refers to the capacity for a drug to produce a positive response upon administration to a patient.
  • a positive response refers to a favorable response to a drug as opposed to a lack of response or an unfavorable response, such as adverse events.
  • a positive response may include, without limitation, (1) inhibition, to some extent, of tumor growth, including slowing down to complete growth arrest; (2) reduction in the number of tumor cells; (3) reduction in tumor size; (4) inhibition (i.e., reduction, slowing down or complete cessation) of tumor ceil infiltration into adjacent peripheral organs and/or tissues; (5) inhibition of metastasis; (6) enhancement of anti-tumor immune response, possibly resulting in regression or rejection of the tumor: (7) relief, to some extent, of one or more symptoms associated with the tumor; (8) increase in the length of survival following treatment; and/or (9) decreased mortality at a given point of time following treatment,
  • a positive response can be expressed in terras 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
  • responder refers to a patient who has cancer, and who exhibits a positive response following treatment with a platinum-based chemotherapy drug.
  • non-resporider refers to a patient who has cancer, and who has not shown a positive response following treatment with a platinum-based chemotherapy drug, [ ⁇ 05 ⁇ ]
  • prediction is used herein to refer to the likelihood that a cancer cell or a cancer patient will have a particular response to treatment, whether positive or negative.
  • prediction refers to a particular response to treatment following surgical removal of the primary tumor.
  • treatment could include chemotherapy.
  • the predictive methods of the present invention can be used clinically to make treatment decisions by choosing the most appropriate treatment modalities for any particular patient.
  • the predictive methods of the present invention are useful tools in predicting if a patient is likely to exhibit a positive response to a treatment regimen, such as chemotherapy, surgical intervention, or both.
  • reference gene refers to a gene whose expression level can be used to compare the expression level of a gene product in a test sample
  • reference genes include housekeeping genes, such as beta-globin, alcohol
  • dehydrogenase or any other gene, the expression of which does not vary depending on the disease status of the cell containing the gene, in another embodiment, all of the assayed genes or a large subset thereof may serve as reference genes.
  • RNA transcript refers to the RNA transcription product of a gene, including, for example, mRNA, an unspliced RNA, a splice variant mRNA. a microRNA, and a fragmented RNA.
  • each gene name used herein corresponds to the Official Symbol assigned to the gene and provided by Eiitrez Gene (URL:
  • tumor sample refers to a sample comprising tumor material obtained from a cancerous patient.
  • the term encompasses tumor tissue 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.
  • tumor sample encompasses a sample comprising tumor cells obtained from sites other than the primary tumor, e.g., circulating tumor cells.
  • 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 further 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.
  • Hybridization generally depends on the ability of denatured DNA to re-anneal when complementary strands are present in an environment below their melting temperature. The higher the degree of desired homology between the probe and hybridizable sequence, the higher the relative temperature that can be used. As a result, it follows that higher relative temperatures would tend to make the reaction conditions more stringent, while lower temperatures less so.
  • stringency of hybridization reactions see Ausubel et ah, Current Protocols in Molecular Biology, Wiley Interscience Publishers, (1995).
  • "Stringent conditions” or “high stringency conditions”, as defined herein, typically: (1) employ low ionic strength and high temperature for washing, for example 0.015 M sodium chloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate at 50°C; (2) employ during hybridization a denaturing agent, such as formamide, for example, 50% (v/v) formamide with 0.1% bovine serum albumin/0.1% Fico!l/0.1 % polyvinyipyrrolidone/SOmM sodium phosphate buffer at pH 6.5 with 750 raM sodium chloride, 75 niM sodium citrate at 42°C; or (3) employ 50% formamide, 5 x SSC (0,75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0, 1% sodium pyrophosphate, 5 x Denhardt's solution, sonicated salmon sperm DNA (50 ,u.g/ml), 0.3% SDS, and 10% de
  • Modely stringent conditions may be identified as described by Sambrook et ah, Molecular Cloning; A Laboratory Manual, New York: Cold Spring Harbor Press, 1989, and include the use of washing solution and hybridization conditions (e.g., temperature, ionic strength and %SDS) less stringent that those described above.
  • moderately stringent conditions is overnight incubation at 37°C in a solution comprising: 20% formamide, 5 x SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5 x Denhardt's solution, 10% dextran sulfate, and 20 mg/ml denatured sheared salmon sperm DNA, followed by washing the filters in 1 x SSC at about 37-50°C, The skilled artisan will recognize how to adjust the temperature, ionic strength, etc. as necessary to accommodate factors such as probe length and the like.
  • subject means a mammal being assessed for treatment and/or being treated.
  • the mammal is a human.
  • the terms "subject,” “individual,” and “patient” thus encompass individuals having cancer (e.g., colorectal cancer), including those who have undergone or are candidates for resection (surgery) to remove cancerous tissue (e.g., cancerous colorectal tissue).
  • the term "surgery” applies to surgical methods undertaken for removal of cancerous tissue, including resection, laparotomy, colectomy (with or without lymphadenectomy), ablative therapy, endoscopic removal, excision, dissection, and tumor biopsy/removal
  • the tumor tissue or sections used for gene expression analysis may have been obtained from any of these methods.
  • threshold or “thresholding” refer to a procedure used to account for non-linear relationships between gene expression measurements and clinical response as well as to further reduce variation in reported patient scores. When thresholding is applied, all measurements below or above a threshold are set to that threshold value.
  • a non-linear relationship between gene expression and outcome could be examined using smoothers or cubic splines to model gene expression and recurrence-free interval in Cox proportional hazards models or gene expression and recurrence status in logistic regression models. Variation in reported patient scores could be examined as a function of variability in gene expression at the limit of quantitation and/or detection for a particular gene.
  • treatment refers to administering or contacting an agent, or carrying out a procedure (e.g., radiation, a surgical procedure, etc.), for the purpose of obtaining an effect.
  • a procedure e.g., radiation, a surgical procedure, etc.
  • 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.
  • the terms cover 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 that 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.
  • tumor refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-eancerous and cancerous cells and tissues.
  • Tumor TL * tumor invades submucosa; T2: tumor invades muscuiaris basement; T3: tumor invades through the muscuiaris basement into the subserose, or into the consic or perirectal tissues;
  • T4 tumor directly invades and/or perforates other organs or structures.
  • Node NO: no regional lymph node metastasis; Nl : metastasis in 1 to 3 regional lymph nodes; N2: metastasis in 4 or more regional lymph nodes.
  • Stage groupings Stage I: Tl, NO, M0 or T2, NO, M0; Stage II: T3, NO, M0 or T4, NO, M0;
  • Stage III any T, Nl-2, M0; Stage IV: any T, any N, Ml .
  • Stage A the tumor penetrates into the mucosa of the bowel wall but not further.
  • Stage B tumor penetrates into and through the muscuiaris basement of the bowel wall.
  • Stage C tumor penetrates into but not through the muscuiaris basement of the bowel wall and there is pathologic evidence of colorectal cancer in the lymph nodes; or tumor penetrates into and through the muscuiaris basement of the bowel wall and there is pathologic evidence of cancer in the lymph nodes.
  • Stage D tumor has spread beyond the confines of the lymph nodes, into other organs, such as the liver, lung, or bone.
  • the term "computer-based system”, as used herein refers to the hardware means, software means, and data storage means used to analyze information.
  • the minimum hardware of a patient computer-based system comprises a central processing unit (CPU), input means, output means, and data storage means.
  • CPU central processing unit
  • input means input means
  • output means output means
  • data storage means data storage means
  • 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 means 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 means” references any hardware and/or software
  • 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).
  • a computer program product such as a portable or fixed computer readable storage medium, whether magnetic, optical or solid state device based.
  • 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,
  • the present invention provides response indicator genes of platinum-based chemotherapy drugs. These genes are listed in Tables 1 and 2.
  • the response indicator genes may be further grouped into gene subsets, depending on their known function. For example, the gene subsets may include an "apoptosis group,” "DNA damage repair group,” “cell cycle control group,”
  • Tables 1 and 2 indicate which gene subset in which each gene may be grouped.
  • the present invention further provides methods for determining genes that co-express with the response indicator genes.
  • the co-expressed genes themselves are useful response indicator genes,
  • the co-expressed genes may be substituted for the response indicator gene with which they co-express, Top 5 Pearson correlations for each of the genes in Tables 1 and 2 are listed in Table 3. In this analysis, median normalized expression for stage 111 patients was utilized.
  • the present invention also provides the top 16 genes in Tables 1 and 2 associated with oxaliplatin benefit controlling for a false discovery rate at 20%, These genes are listed in Table 4,
  • the present invention also provides models, which include 7-10 of the genes listed in Table 4, that are useful for predicting the efficacy of a treatment comprising a platinum-based chemotherapy drug.
  • Table 5 lists 20 such models.
  • the present invention further provides a 9-gene model useful for predicting the efficacy of a treatment comprising a platinum-based chemotherapy drug, and these 9 genes are indicated in Table 6.
  • Table 6 also indicates a biological category in which each of these genes may be grouped.
  • the genes of the 9-gene model may be further grouped into gene groups or pathways, including, for example, "transcription regulation,” “replication and DNA repair,” “cell cycle control/apoptosis,” “proliferation,” “ion/glutamate transport,” and “pyridine metabolism/redox homeostasis” pathways.
  • the present invention provides a number of methods that utilize the response indicator genes and associated information.
  • the present invention provides a method of predicting the efficacy of a treatment comprising a platinum-based chemotherapy drug.
  • the methods of the invention comprise assaying or measuring the expression level of the response indicator gene(s) in a sample comprising cancer cells or in a tumor sample, and predicting the efficacy of the platinum -based chemotherapy drug based on the correlation between the expression level of the response indicator gene(s) and a positive response to the platinura-based chemotherapy drug,
  • the platinum-based chemotherapy drug is oxaliplatin.
  • the response indicator genes and associated information provided by the present invention also have utility in the development of therapies to treat cancers and screening patients for inclusion in clinical trials that test the efficacy of platinum-based chemotherapy drugs.
  • the response indicator genes and associated information may further be used to design or produce a reagent that modulates the level or activity of the expression product.
  • reagents may include, but are not limited to, an antisense RNA, a small inhibitory RNA (siRNA), a ribozyme, a small molecule, a monoclonal antibody, and a polyclonal antibody,
  • a Cox proportional hazards regression model may fit to a particular clinical endpoint (e.g., RFI, DFS, OS).
  • One assumption of the Cox proportional hazards regression model is the proportional hazards assumption, i.e. the assumption that effect parameters multiply the underlying hazard.
  • Assessments of model adequacy may be performed including, but not limited to, examination of the cumulative sum of martingale residuals.
  • One skilled in the art would recognize that there are numerous statistical methods that may be used (e.g., Royston and Parmer (2002), smoothing spline, etc.) to fit a flexible parametric model using the hazard scale and the Weibull distribution with natural spline smoothing of the log cumulative hazards function, with effects for treatment (chemotherapy or observation) and RS allowed to be time-dependent.
  • the relationship between treatment and (1) benefit groups; and (2) clinical/pathologic covariates may also be tested for significance.
  • any of the methods described may group the expression levels of genes,
  • the grouping of genes may be performed at least in part based on knowledge of the contribution of the genes according to physiologic functions or component cellular characteristics, such as in the gene subsets described herein.
  • the formation of groups can facilitate the mathematical weighting of the contribution of various expression levels to the recurrence and/or treatment scores.
  • the weighting of a gene group representing a physiological process or component cellular characteristic can reflect the contribution of that, process or characteristic to the pathology of the cancer and clinical outcome. Accordingly, the present invention provides gene subsets identified herein for use in the methods disclosed herein.
  • increased expression level of one or more genes selected from Table 2 is positively correlated with efficacy of a platinum-based chemotherapy drug in reducing colon cancer recurrence.
  • increased expression level of one or more genes selected from RFC! , MTAL PTMA, HNRNPD, ANAPC2, SPINT2, ZNF143, SPl , EHMTL and MUTYH is negatively correlated with efficacy of a platinum -based chemotherapy drug in reducing colon cancer recurrence
  • increased expression level of one or more genes selected from TXNRDl, MAPREl , ABCC4, SLC l A3, CDK2, and AVEN is positively correlated with efficacy of a platinum-based chemotherapy drug in reducing colon cancer recurrence.
  • increased expression level of one or more genes selected from MTAl, PTMA, SPl, RFC, and SP1NT2 is negatively correlated with efficacy of a platinum-based chemotherapy drug in reducing colon cancer recurrence
  • increased expression level of one or more genes selected from AVEN, CDK2, SLCl A3, and TXNRDl is positively correlated with efficacy of a platinum-based chemotherapy drug in reducing colon cancer recurrence.
  • the platinum-based chemotherapy drug is oxaliplatin and the response indicator gene(s) is assayed or measured in colorectal cancer cells.
  • Oxalipiatin may be provided in combination with one or more anti-cancer agents, such as 5-FU and Seucovorin,
  • the colorectal cancer cells may be a tumor sample obtained from a human patient with colorectal cancer, such as stage II (Dukes B) or stage III (Dukes C) colorectal cancer.
  • the expression level of the response indicator gene(s) is normalized as described in more detail below,
  • increased expression level of one or more genes selected from Table 1 is negatively correlated with efficacy of treatment with oxaliplatin in colorectal cancer cells or in a human patient with colorectal cancer, such as stage II (Dukes B) or stage ill (Dukes C) colorectal cancer,
  • increased expression level of one or more genes selected from Table 2 is positively correlated with efficacy of treatment with oxaliplatin in colorectal cancer cells or in a human patient with colorectal cancer, such as such as stage ⁇ (Dukes B) or stage III (Dukes C) colorectal cancer.
  • cancer cells or a tumor sample is assayed or measured for an expression level of a response indicator gene product(s).
  • the tumor sample can be obtained from a solid tumor, e.g., from a surgical procedure carried out to remove a tumor; or from a tissue or bodily fluid that contains cancer cells.
  • the tumor sample is obtained from a patient with colorectal cancer, such as stage II (Duke's B) or stage III (Duke's C) colorectal cancer.
  • the expression level of a response indicator gene is normalized relative to the level of an expression product of one or more reference genes.
  • the platinum-based chemotherapy drug is oxaliplatin.
  • Oxaliplatin may be provided in combination with one or more anti-cancer agents, such as 5-FU and leucovorin.
  • the efficacy of treatment with a platinum-based chemotherapy drug in an individual patient is predicted by comparing, directly or indirectly, the expression level or normalized expression level of the response indicator gene in the tumor sample from the individual patient to the expression level or normalized expression level of the response indicator gene in a clinically relevant subpopulation of patients.
  • the response indicator 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 individual subject trends toward a level of expression characteristic of a responsive subject, then the gene expression level supports a determination that the individual subject is more likely to be a responder.
  • the expression level or normalized expression level of a response indicator gene from an individual patient can be compared, directly or indirectly, to the expression level or normalized expression level of the response indicator gene in a clinically relevant subpopulation of patients, For example, when compared indirectly, the expression level or normalized expression level of the response indicator gene from the individual patient may be used to calculate a score based on a prediction of the efficacy of treatment with a platinum-based chemotherapy drug,
  • Methods of gene expression profiling include methods based on hybridization analysis of polynucleotides, methods based on sequencing of polynucleotides, and proteomics- based methods.
  • Exemplary methods known in the art for the quantification of mRNA expression in a sample include northern blotting and in situ hybridization (Parker & Barnes, Methods in Molecular Biology 106:247-283 (1999)); RNAse protection assays (Hod, Biotechniques 13:852- 854 (1992)); and PCR-based methods, such as reverse transcription PGR (RT-PCR) (Weis et al., Trends in Genetics 8:263-264 (1992)).
  • RT-PCR reverse transcription PGR
  • Antibodies may be employed that can recognize sequence-specific duplexes, including DNA duplexes, RNA duplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes.
  • Representative methods for sequencing-based gene expression analysis include Serial Analysis of Gene Expression (SAGE), and gene expression analysis by massively parallel signature sequencing (MPSS),
  • mRNA is isolated from a sample.
  • the starting material is typically total RNA isolated from a human tumor, usually from a primary tumor.
  • normal tissues from the same patient can be used as an internal control mRNA can be extracted from a tissue sample, e.g., from a sample that is fresh, frozen (e.g. fresh frozen), or paraffin-embedded and fixed (e.g. formalin-fixed),
  • RNA isolation can be performed using a purification kit, buffer set and protease from commercial manufacturers, such as Qiagen, according to the manufacturer's instructions. For example, total RNA from cells in culture can be isolated using Qiagen RNeasy mini-columns.
  • RNA isolation kits include MasterPureTM Complete DNA and RNA Purification Kit (EPICENTRE®, Madison, WI), and Paraffin Block RNA isolation Kit (Ambion, Inc.).
  • Total RNA from tissue samples can be isolated using RNA Stat-60 (Tel-Test), RNA prepared from a tumor sample can be isolated, for example, by cesium chloride density gradient eentrifugation,
  • the sample containing the RNA is then subjected to reverse transcription to produce e-DNA from the RNA template, followed by exponential amplification in a PCR reaction.
  • the two most commonly used reverse transcriptases are avian myeloblastosis vims reverse transcriptase (AMV-RT) and Moloney murine leukemia virus reverse transcriptase (MMLV-RT),
  • AMV-RT avian myeloblastosis vims reverse transcriptase
  • MMLV-RT Moloney murine leukemia virus reverse transcriptase
  • the reverse transcription step is typically primed using specific primers, random hexaniers, or oligo-dT primers, depending on the circumstances and the goal of expression profiling,
  • extracted RNA can be reverse-transcribed using a GeneAmp RNA PCR kit (Perkin Elmer, CA, USA), following the manufacturer's instructions.
  • PCR-based methods use a thermostable DNA-dependent DNA polymerase, such as a Taq DNA polymerase.
  • TaqMan® PGR typically utilizes the 5 '-nuclease activity of Taq or Tth polymerase to hydroiyze a hybridization probe bound to its target amplicon, but any enzyme with equivalent 5' nuclease activity can be used.
  • a third oligonucleotide, or probe can be designed to facilitate detection of a nucleotide sequence of the amplicon located between the hybridization sites of the two PGR primers.
  • the probe can be detectably labeled, e.g., with a reporter dye, and can further be provided with both a fluorescent dye, and a quencher fluorescent dye, as in a Taqman® probe configuration. Where a Taqman® probe is used, during the amplification reaction, the Taq DNA polymerase enzyme cleaves the probe in a
  • the resultant probe fragments disassociate in solution, and signal from the released reporter dye is free from the quenching effect of the second fluorophore.
  • One molecule of reporter dye is liberated for each new molecule synthesized, and detection of the unquenched reporter dye provides the basis for quantitative interpretation of the data,
  • TaqMan® RT-PCR can be performed using commercially available equipment such as, for example, ⁇ PRISM 7700 or 7900 1 M Sequence Detection System I M (Perkm-Elmer- Appiied Biosystems, Foster City, CA, USA), or Lightcycler (Roche Molecular Biochetnicals, Mannheim, Germany).
  • the 5' nuclease procedure is run on a real-time quantitative PGR device such as the ABI PRISM 7700 iM Sequence Detection SystemTM.
  • the system consists of a thermocycler, laser, charge-coupled device (CCD), camera and computer. The system amplifies samples in a 384-welI format on a thermocycler.
  • the RT-PCR may be performed in triplicate wells with an equivalent of 2ng RNA input per 10 LtL-reaction volume.
  • laser-induced fluorescent signal is collected in real-time through fiber optics cables for ail wells, and detected at the CCD,
  • the system includes software for running the instrument and for analyzing the data,
  • 5'-Nuclease assay data are generally initially expressed as a threshold cycle ("C ") ⁇ Fluorescence values are recorded during every cycle and represent die amount of product amplified to that point in the amplification reaction.
  • the threshold cycle (Q) is generally described as the point when the fluorescent signal is first recorded as statistically significant.
  • RT-PCR is usually performed using an internal standard
  • the ideal internal standard gene (also referred to as a reference gene) is expressed at a constant level among cancerous and non-cancerous tissue of the same origin (i.e., a level that is not significantly different among normal and cancerous tissues), and is not significantly affected by the experimental treatment (i.e., does not exhibit a significant difference in expression level in the relevant tissue as a result of exposure to chemotherapy).
  • reference genes useful in the methods disclosed herein should not exhibit significantly different expression levels in cancerous colon as compared to normal colon tissue.
  • RNAs most frequently used to normalize patterns of gene expression are mRNAs for the housekeeping genes g!yceraidehyde-3-phosphate-dehydrogenase (GAPDH) and ⁇ -actin.
  • Exemplary reference genes used for normalization comprise one or more of the following genes: ATP5E, GPX1, PGK1 , UBB, and VDAC2.
  • Gene expression measurements can he normalized relative to the mean of one or more (e.g., 2, 3, 4, 5, or more) reference genes.
  • Reference-normalized expression measurements can range from 0 to 15, where a one unit increase generally reflects a 2-fold increase in RNA quantity.
  • Real time PGR is compatible both with quantitative competitive PGR, where an internal competitor for each target sequence is used for normalization, and with quantitative comparative PGR using a normalization gene contained within the sample, or a housekeeping gene for RT-PCR.
  • quantitative competitive PGR where an internal competitor for each target sequence is used for normalization
  • quantitative comparative PGR using a normalization gene contained within the sample, or a housekeeping gene for RT-PCR.
  • RNA isolation, purification, primer extension and amplification can be performed according to methods available in the art. (see, e.g., Godfrey et al. J. Molec. Diagnostics 2: 84-91 (2000); Speeht et al., Am. J. Pathol. 158: 41 -29 (2001)). Briefly, a representative process starts with cutting about 10 ⁇ thick sections of paraffin-embedded tumor tissue samples. The RNA is then extracted, and protein and DNA are depleted from the RNA-containing sample. After analysis of the RNA concentration, RNA is reverse transcribed using gene specific primers followed by RT-PCR to provide for cDNA amplification products.
  • PCR primers and probes can be designed based upon exon or intron sequences present in the mRNA transcript of the gene of interest.
  • Primer/probe design can be performed using publicly available software, such as the DNA BLAT software developed by Kent, W.J,, Genome Res. 12(4):656 ⁇ 64 (2002), or by the BLAST software including its variations.
  • repetitive sequences of the target sequence can be masked to mitigate non-specific signals.
  • exemplary tools to accomplish this include the Repeat Masker program available on-line through the Baylor College of Medicine, which screens DNA sequences against a library of repetitive elements and returns a query sequence in which the repetitive, elements are masked.
  • the masked sequences can then be used to design primer and probe sequences using any commercially or otherwise publicly available primer/probe design packages, such as Primer Express (Applied Biosystems); MGB assay ⁇ by ⁇ design (Applied
  • PGR primer design Other factors that can influence PGR primer design include primer length, melting temperature (Tm), and G/C content, specificity, complementary primer sequences, and 3 '-end sequence, in general, optimal PGR primers are generally 17-30 bases in length, and contain about 20-80%, such as, for example, about 50-60% G+C bases, and exhibit Tm's between 50 and 80 °C, e.g. about 50 to 70 °C,
  • Tables A and B provide exemplary primer, probe, and ampiicon sequences for each of the response indicator genes of the invention.
  • the obtained cDNA is spiked with a synthetic DNA molecule (competitor), which matches the targeted cDNA region in all positions, except a single base, and serves as an internal standard.
  • the cDNA/corapetitor mixture is PCR amplified and is subjected to a post-PCR shrimp alkaline phosphatase (SAP) enzyme treatment, which results in the dephosphorylation of the remaining nucleotides.
  • SAP shrimp alkaline phosphatase
  • the PCR products from the competitor and cDNA are subjected to primer extension, which generates distinct mass signals for the competitor- and cDNA-denved PCR. products. After purification, these products are dispensed on a chip array, which is pre-loaded with components needed for analysis with matrix- assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TQF MS) analysis.
  • MALDI-TQF MS matrix- assisted laser desorption ionization time-of-flight mass spectrometry
  • the cDNA present in the reaction is then quantified by analyzing the ratios of the peak areas in the mass spectrum generated, For further details see, e.g. Ding and Cantor, Proc. Nail. Acad. Sci. USA 100:3059-3064 (2003).
  • PCR-based techniques that can find use in the methods disclosed herein include, for example, BeadArray® technology (Illumina, San Diego, CA; Oliphant et al.,
  • Expression levels of a gene of interest can also be assessed using the microarray technique.
  • polynucleotide sequences of interest including cDNAs and
  • oligonucleotides are arrayed on a substrate.
  • the arrayed sequences are then contacted under conditions suitable for specific hybridization with detectably labeled cDNA generated from mRNA of a sample.
  • the source of mRNA typically is total RNA isolated from a tumor sample, and optionally from normal tissue of the same patient as an internal control or cell lines.
  • mRNA can be extracted, for example, from frozen or archived paraffin-embedded and fixed (e.g. forma!in-fixed) tissue samples.
  • PGR amplified inserts of cDNA clones of a gene to be assayed are applied to a substrate in a dense array. Usually at least 10,000 nucleotide sequences are applied to the substrate.
  • the microarrayed genes, immobilized on the microchip at 10,000 elements each are suitable for hybridization under stringent conditions. Fluorescent!y labeled cDNA probes may be generated through incorporation of fluorescent nucleotides by reverse transcription of RN A extracted from tissues of interest. Labeled cDNA probes applied to the chip hybridize with specificity to each spot of DNA on the array.
  • the chip After washing under stringent conditions to remove non-specifically bound probes, the chip is scanned by confocal laser microscopy or by another detection method, such as a CCD camera. Quantitation of hybridization of each arrayed element allows for assessment of corresponding mRNA abundance.
  • Serial analysis of gene expression is a method that allows the simultaneous and quantitative analysis of a large number of gene transcripts, without the need of providing an individual hybridization probe for each transcript.
  • a short sequence tag (about 10-14 bp) is generated that contains sufficient information to uniquely identify a transcript, provided that the tag is obtained from a unique position within each transcript.
  • many transcripts are linked together to form long serial molecules, that can be sequenced, revealing the identity of the multiple tags simultaneously.
  • the expression pattern of any population of transcripts can be quantitatively evaluated by determining the abundance of individual tags, and identifying the gene corresponding to each tag. For more details see, e.g. Velculescu et al., Science 270:484-487 (1995): and
  • Nucleic acid sequencing technologies are suitable methods for analysis of gene expression.
  • the principle underlying these methods is that the number of times a cDNA sequence is detected in a sample is directly related to the relative expression of the raR A corresponding to that sequence.
  • DGE Digital Gene Expression
  • Early methods applying this principle were Serial Analysis of Gene Expression (SAGE) and Massively Parallel Signature Sequencing (MPSS), See, e.g., S. Brenner, et al., Nature Biotechnology 18(6);630-634 (2000). More recently, the advent of "next-generation" sequencing technologies has made DGE simpler, higher throughput, and more affordable. See, e.g., W.
  • RNA for expression analysis from blood, plasma and serum (see for example, Tsui NB et al. (2002) Clin. Chem. 48,1647-53 and references cited therein) and from urine ⁇ see for example, Boom R et al (1990) J Clin Microbiol. 28, 495-503 and reference cited therein) have been described.
  • Inimunohistocheniistry methods are also suitable for detecting the expression levels of genes and applied to the method disclosed herein, Antibodies (e.g., monoclonal antibodies) that specifically bind a gene product of a gene of interest can be used in such methods.
  • the antibodies can be detected by direct labeling of the antibodies themselves, for example, with radioactive labels, fluorescent labels, hapten labels such as biotin, or an enzyme such as horse radish peroxidase or alkaline phosphatase.
  • unlabeled primary antibody can be used in conjunction with a labeled secondary antibody specific for the primary antibody.
  • proteome is defined as the totality of the proteins present in a sample (e.g. tissue, organism, or cell culture) at a certain point of time. Proteomics includes, among other things, study of the global changes of protein expression in a sample (also referred to as
  • Proteomics typically includes the following steps: (1) separation of individual proteins in a sample by 2-D gel electrophoresis (2-D PAGE); (2) identification of the individual proteins recovered from the gel, e.g. my mass spectrometry or N- terminal sequencing, and (3) analysis of the data using bioinformatics.
  • RNA isolation, purification, primer extension and amplification are provided in various published journal articles. ⁇ See, e.g., T.E. Godfrey et a!,. J. Moke. Diagnostics 2: 84-91 (2000); K. Specht et. ah, Am. J. Pathol. 158: 419-29 (2001), M. Cronin, et ah, Am J Pathol 164:35-42 (2004)). Briefly, a representative process starts with cutting a tissue sample section (e.g. about 10 ⁇ thick sections of a paraffin-embedded tumor tissue sample).
  • a tissue sample section e.g. about 10 ⁇ thick sections of a paraffin-embedded tumor tissue sample.
  • RNA is then extracted, and protein and DMA are removed. After analysis of the RN A concentration, RNA repair is performed if desired.
  • the sample can then be subjected to analysis, e.g., by reverse transcription using gene specific promoters followed by PGR,
  • Table 3 provides genes that co-express with particular response indicator genes that have been identified as having a correiation with a positive response to a platinum-based chemotherapy drug.
  • genes often work together in a concerted way, i.e. they are co-expressed, Co-expressed gene groups identified for a disease process like cancer can also serve as response indicator genes.
  • Such co-expressed genes can be assayed in lieu of, or in addition to, assaying of the response indicator gene with which they co- express.
  • a correlation coefficient of equal to or greater than 0.3 is considered to be statistically significant in a sample size of at least 20. ⁇ See e.g., G. Norman, D. Streiner, Biostatistics: The Bare Essentials, 137-138 (3 rd Ed. 2007).)
  • raw expression level data measured for a gene product may be normalized relative to the mean expression level data obtained for one or more reference genes.
  • reference genes include housekeeping genes, such as GAPDH, Alternatively, all of the assayed genes or a large subset thereof may also concurrently serve as reference genes and normalization can be based on the mean or median signal (Ct) of all of the assayed genes or a subset thereof (often referred to as "global normalization" approach).
  • measured normalized amount of a patient tumor mRNA may be compared to the amount found in a cancer tissue reference set. See e.g., Cronin, M, et al., Am. Soc.
  • the normalization may be carried out such that a one unit increase in normalized expression level of a gene product generally reflects a 2-fold increase- in quantity of expression product present in the sample.
  • normalization techniques applicable to qRT-PCR data from tumor tissue see e.g., Silva, S, et al, (2006) BMC Cancer 6, 200; deKok, J. et al. (2005) Laboratory Investigation 85, 154-159.
  • the materials for use in the methods of the present invention are suited for preparation of kits produced in accordance with well known procedures.
  • kits comprising agents, which may include gene-specific or gene-selective probes and/or primers, for quantitating the expression of the disclosed genes for predicting prognostic outcome or response to treatment
  • agents which may include gene-specific or gene-selective probes and/or primers, for quantitating the expression of the disclosed genes for predicting prognostic outcome or response to treatment
  • kits may optionally contain reagents for the extraction of RNA from tumor samples, in particular, fixed paraffin-em bedded tissue samples and/or reagents for RNA amplification.
  • the kits may optionally comprise the reagent(s) with an identifying description or label or instructions relating to their use in the methods of the present invention.
  • kits may comprise containers (including microliter plates suitable for use in an automated implementation of the method), each with one or more of the various reagents (typically in concentrated form) utilized in the methods, including, for example, pre-fabricated mieroarrays, buffers, the appropriate nucleotide triphosphates (e.g., dATP, dCTP, dGTP and dTTP; or rATP, rCTP, rGTP and UTP), reverse transcriptase, DNA polymerase, RNA polymerase, and one or more probes and primers of the present invention (e.g., appropriate length poly(T) or random primers linked to a promoter reactive with the RNA polymerase).
  • the appropriate nucleotide triphosphates e.g., dATP, dCTP, dGTP and dTTP; or rATP, rCTP, rGTP and UTP
  • reverse transcriptase DNA polymerase
  • RNA polymerase e.g.
  • a "report" as described herein is an electronic or tangible document that includes elements that provide information of interest relating to a likelihood assessment and its results
  • a subject report includes at least a likelihood assessment, e.g., an indication as to the likelihood that a cancer patient will experience colon cancer recurrence after a treatment regimen with a platinum -based chemotherapy drug and the likelihood that a cancer patient will experience colon cancer recurrence without treatment with a platinum-based chemotherapy drug.
  • a subject report can be completely or partially
  • a report can further include one or more of: I) information regarding the testing facility; 2) service provider information; 3) patient data: 4) sample data; 5) an interpretive report, which can include various information including: a) indication; b) test data, where test data can include a normalized level of one or more genes of interest, and 6) other features.
  • the present invention therefore provides methods of creating reports and the reports resulting therefrom.
  • the report may include a summary of the expression levels of the RNA transcripts, or the expression products of such RNA transcripts, for certain genes in the cells obtained from the patient's tumor tissue.
  • the report may include a prediction that a particular chemotherapy has an increased efficacy or the report may include a prediction that the
  • the report may include a recommendation for a treatment, modality such as FU-based chemotherapy or FU -based chemotherapy in combination with a platinum-based drug.
  • the report may be presented in electronic format or on paper,
  • the methods of the present invention further include generating a report that includes information regarding efficacy of treatment with a platinum-based chemotherapy drug, such as oxaliplatin.
  • the methods of the present invention can further include a step of generating or outputting a report providing the results of a patient response likelihood assessment, which can be provided in the form of an electronic medium (e.g., an electronic display on a computer monitor), or in the form of a tangible medium (e.g., a report printed on paper or other tangible medium).
  • a report that includes information regarding the efficacy of treatment with a platinum-based chemotherapy drug, such as oxaliplatin, is provided to a user.
  • the report may include information regarding the efficacy of treatment with oxaliplatin in preventing colon cancer recurrence.
  • a person or entity who prepares a report (“report generator”) may also perform the likelihood assessment, The report generator may also perform one or more of sample gathering, sample processing, and data generation, e.g., the report generator may also perform one or more of: a) sample gathering; b) sample processing: c) measuring a level of a response indicator gene expression produces); d) measuring a le vel of a reference gene product(s); and e) determining a normalized level of a response indicator gene expression product(s).
  • an entity other than the report generator can perform one or more sample gathering, sample processing, and data generation.
  • the term "user” or “client” refers to a person or entity to whom a report is transmitted, and may be the same person or entity who does one or more of the following: a) collects a sample; b) processes a sample; c) provides a sample or a processed sample; and d) generates data (e.g., level of a predictive, gene expression product(s); level of a reference gene produet(s); normalized level of a predictive gene expression produci(s)) for use in the likelihood assessment,
  • the person or entity who provides sample collection and/or sample processing and/or data generation, and the person who receives the results and/or report may be different persons, but are both referred to as "users" or “clients.”
  • the user or client provides for data input and review of data output.
  • a "user” can be a health professional (e.g., a clinician, a laboratory technician,
  • the individual who, after computerized data processing according to the methods of the invention, reviews data output is referred to herein as a "reviewer,"
  • the reviewer may be located at a location remote to the user (e.g., at a service provided separate from a healthcare facility where a user may be located).
  • the methods and systems described herein can be implemented in numerous ways. In one embodiment of the invention, the methods involve use of a communications infrastructure, for example, the internet. Several embodiments of the invention are discussed below.
  • the present invention may also be implemented in various forms of hardware, software, firmware, processors, or a combination thereof.
  • the methods and systems described herein can be implemented as a combination of hardware and software.
  • the software can be implemented as an application program tangibly embodied on a program storage device, or different portions of the software implemented in the user's computing environment (e.g., as an applet) and on the reviewer's computing environment, where the reviewer may be located at a remote site (e.g., at a service provider's facility).
  • portions of the data processing can be performed in the user-side computing environment.
  • the user-side computing environment can be programmed to provide for defined test codes to denote a treatment efficacy "score," where the score is transmitted as processed or partially processed responses to the reviewer's computing environment in the form of test code for subsequent execution of one or more algorithms to provide a result and/or generate a report in the reviewer's computing environment.
  • the score can be a numerical score (representative of a numerical value) or a non-numerical score representative of a numerical value or range of numerical values (e.g., "A”: representative of a 90-95% likelihood of a positive response; "High”: representative of a greater than 50% chance of a positive response (or some other selected threshold of likelihood); “Low”: representative of a less than 50% chance of a positive response (or some other selected threshold of likelihood), and the like.
  • the system generally includes a processor unit.
  • the processor unit operates to receive information, which can include test data (e.g., level of a predictive gene product(s); level of a reference gene produet(s); normalized level of a predictive gene product(s); and may also include other data such as patient data.
  • This information received can be stored at least temporarily in a database, and data analyzed to generate a report as described above,
  • Part or ail of the input and output data can also be sent electronically.
  • Certain output data e.g., reports
  • Exemplary output receiving devices can include a display element, a printer, a facsimile device and the like.
  • Electronic forms of transmission and/or display can include email, interactive television, and the like.
  • all or a portion of the input data and/or output data e.g., usually at least the final report
  • the input and output data, including ali or a portion of the final report can be used to populate a patient's medical record that may exist in a confidential database as the healthcare facility.
  • the present invention also contemplates a computer-readable storage medium (e.g., CD-ROM, memory key, flash memory card, diskette, etc.) having stored there on a program which, when executed in a computing environment, provides for implementation of algorithms to carry out all or a portion of the results of a response likelihood assessment as described herein.
  • a computer-readable storage medium e.g., CD-ROM, memory key, flash memory card, diskette, etc.
  • the program includes program instructions for collecting, analyzing and generating output, and generally includes computer readable code devices for interacting with a user as described herein, processing that data in conjunction with analytical information, and generating unique printed or electronic media for that user.
  • the storage medium includes a program that provides for implementation of a portion of the methods described herein (e.g., the user-side aspect of the methods (e.g., data input, report receipt capabilities, etc,)), the program provides for transmission of data input by the user (e.g., via the internet, via an intranet, etc.) to a computing environment at a remote site.
  • the program provides for transmission of data input by the user (e.g., via the internet, via an intranet, etc.) to a computing environment at a remote site.
  • Processing or completion of processing of the data is carried out at the remote site to generate a report. After review of the report, and completion of any needed manual intervention, to provide a complete report, the complete report is then transmitted back to the user as an electronic document or printed document (e.g., fax or mailed paper report).
  • the storage medium containing a program according to the invention can be packaged with instructions (e.g., for program installation, use, etc.) recorded on a suitable substrate or a web address where such instructions may be obtained,
  • the computer-readable storage medium can also be provided in combination with one or more reagents for carrying out a response likelihood assessment (e.g., primers, probes, arrays, or such other kit components).
  • An objective of the study was to determine whether there is a significant relationship between the expression of the genes and clinical benefit from addition of oxalipiatin to adjuvant 5-FU/LV chemotherapy.
  • Improvement in a clinical endpoint such as recurrence-free interval (RFI), overall survival (OS), and disease-free survival (DPS), reflects an increased likelihood of response to treatment with oxalipiatin and a likelihood of a positive response.
  • RFID recurrence-free interval
  • OS overall survival
  • DPS disease-free survival
  • Gene expression of each gene was normalized relative to median expression of the gene candidates for a given patient. Median normalization corrects for degradation due to sample processing (e.g. fixation). The relationship between individual gene expression and clinical benefit from the addition of oxaiipiatin to adjuvant 5-FU/LV chemotherapy was assessed using multivariate Cox Proportional Hazards models which included Qnco pe DX® Colon Cancer Recurrence Score, nodal status (1 -3 vs. 4+ positive nodes), treatment (FU vs. FU
  • the genes were divided in three prespecified classes based on the level of evidence of the relationship between each gene and oxaiipiatin benefit (see, e.g., K, Harradine et al, Mo! Cancer Res. 201 1 9(2): 173-82, in-depth literature searches) and "separate class" analysis was carried out (B. Efron, Ann. Appi. Statist. 2:197-223 (2008)), A false discovery rate was calculated within each class, utilizing information from all the genes to produce efficient estimates of the false discovery rates within each class.
  • Tables 1 and 2 contain 72 genes identified with q-value (probability that a particular gene is a false discovery) ⁇ 0.40. Standardized hazard ratio estimates, p-values, and corresponding 95% confidence intervals for the hazard ratio for interaction of gene expression and treatment are also provided.
  • increased expression level of the one or more genes selected from Table 1 was negatively correlated with efficacy of treatment comprising oxaiipiatin in preventing cancer recurrence in a patient with colorectal cancer
  • increased expression level of one or more genes selected from Table 2 was positively correlated with efficacy of treatment comprising oxaiipiatin in preventing cancer recurrence in a patient with colorectal cancer
  • increased expression level of one or more genes selected from TXNRDI, MAPREL ABCC4, SIX 1 A3, CDK2, and AVEN was positively correlated with efficacy of treatment comprising oxaliplatin in reducing the likelihood of colon cancer recurrence
  • increased expression level of one or more genes selected from RFC, MTA 1 , PTMA, HNRNPD, ANAPC2, SPINT2, ZNF143, EHMTl, SP1 , and MUTYH was negatively- correlated with efficacy of treatment comprising oxaliplatin in reducing the likelihood of colon cancer recurrence
  • a heterogeneity study was conducted to assess between and within patient variability of gene expression.
  • a total of 102 commercially obtained samples from 88 blocks and 38 patients were used, A panel of genes included the 16 predictive genes as well as stromal, immune, cell cycle and reference genes to provide a base for comparison.
  • RT-PCR was used to measure the variability in gene expression between different patients.
  • Figure 1 contains a summary of the within and between patient variability for reference normalized expression of 16 predictive genes and a set of stromal gene known to have high within patient variability.
  • oxaiipiatin benefit prediction genes had about 6 times lower within patient variance compared to stromal genes and thus were found to be robust with respect to heterogeneity,
  • the top 20 multi-gene models without ANAPC2 are presented in Table 5.
  • the models without ZNF143, the gene which was not favored based on the functional form analysis, are boided.
  • the bolded models contained 7 to 9 genes and provided similar performance (4- factor RM-corrected standardized hazard ratios 1.90-1.89).
  • the highlighted model in Table 5, i.e., AVER CDK2, MTA1, PTMA, RFC, SLCI A3, SPI, SPINT2, and TXNRD1 was chosen as the final model to increase the chance of including a potential true predictive gene.
  • Based on the final 9 genes (Table 6). approximately 1 out of 9 genes is expected to be a false discovery (sum of q-values ⁇ 1.07). Simulation analysis of all possible models with 8 predictive genes and 1 false discovery showed that, on average, the log hazard ratio of such models is expected to decrease by about 3% only (data on file) compared to the final model with 9 predictive genes.
  • Table 5 Top 20 models without ANAPC2 and with SCL1A3 threshold
  • Table 6 identifies the individual genes of the 9-gene model (MTAL PTMA, SP1, RF C I, SPINT2, SLC1A3, TXNRD1, AVEN, and CDK2), It additionally provides the standardized HR with 95% confidence intervals, Wald p-vaiue, q-value (FDR), and RM ⁇ corrected ABS STD FIR for each of the genes of the 9-gene model.
  • Table 6 also indicates the prespecified class based on the level of evidence of the relationship between each gene and oxali latin benefit, the biological category, and the relationship between gene expression and oxalipiatin benefit.
  • MTAl The biological functions of MTAl , PTMA, SPl, RFC1 , SPINT2, SLC1A3, TXNR.D1, AVEN, and CDK2 were further considered in order to assign those genes to biological categories.
  • Table 7 shows the bioiogicai category for each of the individual genes in the 9-gene model.
  • MTAl, PTMA, SPl, RFC ] , SPrNT2, S LCI A3, TXNRI3L AVEN, and CDK2 were assigned to one or more bioiogicai pathway, including transcription regulation, replication and DNA repair, cell cycle
  • Table 8 shows the performance of the 9-gene model based that assignment. As shown in Table 8, a given gene may be present in more than one pathway. Accordingly, Table 8 provides the pathway, genes, uncorrected Sid HR and 95% CI, and the relationship between higher expression and oxaiipiatin benefit.
  • increased expression level of one or more genes selected from AVEN and CDK2 was positively correlated with efficacy of treatment comprising oxaiipiatin in reducing the likelihood that a patient will experience colon cancer recurrence
  • increased expression level of SLC 1A3 was positively correlated with efficacy of treatment comprising oxaiipiatin in reducing the likelihood that a patient will experience colon cancer recurrence
  • increased expression level of TXNRDl was positively correlated with efficacy of treatment comprising oxaiipiatin in reducing the likelihood that a patient will experience colon cancel- recurrence .
  • increased expression level of one or more genes selected from MTA1, PTMA, and SP1 was negatively correlated with efficacy of treatment, comprising oxaiipiatin in reducing the likelihood that a patient will experience colon cancer recurrence.
  • Increased expression level of one or more genes selected from PTMA, SP1, and RFC1 was negatively correlated with efficacy of treatment comprising oxaiipiatin in reducing the likelihood that a patient will experience colon cancer recurrence.
  • Increased expression level of one or more genes selected from PTMA, and SPINT2 was negatively correlated with efficacy of treatment comprising oxaiipiatin in reducing the likelihood that a patient will experience colon cancer recurrence.
  • Figure 2 depicts the ability of the 9 ⁇ gene model to identify patients who would substantially benefit from addition of oxaiipiatin to 5FU-based chemotherapy. Specifically. Figure 2 shows a treatment effect hazard ratio predictiveness curve with 95% confidence intervals, with the x-axis showing the population quantile and the y-axis showing the RM- Corrected Treatment Effect Hazard Ratio. Assuming a 'no benefit' group includes patients with treatment effect HR of 0.95 or higher, the final 9-gene model is able to identify 33,2% of patients who would not benefit from addition of oxaliplatin to 5FU based chemotherapy.
  • Figures 3A-3C show time-to-event "survival" curves for the 9-gene model shown in Table 6. Specifically, Figures 1 A-3C show RM-correeted estimates of the proportion of patients remaining free from cancer recurrence (RFI endpoint) at each time after surgery, comparing patients with stage 3 colorectal cancer who were treated with 5FU to patients with stage 3 colorectal cancer who were treated with 5FU+oxalipIatin. The x ⁇ axis shows the time (in years) and the y-axis shows the probability that a patient will remain recurrence free.
  • Figure 3A shows patients in the low tertile of the 9-gene directed sum
  • Figure 3B shows patients in the middle tertile of the 9-gene directed sum
  • Figure 3C shows patients in the upper tertile of the 9-gene directed sum.
  • the survival curves in Figure 3 indicate that patients in the low tertile would not appear to benefit from the addition of oxaliplatin to 5FU treatment (Figure 3A), the patients in the middle tertile appear to derive some benefit from the addition of oxaliplatin to 5FU treatment, and patients in the upper tertile seem to derive more benefit from the addition of oxaliplatin to 5FU treatment (Figure 3C).

Abstract

The present invention provides methods for predicting the efficacy of a treatment with a platinum-based chemotherapy drug in a patient with cancer. The methods generally involve determining an expression level of a gene product that correlates with responsiveness to treatment with a platinum-based chemotherapy drug. In an embodiment of the invention, the platinum-based chemotherapy drug is oxaliplatin, and the cancer is colorectal cancer.

Description

RELATED APPLICATION
[0001] This application claims the benefit of U.S. Provisional Application No, 61/603,697, filed February 27, 2012, which is hereby incorporated by reference in its entirety,
[Θ 02] The present invention relates to genes, the expression levels of which are useful for predicting response of cancer cells and cancer patients to a platinum-based chemotherapy drug.
BACKGROUND
[0003] Platinum-based cancer chemotherapies have had a major clinical impact in the treatment of patients with cancer. Furthermore, an emerging clinical strategy is that the optimal efficacy of novel targeted therapies may be in combination with existing cytotoxic DNA-damaging agents, including oxaiipiatin. Given the expanding role of oxaiipiatin in cancer treatment, it has become increasingly important to understand molecular predictors of oxaiipiatin response in order to provide for more personalized administration of chemotherapy.
[0004] Oxaiipiatin is a third-generation platinum-based ehemotherapeutic agent that has significant activity in colorectal cancer (CRC). Adjuvant therapy with oxaiipiatin, combined with fluoropyrimidine-based chemotherapy, results in significant increases in disease-free survival rates in patients with stage Will colon cancer (Andre, T,, et al.5 "Oxaiipiatin, Fluorouracii, and
Leucovorin as Adjuvant Treatment for Colon Cancer," N. Engl. J. Med., 2004. 350(23): p. 2343- 51 ). In the metastatic setting, combination therapy with 5-FU and oxaiipiatin is the most commonly used front-line regimen, with superior response rates and longer survival than 5-FU alone (Rothenberg, M.L., et ah, "Superiority of Oxaiipiatin and Fiuorouracil-Leucovorin
Compared with Either Therapy Alone in Patients with Progressive Colorectal Cancer After Irinotecan and Fiuorouracil-Leucovorin: interim Results of a Phase ΙΠ Trial," J. Clin. Oncol., 2003. 21(1 1): p. 2059-69; de Gramont, A., et ah, "Rein traduction of Oxaiipiatin is Associated With Improved Survival in Advanced Colorectal Cancer," J. Clin. Oncol., 2007. 25(22): p. 3224- 9). However, it. is apparent that not all patients benefit from oxaiipiatin treatment, and in the face of significant side-effects associated with oxaiipiatin, most notably prolonged neurotoxicity, there is a need for clinical tools to guide use of oxaiipiatin in those patients who are most likely to derive benefit. [0005] Oxa!iplatin induces cytotoxicity through the formation of platinum-DNA adducts, which in turn, activate multiple signaling pathways (Kei!and, L,, "The Resurgence of Platinum-Based Cancer Chemotherapy," Nat. Rev. Cancer, 2007, 7(8): p, 573-84). Alterations in drug efflux and uptake, DNA repair and inactivation of the apoptosis pathways have been hypothesized to promote resistance to platinum agents such as carboplatin and cisplatin (Wang, D. and S.J. Lippard, "Cellular Processing of Platinum Anticancer Drugs," Nat. Rev. Drug Discov., 2005. 4(4): p. 307- 320; Siddick, Z.H., "Cisplatin: Mode of Cytotoxic Action and Molecular Basis of Resistance," Oncogene, 2003, 22(47): p, 7265-79). None of these putative markers of oxaiipiatin sensitivity and resistance have been clinicaily validated, and at present, there are no markers established in clinical use for selecting CRC patients for oxaiipiatin therapy.
[0006] The current clinical practice used for making CRC treatment decisions is determined by clinical and pathological staging. However, these prognostic tools do not predict drug response in an individual patient. Recent insights into the genomics of cancers have enabled development of diagnostic tests that inform clinical decisions for cancer patients (Harris, L„ et ai., "American Society of Clinical Oncology 2007 Update of Recommendations for the Use of Tumor Markers in Breast Cancer," J. Clin. Oncol., 2007. 25(33): p. 5287-312; Dunn., L. and A. Demichele,
"Genomic Predictors of Outcome and Treatment Response in Breast Cancer," Mol. Diagn. Ther., 2009. 13(2): p. 73-90; Paik, S,, et al,, "A Multigene Assay to Predict Recurrence of Tamoxifen- Treated, Node-Negative Breast Cancer," N. Engl. J. Med., 2004. 351(27): p. 2817-25; Paik, S., et al, "Gene Expression and Benefit of Chemotherapy in Women With Node-Negative, Estrogen Receptor-Positive Breast Cancer," J. Clin. Oncol., 2006. 24(23): p. 3726-34), To further advance the personalization of CRC treatment, there is a need for a greater understanding of the genetic alterations in CRC tumors that are associated with patient sensitivity or resistance to oxaiipiatin.
[0007] The present invention provides response indicator genes for platinum-based chemotherapy drugs. These genes are provided in Tables 1 and 2, The present invention also provides gene subsets of" the response indicator genes based on their known function. These gene subsets include, but are not limited Co, invasion/migration, proliferation, DNA damage repair, apoptosis, cell cycle control, MAPK signaling, FGF Signaling, PI3K/AKT, antioxidant defense, Wnt signaling, thyroxine signaling, ion transport, oxidative stress response, stress induced, G-protein signaling, heat shock, pyridine metabolism, ABC transporters, glutathione metabolism, methionine metabolism, TGF beta, immune inflammation, amino acid metabolism, and folic acid metabolism gene subsets. Tables 1 and 2 provide gene subsets in which each gene may be grouped. The present invention also provides methods of identifying gene cliques, i.e. genes that co-express with a response indicator gene and exhibit correlation of expression with the response indicator gene, and thus may be substituted for that response indicator gene in an assay. Table 3 provides a list of the top 5 genes that co-express with the genes listed in Tables 1 and 2.
[0008] In an embodiment of the invention, increased expression level of one or more response indicator genes selected from Table 1 is negatively correlated with the efficacy of a platinum- based chemotherapy drug.
[Ο0Θ9] In another embodiment, of the invention, increased expression level of one or more response indicator genes selected from Table 2 is positively correlated with the efficacy of a platinum-based chemotherapy drug.
[0010 ] in a specific embodiment of the invention, increased expression level of one or more genes selected from RFC1 , MTA1 , PTMA, HNRNPD, ANAPC2, SPINT2, ZNF143, SP1, EHMT1, and MUTYH is negatively correlated with the efficacy of a platinum-based chemotherapy drug in reducing the likelihood of colon cancer recurrence, and increased expression level of one or more genes selected from TXNRD1, MAPRE1, ABCC4, SIX 1 A3, CDK2, and AVEN is positively correlated with the efficacy of a platinum-based chemotherapy drug in reducing the likelihood of colon cancer recurrence.
[0011 ] The present invention further provides methods and compositions for predicting the efficacy of a treatment comprising a platinum-based chemotherapy drug based on the expression level of one or more response indicator genes in a tumor sample obtained from the patient.
Specifically, the method comprises assaying or measuring an expression level of one or more response indicator gene products. The response indicator gene is selected from any one of the genes listed in Tables 1 and 2. In a further embodiment, the expression level of the response indicator gene is normalized. The expression level or the normalized expression level is used to predict the efficacy of a platinum-based chemotherapy drug, wherein increased expression level or increased normalized expression level of one or more response indicator genes selected from Table I is negatively correlated with the efficacy of a platinum -based chemotherapy drug, and increased expression level or increased normalized expression level of one or more response indicator genes selected from Table 2 is positively correlated with the efficacy of a platinum-based chemotherapy drug. In yet another embodiment of the invention, increased expression level of one or more genes selected from RFC 1 , MTA1 , PTMA, HNRNPD, ANAPC2, SP1NT2, Z F143, SPL EHMTI, and MUTYH is negatively correlated with the efficacy of a platinum-based chemotherapy drag, and increased expression level of one or more genes selected from TXNRD1 , MAPREJ , ABCC4, SLC 1A3, CDK2, and AVEN is positively correlated with the efficacy of a platinum-based chemotherapy drug. In a further embodiment of the invention, a report is generated based on the predicted efficacy of a platinum-based chemotherapy drug. [0012] The methods of the present invention contemplate determining the expression level of at least one response indicator gene or its gene product. For all aspects of the present invention, the methods may further include determining the expression levels of at least two response indicator 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 response indicator 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 response indicator 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 response indicator 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 response indicator 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 response indicator 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 response indicator 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 response indicator genes, or their expression products. The methods may involve determination of the expression levels of at least ten (10), at least eleven (1 1), at least twelve (12). at least thirteen (13), at least fourteen (14), at least fifteen (15), or at least sixteen (16) of the response indicator genes, or their expression products.
[0013] The expression level, or normalized expression level, of the response indicator gene, or its expression product, is used to predict the efficacy of a platinum-based chemotherapy drug. In an embodiment of the invention, a likelihood score (e.g., a score predicting the likelihood of cancer recurrence with and without adjuvant treatment with a platinum-based chemotherapy drug) can be caicuiated based on the expression level or normalized expression level, A score may be calculated using weighted values based on the expression level or normalized expression level of a response indicator gene and its contribution to response to a platinum-based chemotherapy drug, [0014] In an embodiment of the invention, the expression product of the response indicator gene to be assayed or measured is an RNA transcript. In one aspect, the RNA transcripts are fragmented. In another embodiment, the expression product is a polypeptide. Determining the expression level of one or more response indicator gene products 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 T- PCR (reverse transcriptase PGR), quantitative RT-PCR (qRT-PCR), or other PCR-based methods. inimunohistochemistry, proteomics techniques, an array-based method, polynucleotide sequencing, or any other methods known in the art or their combination.
[0015] 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, fresh, or frozen tissue. For example, the tissue may be obtained from a surgical resection or by body fluid containing a cancer cell, e.g. urine, blood, etc. In an embodiment of the invention, the tumor sample is obtained from a patient with colorectal cancer. In a specific embodiment of the invention, the patient has stage II (Dukes B) or stage III (Dukes C) colorectal cancer.
[0016] In another embodiment of the invention, the platinum-based chemotherapy drug is se lected from cisplatin. carboplatin, and oxaiiplatin. In a particular embodiment, the platinum-based chemotherapy drug is oxaiiplatin. Oxaiiplatin may be provided alone, or in combination, with one or more additional anti-cancer agents, in a specific embodiment, oxaiiplatin is provided in combination with fluorouracil (5-FU) and leucovorin.
BRIEF DESCRIPTION OF THE DRAWINGS
(0017] FIGS. 1A-1B show the within and between patient standard deviations for 16 genes for predicting a benefit from the addition of oxaiiplatin to 5FU treatment (FIG, I A) and stromal genes (FIG. IB).
[0018] FIG. 2 shows a regression to the mean corrected predictiveness curve for the 9-gene mode! for predicting a benefit from the addition of oxaiiplatin to treatment with 5FU~hased
chemotherapy.
[0019] FIGS. 3A-3C show survival curves for the tertiles of the score from the 9-gene model. FIG. 3 A shows the regression to the mean corrected estimates of survival curves for recurrence- free interval, stage 3 patients only for the first tertile. FIG 3B shows the regression to the mean corrected estimates of survival curves for recurrence-free interval, stage 3 patients only for the second tertile. FIG 3C shows the regression to the mean corrected estimates of survival curves for recurrence-free interval, stage 3 patients only for the third tertile.
[ΘΘ20] 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 ah. Dictionary of Microbiology and Molecular Biology, 2,,d ed., J. Wiley & Sons (New York, NY 1994), and March, Advanced Organic Chemistry Reactions, Mechanisms and Structure, 4th ed., J. Wiley & Sons (New York, NY 1992), provide one skilled in the art with a general guide to many of the terms used in the present application. |002i| One skilled in the art will recognize many methods and materials similar or equivalent to those described herein that may be used in the practice of the present invention. Indeed, the present invention is in no way limited to the methods and materials described herein. For purposes of the invention, the following terms are defined below.
[0022] As used herein, the term "amplicon" refers to a piece ofDNA that has been synthesized using an amplification technique, such as the polymerase chain reaction (PGR) and ligase chain reaction.
[0023] The term "anti-eaneer agent" as used herein refers to any molecule, compound, chemical or composition that has an anti-cancer effect, such as a "positive response" as defined below. Anti-cancer agents include, without limitation, chemotherapeutic agents, radtotherapeutic agents, cytokines, anti-angiogemc agents, apoptosis-inducing agents or anti-cancer immunotoxins, such as antibodies, Examples of anti-cancer agents include, without limitation, methotrexate, taxol, mercaptopurine, thioguanine. hydroxyurea, cytarabine, cyclophosphamide, ifosfamsde, nitrosoureas, mitomycin, dacarbazine, procarbazine, etoposides, eampathecins, bleomycin, doxorubicin, idaruhiein, dai orubicin, dactinomyciii, plicamycin, mitoxantrone, asparaginase, vinblastine, vincristine, vinorelbine, paciitaxel, docetaxel, fluorouracii (5-FU), and ieucovorin. Other anti-cancer agents are known in the art. In an embodiment of the invention, the anti-cancer agent is 5-FU and Ieucovorin,
[0024] The terms "assay" or "assaying" as used herein refer to performing a quantitative or qualitative analysis of a component in a sample. The terms include laboratory or clinical observations, and/or measuring the level of the component in the sample.
[0025] The terms "cancer" and "cancerous" as used herein, refer to or describe the physiological condition that is typically characterized by unregulated cell growth. Examples of cancer in the present application include cancer of the gastrointestinal tract, such as invasive colorectal cancer or Stage ΪΙ (Dukes B) or Stage Til (Dukes C) colorectal cancer,
[0026] The term "co-expressed" as used herein refers to a statistical correlation between the expression level of one gene and the expression level of another gene. Pairwise co-expression may be calculated by various methods known in the art, e.g., by calculating Pearson correlation coefficients or Spearman correlation coefficient. Co-expressed gene cliques may also be identified using a graph theory. An analysis of co-expression may be calculated using normalized expression data.
[0027] The terms "colon cancer" and "colorectal cancer" are used interchangeably herein and refer in the broadest sense to (1 ) all stages and a!! forms of cancer arising from epithelial ceils of the large intestine and/or rectum and/or (2) ail stages and all forms of cancer affecting the lining of the large intestine and/or rectum. In the staging systems used for classification of colorectal cancer, the colon and rectum are treated as one organ,
[0028] The term "correlates" or "correlating" as used herein refers to a statistical association between instances of two events, where events may 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 present invention provides genes and gene subsets, the expression levels of which are correlated with a particular outcome measure, such as between the expression level of a gene and the efficacy of treating a patient with a drug. For example, the increased expression level of a gene product may be positively correlated with efficacy of a chemotherapy drug.. Such a positive correlation may be demonstrated statistically in various ways, e.g, by a gene expression- treatment interaction hazard ratio for cancer recurrence < 1. In another example, the increased expression level of a gene product may be negatively correlated with efficacy of a chemotherapy drug. In this case, for example, chemotherapy may have decreased efficacy. Such a negative correlation indicates that chemotherapy will likely be less efficacious for the patient, and this may be demonstrated statistically in various ways, e.g., a gene expression-treatment interaction hazard ratio for cancer recurrence > 1.
[0029] The expression data used in the methods disclosed herein can be standardized.
Standardization refers to a process to effectively put all the genes on a comparable scale. This is performed because some genes will exhibit more variation (a broader range of expression) than others. Standardization is performed by dividing each expression value by its standard deviation across ail samples for that gene. Hazard ratios are then interpreted as the relative risk of recurrence per 1 standard deviation increase in gene expression.
[0030] The term "Ct" as used herein refers to threshold cycle, the cycle number in quantitative polymerase chain reaction (qPCR) at which the fluorescence generated within a reaction well exceeds the defined threshold, i.e. the point during the reaction at which a sufficient number of amplicons have accumulated to meet the defined threshold.
[0031] The term "expression level" as used herein refers to qualitative or quantitative
determination of an expression product, or gene product. Expression level may be determined for the UNA expression level of a gene or for the polypeptide expression level of a gene. The term "normalized" expression level as used herein refers to an expression level of a response indicator gene relative to the level of an expression product of a reference gene(s), which might be all measured expression products in the sample, a single reference expression product, or a particular set of expression products. A gene exhibits an "increased expression level" when the expression level of an expression product is higher in a first sample, such as in a clinically relevant subpopulation of patients (e.g., patients who are responsive to a platinum-based chemotherapy drug), than in a second sample, such as in a related subpopulation (e.g., patients who are not responsive to the platinum-based chemotherapy drug).
[0Θ32] in the context of an analysis of an expression level of a gene in tissue obtained from an individual subject, a gene exhibits "increased expression," or "increased normalized expression," when the expression level or normalized expression level of the gene in the subject trends toward, or more closely approximates, the expression level or normalized expression level characteristic of a clinically relevant subpopulation of patients.
[0033] Thus, for example, when the gene analyzed is a gene that shows increased expression in responsive subjects as compared to non-responsive subjects, then "increased expression" or "Increased normalized" expression level of a given gene can be described as being positively correlated with efficacy of a platinum-based chemotherapy drug. If the expression level of the 'gene in the individual subject trends toward a level of expression characteristic of a responsive subject, then the gene expression level supports a determination that the individual subject ss more likely to be a responder. If the expression level of the gene in the individual subject trends toward a level of expression characteristic of a non-responsive subject then the gene expression level supports a determination that the individual subject is more likely to be a inon-responder,
[0034] The term "expression product" or "gene product" are used herein to refer to the RNA transcription products (transcripts) of a gene, including rnRNA, and the polypeptide translation products of such RNA transcripts. An expression product may be, for example, an unspliced RNA, an rnRNA, a splice variant mRNA, a microRNA, a fragmented RNA, a polypeptide, a post- translationally modified polypeptide, a splice variant polypeptide, etc.
[0035] The term "long-term" survival is used herein to refer to survival for a particular time period. In an embodiment of the invention, the time period of long-term survival is for at least 3 years. In another embodiment, the time period of long-term survival is for at least 5 years.
[0036] The term "measuring" as used herein refers to performing a physical act of determining the dimension, quantity, or capacity of a component in a sample.
[0037] The term "microarray" as used herein refers to an ordered arrangement of hybrid izable array elements, e.g., oligonucleotide or polynucleotide probes, on a substrate.
[0038] The term "polynucleotide" generally refers to any polyribonucleotide or
polydeoxribonucieotide, which may be unmodified RNA or DNA or modified RNA or DNA, Thus, for instance, polynucleotides as used herein include, without limitation, single- and double- stranded DNA, DNA including single- and double-stranded regions, single- and double-stranded RNA, and RNA including single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or. more typically, double-stranded or include single- and double-stranded regions. In addition, the term "polynucleotide" as used herein refers to triple- stranded regions comprising RNA or DNA or both RNA and DNA. The strands in such regions may be from the same molecule or from different molecules. The regions may include all of one or more of the molecules, but more typically involve only a region of some of the moiecuies, One of the moiecuies of a triple-helical region often is an oligonucleotide. The term "polynucleotide" also includes DNAs (including cDNAs) and RNAs and those that contain one or more modified bases, Thus, DNAs or RNAs with backbones modified for stability or for other reasons, are "polynucleotides" as that term is used herein. Moreover, DNAs or RNAs comprising unusual bases, such as inosine, or modified bases, such as tritiated bases, are included within the term "polynucleotides" as used herein, in general, the term "polynucleotide" embraces all chemically, enzymatieally and/or metabolically modified forms of unmodified polynucleotides, as well as the chemical forms of DNA and RNA characteristic of viruses and cells, including simple and complex ceils.
[0039] The term "oligonucleotide" refers to a relatively short polynucleotide, including, without limitation, single-stranded deoxyribonucleotides, single- or double-stranded ribonucleotides, RNA/DNA hybrids and double-stranded DNAs, Oligonucleotides, such as single-stranded DNA probe oligonucleotides, are often synthesized by chemical methods, for example using automated oligonucleotide synthesizers that are commercially available. However, oligonucleotides can be made by a variety of other methods, including in vitro recombinant DNA-mediated techniques and by expression of DNAs in cells and organisms.
[0040] The term "primer" or "oligonucleotide primer" as used herein, refers to an oligonucleotide that 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, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19 or 20 more nucleotides either 5' or 3' from either termini or from both termini.
[0041] 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 affected 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 covaient addition of bases at its 3' end,
[0042] 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.
[0043] As used herein, the term "probe" or "oligonucleotide probe", used interchangeably herein, refers to a structure comprised of a polynucleotide, as defined above, that contains a nucleic acid sequence complementary to a nucleic acid sequence present in the target nucleic acid anaiyte (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 ail 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 1 5 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 slated ranges, in some embodiments, the probes 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 terra "about" may be construed to mean 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19 or 20 more nucleotides either 5' or 3' from either termini or from both termini.
[0044] As used herein, the term "pathology" of cancer includes all phenomena that comprise 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.
[0045] The term "platinum-based chemotherapy drug" as used herein refers to a molecule or a composition comprising a molecule containing a coordination complex comprising the chemical element platinum and useful as a chemotherapy drug. Platinum-based chemotherapy drugs generally act by inhibiting DNA synthesis and have some alkylating activity. Examples of platinum-based chemotherapy drugs include cispiatin, carbopiatin, and oxaiipiatin. Platinum- based chemotherapy drugs encompass those that are currently being used as part of a
chemotherapy regimen, those that are currently in development, and those that may be developed in the future. The platinum-based chemotherapy drug may be administered as a monotherapy, or in combination with other anti-cancer agents, or as prodrugs, or together with local therapies such as surgery and radiation, or as adjuvant or neoadjuvant chemotherapy, or as part of a multimodal approach to the treatment of neoplastic disease. For example, oxaiipiatin may be administered alone, or in combination with fluorouracii (5-FU) and/or leucovorin for the treatment of colorectal cancer.
[0046] The term "efficacy" as used herein refers to the capacity for a drug to produce a positive response upon administration to a patient.
[0047] The term "positive response" as used herein refers to a favorable response to a drug as opposed to a lack of response or an unfavorable response, such as adverse events. A positive response may include, without limitation, (1) inhibition, to some extent, of tumor growth, including slowing down to complete growth arrest; (2) reduction in the number of tumor cells; (3) reduction in tumor size; (4) inhibition (i.e., reduction, slowing down or complete cessation) of tumor ceil infiltration into adjacent peripheral organs and/or tissues; (5) inhibition of metastasis; (6) enhancement of anti-tumor immune response, possibly resulting in regression or rejection of the tumor: (7) relief, to some extent, of one or more symptoms associated with the tumor; (8) increase in the length of survival following treatment; and/or (9) decreased mortality at a given point of time following treatment, In individual patients, a positive response can be expressed in terras 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 time without recurrence, increase in the length of survival fol lowing 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 a positive response to treatment. A positive response to a platinum-based chemotherapy drug may include reducing the likelihood of colon cancer recurrence,
[0048] The term "responder" as used herein refers to a patient who has cancer, and who exhibits a positive response following treatment with a platinum-based chemotherapy drug.
[0049] The term "non-resporider" as used herein refers to a patient who has cancer, and who has not shown a positive response following treatment with a platinum-based chemotherapy drug, [Θ05Ο] The term "prediction" is used herein to refer to the likelihood that a cancer cell or a cancer patient will have a particular response to treatment, whether positive or negative. In an
embodiment of the invention, "prediction" refers to a particular response to treatment following surgical removal of the primary tumor. For example, treatment could include chemotherapy.
[0051] The predictive methods of the present invention can be used clinically to make treatment decisions by choosing the most appropriate treatment modalities for any particular patient. The predictive methods of the present invention are useful tools in predicting if a patient is likely to exhibit a positive response to a treatment regimen, such as chemotherapy, surgical intervention, or both.
[0052] 'The term "reference gene" as used herein refers to a gene whose expression level can be used to compare the expression level of a gene product in a test sample, in an embodiment of the invention, reference genes include housekeeping genes, such as beta-globin, alcohol
dehydrogenase, or any other gene, the expression of which does not vary depending on the disease status of the cell containing the gene, in another embodiment, all of the assayed genes or a large subset thereof may serve as reference genes.
[0053] The term "response indicator gene" as used herein refers to a gene, the expression of which correlates positively or negatively with a positive response to a platinum-based chemotherapy drug, such as oxaliplatin, The expression of a response indicator gene may be determined by assaying or measuring the expression level of an expression product of the response indicator gene. (0054] The term "RNA transcript" as used herein refers to the RNA transcription product of a gene, including, for example, mRNA, an unspliced RNA, a splice variant mRNA. a microRNA, and a fragmented RNA.
[0055] Unless indicated otherwise, each gene name used herein corresponds to the Official Symbol assigned to the gene and provided by Eiitrez Gene (URL:
www.ncbi.nlm.nih.gov/sites/entrez) as of the filing date of this appiication.
[0056] The term "tumor sample" as used herein refers to a sample comprising tumor material obtained from a cancerous patient. The term encompasses tumor tissue 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. Additionally, the term "tumor sample" encompasses a sample comprising tumor cells obtained from sites other than the primary tumor, e.g., circulating tumor cells. The term also 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 further 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.
[0057] "Stringency" of hybridization reactions is readily determinable by one of ordinary skill in the art, and generally is an empirical calculation dependent upon probe length, washing temperature, and salt concentration. In general, longer probes require higher temperatures for proper annealing, while shorter probes need lower temperatures. Hybridization generally depends on the ability of denatured DNA to re-anneal when complementary strands are present in an environment below their melting temperature. The higher the degree of desired homology between the probe and hybridizable sequence, the higher the relative temperature that can be used. As a result, it follows that higher relative temperatures would tend to make the reaction conditions more stringent, while lower temperatures less so. For additional details and explanation of stringency of hybridization reactions, see Ausubel et ah, Current Protocols in Molecular Biology, Wiley Interscience Publishers, (1995).
[0058] "Stringent conditions" or "high stringency conditions", as defined herein, typically: (1) employ low ionic strength and high temperature for washing, for example 0.015 M sodium chloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate at 50°C; (2) employ during hybridization a denaturing agent, such as formamide, for example, 50% (v/v) formamide with 0.1% bovine serum albumin/0.1% Fico!l/0.1 % polyvinyipyrrolidone/SOmM sodium phosphate buffer at pH 6.5 with 750 raM sodium chloride, 75 niM sodium citrate at 42°C; or (3) employ 50% formamide, 5 x SSC (0,75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0, 1% sodium pyrophosphate, 5 x Denhardt's solution, sonicated salmon sperm DNA (50 ,u.g/ml), 0.3% SDS, and 10% dextran sulfate at 42°C, with washes at 42°C in 0.2 x SSC (sodium chloride/sodium citrate) and 50% formamide, followed by a high-stringency wash consistirsg of 0.1 x SSC containing EDTA at 55°C.
[0059] "Moderately stringent conditions" may be identified as described by Sambrook et ah, Molecular Cloning; A Laboratory Manual, New York: Cold Spring Harbor Press, 1989, and include the use of washing solution and hybridization conditions (e.g., temperature, ionic strength and %SDS) less stringent that those described above. An example of moderately stringent conditions is overnight incubation at 37°C in a solution comprising: 20% formamide, 5 x SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5 x Denhardt's solution, 10% dextran sulfate, and 20 mg/ml denatured sheared salmon sperm DNA, followed by washing the filters in 1 x SSC at about 37-50°C, The skilled artisan will recognize how to adjust the temperature, ionic strength, etc. as necessary to accommodate factors such as probe length and the like.
[0060] 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), including those who have undergone or are candidates for resection (surgery) to remove cancerous tissue (e.g., cancerous colorectal tissue).
[0061] As used herein, the term "surgery" applies to surgical methods undertaken for removal of cancerous tissue, including resection, laparotomy, colectomy (with or without lymphadenectomy), ablative therapy, endoscopic removal, excision, dissection, and tumor biopsy/removal The tumor tissue or sections used for gene expression analysis may have been obtained from any of these methods.
[0062] The terms "threshold" or "thresholding" refer to a procedure used to account for non-linear relationships between gene expression measurements and clinical response as well as to further reduce variation in reported patient scores. When thresholding is applied, all measurements below or above a threshold are set to that threshold value. A non-linear relationship between gene expression and outcome could be examined using smoothers or cubic splines to model gene expression and recurrence-free interval in Cox proportional hazards models or gene expression and recurrence status in logistic regression models. Variation in reported patient scores could be examined as a function of variability in gene expression at the limit of quantitation and/or detection for a particular gene. [0063] The terms "treatment" and "treating" refer to administering or contacting an agent, or carrying out a procedure (e.g., radiation, a surgical procedure, etc.), for the purpose of obtaining an effect. In a subject, 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. The terms cover 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 that 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.
[0064] The term "tumor" as used herein, refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-eancerous and cancerous cells and tissues.
[0065] Two main staging systems are known in the art for colorectal cancer. According to the tumor, node, metastasis (TNM) staging system of the American Joint Committee on Cancer (AJCC) (Green et ah (eds.), AJCC Cancer Staging Manual, 6th ed., Springer: New York, NY, 2002), the various stages of colorectal cancer are defined as follows:
[0066] Tumor: TL* tumor invades submucosa; T2: tumor invades muscuiaris propria; T3: tumor invades through the muscuiaris propria into the subserose, or into the pericolic or perirectal tissues;
T4: tumor directly invades and/or perforates other organs or structures.
[ΘΘ67] Node: NO: no regional lymph node metastasis; Nl : metastasis in 1 to 3 regional lymph nodes; N2: metastasis in 4 or more regional lymph nodes.
[ΘΘ68] Metastasis: M0: no distant metastasis; Mi : distant metastasis present.
[0Θ69] Stage groupings: Stage I: Tl, NO, M0 or T2, NO, M0; Stage II: T3, NO, M0 or T4, NO, M0;
Stage III: any T, Nl-2, M0; Stage IV: any T, any N, Ml .
[0070] According to the Modified Duke Staging System, the various stages of colorectal cancer are defined as follows:
[0071] Stage A: the tumor penetrates into the mucosa of the bowel wall but not further. Stage B: tumor penetrates into and through the muscuiaris propria of the bowel wall. Stage C: tumor penetrates into but not through the muscuiaris propria of the bowel wall and there is pathologic evidence of colorectal cancer in the lymph nodes; or tumor penetrates into and through the muscuiaris propria of the bowel wall and there is pathologic evidence of cancer in the lymph nodes. Stage D: tumor has spread beyond the confines of the lymph nodes, into other organs, such as the liver, lung, or bone.
[0072] The term "computer-based system", as used herein refers to the hardware means, software means, and data storage means used to analyze information. The minimum hardware of a patient computer-based system comprises a central processing unit (CPU), input means, output means, and data storage means. 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 present invention,
[0073] 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 means 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,
[0074] A "processor" or "computing means" 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,
[0075] 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.
[0076] 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.
[0077] 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 "a platinum-based chemotherapy drug" includes reference to one or more platinum-based chemotherapy drug, and so forth.
[0078] 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 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. 1. Freshney, ed,, 1987); "Methods in Enzymolog " (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. Caios, 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).
[0079] The present invention provides response indicator genes of platinum-based chemotherapy drugs. These genes are listed in Tables 1 and 2. The response indicator genes may be further grouped into gene subsets, depending on their known function. For example, the gene subsets may include an "apoptosis group," "DNA damage repair group," "cell cycle control group,"
"invasion/migration group," "proliferation group," "ABC transporter group," and "nucleotide excision repair (NER) group," Tables 1 and 2 indicate which gene subset in which each gene may be grouped. The present invention further provides methods for determining genes that co-express with the response indicator genes. The co-expressed genes themselves are useful response indicator genes, The co-expressed genes may be substituted for the response indicator gene with which they co-express, Top 5 Pearson correlations for each of the genes in Tables 1 and 2 are listed in Table 3. In this analysis, median normalized expression for stage 111 patients was utilized.
[0080] The present invention also provides the top 16 genes in Tables 1 and 2 associated with oxaliplatin benefit controlling for a false discovery rate at 20%, These genes are listed in Table 4, The present invention also provides models, which include 7-10 of the genes listed in Table 4, that are useful for predicting the efficacy of a treatment comprising a platinum-based chemotherapy drug. Table 5 lists 20 such models.
[0081] The present invention further provides a 9-gene model useful for predicting the efficacy of a treatment comprising a platinum-based chemotherapy drug, and these 9 genes are indicated in Table 6. Table 6 also indicates a biological category in which each of these genes may be grouped. As indicated in Table 8, the genes of the 9-gene model may be further grouped into gene groups or pathways, including, for example, "transcription regulation," "replication and DNA repair," "cell cycle control/apoptosis," "proliferation," "ion/glutamate transport," and "pyridine metabolism/redox homeostasis" pathways. [0082] The present invention provides a number of methods that utilize the response indicator genes and associated information. In one aspect, the present invention provides a method of predicting the efficacy of a treatment comprising a platinum-based chemotherapy drug. The methods of the invention comprise assaying or measuring the expression level of the response indicator gene(s) in a sample comprising cancer cells or in a tumor sample, and predicting the efficacy of the platinum -based chemotherapy drug based on the correlation between the expression level of the response indicator gene(s) and a positive response to the platinura-based chemotherapy drug, In a particular embodiment of the invention, the platinum-based chemotherapy drug is oxaliplatin.
[©0S3] The response indicator genes and associated information provided by the present invention also have utility in the development of therapies to treat cancers and screening patients for inclusion in clinical trials that test the efficacy of platinum-based chemotherapy drugs. The response indicator genes and associated information may further be used to design or produce a reagent that modulates the level or activity of the expression product. Such reagents may include, but are not limited to, an antisense RNA, a small inhibitory RNA (siRNA), a ribozyme, a small molecule, a monoclonal antibody, and a polyclonal antibody,
[0084] In various embodiments of the methods of the present invention, various technological approaches are available for assaying or measuring the expression levels of the response indicator genes, including, without limitation, RT-PCR, microarrays, serial analysis of gene expression (SAGE), and nucleic acid sequencing, which are described in more detail below.
Figure imgf000019_0001
OF A Ρΐ,Λ S'j NU tV.-BASHD CHE OMgg XijRJJg.
[ΘΘ85] One skilled in the art will recognize that there are many statistical methods that may be used to determine whether there is a correlation between an outcome of interest (e.g., efficacy of chemotherapy) and expression levels of a gene product as described here. This relationship can be presented as a continuous treatment score (TS), or patients may be stratified into benefit groups (e.g., low, intermediate, high). For example, a Cox proportional hazards regression model may fit to a particular clinical endpoint (e.g., RFI, DFS, OS). One assumption of the Cox proportional hazards regression model is the proportional hazards assumption, i.e. the assumption that effect parameters multiply the underlying hazard. Assessments of model adequacy may be performed including, but not limited to, examination of the cumulative sum of martingale residuals. One skilled in the art would recognize that there are numerous statistical methods that may be used (e.g., Royston and Parmer (2002), smoothing spline, etc.) to fit a flexible parametric model using the hazard scale and the Weibull distribution with natural spline smoothing of the log cumulative hazards function, with effects for treatment (chemotherapy or observation) and RS allowed to be time-dependent. (See, e.g., P. Royston, M. Parmer, Statistics in Medicine 21(15:2175-2197 (2002).). The relationship between treatment and (1) benefit groups; and (2) clinical/pathologic covariates may also be tested for significance. For example, one skilled in the art could identify significant trends in absolute oxaliplatin benefit for recurrence at 5 years across the low, intermediate, and high oxaliplatin benefit groups for FU-based chemotherapy or FU-based chemotherapy & oxaliplatin groups. An absolute benefit of at least 5-6% in the high
chemotherapy benefit group would be considered clinically significant.
[0086] Any of the methods described may group the expression levels of genes, The grouping of genes may be performed at least in part based on knowledge of the contribution of the genes according to physiologic functions or component cellular characteristics, such as in the gene subsets described herein. The formation of groups, in addition, can facilitate the mathematical weighting of the contribution of various expression levels to the recurrence and/or treatment scores. The weighting of a gene group representing a physiological process or component cellular characteristic can reflect the contribution of that, process or characteristic to the pathology of the cancer and clinical outcome. Accordingly, the present invention provides gene subsets identified herein for use in the methods disclosed herein.
[ΡΘ87] The genes associated with platinum-based chemotherapy response, or response indicator genes, of the present invention are listed in Tables 1 and 2. In an embodiment of the invention, increased expression level of one or more genes selected from Table 1 is negatively correlated with efficacy of a platinum-based chemotherapy drug in reducing colon cancer recurrence.
[0088] in another embodiment of the invention, increased expression level of one or more genes selected from Table 2 is positively correlated with efficacy of a platinum-based chemotherapy drug in reducing colon cancer recurrence.
[0089] In a specific embodiment of the invention, increased expression level of one or more genes selected from RFC! , MTAL PTMA, HNRNPD, ANAPC2, SPINT2, ZNF143, SPl , EHMTL and MUTYH is negatively correlated with efficacy of a platinum -based chemotherapy drug in reducing colon cancer recurrence, and increased expression level of one or more genes selected from TXNRDl, MAPREl , ABCC4, SLC l A3, CDK2, and AVEN is positively correlated with efficacy of a platinum-based chemotherapy drug in reducing colon cancer recurrence.
[0090] In another embodiment of the invention, increased expression level of one or more genes selected from MTAl, PTMA, SPl, RFC, and SP1NT2 is negatively correlated with efficacy of a platinum-based chemotherapy drug in reducing colon cancer recurrence, and increased expression level of one or more genes selected from AVEN, CDK2, SLCl A3, and TXNRDl is positively correlated with efficacy of a platinum-based chemotherapy drug in reducing colon cancer recurrence.
[0091] In a particular embodiment of the invention, the platinum-based chemotherapy drug is oxaliplatin and the response indicator gene(s) is assayed or measured in colorectal cancer cells. Oxalipiatin may be provided in combination with one or more anti-cancer agents, such as 5-FU and Seucovorin, The colorectal cancer cells may be a tumor sample obtained from a human patient with colorectal cancer, such as stage II (Dukes B) or stage III (Dukes C) colorectal cancer. In another embodiment, the expression level of the response indicator gene(s) is normalized as described in more detail below,
[0092] Thus, in an embodiment of the invention, increased expression level of one or more genes selected from Table 1 is negatively correlated with efficacy of treatment with oxaliplatin in colorectal cancer cells or in a human patient with colorectal cancer, such as stage II (Dukes B) or stage ill (Dukes C) colorectal cancer,
[0093] In another embodiment of the invention, increased expression level of one or more genes selected from Table 2 is positively correlated with efficacy of treatment with oxaliplatin in colorectal cancer cells or in a human patient with colorectal cancer, such as such as stage Π (Dukes B) or stage III (Dukes C) colorectal cancer.
METHODS S O PUKDICT EFFICACY OF A PLATS NUM-BASF.?) CH EMO H ERAPY mux;
[0094] As described above, a number of response indicator genes were identified. Expression levels or normalized expression levels of these indicator gene products can then be determined in cancer cells or in a tumor sample obtained from an individual patient who has cancer and for whom treatment with a platinum-based chemotherapy drug is being contemplated. Depending on the outcome of the assessment, treatment with a platinum-based chemotherapy drug may be indicated, or an alternative treatment regimen may be indicated,
[0095] In carrying out the method of the present invention, cancer cells or a tumor sample is assayed or measured for an expression level of a response indicator gene product(s). The tumor sample can be obtained from a solid tumor, e.g., from a surgical procedure carried out to remove a tumor; or from a tissue or bodily fluid that contains cancer cells. In an embodiment of the invention, the tumor sample is obtained from a patient with colorectal cancer, such as stage II (Duke's B) or stage III (Duke's C) colorectal cancer. In another embodiment, the expression level of a response indicator gene is normalized relative to the level of an expression product of one or more reference genes. In a particular embodiment of the invention, the platinum-based chemotherapy drug is oxaliplatin. Oxaliplatin may be provided in combination with one or more anti-cancer agents, such as 5-FU and leucovorin. [0096] The efficacy of treatment with a platinum-based chemotherapy drug in an individual patient is predicted by comparing, directly or indirectly, the expression level or normalized expression level of the response indicator gene in the tumor sample from the individual patient to the expression level or normalized expression level of the response indicator gene in a clinically relevant subpopulation of patients. Thus, as explained above, when the response indicator 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 individual subject trends toward a level of expression characteristic of a responsive subject, then the gene expression level supports a determination that the individual subject is more likely to be a responder.
[0097] It is understood that the expression level or normalized expression level of a response indicator gene from an individual patient can be compared, directly or indirectly, to the expression level or normalized expression level of the response indicator gene in a clinically relevant subpopulation of patients, For example, when compared indirectly, the expression level or normalized expression level of the response indicator gene from the individual patient may be used to calculate a score based on a prediction of the efficacy of treatment with a platinum-based chemotherapy drug,
[0098] It is also understood that it can be useful to measure the expression level of a response indicator gene product at multiple time points, for example, prior to and during the course of treatment with a platinum-based chemotherapy drug. For example, an initial assessment of the efficacy of treatment with a platinum-based chemotherapy drug can be made prior to initiation of treatment in order to optimize treatment choice,
[0099J Development of drug resistance is a well-known phenomenon in chemotherapeutic treatment of cancer patients. As they proliferate, tumor cells can accumulate mutations that confer drug resistance through a variety of mechanisms, including resistance to a platinum-based chemotherapy drug. Tests that utilize the measurement of response indicator genes to assess the efficacy of treatment with a platinum-based chemotherapy drug can be carried out at time intervals to monitor changes indicative of the onset of drug resistance that may arise from changes in the tumor over time. It is not necessary to know what mutations or changes have taken place in the tumor in order to monitor consequent changes in the gene expression level of response indicator genes and assess the efficacy of treatment with a platinum-based chemotherapy drug.
[00100] Methods of gene expression profiling include methods based on hybridization analysis of polynucleotides, methods based on sequencing of polynucleotides, and proteomics- based methods. Exemplary methods known in the art for the quantification of mRNA expression in a sample include northern blotting and in situ hybridization (Parker & Barnes, Methods in Molecular Biology 106:247-283 (1999)); RNAse protection assays (Hod, Biotechniques 13:852- 854 (1992)); and PCR-based methods, such as reverse transcription PGR (RT-PCR) (Weis et al., Trends in Genetics 8:263-264 (1992)). Antibodies may be employed that can recognize sequence- specific duplexes, including DNA duplexes, RNA duplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes. Representative methods for sequencing-based gene expression analysis include Serial Analysis of Gene Expression (SAGE), and gene expression analysis by massively parallel signature sequencing (MPSS),
Reverse Tra grigtion PCR (RT-PCR)
[00101] Typically, mRNA is isolated from a sample. The starting material is typically total RNA isolated from a human tumor, usually from a primary tumor. Optionally, normal tissues from the same patient can be used as an internal control mRNA can be extracted from a tissue sample, e.g., from a sample that is fresh, frozen (e.g. fresh frozen), or paraffin-embedded and fixed (e.g. formalin-fixed),
[00102] General methods for mRNA extraction are well known in the art and are disclosed in standard textbooks of molecular biology, including Ausubef et al., Current Protocols of Mojgg MLBjQlQgy-. John Wiley and Sons (1997). Methods for RNA extraction from paraffin embedded tissues are disclosed, for example, in Rupp and Locker, Lab Invest 56:A67 (1987), and De Andres et al, BioTechniques 18:42044 (1995). In particular, RNA isolation can be performed using a purification kit, buffer set and protease from commercial manufacturers, such as Qiagen, according to the manufacturer's instructions. For example, total RNA from cells in culture can be isolated using Qiagen RNeasy mini-columns. Other commercially available RNA isolation kits include MasterPure™ Complete DNA and RNA Purification Kit (EPICENTRE®, Madison, WI), and Paraffin Block RNA isolation Kit (Ambion, Inc.). Total RNA from tissue samples can be isolated using RNA Stat-60 (Tel-Test), RNA prepared from a tumor sample can be isolated, for example, by cesium chloride density gradient eentrifugation,
[00103] The sample containing the RNA is then subjected to reverse transcription to produce e-DNA from the RNA template, followed by exponential amplification in a PCR reaction. The two most commonly used reverse transcriptases are avian myeloblastosis vims reverse transcriptase (AMV-RT) and Moloney murine leukemia virus reverse transcriptase (MMLV-RT), The reverse transcription step is typically primed using specific primers, random hexaniers, or oligo-dT primers, depending on the circumstances and the goal of expression profiling, For example, extracted RNA can be reverse-transcribed using a GeneAmp RNA PCR kit (Perkin Elmer, CA, USA), following the manufacturer's instructions. The derived cDNA can then be used as a template in the subsequent PGR reaction. [ΘΘ1.04] PCR-based methods use a thermostable DNA-dependent DNA polymerase, such as a Taq DNA polymerase. For example, TaqMan® PGR typically utilizes the 5 '-nuclease activity of Taq or Tth polymerase to hydroiyze a hybridization probe bound to its target amplicon, but any enzyme with equivalent 5' nuclease activity can be used. Two oligonucleotide primers are used to generate an amplicon typical of a PGR reaction product, A third oligonucleotide, or probe, can be designed to facilitate detection of a nucleotide sequence of the amplicon located between the hybridization sites of the two PGR primers. The probe can be detectably labeled, e.g., with a reporter dye, and can further be provided with both a fluorescent dye, and a quencher fluorescent dye, as in a Taqman® probe configuration. Where a Taqman® probe is used, during the amplification reaction, the Taq DNA polymerase enzyme cleaves the probe in a
template-dependent manner. The resultant probe fragments disassociate in solution, and signal from the released reporter dye is free from the quenching effect of the second fluorophore. One molecule of reporter dye is liberated for each new molecule synthesized, and detection of the unquenched reporter dye provides the basis for quantitative interpretation of the data,
[00105] TaqMan® RT-PCR can be performed using commercially available equipment such as, for example, ΑΒΪ PRISM 7700 or 79001 M Sequence Detection System I M (Perkm-Elmer- Appiied Biosystems, Foster City, CA, USA), or Lightcycler (Roche Molecular Biochetnicals, Mannheim, Germany). In a preferred embodiment, the 5' nuclease procedure is run on a real-time quantitative PGR device such as the ABI PRISM 7700 iM Sequence Detection System™. The system consists of a thermocycler, laser, charge-coupled device (CCD), camera and computer. The system amplifies samples in a 384-welI format on a thermocycler. The RT-PCR may be performed in triplicate wells with an equivalent of 2ng RNA input per 10 LtL-reaction volume. During amplification, laser-induced fluorescent signal is collected in real-time through fiber optics cables for ail wells, and detected at the CCD, The system includes software for running the instrument and for analyzing the data,
[00106] 5'-Nuclease assay data are generally initially expressed as a threshold cycle ("C ")■ Fluorescence values are recorded during every cycle and represent die amount of product amplified to that point in the amplification reaction. The threshold cycle (Q) is generally described as the point when the fluorescent signal is first recorded as statistically significant.
[00107] To minimize errors and the effect of sample-to-sampie variation, RT-PCR is usually performed using an internal standard, The ideal internal standard gene (also referred to as a reference gene) is expressed at a constant level among cancerous and non-cancerous tissue of the same origin (i.e., a level that is not significantly different among normal and cancerous tissues), and is not significantly affected by the experimental treatment (i.e., does not exhibit a significant difference in expression level in the relevant tissue as a result of exposure to chemotherapy). For example, reference genes useful in the methods disclosed herein should not exhibit significantly different expression levels in cancerous colon as compared to normal colon tissue. RNAs most frequently used to normalize patterns of gene expression are mRNAs for the housekeeping genes g!yceraidehyde-3-phosphate-dehydrogenase (GAPDH) and β-actin. Exemplary reference genes used for normalization comprise one or more of the following genes: ATP5E, GPX1, PGK1 , UBB, and VDAC2. Gene expression measurements can he normalized relative to the mean of one or more (e.g., 2, 3, 4, 5, or more) reference genes. Reference-normalized expression measurements can range from 0 to 15, where a one unit increase generally reflects a 2-fold increase in RNA quantity.
[00108] Real time PGR is compatible both with quantitative competitive PGR, where an internal competitor for each target sequence is used for normalization, and with quantitative comparative PGR using a normalization gene contained within the sample, or a housekeeping gene for RT-PCR. For further details see, e.g. Held et al, Genome Research 6:986-994 (1996),
[00109] The steps of a representative protocol tor use in the methods of the present disclosure use fixed, paraffin-embedded tissues as the RNA source. mRNA isolation, purification, primer extension and amplification can be performed according to methods available in the art. (see, e.g., Godfrey et al. J. Molec. Diagnostics 2: 84-91 (2000); Speeht et al., Am. J. Pathol. 158: 41 -29 (2001)). Briefly, a representative process starts with cutting about 10 μτ thick sections of paraffin-embedded tumor tissue samples. The RNA is then extracted, and protein and DNA are depleted from the RNA-containing sample. After analysis of the RNA concentration, RNA is reverse transcribed using gene specific primers followed by RT-PCR to provide for cDNA amplification products.
Design of PCR Primers and Probes
[00110] PCR primers and probes can be designed based upon exon or intron sequences present in the mRNA transcript of the gene of interest. Primer/probe design can be performed using publicly available software, such as the DNA BLAT software developed by Kent, W.J,, Genome Res. 12(4):656~64 (2002), or by the BLAST software including its variations.
[OOlllj Where necessary or desired, repetitive sequences of the target sequence can be masked to mitigate non-specific signals. Exemplary tools to accomplish this include the Repeat Masker program available on-line through the Baylor College of Medicine, which screens DNA sequences against a library of repetitive elements and returns a query sequence in which the repetitive, elements are masked. The masked sequences can then be used to design primer and probe sequences using any commercially or otherwise publicly available primer/probe design packages, such as Primer Express (Applied Biosystems); MGB assay~by~design (Applied
Biosystems); Primer3 (Steve Rozen and Helen J. Skaletsky (2000) Primer3 on the WWW for general users and for biologist, programmers, In: Rrawetz S, Misener S (eds) Bioinformatics Methods and Protocols: Methods in Molecular Biology. Humana Press, Totowa, NJ, pp 365-386).
[00112] Other factors that can influence PGR primer design include primer length, melting temperature (Tm), and G/C content, specificity, complementary primer sequences, and 3 '-end sequence, in general, optimal PGR primers are generally 17-30 bases in length, and contain about 20-80%, such as, for example, about 50-60% G+C bases, and exhibit Tm's between 50 and 80 °C, e.g. about 50 to 70 °C,
[00113] For further guidelines for PGR primer and probe design see, e.g. Dieffenbach, CW, et al, "General Concepts for PGR Primer Design" in: PCR Primer, A Laboratory Manual, Cold Spring Harbor Laboratory Press,, New York, 1995, pp. 133-155; Innis and Ge!fand, "Optimization ofPCRs" in: PCR Protocols, A Guide to Methods and Applications, CRC Press, London, 1994, pp. 5-1 1 ; and Plasterer, T.N. Primerselect: Primer and probe design. Methods Mol. Biol. 70:520-527 (1997), the entire disclosures of which are hereby expressly incorporated by reference,
[00114] Tables A and B provide exemplary primer, probe, and ampiicon sequences for each of the response indicator genes of the invention.
Mass ARRAY® System
[00115] In MassARRAY-hased methods, such as the exemplary method developed by Sequenom, Inc. (San Diego, CA) following the isolation of R.NA and reverse transcription, the obtained cDNA is spiked with a synthetic DNA molecule (competitor), which matches the targeted cDNA region in all positions, except a single base, and serves as an internal standard. The cDNA/corapetitor mixture is PCR amplified and is subjected to a post-PCR shrimp alkaline phosphatase (SAP) enzyme treatment, which results in the dephosphorylation of the remaining nucleotides. After inactivation of the alkaline phosphatase, the PCR products from the competitor and cDNA are subjected to primer extension, which generates distinct mass signals for the competitor- and cDNA-denved PCR. products. After purification, these products are dispensed on a chip array, which is pre-loaded with components needed for analysis with matrix- assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TQF MS) analysis. The cDNA present in the reaction is then quantified by analyzing the ratios of the peak areas in the mass spectrum generated, For further details see, e.g. Ding and Cantor, Proc. Nail. Acad. Sci. USA 100:3059-3064 (2003).
Other PCR- bas .Mfcj. fe
[00116] Further PCR-based techniques that can find use in the methods disclosed herein include, for example, BeadArray® technology (Illumina, San Diego, CA; Oliphant et al.,
Discovery of Markers for Disease (Supplement to Biotechniques), June 2002; Ferguson et al., Analytical Chemistry 72:5618 (2000)); BeadsArray for Detection of Gene Expression® (BADGE), using the commercially available LuminexlOO LabMAP® system and multiple color-coded microspheres (Luminex Corp., Austin, TX) in a rapid assay for gene expression (Yang et al, Genome Res. 11 : 1888-1898 (2001)); and high coverage expression profiling (HiCEP) analysis (Fukumura et ah, Nucl. Acids. Res. 31(16) e94 (2003).
Mkro^rr ys
[00117] Expression levels of a gene of interest can also be assessed using the microarray technique. In this method, polynucleotide sequences of interest (including cDNAs and
oligonucleotides) are arrayed on a substrate. The arrayed sequences are then contacted under conditions suitable for specific hybridization with detectably labeled cDNA generated from mRNA of a sample. As in the RT-PCR method, the source of mRNA typically is total RNA isolated from a tumor sample, and optionally from normal tissue of the same patient as an internal control or cell lines. mRNA can be extracted, for example, from frozen or archived paraffin-embedded and fixed (e.g. forma!in-fixed) tissue samples.
[00118] For example, PGR amplified inserts of cDNA clones of a gene to be assayed are applied to a substrate in a dense array. Usually at least 10,000 nucleotide sequences are applied to the substrate. For example, the microarrayed genes, immobilized on the microchip at 10,000 elements each, are suitable for hybridization under stringent conditions. Fluorescent!y labeled cDNA probes may be generated through incorporation of fluorescent nucleotides by reverse transcription of RN A extracted from tissues of interest. Labeled cDNA probes applied to the chip hybridize with specificity to each spot of DNA on the array. After washing under stringent conditions to remove non-specifically bound probes, the chip is scanned by confocal laser microscopy or by another detection method, such as a CCD camera. Quantitation of hybridization of each arrayed element allows for assessment of corresponding mRNA abundance.
[001.19] With dual color fluorescence, separately labeled cD A probes generated from two sources of RNA are hybridized pair wise to the array. The relative abundance of the transcripts from the two sources corresponding to each specified gene is thus determined simultaneously. The miniaturized scale of the hybridization affords a convenient and rapid evaluation of the expression pattern for large numbers of genes. Such methods have been shown to have the sensitivity required to detect rare transcripts, which are expressed at a few copies per cell, and to reproducibly detect at least approximately two-fold differences in the expression levels (Schena et at, Prac. Natl. Acad. Sci. USA 93(2):106-149 (1996)). Microarray analysis can be performed on commercially available equipment, following the manufacturer's protocols, such as by using the Affymetrix GenChip© technology, or Incyte's microarray technology. Serial Analysts of Gene Expression (SAGE)
[00120] Serial analysis of gene expression (SAGE) is a method that allows the simultaneous and quantitative analysis of a large number of gene transcripts, without the need of providing an individual hybridization probe for each transcript. First, a short sequence tag (about 10-14 bp) is generated that contains sufficient information to uniquely identify a transcript, provided that the tag is obtained from a unique position within each transcript. Then, many transcripts are linked together to form long serial molecules, that can be sequenced, revealing the identity of the multiple tags simultaneously. The expression pattern of any population of transcripts can be quantitatively evaluated by determining the abundance of individual tags, and identifying the gene corresponding to each tag. For more details see, e.g. Velculescu et al., Science 270:484-487 (1995): and
Velculescu et al., Cell 88:243-51 (1997),
[00121] Nucleic acid sequencing technologies are suitable methods for analysis of gene expression. The principle underlying these methods is that the number of times a cDNA sequence is detected in a sample is directly related to the relative expression of the raR A corresponding to that sequence. These methods are sometimes referred to by the term Digital Gene Expression (DGE) to reflect the discrete numeric property of the resulting data. Early methods applying this principle were Serial Analysis of Gene Expression (SAGE) and Massively Parallel Signature Sequencing (MPSS), See, e.g., S. Brenner, et al., Nature Biotechnology 18(6);630-634 (2000). More recently, the advent of "next-generation" sequencing technologies has made DGE simpler, higher throughput, and more affordable. See, e.g., W. Ansorge, New Biotechnology 25(4): 195- 203 (2009): M. Meizger, Nature Rev. Genetics 1 1 :31-46 (2010); S. Morrissy, et al„ Curr Protoc Hum Genet. 11(11. l l):l-36 (Apr, 2010). As a result, more laboratories are able to utilize DGE to screen the expression of more genes in more individual patient samples than previously possible. See, e.g., J, Marioni, Genome Research 18(9): 1509-1517 (2008): R, Morin, Genome Research 18(4):610-621 (2008); A. Mortazavi, Nature Methods 5(7):621-628 (2008); N. Cloonan, Nature Methods 5(7):613~619 (2008),
Isolating RNA from Body Fj gds
[00122] Methods of isolating RNA for expression analysis from blood, plasma and serum (see for example, Tsui NB et al. (2002) Clin. Chem. 48,1647-53 and references cited therein) and from urine {see for example, Boom R et al (1990) J Clin Microbiol. 28, 495-503 and reference cited therein) have been described. [00123] Inimunohistocheniistry methods are also suitable for detecting the expression levels of genes and applied to the method disclosed herein, Antibodies (e.g., monoclonal antibodies) that specifically bind a gene product of a gene of interest can be used in such methods. The antibodies can be detected by direct labeling of the antibodies themselves, for example, with radioactive labels, fluorescent labels, hapten labels such as biotin, or an enzyme such as horse radish peroxidase or alkaline phosphatase. Alternatively, unlabeled primary antibody can be used in conjunction with a labeled secondary antibody specific for the primary antibody.
Immunohistoehernistry protocols and kits are well known in the art and are commercially available.
Froteomks
[00124] The term "proteome" is defined as the totality of the proteins present in a sample (e.g. tissue, organism, or cell culture) at a certain point of time, Proteomics includes, among other things, study of the global changes of protein expression in a sample (also referred to as
"expression proteomics"). Proteomics typically includes the following steps: (1) separation of individual proteins in a sample by 2-D gel electrophoresis (2-D PAGE); (2) identification of the individual proteins recovered from the gel, e.g. my mass spectrometry or N- terminal sequencing, and (3) analysis of the data using bioinformatics.
[00125] The steps of a representative protocol for profiling gene expression using fixed, paraffin-embedded tissues as the RNA source, including mRNA isolation, purification, primer extension and amplification are provided in various published journal articles. {See, e.g., T.E. Godfrey et a!,. J. Moke. Diagnostics 2: 84-91 (2000); K. Specht et. ah, Am. J. Pathol. 158: 419-29 (2001), M. Cronin, et ah, Am J Pathol 164:35-42 (2004)). Briefly, a representative process starts with cutting a tissue sample section (e.g. about 10 μηι thick sections of a paraffin-embedded tumor tissue sample). The RNA is then extracted, and protein and DMA are removed. After analysis of the RN A concentration, RNA repair is performed if desired. The sample can then be subjected to analysis, e.g., by reverse transcription using gene specific promoters followed by PGR,
£oixraEi¾sioN ANALYSIS
[00126] Table 3 provides genes that co-express with particular response indicator genes that have been identified as having a correiation with a positive response to a platinum-based chemotherapy drug. To perform particular biological processes, genes often work together in a concerted way, i.e. they are co-expressed, Co-expressed gene groups identified for a disease process like cancer can also serve as response indicator genes. Such co-expressed genes can be assayed in lieu of, or in addition to, assaying of the response indicator gene with which they co- express.
[00127] One skilled in the art will recognize that many co-expression analysis methods now known or later developed will fail within the scope and spirit of the present invention. These methods may incorporate, for example, correlation coefficients, co-expression network analysis, clique analysis, etc., and may be based on expression data from RT-PCR, microarrays, sequencing, and other similar technologies. For example, gene expression clusters can be identified using pair- wise analysis of correlation based on Pearson or Spearman correlation coefficients. {See e.g, Pearson K. and Lee A., Biomeirika 2:357 (1902); C. Spearman, Amer. J. Psychol, 15:72-101 (1904); J. Myers, A. Well, Research Design and Statistical Analysis, p. 508 (2nd Ed., 2003).) In general, a correlation coefficient of equal to or greater than 0.3 is considered to be statistically significant in a sample size of at least 20. {See e.g., G. Norman, D. Streiner, Biostatistics: The Bare Essentials, 137-138 (3rd Ed. 2007).)
[00128] In order to minimize expression measurement variations due to non-biological variations in samples, e.g., the amount and quality of expression product to be measured, raw expression level data measured for a gene product (e.g., cycle threshold (Ct) measurements obtained by qRT-PCR) may be normalized relative to the mean expression level data obtained for one or more reference genes. Examples of reference genes include housekeeping genes, such as GAPDH, Alternatively, all of the assayed genes or a large subset thereof may also concurrently serve as reference genes and normalization can be based on the mean or median signal (Ct) of all of the assayed genes or a subset thereof (often referred to as "global normalization" approach). On a gene-by-gene basis, measured normalized amount of a patient tumor mRNA may be compared to the amount found in a cancer tissue reference set. See e.g., Cronin, M, et al., Am. Soc.
Investigative Pathology 164:35-42 (2004), The normalization may be carried out such that a one unit increase in normalized expression level of a gene product generally reflects a 2-fold increase- in quantity of expression product present in the sample. For further information on normalization techniques applicable to qRT-PCR data from tumor tissue, see e.g., Silva, S, et al, (2006) BMC Cancer 6, 200; deKok, J. et al. (2005) Laboratory Investigation 85, 154-159. [00129] The materials for use in the methods of the present invention are suited for preparation of kits produced in accordance with well known procedures. The present invention thus provides kits comprising agents, which may include gene-specific or gene-selective probes and/or primers, for quantitating the expression of the disclosed genes for predicting prognostic outcome or response to treatment, Such kits may optionally contain reagents for the extraction of RNA from tumor samples, in particular, fixed paraffin-em bedded tissue samples and/or reagents for RNA amplification. In addition, the kits may optionally comprise the reagent(s) with an identifying description or label or instructions relating to their use in the methods of the present invention. The kits may comprise containers (including microliter plates suitable for use in an automated implementation of the method), each with one or more of the various reagents (typically in concentrated form) utilized in the methods, including, for example, pre-fabricated mieroarrays, buffers, the appropriate nucleotide triphosphates (e.g., dATP, dCTP, dGTP and dTTP; or rATP, rCTP, rGTP and UTP), reverse transcriptase, DNA polymerase, RNA polymerase, and one or more probes and primers of the present invention (e.g., appropriate length poly(T) or random primers linked to a promoter reactive with the RNA polymerase). Mathematical algorithms used to estimate or quantify prognostic or predictive information are also properly potential components of kits.
REPORTS
[ΘΘ130] The methods of this invention are suited for the preparation of reports summarizing the predictions resulting from the methods of the present invention. A "report" as described herein, is an electronic or tangible document that includes elements that provide information of interest relating to a likelihood assessment and its results, A subject report includes at least a likelihood assessment, e.g., an indication as to the likelihood that a cancer patient will experience colon cancer recurrence after a treatment regimen with a platinum -based chemotherapy drug and the likelihood that a cancer patient will experience colon cancer recurrence without treatment with a platinum-based chemotherapy drug. A subject report can be completely or partially
electronically generated, e.g., presented on an electronic display (e.g., computer monitor). A report can further include one or more of: I) information regarding the testing facility; 2) service provider information; 3) patient data: 4) sample data; 5) an interpretive report, which can include various information including: a) indication; b) test data, where test data can include a normalized level of one or more genes of interest, and 6) other features.
[00131] The present invention therefore provides methods of creating reports and the reports resulting therefrom. The report may include a summary of the expression levels of the RNA transcripts, or the expression products of such RNA transcripts, for certain genes in the cells obtained from the patient's tumor tissue. The report may include a prediction that a particular chemotherapy has an increased efficacy or the report may include a prediction that the
chemotherapy has a decreased efficacy, The report may include a recommendation for a treatment, modality such as FU-based chemotherapy or FU -based chemotherapy in combination with a platinum-based drug. The report may be presented in electronic format or on paper,
[00132] Thus, in some embodiments, the methods of the present invention further include generating a report that includes information regarding efficacy of treatment with a platinum-based chemotherapy drug, such as oxaliplatin. For example, the methods of the present invention can further include a step of generating or outputting a report providing the results of a patient response likelihood assessment, which can be provided in the form of an electronic medium (e.g., an electronic display on a computer monitor), or in the form of a tangible medium (e.g., a report printed on paper or other tangible medium).
[00133] A report that includes information regarding the efficacy of treatment with a platinum-based chemotherapy drug, such as oxaliplatin, is provided to a user. The report may include information regarding the efficacy of treatment with oxaliplatin in preventing colon cancer recurrence. An assessment as to the likelihood that a cancer patient will experience cancer recurrence with or without a platinum-based chemotherapy drug, such as oxaliplatin, is referred to as a "response likelihood assessment" or "likelihood assessment," A person or entity who prepares a report ("report generator") may also perform the likelihood assessment, The report generator may also perform one or more of sample gathering, sample processing, and data generation, e.g., the report generator may also perform one or more of: a) sample gathering; b) sample processing: c) measuring a level of a response indicator gene expression produces); d) measuring a le vel of a reference gene product(s); and e) determining a normalized level of a response indicator gene expression product(s). Alternatively, an entity other than the report generator can perform one or more sample gathering, sample processing, and data generation.
[ΘΘ134] The term "user" or "client" refers to a person or entity to whom a report is transmitted, and may be the same person or entity who does one or more of the following: a) collects a sample; b) processes a sample; c) provides a sample or a processed sample; and d) generates data (e.g., level of a predictive, gene expression product(s); level of a reference gene produet(s); normalized level of a predictive gene expression produci(s)) for use in the likelihood assessment, In some cases, the person or entity who provides sample collection and/or sample processing and/or data generation, and the person who receives the results and/or report may be different persons, but are both referred to as "users" or "clients." In certain embodiments, e.g., where the methods are completely executed on a single computer, the user or client provides for data input and review of data output. A "user" can be a health professional (e.g., a clinician, a laboratory technician, a physician (e.g., an oncologist, surgeon, pathologist), etc.),
[ΘΘ135] in embodiments where the user only executes a portion of the method, the individual who, after computerized data processing according to the methods of the invention, reviews data output (e.g.. results prior to release to provide a complete report, a complete, or reviews an "incomplete" report and provides for manual intervention and completion of an interpretive report) is referred to herein as a "reviewer," The reviewer may be located at a location remote to the user (e.g., at a service provided separate from a healthcare facility where a user may be located).
[00136] Where government regulations or other restrictions apply (e.g., requirements by health, malpractice, or liability insurance), all results, whether generated wholly or partially electronically, are subjected to a quality control routine prior to release to the user,
[00137] The methods and systems described herein can be implemented in numerous ways. In one embodiment of the invention, the methods involve use of a communications infrastructure, for example, the internet. Several embodiments of the invention are discussed below. The present invention may also be implemented in various forms of hardware, software, firmware, processors, or a combination thereof. The methods and systems described herein can be implemented as a combination of hardware and software. The software can be implemented as an application program tangibly embodied on a program storage device, or different portions of the software implemented in the user's computing environment (e.g., as an applet) and on the reviewer's computing environment, where the reviewer may be located at a remote site (e.g., at a service provider's facility).
|00138] In an embodiment of the invention, during or after data input by the user, portions of the data processing can be performed in the user-side computing environment. For example, the user-side computing environment can be programmed to provide for defined test codes to denote a treatment efficacy "score," where the score is transmitted as processed or partially processed responses to the reviewer's computing environment in the form of test code for subsequent execution of one or more algorithms to provide a result and/or generate a report in the reviewer's computing environment. The score can be a numerical score (representative of a numerical value) or a non-numerical score representative of a numerical value or range of numerical values (e.g., "A": representative of a 90-95% likelihood of a positive response; "High": representative of a greater than 50% chance of a positive response (or some other selected threshold of likelihood); "Low": representative of a less than 50% chance of a positive response (or some other selected threshold of likelihood), and the like.
[00139] As a computer system, the system generally includes a processor unit. The processor unit operates to receive information, which can include test data (e.g., level of a predictive gene product(s); level of a reference gene produet(s); normalized level of a predictive gene product(s); and may also include other data such as patient data. This information received can be stored at least temporarily in a database, and data analyzed to generate a report as described above,
[00140] Part or ail of the input and output data can also be sent electronically. Certain output data (e.g., reports) can be sent electronically or teiephoniealiy (e.g., by facsimile, using devices such as fax hack). Exemplary output receiving devices can include a display element, a printer, a facsimile device and the like. Electronic forms of transmission and/or display can include email, interactive television, and the like. In an embodiment of the invention, all or a portion of the input data and/or output data (e.g., usually at least the final report) are maintained on a web server for access, preferably confidential access, with typical browsers. The data may be accessed or sent to health professionals as desired. The input and output data, including ali or a portion of the final report, can be used to populate a patient's medical record that may exist in a confidential database as the healthcare facility.
[00141] The present invention also contemplates a computer-readable storage medium (e.g., CD-ROM, memory key, flash memory card, diskette, etc.) having stored there on a program which, when executed in a computing environment, provides for implementation of algorithms to carry out all or a portion of the results of a response likelihood assessment as described herein. Where the computer-readable medium contains a complete program for carrying out the methods described herein, the program includes program instructions for collecting, analyzing and generating output, and generally includes computer readable code devices for interacting with a user as described herein, processing that data in conjunction with analytical information, and generating unique printed or electronic media for that user.
[00142] Where the storage medium includes a program that provides for implementation of a portion of the methods described herein (e.g., the user-side aspect of the methods (e.g., data input, report receipt capabilities, etc,)), the program provides for transmission of data input by the user (e.g., via the internet, via an intranet, etc.) to a computing environment at a remote site.
Processing or completion of processing of the data is carried out at the remote site to generate a report. After review of the report, and completion of any needed manual intervention, to provide a complete report, the complete report is then transmitted back to the user as an electronic document or printed document (e.g., fax or mailed paper report). The storage medium containing a program according to the invention can be packaged with instructions (e.g., for program installation, use, etc.) recorded on a suitable substrate or a web address where such instructions may be obtained, The computer-readable storage medium can also be provided in combination with one or more reagents for carrying out a response likelihood assessment (e.g., primers, probes, arrays, or such other kit components).
[00143] Having described the invention, the same will be more readily understood through reference to the following Examples, which are provided by way of illustration, and are not intended to limit the invention in any way. All citations through the disclosure are hereby expressly incorporated by reference.
EXAMPLES
Example 1: Gene Identification Studi s
[00144] Expression levels of genes, including those listed in Tables 1-2, were studied on tissue samples obtained from human patients with colorectal cancer enrolled in the National Surgical Adjuvant Breast and Bowel Project (NSABP) protocol C-07 (NSABP C-07) phase fll clinical trial. See Kuebier J.P. et al,, "Oxalipiatin Combined with Weekly Bolus Fiuorouracii and Leueovorin as Surgical Adjuvant Chemotherapy for Stage II and 11 Colon Cancer: Results from NSABP C-07," J. Clin. Oncol, 25:2198-2204 (2007). An objective of the study was to determine whether there is a significant relationship between the expression of the genes and clinical benefit from addition of oxalipiatin to adjuvant 5-FU/LV chemotherapy. Improvement in a clinical endpoint, such as recurrence-free interval (RFI), overall survival (OS), and disease-free survival (DPS), reflects an increased likelihood of response to treatment with oxalipiatin and a likelihood of a positive response.
[00145] Patients in the NSABP C-07 study had either stage II or stage III colorectal cancer and had undergone a potentially curative resection. Their tissue samples were archived, formalin- fixed, and paraffin-embedded prior to treatment. Patients were then randomly assigned to one of the following treatment regimens: (1) FULV: 5-fluorouracil (5-FU) 500 mg/m"4 intravenous (IV) bolus weekly for 6 weeks plus leueovorin 500 mg/m2 IV weekly for 6 weeks during each 8-week cycle for three cycles; or (2) FLOX: the same FULV regimen with oxalipiatin 85 mg/m2 IV administered on weeks 1, 3, and 5 of each 8-week cycle for three cycles. Data regarding the clinical responses of each patient are available. See id.
[0Θ146] The expression of each gene was quantitatively measured for each of
approximately 900 patients from the archived, formalin-fixed paraffin-embedded tissue (FPET) samples by RT-PCR. The primers and probes for each of the genes are set forth in Table A and amp!icons of each gene are set forth in Table B, The Accession Number as given in the Entrez Gene online database by the National Center for Biotechnology information for each gene is provided in Table A.
[00147] Gene expression of each gene was normalized relative to median expression of the gene candidates for a given patient. Median normalization corrects for degradation due to sample processing (e.g. fixation). The relationship between individual gene expression and clinical benefit from the addition of oxaiipiatin to adjuvant 5-FU/LV chemotherapy was assessed using multivariate Cox Proportional Hazards models which included Qnco pe DX® Colon Cancer Recurrence Score, nodal status (1 -3 vs. 4+ positive nodes), treatment (FU vs. FU
+Oxalipiatin), gene expression and an interaction of gene expression with treatment. These models were fit to recurrence free interval (RFI) endpoint defined as time from randomization to first colon cancer recurrence or death due to recurrence of colon cancer. The robust variance estimator (D, Lin, L.Wei, JASA Vol. 84, #408, 1989) was utilized to control for possible departures from model assumptions. These models were fit in stage III colon cancer patients, a subset of the C-07 study population which benefitted the most from oxaiipiatin. The analyses controlled the false discovery rate using Storey's procedure. (J. Storey, Journal of the Royal Statistical Soeiety:479- 98 (2002)). In addition, the genes were divided in three prespecified classes based on the level of evidence of the relationship between each gene and oxaiipiatin benefit (see, e.g., K, Harradine et al, Mo! Cancer Res. 201 1 9(2): 173-82, in-depth literature searches) and "separate class" analysis was carried out (B. Efron, Ann. Appi. Statist. 2:197-223 (2008)), A false discovery rate was calculated within each class, utilizing information from all the genes to produce efficient estimates of the false discovery rates within each class. Tables 1 and 2 contain 72 genes identified with q-value (probability that a particular gene is a false discovery) < 0.40. Standardized hazard ratio estimates, p-values, and corresponding 95% confidence intervals for the hazard ratio for interaction of gene expression and treatment are also provided.
[00148] Accordingly, increased expression level of the one or more genes selected from Table 1 was negatively correlated with efficacy of treatment comprising oxaiipiatin in preventing cancer recurrence in a patient with colorectal cancer, and increased expression level of one or more genes selected from Table 2 was positively correlated with efficacy of treatment comprising oxaiipiatin in preventing cancer recurrence in a patient with colorectal cancer,
Table 1, Genes with higher expression associated with less benefit from oxaiipiatin
interaction of gene expression and treatment
J ~\ Gene Official j Standardized Wa!d p- J q-value Biological category j Symbol j H {95% CI) vaiue 1 (FDR)
[ 1 I MTA1_ 1 MTAl j 1,63 ] (1.28,2.08) i 0.00007 j 0.031 invasion/Migration
nvasion/Migration
DNA Damage Repair G-protein signaling
Apoptosis
Table 2, Geaes with high expression associated with more benefit from oxaiipiatm.
Figure imgf000037_0001
Figure imgf000038_0001
ABCC2 0.78 j (0.59,1.03) [ 0.083 | 0.397 j ABC transporters
Table 3. Top 5 Pearsoe eorrelatioiis for genes associated with oxalip!aiin besefit.
Figure imgf000038_0002
Figure imgf000039_0001
Figure imgf000040_0001
Figure imgf000041_0001
Kxamplc 2: Development of a V!»iH-C>y8¾
[00149] For development of a multi-gene model, the list of potential candidates for oxaliplatin benefit prediction was restricted to the set of 16 genes associated with benefit from addition of oxaliplaiin to 5~FU based chemotherapy controlling false discovery rate at 20% (Table 4). The contributions of these genes in a single-gene mode! were similar and ranged from 1.10 to 1.21 in terms of standardized regression to the mean corrected hazard ratio (Crager M,. "Gene identification using true discovery rate degree of association sets and estimates corrected for regression to the mean," Stat, Med., 2010, 29(1): p, 33-45) for interaction of gene expression and treatment. Both directions of association were represented: higher expression of some genes was associated with more benefit and higher expression of other genes was associated with less benefit from oxaliplatin (Table 4). Based on these observations, the same weights (+1 or -1) were assigned to standardized expression of each gene, accounting for direction of association, to form "directed sums."
[00150] For example, increased expression level of one or more genes selected from TXNRDI, MAPREL ABCC4, SIX 1 A3, CDK2, and AVEN was positively correlated with efficacy of treatment comprising oxaliplatin in reducing the likelihood of colon cancer recurrence, and increased expression level of one or more genes selected from RFC, MTA 1 , PTMA, HNRNPD, ANAPC2, SPINT2, ZNF143, EHMTl, SP1 , and MUTYH was negatively- correlated with efficacy of treatment comprising oxaliplatin in reducing the likelihood of colon cancer recurrence,
Table 4. Genes associated with oxaliplatin benefit controlling false discovery rate at
20%
Higher 1 expression ~
Gene more (÷)/ ABS
Official less (-) STD q- Value RM-Corrected
N Symbol benefst p-valne H (FDR) ABS STD HR Pathway j
1 RFC1 (··) 0.000 1.88 0.05 1.14 DNA Damage Repair
1 2 ΜΊΆ1 (-) 0.000 1 ,63 0.03 1.18 invasion/Migration
1 3 PTMA i~) 0.000 1.74 0.05 1.21 Proliferation j
TXNRDI (+) 0.000 1.71 0.07 1.17 Pyridine Metabolism j
5 HNRNPD (-) 0.001 1.50 0.12 1.14 Apoptosis s
JL MAPRE1 (+) 0.002 : 1 .56 0.13 1.10 Cell Cycle Control i
7 ANAPC2 (-) 0.002 1.50 0.13 1.14 Cell Cycle Control : l
8 SPINT2 (-) 0.002 3.54 0.13 1.14 Proliferation j
9 ZNF143 (-) 0.001 1.49 0.14 1.18 Ceil Cycle Control |
10 ! ABCC4 (+) 0.003 1 .57 0.15 1.15 ABC transporters ]
11 S LCI A3 (+) 0.003 1.48 0.17 1.10 ion Transport |
12 EHMTl (-) 0.004 1.54 0.180 1.12 DNA Damage Repair ]
13 CDK2 (+) 0.005 1.52 0. 1 85 1 J 4 Ceil Cycle Control
! l4 SP1 (-) 0.007 1.62 0.19 1.13 DNA Damage Repair | l i s AVEN (+) 0.006 , 1.44 0.192 1 .14 Apoptosis 1 1 ,39 0.193 5 .1 3 IDNA Damage Repair
[ΘΘ151] Phe following multivariate algorithm development method was then used to examine the results of these multi-gene models: (1) consider all combinations of the 16 identified genes and (2) sort by standardized hazard ratio corrected for regression to the mean (RM). This method corrects for "cherry picking" among combinations from all 735 genes investigated in the gene identification study. The identified maximum RM-corrected hazard ratio is unbiased (Crager M., "Gene identification using true discovery rate degree of association sets and estimates corrected for regression to the mean," Stat. Med,, 2010. 29(1): p. 33-45) and provides a realistic estimate of the performance of the given multi-gene model on an independent dataset. To provide a more conservative estimate of performance, the 4-factor RM-correction was utilized which considered a given mode! in the context of all possible models with up to 4 genes. As the number of genes (factors) decreases, the num ber of models which predict benefit from oxaiipiatin decreases as well, so the estimate of performance for a given model would be adjusted (pulled down) by a more substantial amount. The following additional considerations were incorporated in the analysis:
Assay performance of genes,
[00152] During the linearity study of analytical performance of the 16 genes, the assay for ANAPC2 was found to have non-optimal performance and was excluded from further consideration,
Hgteroggngity_ sen .expression.
[00153] A heterogeneity study was conducted to assess between and within patient variability of gene expression. A total of 102 commercially obtained samples from 88 blocks and 38 patients were used, A panel of genes included the 16 predictive genes as well as stromal, immune, cell cycle and reference genes to provide a base for comparison. To determine between patient variability, RT-PCR was used to measure the variability in gene expression between different patients. Within patient variability, on the other hand, compared the variability in gene expression measurements from the same patient, for example between different blocks from the same tumor and between different sections witiiin the same block from the same tumor. Figure 1 contains a summary of the within and between patient variability for reference normalized expression of 16 predictive genes and a set of stromal gene known to have high within patient variability. On average, oxaiipiatin benefit prediction genes had about 6 times lower within patient variance compared to stromal genes and thus were found to be robust with respect to heterogeneity,
[00154 ] The presence of low within patient variability for identified predictive genes is an important factor which increases the probability of detecting a true predictive effect. Thus predictive genes which possess this quality would be most desirable for development of predictive assays.
Level and variability of gene expression.
[00155] RT-PCR examination of the level and variability of expression led to decision to threshoid the expression of SLC i A3, a gerse which was relative low expressed at 35 Ct. Thus, ail values with gene expression above 35 Ct on aggregate scale were set to 35.
[00156] Evaluation of the form of the relationship between gene expression and outcome suggested that ZNF143 might be more variable and hence less likely to be reliable in future studies.
[00157] The top 20 multi-gene models without ANAPC2 are presented in Table 5. The models without ZNF143, the gene which was not favored based on the functional form analysis, are boided. The bolded models contained 7 to 9 genes and provided similar performance (4- factor RM-corrected standardized hazard ratios 1.90-1.89). The highlighted model in Table 5, i.e., AVER CDK2, MTA1, PTMA, RFC, SLCI A3, SPI, SPINT2, and TXNRD1 , was chosen as the final model to increase the chance of including a potential true predictive gene. Based on the final 9 genes (Table 6). approximately 1 out of 9 genes is expected to be a false discovery (sum of q-values ~ 1.07). Simulation analysis of all possible models with 8 predictive genes and 1 false discovery showed that, on average, the log hazard ratio of such models is expected to decrease by about 3% only (data on file) compared to the final model with 9 predictive genes.
Table 5: Top 20 models without ANAPC2 and with SCL1A3 threshold
Figure imgf000044_0001
Figure imgf000045_0001
[00158] Data regarding performance of the 9-gene model is shown in Tables 6-7 and Figures 2-3. Table 6 identifies the individual genes of the 9-gene model (MTAL PTMA, SP1, RF C I, SPINT2, SLC1A3, TXNRD1, AVEN, and CDK2), It additionally provides the standardized HR with 95% confidence intervals, Wald p-vaiue, q-value (FDR), and RM~ corrected ABS STD FIR for each of the genes of the 9-gene model. Table 6 also indicates the prespecified class based on the level of evidence of the relationship between each gene and oxali latin benefit, the biological category, and the relationship between gene expression and oxalipiatin benefit.
Table 6. Individual genes sis the 9-gesie model
Figure imgf000045_0002
[00159] Considering the 9-gene model as a whole, the standardized HR (95% CI) without RM-correction is 2.76 (2.08, 3.65), p=1.40E-12, and the 4-factor RM-corrected standardized HR (95% CI) is 1.89 (1.61 , 2.2.3), q=0.5%.
[00160] The biological functions of MTAl , PTMA, SPl, RFC1 , SPINT2, SLC1A3, TXNR.D1, AVEN, and CDK2 were further considered in order to assign those genes to biological categories. Table 7 shows the bioiogicai category for each of the individual genes in the 9-gene model.
Table 7. Biological categories of the individual genes in the 9~gene model
Figure imgf000046_0001
;CDK2 Ceil Cycle Control; Cell Cycle Arrest
{00161] Based on the biological categories shown in Table 7, MTAl, PTMA, SPl, RFC ] , SPrNT2, S LCI A3, TXNRI3L AVEN, and CDK2 were assigned to one or more bioiogicai pathway, including transcription regulation, replication and DNA repair, cell cycle
eontrol/apoptosis, ion/glutamate transport, pyridine metabolism/redox homeostasis, and proliferation. Table 8 shows the performance of the 9-gene model based that assignment. As shown in Table 8, a given gene may be present in more than one pathway. Accordingly, Table 8 provides the pathway, genes, uncorrected Sid HR and 95% CI, and the relationship between higher expression and oxaiipiatin benefit.
[00162] For example, increased expression level of one or more genes selected from AVEN and CDK2 was positively correlated with efficacy of treatment comprising oxaiipiatin in reducing the likelihood that a patient will experience colon cancer recurrence, increased expression level of SLC 1A3 was positively correlated with efficacy of treatment comprising oxaiipiatin in reducing the likelihood that a patient will experience colon cancer recurrence, increased expression level of TXNRDl was positively correlated with efficacy of treatment comprising oxaiipiatin in reducing the likelihood that a patient will experience colon cancel- recurrence ,
[00163] Furthermore, for example, increased expression level of one or more genes selected from MTA1, PTMA, and SP1 was negatively correlated with efficacy of treatment, comprising oxaiipiatin in reducing the likelihood that a patient will experience colon cancer recurrence. Increased expression level of one or more genes selected from PTMA, SP1, and RFC1 was negatively correlated with efficacy of treatment comprising oxaiipiatin in reducing the likelihood that a patient will experience colon cancer recurrence. Increased expression level of one or more genes selected from PTMA, and SPINT2 was negatively correlated with efficacy of treatment comprising oxaiipiatin in reducing the likelihood that a patient will experience colon cancer recurrence.
Tabl e 8. Performance o I models based oe the p •st ways
Figure imgf000047_0001
[00164] Figure 2 depicts the ability of the 9~gene model to identify patients who would substantially benefit from addition of oxaiipiatin to 5FU-based chemotherapy. Specifically. Figure 2 shows a treatment effect hazard ratio predictiveness curve with 95% confidence intervals, with the x-axis showing the population quantile and the y-axis showing the RM- Corrected Treatment Effect Hazard Ratio. Assuming a 'no benefit' group includes patients with treatment effect HR of 0.95 or higher, the final 9-gene model is able to identify 33,2% of patients who would not benefit from addition of oxaliplatin to 5FU based chemotherapy.
[00165] Figures 3A-3C show time-to-event "survival" curves for the 9-gene model shown in Table 6. Specifically, Figures 1 A-3C show RM-correeted estimates of the proportion of patients remaining free from cancer recurrence (RFI endpoint) at each time after surgery, comparing patients with stage 3 colorectal cancer who were treated with 5FU to patients with stage 3 colorectal cancer who were treated with 5FU+oxalipIatin. The x~axis shows the time (in years) and the y-axis shows the probability that a patient will remain recurrence free. Figure 3A shows patients in the low tertile of the 9-gene directed sum; Figure 3B shows patients in the middle tertile of the 9-gene directed sum; and Figure 3C shows patients in the upper tertile of the 9-gene directed sum. The survival curves in Figure 3 indicate that patients in the low tertile would not appear to benefit from the addition of oxaliplatin to 5FU treatment (Figure 3A), the patients in the middle tertile appear to derive some benefit from the addition of oxaliplatin to 5FU treatment, and patients in the upper tertile seem to derive more benefit from the addition of oxaliplatin to 5FU treatment (Figure 3C).
[00166] While the present invention has been described with reference to the specific embodiments thereof, it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process, process step or steps, to the objective, spirit and scope of the present invention. Ail such modifications are intended to be within the scope of the claims appended hereto.
Figure imgf000049_0001
Figure imgf000050_0001
Figure imgf000051_0001
Figure imgf000052_0001
Figure imgf000053_0001

Claims

1. A method of predicting efficacy of a treatment comprising oxaliplatin in a hum an patient with colorectal cancer, comprising:
a. assaying an expression level of one or more genes selected from Tables 1 and 2, in a tumor sample obtained from the patient; and
b. predicting efficacy of the treatment comprising oxaliplatin, wherein:
increased expression level of the one or more genes selected from Table 1 is negatively correlated with efficacy of treatment comprising oxaliplatin, and increased expression level of one of more genes selected from Table 2 is positively correlated with efficacy of treatment comprising oxaliplatin.
2. The method of claim 1, wherein the expression level of two or more genes selected from RFC 1, MTA 1, PTMA, HNRNPD, ANAPC2, SPINT2, ZNF143, SP1, EHMT1, MUTYH,
TXNRD1, MAPRE1 , ABCC4, SLC1A3, CDK2, and AVEN is assayed,
3. The method of claim 1 , wherein the expression level of one or more genes selected from MTA1 , PTMA, SP1 , RFC] , SPINT2, SLC1A3, TXNRD1, AVEN, and CDK2 is assayed,
4. The method of claims 1 or 3, wherein the expression level of one or more genes selected from MTA, PTMA, and SPi is assayed,
5. The method of claims 1 or 3, wherein the expression level of one or more genes selected from PTMA, SPI, and RFC is assayed.
6. The method of claims 1 or 3, wherein the expression level of one or more genes selected from AVEN and CD 2 is assayed.
7. The method of claims 1 or 3, wherein the expression level of SLC1A3 is assayed.
8. The method of claims 1 or 3, wherein the expression level of TXNRD1 is assayed.
9. The method of any one of claims 1 -8, wherein the expression level of the one or more genes is normalized relative to median expression of that gene for a given patient to obtain a normalized expression leve l of the one or more genes.
10. The method of any one of claims 1-9, wherein the expression level of the one or more genes is a level of an RNA transcript of the one or more genes.
1 1. The method of any one of claims 1-9, wherein the expression level of the one or more genes is a polypeptide level of the one or more genes,
12. The method of claim 10, wherem the level of the RNA transcript of the one or more genes is assayed using reverse transcription polymerase chain reaction (RT-PCR).
13. The method of any one of claims 1-12, wherein the tumor sample is a fixed, wax-embedded tissue sample.
14. The method of any one of claims 1-13, wherein the treatment further comprises one more or additional anti-cancer agents,
15. The method of claim 14, wherein the one or more additional anti-cancer agents is 5- fluorouracil (5~FU) and leucovorin (LV).
16. The method of any one of claims 1-15, wherein the colorectal cancer is stage 11 (Dukes B) or stage III (Dukes C) colorectal cancer.
17. The method of any one of claims 1-16, further comprising creating a report based on the normalized expression level of the one or more genes.
18. The method of any one of claims 1 -17, wherein the one or more genes is substituted with one or more co-expressed genes thereof, and wherein the one or more co-expressed genes is selected from Table 3.
19. The method of any one of claims 1-18, wherein efficacy of a treatment comprising oxalip latin is reducing the likelihood of colon cancer recurrence.
PCT/US2013/027789 2012-02-27 2013-02-26 Gene expression markers for prediction of efficacy of platinum-based chemotherapy drugs WO2013130465A2 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US201261603697P 2012-02-27 2012-02-27
US61/603,697 2012-02-27

Publications (2)

Publication Number Publication Date
WO2013130465A2 true WO2013130465A2 (en) 2013-09-06
WO2013130465A3 WO2013130465A3 (en) 2015-07-02

Family

ID=49083435

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2013/027789 WO2013130465A2 (en) 2012-02-27 2013-02-26 Gene expression markers for prediction of efficacy of platinum-based chemotherapy drugs

Country Status (1)

Country Link
WO (1) WO2013130465A2 (en)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3198035A2 (en) * 2014-09-26 2017-08-02 Medical Prognosis Institute A/S Methods for predicting drug responsiveness
US10907214B2 (en) 2016-12-30 2021-02-02 Oncology Venture ApS Methods for predicting drug responsiveness in cancer patients
US11207269B2 (en) 2009-09-17 2021-12-28 Bio-Bedst Aps Medical use of sPLA2 hydrolysable liposomes
US11421284B2 (en) 2016-10-07 2022-08-23 Allarity Therapeutics Europe ApS Methods for predicting drug responsiveness in cancer patients

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DK2294215T3 (en) * 2008-05-12 2013-04-22 Genomic Health Inc Tests to predict cancer patients' response to various chemotherapeutic treatment options
US20120046186A1 (en) * 2010-08-20 2012-02-23 Pelham Robert J Gene Expression Markers for Prediction of Response to Platinum-Based Chemotherapy Drugs

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11207269B2 (en) 2009-09-17 2021-12-28 Bio-Bedst Aps Medical use of sPLA2 hydrolysable liposomes
EP3198035A2 (en) * 2014-09-26 2017-08-02 Medical Prognosis Institute A/S Methods for predicting drug responsiveness
US10570457B2 (en) 2014-09-26 2020-02-25 Medical Prognosis Institute A/S Methods for predicting drug responsiveness
EP3198035B1 (en) * 2014-09-26 2022-11-02 Allarity Therapeutics Europe ApS Methods for predicting drug responsiveness
US11421284B2 (en) 2016-10-07 2022-08-23 Allarity Therapeutics Europe ApS Methods for predicting drug responsiveness in cancer patients
US10907214B2 (en) 2016-12-30 2021-02-02 Oncology Venture ApS Methods for predicting drug responsiveness in cancer patients

Also Published As

Publication number Publication date
WO2013130465A3 (en) 2015-07-02

Similar Documents

Publication Publication Date Title
JP6246845B2 (en) Methods for quantifying prostate cancer prognosis using gene expression
JP5486718B2 (en) Gene expression markers for prognosis of colorectal cancer
JP6351112B2 (en) Gene expression profile algorithms and tests to quantify the prognosis of prostate cancer
JP2016104014A (en) Gene expression profile algorithm and test for likelihood of recurrence of colorectal cancer and response to chemotherapy
EP3556867A1 (en) Methods to predict clinical outcome of cancer
JP7301798B2 (en) Gene Expression Profile Algorithm for Calculating Recurrence Scores for Patients with Kidney Cancer
WO2013130465A2 (en) Gene expression markers for prediction of efficacy of platinum-based chemotherapy drugs
WO2018148642A1 (en) Algorithms and methods for assessing late clinical endpoints in prostate cancer
WO2014130617A1 (en) Method of predicting breast cancer prognosis
US20110287958A1 (en) Method for Using Gene Expression to Determine Colorectal Tumor Stage
WO2014130444A1 (en) Method of predicting breast cancer prognosis
NZ752676B2 (en) Gene expression profile algorithm for calculating a recurrence score for a patient with kidney cancer

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 13754026

Country of ref document: EP

Kind code of ref document: A2

NENP Non-entry into the national phase in:

Ref country code: DE

122 Ep: pct app. not ent. europ. phase

Ref document number: 13754026

Country of ref document: EP

Kind code of ref document: A2