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
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P11/00—Drugs for disorders of the respiratory system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q2600/00—Oligonucleotides characterized by their use
  • C12Q2600/118—Prognosis of disease development
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q2600/00—Oligonucleotides characterized by their use
  • C12Q2600/158—Expression markers

Definitions

• the invention generally relates to a molecular classification of disease and particularly to molecular markers for lung cancer prognosis and therapy selection and methods of use thereof.
• Cancer is a major public health problem, accounting for roughly 25% of all deaths in the United States. Though many treatments have been devised for various cancers, these treatments often vary in severity of side effects. It is useful for clinicians to know how aggressive a patient's cancer is in order to determine how aggressively to treat the cancer.
• NSCLC Early stage non small cell lung cancer
• the present invention is based in part on the surprising discovery that the expression of those genes whose expression closely tracks the cell cycle (“cell-cycle genes,” “CCGs,” or “CCP genes” as further defined below) is particularly useful in selecting appropriate therapy for and determining prognosis in lung cancer.
• one aspect of the present invention provides a method for determining the prognosis and/or the likelihood of response to a particular treatment regimen in a patient having lung cancer, which comprises: determining in a sample from the patient the expression of a plurality of test genes comprising at least 6, 8 or 10 cell-cycle genes (e.g., genes in any of Tables 1-11 or Panels A-H, J, or K; "sub-panels" of Panel F in Tables 21 to 25), and correlating increased expression of said plurality of test genes to a poor prognosis and/or an increased likelihood of response to the particular treatment regimen (e.g.
• a treatment regimen comprising chemotherapy) or, optionally, (b) correlating no increased expression of said plurality of test genes to a good prognosis and/or no increased likelihood of response to the treatment regimen.
• the lung cancer is adenocarcinoma.
• the lung cancer is typical lung carcinoid.
• the lung cancer is atypical lung carcinoid.
• the plurality of test genes includes at least 8 cell- cycle genes, or at least 10, 15, 20, 25 or 30 cell-cycle genes (e.g., genes in any of Tables 1-11 or Panels A-H, J, or K; "sub-panels" of Panel F in Tables 21 to 25).
• at least some proportion of the test genes e.g., at least 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 99%
• all of the test genes are cell-cycle genes.
• the step of determining the expression of the plurality of test genes in the tumor sample comprises measuring the amount of mRNA in the tumor sample transcribed from each of from 6 to about 200 cell-cycle genes; and measuring the amount of mRNA of one or more housekeeping genes in the tumor sample.
• the method of determining the prognosis and/or the likelihood of response to a particular treatment regimen comprises (1) determining in a tumor sample from a patient having lung cancer the expression of a panel of genes in said tumor sample including at least 4 or at least 8 cell-cycle genes (e.g., genes in any of Tables 1-11 or Panels A-H, J, or K; "sub-panels" of Panel F in Tables 21 to 25); (2) providing a test value by (a) weighting the determined expression of each of a plurality of test genes selected from the panel of genes with a predefined coefficient, and (b) combining the weighted expression to provide the test value, wherein at least 50%, at least 75% or at least 85% of the plurality of test genes are cell-cycle genes; and (3)(a) correlating an increased level of overall expression of the plurality of test genes to a poor prognosis and/or an increased likelihood of response to the particular treatment regimen (e.g., a treatment regimen comprising chemotherapy), or (b)
• the particular treatment regimen
• the methods of the invention further include a step of comparing the test value provided in step (2) above to one or more reference values, and correlating the test value to an increased likelihood of response to the particular treatment regimen.
• a test value greater than the reference value is correlated to an increased likelihood of response to treatment comprising chemotherapy.
• the test value is correlated to an increased likelihood of response to treatment (e.g., treatment comprising chemotherapy) if the test value exceeds the reference value by at least some amount (e.g., at least 0.5, 0.75, 0.85, 0.90, 0.95, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more fold or standard deviations).
• the method of determining the likelihood of response to a particular treatment regimen comprises (1) determining in a tumor sample from a patient having lung cancer the expression of a panel of genes in said tumor sample including at least 4 or at least 8 cell-cycle genes (e.g., genes in any of Tables 1-1 1 or Panels A-H, J, or K; "sub-panels" of Panel F in Tables 21 to 25); (2) providing a test value by (a) weighting the determined expression of each of a plurality of test genes selected from the panel of genes with a predefined coefficient, and (b) combining the weighted expression to provide the test value, wherein the cell-cycle genes are weighted to contribute at least 50%, at least 75% or at least 85% of the test value; and (3)(a) correlating a test value that is greater than some reference to a poor prognosis and/or an increased likelihood of response to the particular treatment regimen (e.g., a treatment regimen comprising chemotherapy), or (b) correlating a test value
• the particular treatment regimen e
• the present invention provides a method of treating cancer in a patient identified as having lung cancer, comprising: (1) determining in a tumor sample from the patient the expression of a panel of genes in the tumor sample including at least 4 or at least 8 cell-cycle genes (e.g., genes in any of Tables 1-1 1 or Panels A-H, J, or K; "sub-panels" of Panel F in Tables 21 to 25); (2) providing a test value by (a) weighting the determined expression of each of a plurality of test genes selected from said panel of genes with a predefined coefficient, and (b) combining the weighted expression to provide said test value, wherein the cell-cycle genes are weighted to contribute at least 50%, at least 75% or at least 85% of the test value; (3)(a) correlating an increased level of overall expression of the plurality of test genes to a poor prognosis and/or an increased likelihood of response to a particular treatment regimen (e.g., a treatment regimen comprising chemotherapy), or (b) cor
• a particular treatment regimen
• the present invention further provides a diagnostic kit for determining the prognosis in a patient having lung cancer and/or predicting the likelihood of response to a particular treatment regimen (e.g., a treatment regimen comprising chemotherapy) in a patient having lung cancer, comprising, in a compartmentalized container, a plurality of
• oligonucleotides hybridizing to at least 8 test genes, wherein less than 10%, 30% or less than 40% of all of the at least 8 test genes are non-cell-cycle genes; and one or more
• the kit consists essentially of, in a compartmentalized container, a first plurality of PCR reaction mixtures for PCR amplification of from 5 or 10 to about 300 test genes, wherein at least 30% or 50%, at least 60% or at least 80% of such test genes are cell-cycle genes (e.g., genes in any of Tables 1-11 or Panels A-H, J, or K; "sub-panels" of Panel F in Tables 21 to 25), and wherein each reaction mixture comprises a PCR primer pair for PCR amplifying one of the test genes; and a second plurality of PCR reaction mixtures for PCR amplification of at least one control (e.g., housekeeping) gene.
• a first plurality of PCR reaction mixtures for PCR amplification of from 5 or 10 to about 300 test genes, wherein at least 30% or 50%, at least 60% or at least 80% of such test genes are cell-cycle genes (e.g., genes in any of Tables 1-11 or Panels A-H, J, or K; "sub-
• the kit comprises one or more computer software programs for calculating a test value representing the expression of the test genes (either the overall expression of all test genes or of some subset) and for comparing this test value to some reference value.
• such computer software is programmed to weight the test genes such that cell-cycle genes are weighted to contribute at least 50%, at least 75% or at least 85% of the test value.
• such computer software is programmed to communicate (e.g., display) that the patient has an increased likelihood of response to a treatment regimen comprising chemotherapy if the test value is greater than the reference value (e.g., by more than some predetermined amount).
• the present invention also provides the use of (1) a plurality of oligonucleotides hybridizing to at least 4 or at least 8 cell-cycle genes (e.g., genes in any of Tables 1-1 1 or Panels A-H, J, or K; "sub-panels" of Panel F in Tables 21 to 25); and (2) one or more oligonucleotides hybridizing to at least one control (e.g., housekeeping) gene, for the manufacture of a diagnostic product for determining the expression of the test genes in a tumor sample from a patient having lung cancer, to determine prognosis in said patient and/or to predict the likelihood of responding to a treatment regimen comprising chemotherapy, wherein an increased level of the overall expression of the test genes indicates an increased likelihood, whereas no increase in the overall expression of the test genes indicates no increased likelihood.
• a control e.g., housekeeping
• the oligonucleotides are PCR primers suitable for PCR amplification of the test genes. In other embodiments, the oligonucleotides are probes hybridizing to the test genes under stringent conditions. In some embodiments, the plurality of oligonucleotides are probes for hybridization under stringent conditions to, or are suitable for PCR amplification of, from 4 to about 300 test genes, at least 50%, 70% or 80% or 90% of the test genes being cell-cycle genes.
• the plurality of oligonucleotides are hybridization probes for, or are suitable for PCR amplification of, from 20 to about 300 test genes, at least 30%, 40%, 50%, 70% or 80% or 90% of the test genes being cell-cycle genes.
• the present invention further provides a system for determining the prognosis in a patient having lung cancer and/or the likelihood of response to a particular treatment regimen in a patient having lung cancer, comprising: (1) a sample analyzer for determining the expression levels of a panel of genes in a tumor sample including at least 4 cell-cycle genes (e.g., genes in any of Tables 1-1 1 or Panels A-H, J, or K; "sub-panels" of Panel F in Tables 21 to 25), wherein the sample analyzer contains the tumor sample, mRNA molecules expressed from the panel of genes and extracted from the sample, or cDNA molecules from said mRNA molecules; (2) a first computer program for (a) receiving gene expression data on at least 4 test genes selected from the panel of genes, (b) weighting the determined expression of each of the test genes with a predefined coefficient, and (c) combining the weighted expression to provide a test value, wherein at least 50%, at least at least 75% of at least 4 test genes are cell-cycle genes; and
• the invention provides a system for determining the prognosis in a patient having lung cancer and/or the likelihood of response to a particular treatment regimen in a patient having lung cancer, comprising: (1) a sample analyzer for determining the expression levels of a panel of genes in a tumor sample including at least 4 cell-cycle genes (e.g., genes in any of Tables 1-1 1 or Panels A-H, J, or K; "sub-panels" of Panel F in Tables 21 to 25), wherein the sample analyzer contains the tumor sample, mRNA molecules expressed from the panel of genes and extracted from the sample, or cDNA molecules from said mRNA molecules; (2) a first computer program for (a) receiving gene expression data on at least 4 test genes selected from the panel of genes, (b) weighting the determined expression of each of the test genes with a predefined coefficient, and (c) combining the weighted expression to provide a test value, wherein the cell-cycle genes are weighted to contribute at least 50%, at least 75% or at
• Figure 1 is a Kaplan Meier plot of clinical sample set 1, stage I and II, using CCP score quartiles and disease survival as outcome measure.
• Figure 2 is Kaplan Meier plot of clinical sample set 1 stage IB only, using the CCP mean to separate a high CCP from a low CCP group and disease survival as outcome measure.
• FIG. 3 shows the distribution of CCP scores in two independent stage
• Figure 4 is a Kaplan Meier survival analysis of CCP score in the combined stage IB samples of set 1 and set 2.
• Figure 5 is a Kaplan Meier survival analysis of CCP and treatment in combined stage IB samples.
• Figure 6 is an illustration of an example of a system useful in certain aspects and embodiments of the invention.
• Figure 7 is a flowchart illustrating an example of a computer- implemented method of the invention.
• Figure 8 is an illustration of the predictive power for CCG panels of different sizes.
• Figure 9 shows the distribution of CCP scores in the Combined Cohort of Example 2.
• Figure 10 is a Kaplan Meier survival analysis of CCP score in the
• Figure 1 1 shows how CCP score predicts treatment benefit in Example 3.
• Figure 12 shows the consistency of hazard ratios for CCP score across cohorts.
• Figure 13 shows the consistency of hazard ratios for pathological stage across cohorts.
• Figure 14 shows predicted 5-year disease mortality risk as a function of
• Figure 15 shows 5-year disease mortality risk as predicted by Prognostic
• Figure 16 shows predicted 5-year disease mortality risk as a function of
• Figure 17 is a Kaplan Meier survival analysis of Prognostic Score (as shown in the validation study in Example 4).
• Figure 18 shows 5-year disease mortality risk as predicted by Prognostic
• the present invention is based in part on the discovery that genes whose expression closely tracks the cell cycle (“cell-cycle genes” or “CCGs”) are particularly powerful genes for classifying lung cancer, including determining prognosis and/or the likelihood a particular patient will respond to a particular treatment regimen (e.g., a regimen comprising chemotherapy).
• a particular treatment regimen e.g., a regimen comprising chemotherapy.
• CCG gene and “CCG” herein refer to a gene whose expression level closely tracks the progression of the cell through the cell-cycle. See, e.g., Whitfield et al, MOL. BIOL. CELL (2002) 13 : 1977-2000.
• the term "cell-cycle progression" or “CCP” will also be used in this application and will generally be interchangeable with CCG (i.e., a CCP gene is a CCG; a CCP score is a CCG score). More specifically, CCGs show periodic increases and decreases in expression that coincide with certain phases of the cell cycle— e.g., STK15 and PLK show peak expression at G2/M. Id.
• CCGs have clear, recognized cell-cycle related function—e.g., in DNA synthesis or repair, in chromosome condensation, in cell-division, etc.
• some CCGs have expression levels that track the cell-cycle without having an obvious, direct role in the cell-cycle— e.g., UBE2S encodes a ubiquitin-conjugating enzyme, yet its expression closely tracks the cell-cycle.
• a CCG according to the present invention need not have a recognized role in the cell-cycle.
• Exemplary CCGs are listed in Tables 1, 2, 3, 5, 6, 7, 8 & 9. A fuller discussion of CCGs, including an extensive (though not exhaustive) list of CCGs, can be found in International Application No. PCT /US2010/020397 (pub. no.
• WO/2010/080933 (see, e.g., Table 1 in WO/2010/080933).
• International Application No. PCT/US2010/020397 (pub. no. WO/2010/080933 (see also corresponding U.S. Application No. 13/177,887)) and International Application No. PCT/ US2011/043228 (pub no.
• Whether a particular gene is a CCG may be determined by any technique known in the art, including those taught in Whitfield et al, MoL. BIOL. CELL (2002) 13: 1977- 2000; Whitfield et al, MOL. CELL. BIOL. (2000) 20:4188-4198; WO/2010/080933 fl[ [0039]). All of the CCGs in Table 1 below form a panel of CCGs ("Panel A") useful in the invention. As will be shown detail throughout this document, individual CCGs (e.g., CCGs in Table 1) and subsets of these genes can also be used in the invention.
• PAICS* 10606 Hs00272390_ml NM_001079524.1; NM 001079525.1;
• ABI Assay ID means the catalogue ID number for the gene expression assay commercially available from Applied Biosystems Inc. (Foster City, CA) for the particular gene.
• one aspect of the present invention provides a method for determining the prognosis in a patient having lung cancer and/or the likelihood of response to a particular treatment regimen in a patient having lung cancer, which comprises: determining in a tumor sample from the patient the expression of a plurality of test genes comprising at least 2, 3, 4, 5, 6, 7 or at least 8, 9, 10 or 12 cell-cycle genes (e.g., genes in any of Tables 1-11 or Panels A-H, J, or K; "sub-panels" of Panel F in Tables 21 to 25), and correlating increased expression of said plurality of test genes to a poor prognosis and/or an increased likelihood of response to the particular treatment regimen (e.g., a treatment regimen comprising chemotherapy).
• a treatment regimen comprising chemotherapy
• Lung cancer as used herein includes at least adenocarcinoma, atypical lung carcinoids, and typical lung carcinoids.
• Several embodiments of the invention described herein involve a step of correlating high CCP gene expression according to the present invention (e.g., high expression of a panel of CCP genes as described in various embodiments throughout this application; a test value derived from or reflecting high expression of such a panel; etc.) to a particular clinical feature (e.g., a poor prognosis; an increased likelihood of lung cancer recurrence; an increased likelihood of response to chemotherapy; etc.) if the CCP gene expression is greater than some reference (or optionally to another feature, e.g., good prognosis, if the expression is less than some reference).
• a particular clinical feature e.g., a poor prognosis; an increased likelihood of lung cancer recurrence; an increased likelihood of response to chemotherapy; etc.
• a further, related embodiment of the invention may involve, in addition to or instead of a correlating step, one or both of the following steps: (a) concluding that the patient has (or classifying the patient as having) the clinical feature based at least in part on high CCP expression (or a test value derived from or reflecting such); or (b) communicating that the patient has the clinical feature based at least in part on high CCP expression (or a test value derived from or reflecting such).
• one embodiment described in this document is a method for determining in a patient the prognosis of lung cancer or the likelihood of such a patient to respond to chemotherapy, comprising: (1) determining the expression of a plurality of test genes comprising at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or 15 or more cell-cycle genes (e.g., CCGs in Panel F; in any of Panels H, I, J, L, M, N & O; or in any sub- panel of Panel F in any of Tables 21 through 25; etc.), and (2) correlating high expression of said plurality of test genes to poor prognosis of the lung cancer in the patient or an increased likelihood of response to chemotherapy.
• a plurality of test genes comprising at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or 15 or more cell-cycle genes (e.g., CCGs in Panel F; in any of Panels H, I, J, L, M, N & O; or in any sub- panel of Panel F in any of Tables 21 through 25; etc.
• this description of this embodiment is understood to include a description of two further, related embodiments, i.e., a method for determining in a patient the prognosis of lung cancer or the likelihood of such a patient to respond to chemotherapy, comprising: (1) determining the expression of a plurality of test genes comprising at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or 15 or more cell-cycle genes (e.g., CCGs in Panel F; in any of Panels H, I, J, L, M, N & O; or in any sub-panel of Panel F in any of Tables 21 through 25; etc.), and (2)(a) concluding that said patient has a poor prognosis of the lung cancer in the patient or an increased likelihood of response to chemotherapy based at least in part on high expression of said plurality of test genes; or (2)(b) communicating that said patient has a poor prognosis of the lung cancer in the patient or an increased likelihood of response to chemotherapy based at least in part on high expression of said plurality of test genes.
• correlating a particular assay or analysis output e.g., high CCG expression, test value incorporating CCG expression greater than some reference value, etc.
• some likelihood e.g., increased, not increased, decreased, etc.
• some clinical event or outcome e.g., recurrence, progression, cancer-specific death, etc.
• such correlating may comprise assigning a risk or likelihood of the clinical event or outcome occurring based at least in part on the particular assay or analysis output.
• risk is a percentage probability of the event or outcome occurring.
• the patient is assigned to a risk group (e.g., low risk, intermediate risk, high risk, etc.).
• low risk is any percentage probability below 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50%.
• intermediate risk is any percentage probability above 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% and below 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75%.
• high risk is any percentage probability above 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%.
• communicating means to make such information known to another person or transfer such information to a thing (e.g., a computer).
• a patient's prognosis or risk of recurrence is communicated.
• the information used to arrive at such a prognosis or risk prediction e.g., expression levels of a panel of biomarkers comprising a plurality of CCGs, clinical or pathologic factors, etc.
• This communication may be auditory (e.g., verbal), visual (e.g., written), electronic (e.g., data transferred from one computer system to another), etc.
• communicating a cancer e.g., a cancer
• the classification comprises generating a report that communicates the cancer classification.
• the report is a paper report, an auditory report, or an electronic record.
• the report is displayed and/or stored on a computing device (e.g., handheld device, desktop computer, smart device, website, etc.).
• the cancer classification is communicated to a physician (e.g., a report communicating the classification is provided to the physician).
• the cancer classification is communicated to a patient (e.g., a report communicating the classification is provided to the patient).
• Communicating a cancer classification can also be accomplished by transferring information (e.g., data) embodying the classification to a server computer and allowing an intermediary or end-user to access such information (e.g., by viewing the information as displayed from the server, by downloading the information in the form of one or more files transferred from the server to the intermediary or end-user's device, etc.).
• information e.g., data
• intermediary or end-user e.g., by viewing the information as displayed from the server, by downloading the information in the form of one or more files transferred from the server to the intermediary or end-user's device, etc.
• an embodiment of the invention comprises concluding some fact (e.g., a patient's prognosis or a patient's likelihood of recurrence)
• this may include a computer program concluding such fact, typically after performing some algorithm that incorporates information on the status of CCGs in a patient sample (e.g., as shown in Figure 7).
• determining the expression of a plurality of genes comprises receiving a report communicating such expression.
• this report communicates such expression in a qualitative manner (e.g., "high” or “increased”).
• this report communicates such expression indirectly by communicating a score (e.g., prognosis score, recurrence score, etc.) that incorporates such expression.
• the method includes (1) obtaining a sample from a patient having lung cancer; (2) determining the expression of a panel of genes in the tumor sample including at least 2, 4, 5, 6, 7 or at least 8, 9, 10 or 12 cell-cycle genes (e.g., genes in any of Tables 1-11 or Panels A-H, J, or K; "sub-panels" of Panel F in Tables 21 to 25); (3) providing a test value by (a) weighting the determined expression of each of a plurality of test genes selected from the panel of genes with a predefined coefficient, and (b) combining the weighted expression to provide said test value, wherein at least 20%, at least 50%, at least 75% or at least 90% of said plurality of test genes are cell-cycle genes (e.g., genes in any of Tables 1-1 1 or Panels A-H, J, or K; "sub-panels” of Panel F in Tables 21 to 25); and (4)(a) correlating an increased level of expression of the plurality of test genes to a poor
• the method comprises (4)(a) concluding that the patient has a poor prognosis and/or an increased likelihood of response to the particular treatment regimen based at least in part on increased expression of said plurality of test genes or (b) concluding that the patient has a good prognosis and/or no increased likelihood of response to the particular treatment regimen based at least in part on no increased expression of said plurality of test genes; and/or (4)(a) communicating that the patient has a poor prognosis and/or an increased likelihood of response to the particular treatment regimen based at least in part on increased expression of said plurality of test genes or (b) communicating that the patient has a good prognosis and/or no increased likelihood of response to the particular treatment regimen based at least in part on no increased expression of said plurality of test genes.
• test genes are weighted such that the cell- cycle genes are weighted to contribute at least 50%, at least 55%, at least 60%, at least 65%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% of the test value.
• 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 75%, 80%, 85%, 90%, 95%, or at least 99% or 100% of the plurality of test genes are cell-cycle genes. Unless otherwise indicated, "obtaining a sample” herein means “providing or obtaining.”
• the method comprises: (1) obtaining a tumor sample from a patient identified as having lung cancer; (2) determining the expression of a panel of genes in the tumor sample including at least 2, 4, 6, 8 or 10 cell-cycle genes (e.g., genes in any of Tables 1-1 1 or Panels A-H, J, or K; "sub-panels" of Panel F in Tables 21 to 25); and (3) providing a test value by (a) weighting the determined expression of each of a plurality of test genes selected from said panel of genes with a predefined coefficient, and (b) combining the weighted expression to provide said test value, wherein the cell-cycle genes are weighted to contribute at least 20%, 50%, at least 75% or at least 90% of the test value; and (4)(a) correlating an increased level of expression of the plurality of test genes to a poor prognosis and/or an increased likelihood of response to the particular treatment regimen (e.g., a treatment regimen comprising chemotherapy) or (b) correlating no increased level
• the particular treatment regimen e.g
• the method comprises (4)(a) concluding that the patient has a poor prognosis and/or an increased likelihood of response to the particular treatment regimen based at least in part on increased expression of said plurality of test genes or (b) concluding that the patient has a good prognosis and/or no increased likelihood of response to the particular treatment regimen based at least in part on no increased expression of said plurality of test genes; and/or (4)(a) communicating that the patient has a poor prognosis and/or an increased likelihood of response to the particular treatment regimen based at least in part on increased expression of said plurality of test genes or (b) communicating that the patient has a good prognosis and/or no increased likelihood of response to the particular treatment regimen based at least in part on no increased expression of said plurality of test genes.
• the present invention encompasses a further, related embodiment involving a test value or score (e.g., CCP score, etc.) derived from, incorporating, and/or, at least to some degree, reflecting such expression levels.
• a test value or score e.g., CCP score, etc.
• the bare CCP gene expressions data or levels need not be used in the various methods, systems, etc. of the invention; a test value or score derived from such numbers or lengths may be used.
• test value will be compared to a reference value (as described at length in this document) and the method will end by correlating a high test value (or a test value derived from, incorporating, and/or, at least to some degree, reflecting high CCP gene expression) to a poor prognosis.
• a high test value or a test value derived from, incorporating, and/or, at least to some degree, reflecting high CCP gene expression
• the invention encompasses, mutatis mutandis, corresponding embodiments where the test value or score is used to determine the patient's prognosis, the patient's likelihood of response to a particular treatment regimen, the patient's or patient's sample's likelihood of having a breast cancer recurrence, etc.
• the invention generally comprises determining the status of a panel of genes comprising at least two CCGs, in tissue or cell sample, particularly a tumor sample, from a patient.
• determining the status of a gene refers to determining the presence, absence, or extent/level of some physical, chemical, or genetic characteristic of the gene or its expression product(s). Such characteristics include, but are not limited to, expression levels, activity levels, mutations, copy number, methylation status, etc.
• CCGs in the context of CCGs as used to determine likelihood of response to a particular treatment regimen (e.g., a treatment regimen comprising chemotherapy), particularly useful characteristics include expression levels (e.g., mRNA, cDNA or protein levels) and activity levels. Characteristics may be assayed directly (e.g., by assaying a CCG's expression level) or determined indirectly (e.g., assaying the level of a gene or genes whose expression level is correlated to the expression level of the CCG).
• expression levels e.g., mRNA, cDNA or protein levels
• activity levels e.g., activity levels
• Characteristics may be assayed directly (e.g., by assaying a CCG's expression level) or determined indirectly (e.g., assaying the level of a gene or genes whose expression level is correlated to the expression level of the CCG).
• ABSORS means a marker's status in a particular sample differs from the status generally found in average samples (e.g., healthy samples, average diseased samples). Examples include mutated, elevated, decreased, present, absent, etc.
• An "elevated status” means that one or more of the above characteristics (e.g., expression or mRNA level) is higher than normal levels. Generally this means an increase in the characteristic (e.g., expression or mRNA level) as compared to an index value as discussed below.
• a “low status” means that one or more of the above characteristics (e.g., gene expression or mRNA level) is lower than normal levels. Generally this means a decrease in the characteristic (e.g., expression) as compared to an index value as discussed below.
• a threshold status means that one or more of the above characteristics (e.g., gene expression or mRNA level) is lower than normal levels. Generally this means a decrease in the characteristic (e.g., expression) as compared to an index value as discussed below
• negative status generally means the characteristic is absent or undetectable or, in the case of sequence analysis, there is a deleterious sequence variant (including full or partial gene deletion).
• Gene expression can be determined either at the RNA level (i.e., mRNA or noncoding RNA (ncRNA)) (e.g., miRNA, tRNA, rRNA, snoRNA, siRNA and piRNA) or at the protein level. Measuring gene expression at the mRNA level includes measuring levels of cDNA corresponding to mRNA. Levels of proteins in a tumor sample can be determined by any known technique in the art, e.g., HPLC, mass spectrometry, or using antibodies specific to selected proteins (e.g., IHC, ELISA, etc.).
• the amount of RNA transcribed from the panel of genes including test genes is measured in the tumor sample.
• the amount of RNA of one or more housekeeping genes in the tumor sample is also measured, and used to normalize or calibrate the expression of the test genes.
• normalizing genes and “housekeeping genes” are defined herein below.
• the plurality of test genes may include at least 2, 3 or 4 cell-cycle genes, which constitute at least 50%, 75% or 80% of the plurality of test genes, and preferably 100% of the plurality of test genes.
• the plurality of test genes includes at least 5, 6, 7, or at least 8 cell-cycle genes, which constitute at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80% or 90% of the plurality of test genes, and preferably 100% of the plurality of test genes.
• a panel of genes is a plurality of genes. In some embodiments these genes are assayed together in one or more samples from a patient.
• the plurality of test genes includes at least 8, 10,
• cell-cycle genes which constitute at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80% or 90% of the plurality of test genes, and preferably 100% of the plurality of test genes.
• tumor sample means any biological sample containing one or more tumor cells, or one or more tumor-derived DNA, RNA or protein, and obtained from a cancer patient.
• a tissue sample obtained from a tumor tissue of a cancer patient is a useful tumor sample in the present invention.
• the tissue sample can be an FFPE sample, or fresh frozen sample, and preferably contain largely tumor cells.
• a single malignant cell from a cancer patient's tumor is also a useful tumor sample. Such a malignant cell can be obtained directly from the patient's tumor, or purified from the patient's bodily fluid (e.g., blood, urine).
• a bodily fluid such as blood, urine, sputum and saliva containing one or tumor cells, or tumor-derived RNA or proteins, can also be useful as a tumor sample for purposes of practicing the present invention.
• the patient having a cancer e.g., lung cancer
• telomere length e.g., telomere length, telomere length, telomere length, telomere length, etc.
• qRT-PCRTM quantitative real-time PCRTM
• immunoanalysis e.g., ELISA, immunohistochemistry
• sequencing e.g., quantitative sequencing
• the activity level of a polypeptide encoded by a gene may be used in much the same way as the expression level of the gene or polypeptide. Often higher activity levels indicate higher expression levels and while lower activity levels indicate lower expression levels.
• the invention provides any of the methods discussed above, wherein the activity level of a polypeptide encoded by the CCG is determined rather than or in addition to the expression level of the CCG.
• the activity level of a polypeptide encoded by the CCG is determined rather than or in addition to the expression level of the CCG.
• Those skilled in the art are familiar with techniques for measuring the activity of various such proteins, including those encoded by the genes listed in Exemplary CCGs are listed in Tables 1, 2, 3, 5, 6, 7, 8, 9, 10 & 1 1.
• the methods of the invention may be practiced independent of the particular technique used.
• the expression of one or more normalizing amino acids [0060] In preferred embodiments, the expression of one or more normalizing amino acids
• normalizing genes referred to the genes whose expression is used to calibrate or normalize the measured expression of the gene of interest (e.g., test genes). Importantly, the expression of normalizing genes should be independent of cancer
• housekeeping genes known in the art can be used.
• Housekeeping genes are well known in the art, with examples including, but are not limited to, GUSB (glucuronidase, beta), HMBS (hydroxymethylbilane synthase), SDHA (succinate dehydrogenase complex, subunit A, flavoprotein), UBC (ubiquitin C) and YWHAZ (tyrosine 3- monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide).
• GUSB glucose curonidase, beta
• HMBS hydroxymethylbilane synthase
• SDHA succinate dehydrogenase complex, subunit A, flavoprotein
• UBC ubiquitin C
• YWHAZ tyrosine 3- monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide.
• At least 2, 3, 4, 5, 6, 7, 8, 9, 10 or 15 housekeeping genes are used to provide a combined normalizing gene set.
• the amount of gene expression of such normalizing genes can be averaged, combined together by straight additions or by a defined algorithm.
• Some examples of particularly useful housekeeper genes for use in the methods and compositions of the invention include those listed in Table A below.
• RNA levels for the genes In the case of measuring RNA levels for the genes, one convenient and sensitive approach is real-time quantitative PCRTM (qPCR) assay, following a reverse transcription reaction.
• qPCR real-time quantitative PCRTM
• a cycle threshold C t is determined for each test gene and each normalizing gene, i.e., the number of cycles at which the fluorescence from a qPCR reaction above background is detectable.
• the overall expression of the one or more normalizing genes can be represented by a "normalizing value" which can be generated by combining the expression of all normalizing genes, either weighted eaqually (straight addition or averaging) or by different predefined coefficients.
• the normalizing value Cm can be the cycle threshold (C t ) of one single normalizing gene, or an average of the C t values of 2 or more, preferably 10 or more, or 15 or more normalizing genes, in which case, the predefined coefficient is 1/N, where N is the total number of normalizing genes used.
• Cm (Can + C t H2 + " C t Hn) N.
• the methods of the invention generally involve determining the level of expression of a panel of CCGs. With modern high-throughput techniques, it is often possible to determine the expression level of tens, hundreds or thousands of genes. Indeed, it is possible to determine the level of expression of the entire transcriptome (i.e., each transcribed sequence in the genome). Once such a global assay has been performed, one may then informatically analyze one or more subsets of transcripts (i.e., panels or, as often used herein, pluralities of test genes).
• test genes comprising primarily CCGs according to the present invention by combining the expression level values of the individual test genes to obtain a test value.
• the test value provided in the present invention can represent the overall expression level of the plurality of test genes composed substantially of (or weighted to be represented substantially by) cell-cycle genes.
• the test value incorporating the overall expression of the plurality of test genes can be provided by combining the normalized expression of all test genes, either by straight addition or averaging (i.e., weighted equally) or by a different predefined coefficient.
• test value (AC t i + AC t 2 + " ' + AC tn )/n.
• this document discloses using the expression of a plurality of genes (e.g., "determining [in a tumor sample from the patient] the expression of a plurality of test genes” or “correlating increased expression of said plurality of test genes to an increased likelihood of response")
• this includes in some embodiments using a test value incorporating, representing or corresponding to the overall expression of this plurality of genes (e.g., "determining [in a tumor sample from the patient] a test value representing the expression of a plurality of test genes” or “correlating an increased test value [or a test value above some reference value] representing the expression of said plurality of test genes to an increased likelihood of response”).
• CCGs do not correlate well with the mean.
• such genes may be grouped, assayed, analyzed, etc. separately from those that correlate well. This is especially useful if these non-correlated genes are
• non-correlated genes are analyzed together with correlated genes.
• a CCG is non-correlated if its correlation to the CCG mean is less than 0.5, 0.4, 0.3, 0.2, 0.10, 0.09, 0.08, 0.07, 0.06, 0.05, 0.04, 0.03, 0.02, 0.01 or less.
• the individual predictive power of each gene may be used to rank them in importance.
• the inventors have determined that the CCGs in Panel C can be ranked as shown in Table 6 below according to the predictive power of each individual gene.
• the CCGs in Panel F can be similarly ranked as shown in Table 7 below.
• the plurality of test genes comprises the top 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40 or more CCGs listed in Table 2, 3, 5, 6, 7, 12, 13, 14, 15, 16, 17, 18 or 19.
• the plurality of test genes comprises at least some number of CCGs (e.g., at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more CCGs) and this plurality of CCGs comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 of the following genes: ASPM, BIRC5, BUB IB, CCNB2, CDC2, CDC20, CDCA8, CDKN3, CENPF, DLGAP5, FOXM1, KIAAOlOl, KIFll, KIF2C, KIF4A, MCMIO, NUSAPl, PRCl, RACGAPl, and TPX2.
• CCGs e.g., at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more CCGs
• ASPM BIRC5, BUB IB, CCNB2, CDC2, CDC20, CDCA8, CDKN3, CENPF, DLGAP5, FOXM1, KIAAOlOl, K
• the plurality of test genes comprises at least some number of CCGs (e.g., at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more CCGs) and this plurality of CCGs comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 of the following genes: TPX2, CCNB2, KIF4A, KIF2C, BIRC5, RACGAPl, CDC2, PRCl, DLGAP5/DLG7, CEP55, CCNB1, TOP2A, CDC20, KIF20A, BUB1B, CDKN3, NUSAPl, CCNA2, KIFll, and CDCA8.
• CCGs e.g., at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more CCGs
• this plurality of CCGs comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 of the following genes: TPX2, CCNB2, KIF4A, KIF2C,
• the plurality of test genes comprises at least some number of CCGs (e.g., at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more CCGs) and this plurality of CCGs comprises any one, two, three, four, five, six, seven, eight, nine, or ten or all of gene numbers 1 & 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7, 1 to 8, 1 to 9, or 1 to 10 of any of Table 2, 3, 5, 6, 7, 12, 13, 14, 15, 16, 17, 18 or 19.
• CCGs e.g., at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more CCGs
• this plurality of CCGs comprises any one, two, three, four, five, six, seven, eight, nine, or ten or all of gene numbers 1 & 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7, 1 to 8, 1 to 9, or 1 to 10 of any of Table 2, 3, 5, 6, 7, 12, 13, 14, 15, 16, 17, 18 or 19.
• the plurality of test genes comprises at least some number of CCGs (e.g., at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more CCGs) and this plurality of CCGs comprises any one, two, three, four, five, six, seven, eight, or nine or all of gene numbers 2 & 3, 2 to 4, 2 to 5, 2 to 6, 2 to 7, 2 to 8, 2 to 9, or 2 to 10 of any of Table 2, 3, 5, 6, 7, 12, 13, 14, 15, 16, 17, 18 or 19.
• CCGs e.g., at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more CCGs
• this plurality of CCGs comprises any one, two, three, four, five, six, seven, eight, or nine or all of gene numbers 2 & 3, 2 to 4, 2 to 5, 2 to 6, 2 to 7, 2 to 8, 2 to 9, or 2 to 10 of any of Table 2, 3, 5, 6, 7, 12, 13, 14, 15, 16, 17, 18 or 19.
• the plurality of test genes comprises at least some number of CCGs (e.g., at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more CCGs) and this plurality of CCGs comprises any one, two, three, four, five, six, seven, or eight or all of gene numbers 3 & 4, 3 to 5, 3 to 6, 3 to 7, 3 to 8, 3 to 9, or 3 to 10 of any of Table 2, 3, 5, 6, 7, 12, 13, 14, 15, 16, 17, 18 or 19.
• CCGs e.g., at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more CCGs
• this plurality of CCGs comprises any one, two, three, four, five, six, seven, or eight or all of gene numbers 3 & 4, 3 to 5, 3 to 6, 3 to 7, 3 to 8, 3 to 9, or 3 to 10 of any of Table 2, 3, 5, 6, 7, 12, 13, 14, 15, 16, 17, 18 or 19.
• the plurality of test genes comprises at least some number of CCGs (e.g., at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more CCGs) and this plurality of CCGs comprises any one, two, three, four, five, six, or seven or all of gene numbers 4 & 5, 4 to 6, 4 to 7, 4 to 8, 4 to 9, or 4 to 10 of any of Table 2, 3, 5, 6, 7, 12, 13, 14, 15, 16, 17, 18 or 19.
• CCGs e.g., at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more CCGs
• this plurality of CCGs comprises any one, two, three, four, five, six, or seven or all of gene numbers 4 & 5, 4 to 6, 4 to 7, 4 to 8, 4 to 9, or 4 to 10 of any of Table 2, 3, 5, 6, 7, 12, 13, 14, 15, 16, 17, 18 or 19.
• the plurality of test genes comprises at least some number of CCGs (e.g., at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more CCGs) and this plurality of CCGs comprises any one, two, three, four, five, six, seven, eight, nine, 10, 11, 12, 13, 14, or 15 or all of gene numbers 1 & 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7, 1 to 8, 1 to 9, 1 to 10, 1 to 11, 1 to 12, 1 to 13, 1 to 14, or 1 to 15 of any of Table 2, 3, 5, 6, 7, 12, 13, 14, 15, 16, 17, 18 or 19.
• CCGs e.g., at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more CCGs
• this plurality of CCGs comprises any one, two, three, four, five, six, seven, eight, nine, 10, 11, 12, 13, 14, or 15 or all of gene numbers 1 & 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7, 1 to 8, 1 to 9, 1
• the plurality of test genes comprises at least some number of CCGs (e.g., at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more CCGs) and this plurality of CCGs comprises gene numbers 1 & 2; 1 & 2-3; 1 & 3-4; 1 & 4-5; 1 & 5-6; 1 & 6-7; 1 & 7-8; 1 & 8-9; 1 & 9 & 10; 1 & 10 & 1 1; 1 & 3; 1 & 2-4; 1 & 3-5; 1 & 4-6; 1 & 5-7; 1 & 6-8; 1 & 7-9; 1 & 8-10; 1 & 9 & 1 1 ; 1 & 4; 1 & 2-5; 1 & 3-6; 1 & 4-7; 1 & 5-8; 1 & 6-9; 1 & 7-10; 1 & 8-11 ; 1 & 5; 1 & 2-6; 1 & 3-7; 1 &
• the test value incorporating or representing the overall expression of the plurality of test genes is compared to one or more reference values (or index values), and optionally correlated to a poor or good prognosis (e.g., shorter expected post-surgery metastasis-free survival) or an increased or no increased likelihood of response to treatment comprising chemotherapy.
• a test value greater than the reference value(s) or a test value that, relative to the reference value, represents increased expression of the test genes
• the test value is deemed “greater than” the reference value (e.g., the threshold index value), and thus correlated to a poor prognosis and/or an increased likelihood of response to treatment comprising chemotherapy, if the test value exceeds the reference value by at least some amount (e.g., at least 0.5, 0.75, 0.85, 0.90, 0.95, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more fold or standard deviations).
• the reference value e.g., the threshold index value
• the index value may incorporate or represent the gene expression levels found in a normal sample obtained from the patient of interest (including tissue surrounding the cancerous tissue in a biopsy), in which case an expression level in the tumor sample significantly higher than this index value would indicate, e.g., increased likelihood of response to a particular treatment regimen (e.g., a treatment regimen comprising chemotherapy).
• the index value may incorporate or represent the average expression level for a set of individuals from a diverse cancer population or a subset of the population. For example, one may determine the average expression level of a gene or gene panel in a random sampling of patients with cancer (e.g., lung cancer).
• This average expression level may be termed the "threshold index value,” with patients having a test value higher than this value or a test value representing expression higher than the expression represented by the threshold index value (or at least some amount higher than this value) expected to have a better prognosis and/or a greater likelihood of response to a particular treatment regimen (e.g., a treatment regimen comprising chemotherapy) than those having a test value lower than this value.
• a particular treatment regimen e.g., a treatment regimen comprising chemotherapy
• the index value may incorporate or represent the average expression level of a particular gene or gene panel in a plurality of training patients (e.g., lung cancer patients) with similar outcomes whose clinical and follow-up data are available and sufficient to define and categorize the patients by disease outcome, e.g., response to a particular treatment regimen (e.g., a treatment regimen comprising chemotherapy). See, e.g., Examples, infra.
• a particular treatment regimen e.g., a treatment regimen comprising chemotherapy.
• a "poor prognosis index value” or a "good response index value” can be generated from a plurality of training cancer patients characterized as having "poor prognosis” or a "good prognosis/response", e.g., relatively short expected survival (e.g., overall survival, disease-free survival, distant metastasis-free survival, etc.); complete response, partial response, or stable disease (e.g., by RECIST criteria) after treatment comprising chemotherapy.
• relatively short expected survival e.g., overall survival, disease-free survival, distant metastasis-free survival, etc.
• complete response, partial response, or stable disease e.g., by RECIST criteria
• a "good response index value” or a"poor response index value” can be generated from a plurality of training cancer patients defined as having "good prognosis” or “poor response”, e.g., absence of complete response, partial response, or stable disease (e.g., by RECIST criteria) after treatment comprising chemotherapy.
• a good response index value of a particular gene or gene panel may represent the average level of expression of the particular gene or gene panel in patients having a "good response”
• a poor response index value of a particular gene or gene panel represents the average level of expression of the particular gene or gene panel in patients having a "poor response.”
• index values may be determined thusly:
• a threshold value may be set for the cell cycle mean.
• the optimal threshold value is selected based on the receiver operating characteristic (ROC) curve, which plots sensitivity vs (1 - specificity). For each increment of the cell cycle mean, the sensitivity and specificity of the test is calculated using that value as a threshold.
• the actual threshold will be the value that optimizes these metrics according to the artisan's requirements (e.g., what degree of sensitivity or specificity is desired, etc.).
• FIG. l and the accompanying discussion herein demonstrate determination of a threshold value determined and validated
• Panels of CCGs can accurately predict response, as shown in FIG. l and Table 20.
• Those skilled in the art are familiar with various ways of determining the expression of a panel of genes (i. e. , a plurality of genes).
• Increased expression in this context will mean the average expression is higher than the average expression level of these genes in some reference (e.g., higher than in normal patients; higher than some index value that has been determined to represent the average expression level in a reference population, such as patients with the same cancer; etc.).
• a certain number e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30 or more
• a certain proportion e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 100%
• the expression of a panel of genes by determining the absolute copy number of the analyte representing each gene in the panel (e.g., mR A, cDNA, protein) and either total or average these across the genes.
• Response (e.g., response to a particular treatment regimen) is a well- known term in the art and is used herein according to its known meaning.
• the meaning of "response” may be cancer-type dependent, with response in lung cancer meaning something different from response in prostate cancer.
• some objective criteria of response include Response Evaluation Criteria In Solid Tumors (RECIST), a set of published rules (e.g., changes in tumor size, etc.) that define when cancer patients improve (“respond”), stay the same (“stabilize”), or worsen ("progression”) during treatments.
• RECIST Response Evaluation Criteria In Solid Tumors
• Response can also include survival metrics (e.g., “disease-free survival” (DFS), “overall survival” (OS), etc).
• DFS disease-free survival
• OS overall survival
• RECIST criteria can include: (a) Complete response (CR): disappearance of all metastases; (b) Partial response (PR): at least a 30% decrease in the sum of the largest diameter (LD) of the metastatic lesions, taking as reference the baseline sum LD; (c) Stable disease (SD): neither sufficient shrinkage to qualify for PR nor sufficient increase to qualify for PD taking as references the smallest sum LD since the treatment started; (d) Progression (PD): at least a 20% increase in the sum of the LD of the target metastatic lesions taking as reference the smallest sum LD since the treatment started or the appearance of one or more new lesions.
• CR Complete response
• PR Partial response
• SD Stable disease
• PD Progression
• a medical management regimen with at least some defined parameters. These may include administration (including prescription) of particular therapeutic agent alone; a specific combination of agents (e.g., FOLFOX, FOLFIRI); a combination of agents at least comprising a particular agent (e.g., 5- fluorouracil) or subcombination of agents (e.g., platinum compounds with taxanes) together with any other agents or interventions (e.g., surgery, radiation); a surgical or other intervention (e.g., surgical resection of the tumor, radiation therapy); or any combination of these (e.g., surgical resection of the tumor followed by chemotherapy, also known as "adjuvant" chemotherapy).
• agents e.g., FOLFOX, FOLFIRI
• a combination of agents at least comprising a particular agent e.g., 5- fluorouracil
• subcombination of agents e.g., platinum compounds with taxanes
• any other agents or interventions e.g., surgery, radiation
• the particular treatment e.g., a treatment regimen comprising chemotherapy
• comprises a platinum-based compound e.g., cisplatin, carboplatin, oxaliplatin
• a taxane e.g., docetaxel, paclitaxel
• CCGs For many lung cancer patients and their physicians surgery to remove the tumor (sometimes including surrounding healthy tissue) is the standard of care. Because surgery can cure some patients and adjuvant chemotherapy is debilitating and expensive, the decision whether to undertake adjuvant chemotherapy is more difficult. In some embodiments, increased expression of CCGs correlates with increased likelihood of response to adjuvant chemotherapy (and thus in some embodiments adjuvant chemotherapy is administered, recommended or prescribed if expression of CCGs is increased).
• increased expression of a plurality of test genes comprising CCGs, where CCGs are weighted to contribute at least 50% or more to a test value incorporating or representing the expression of the plurality of test genes, correlates with increased likelihood of response to adjuvant chemotherapy (and thus in some embodiments adjuvant chemotherapy is administered, recommended or prescribed if expression of the plurality of test genes is increased).
• a patient has an "increased likelihood" of some clinical feature or outcome (e.g., response) if the probability of the patient having the feature or outcome exceeds some reference probability or value.
• the reference probability may be the probability of the feature or outcome across the general relevant patient population. For example, if the probability of response (e.g., to treatment comprising chemotherapy) in the general lung cancer patient population (or some specific subpopulation, e.g., in stage la, lb, or II lung cancer patients) is X% and a particular patient has been determined by the methods of the present invention to have a probability of response of Y%, and if Y > X, then the patient has an "increased likelihood" of response.
• a threshold or reference value may be determined and a particular patient's probability of response may be compared to that threshold or reference. Because predicting response is a prognostic endeavor, "predicting prognosis" will sometimes be used herein to refer to predicting response.
• prognosis is often used in a relative sense. Often when it is said that a patient has a poor prognosis, this means the patient has a worse prognosis than other (e.g., average) patients (or worse than the patient would have had if the patient had different clinical indications). Thus, unless expressly stated otherwise or the context clearly indicates otherwise, “poor prognosis” includes “poorer prognosis” and “good prognosis” includes “better prognosis.” As discussed elsewhere in this document, prognosis can include a patient's likelihood of cancer recurrence, cancer metastasis, or new primary cancer(s).
• “poor prognosis” means the patient has an "increased likelihood” (as discussed in the preceding paragraph) of one of these clinical outcomes.
• Prognosis can also include the likelihood of survival (e.g., overall survival, disease-free survival, distant metastasis-free survival, etc.).
• “poor prognosis” means either (a) the patient's (estimated) expected survival time is some certain amount (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 years), which is lower than some reference amount; or (b) the patient has a "decreased likelihood” (as discussed in the preceding paragraph) of survival beyond a certain amount of time (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or more years). The opposite would of course be true for a "good prognosis.”
• some embodiments of the invention comprise determining the expression of a single CCG listed in any of Table 1, 2, 3, 5, 6, 7, 8, 9, 10 or 1 1 or Panel A, B, C, D, E, F, G, H, J or K and correlating increased expression to increased likelihood of response.
• FIG. l and Table 20 show that panels of CCGs (e.g., 2, 3, 4, 5, or 6
• the invention provides a method of classifying a cancer comprising determining the status of a panel of genes (e.g., a plurality of test genes) comprising a plurality of CCGs.
• a panel of genes e.g., a plurality of test genes
• increased expression in a panel of genes may refer to the average expression level of all panel or test genes in a particular patient being higher than the average expression level of these genes in normal patients (or higher than some index value that has been determined to represent the normal average expression level).
• increased expression in a panel of genes may refer to increased expression in at least a certain number (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30 or more) or at least a certain proportion (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 100%) of the genes in the panel as compared to the average normal expression level.
• a certain number e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30 or more
• a certain proportion e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 100%
• the panel comprises at least 3, 4, 5, 6, 7, 8, 9, 10,
• the panel comprises at least 10, 15, 20, or more CCGs. In some embodiments the panel comprises between 5 and 100 CCGs, between 7 and 40 CCGs, between 5 and 25 CCGs, between 10 and 20 CCGs, or between 10 and 15 CCGs. In some embodiments CCGs comprise at least a certain proportion of the panel. Thus in some embodiments the panel comprises at least 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% CCGs.
• the panel comprises at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 70, 80, 90, 100, 200, or more CCGs, and such CCGs constitute of at least 50%, 60%, 70%, preferably at least 75%, 80%, 85%, more preferably at least 90%, 95%, 96%, 97%, 98%, or 99% or more of the total number of genes in the panel.
• the panel of CCGs comprises the genes in Table 1, 2, 3, 5, 6, 7, 8, 9, 10 or 11 ; Panel A, B, C, D, E, F, G, H, J or K; or "sub- panels" of Panel F in Tables 21 to 25.
• the panel comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 20, 25, 30, or more of the genes in Table 1, 2, 3, 5, 6, 7, 8, 9, 10 or 11 ; Panel A, B, C, D, E, F, G, H, J or K; or "sub-panels" of Panel F in Tables 21 to 25.
• the invention provides a method of determining prognosis and/or predicting response to a particular treatment regimen (e.g., a regimen comprising
• the method comprising determining the status of the CCGs in any one of Table 1, 2, 3, 5, 6, 7, 8, 9, 10 or 1 1; Panel A, B, C, D, E, F, G, H, J or K; or "sub-panels" of Panel F in Tables 21 to 25 and correlating increased expression of the panel to a poor prognosis and/or increased likelihood of response to the treatment regimen.
• Panel A, B, C, D, E, F, G, H, J or K; or "sub-panels" of Panel F in Tables 21 to 25) are useful in determining prognosis and/or predicting response to particular treatment.
• Table 18 ranks the CCP genes of Table 10 according to the highest weighted combination score (which gives greater weight to p-value over correlation to the mean) calculated by the following formula:
• Panel F of Table 9 were ranked according to independent predictive value (p-value) in the study reported as Example 3 below. These rankings are shown in Table 19 below.
• CCG signatures the particular CCGs assayed is often not as important as the total number of CCGs.
• the number of CCGs assayed can vary depending on many factors, e.g., technical constraints, cost considerations, the classification being made, the cancer being tested, the desired level of predictive power, etc.
• Increasing the number of CCGs assayed in a panel according to the invention is, as a general matter, advantageous because, e.g., a larger pool of mRNAs to be assayed means less "noise" caused by outliers and less chance of an assay error throwing off the overall predictive power of the test.
• cost and other considerations will generally limit this number and finding the optimal number of CCGs for a signature is desirable.
• each 2-gene and 3-gene sub-panel chosen from Panel F is significantly predictive of lung cancer prognosis in the cohorts described in Examples 1-3.
• the panel of genes comprises a sub-panel of any of Tables 21 to 25.
• the invention provides a method of determining the prognosis of a patient having lung cancer or the likelihood of cancer recurrence in said patient, comprising: (1) obtaining a sample from said patient; (2) determining the expression levels of a panel of genes in said sample, wherein said panel comprises a sub-panel of Panel F chosen from any of Tables 21 to 25; (3) providing a test value by (i) weighting the determined expression of each of a plurality of test genes selected from said panel of genes with a predefined coefficient, and (ii) combining the weighted expression to provide said test value, wherein the genes of said sub-panel are weighted (e.g., collectively) to contribute at least 25% of the test value; and (4) classifying said patient as having a poor or a good prognosis
• the optimal number of CCGs in a signature (no) can be found wherever the following is true
• P is the predictive power (i.e., P Sil is the predictive power of a signature with n genes and P Plastic + i is the predictive power of a signature with n genes plus one) and Co is some optimization constant.
• Predictive power can be defined in many ways known to those skilled in the art including, but not limited to, the signature's p-value.
• Co can be chosen by the artisan based on his or her specific constraints. For example, if cost is not a critical factor and extremely high levels of sensitivity and specificity are desired, Co can be set very low such that only trivial increases in predictive power are disregarded. On the other hand, if cost is decisive and moderate levels of sensitivity and specificity are acceptable, Co can be set higher such that only significant increases in predictive power warrant increasing the number of genes in the signature.
• a graph of predictive power as a function of gene number may be plotted (as in FIG.1) and the second derivative of this plot taken.
• the point at which the second derivative decreases to some predetermined value (Co') may be the optimal number of genes in the signature.
• FIG.1 illustrates the empirical determination of optimal numbers of
• some embodiments of the invention provide a method of predicting prognosis (or likelihood of response to a particular treatment regimen) in a patient having lung cancer comprising determining the status of a panel of genes, wherein the panel comprises between about 10 and about 15 CCGs and increased expression of the CCGs indicates a poor prognosis (or an increased likelihood of response to the particular treatment, e.g., treatment comprising chemotherapy).
• the panel comprises between about 10 and about 15 CCGs and the CCGs constitute at least 90% of the panel (or are weighted to contribute at least 75%).
• the panel comprises CCGs plus one or more additional markers that significantly increase the predictive power of the panel (i.e., make the predictive power significantly better than if the panel consisted of only the CCGs).
• CCGs including any of those listed in Table 1, 2, 3, 5, 6, 7, 8, 9, 10 or 1 1; Panel A, B, C, D, E, F, G, H, J or K; or "sub-panels" of Panel F in Tables 21 to 25) can be used to practice the invention.
• the panel comprises at least 3, 4, 5, 6, 7, 8, 9, 10,
• the panel comprises between 5 and 100 CCGs, between 7 and 40 CCGs, between 5 and 25 CCGs, between 10 and 20 CCGs, or between 10 and 15 CCGs.
• CCGs comprise at least a certain proportion of the panel.
• the panel comprises at least 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% CCGs.
• the CCGs are any of the genes listed in Table 1, 2, 3, 5, 6, 7, 8, 9, 10 or 1 1; Panel A, B, C, D, E, F, G, H, J or K; or "sub-panels" of Panel F in Tables 21 to 25.
• the panel comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more genes in any of Table 1, 2, 3, 5, 6, 7, 8, 9, 10 or 1 1 ; Panel A, B, C, D, E, F, G, H, J or K; or "sub-panels" of Panel F in Tables 21 to 25.
• the panel comprises all of the genes in any of Table 1, 2, 3, 5, 6, 7, 8, 9, 10 or 1 1; Panel A, B, C, D, E, F, G, H, J or K; or "sub-panels" of Panel F in Tables 21 to 25.
• the plurality of test genes comprises at least some number of CCGs (e.g., at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more CCGs) and this plurality of CCGs comprises the top 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 20, 25, 30, 35, 40 or more CCGs listed in Table 2, 3, 5, 6, 7, 12, 13, 14, 15, 16, 17, 18 or 19.
• the plurality of test genes comprises at least some number of CCGs (e.g., at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more CCGs) and this plurality of CCGs comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 of the following genes: ASPM, BIRC5, BUB1B, CCNB2, CDC2, CDC20, CDCA8, CDKN3, CENPF, DLGAP5, FOXM1, KIAA0101, KIF11, KIF2C, KIF4A, MCM10, NUSAP1, PRC1, RACGAP1, and TPX2.
• CCGs e.g., at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more CCGs
• ASPM BIRC5, BUB1B, CCNB2, CDC2, CDC20, CDCA8, CDKN3, CENPF, DLGAP5, FOXM1, KIAA0101, KIF11, KIF2C
• the plurality of test genes comprises at least some number of CCGs (e.g., at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more CCGs) and this plurality of CCGs comprises any one, two, three, four, five, six, seven, eight, nine, or ten or all of gene numbers 1 & 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7, 1 to 8, 1 to 9, or 1 to 10 of any of Table 2, 3, 5, 6, 7, 12, 13, 14, 15, 16, 17, 18 or 19.
• CCGs e.g., at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more CCGs
• this plurality of CCGs comprises any one, two, three, four, five, six, seven, eight, nine, or ten or all of gene numbers 1 & 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7, 1 to 8, 1 to 9, or 1 to 10 of any of Table 2, 3, 5, 6, 7, 12, 13, 14, 15, 16, 17, 18 or 19.
• the plurality of test genes comprises at least some number of CCGs (e.g., at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more CCGs) and this plurality of CCGs comprises any one, two, three, four, five, six, seven, eight, or nine or all of gene numbers 2 & 3, 2 to 4, 2 to 5, 2 to 6, 2 to 7, 2 to 8, 2 to 9, or 2 to 10 of any of Table 2, 3, 5, 6, 7, 12, 13, 14, 15, 16, 17, 18 or 19.
• CCGs e.g., at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more CCGs
• this plurality of CCGs comprises any one, two, three, four, five, six, seven, eight, or nine or all of gene numbers 2 & 3, 2 to 4, 2 to 5, 2 to 6, 2 to 7, 2 to 8, 2 to 9, or 2 to 10 of any of Table 2, 3, 5, 6, 7, 12, 13, 14, 15, 16, 17, 18 or 19.
• the plurality of test genes comprises at least some number of CCGs (e.g., at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more CCGs) and this plurality of CCGs comprises any one, two, three, four, five, six, seven, or eight or all of gene numbers 3 & 4, 3 to 5, 3 to 6, 3 to 7, 3 to 8, 3 to 9, or 3 to 10 of any of Table 2, 3, 5, 6, 7, 12, 13, 14, 15, 16, 17, 18 or 19.
• CCGs e.g., at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more CCGs
• this plurality of CCGs comprises any one, two, three, four, five, six, seven, or eight or all of gene numbers 3 & 4, 3 to 5, 3 to 6, 3 to 7, 3 to 8, 3 to 9, or 3 to 10 of any of Table 2, 3, 5, 6, 7, 12, 13, 14, 15, 16, 17, 18 or 19.
• the plurality of test genes comprises at least some number of CCGs (e.g., at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more CCGs) and this plurality of CCGs comprises any one, two, three, four, five, six, or seven or all of gene numbers 4 & 5, 4 to 6, 4 to 7, 4 to 8, 4 to 9, or 4 to 10 of any of Table 2, 3, 5, 6, 7, 12, 13, 14, 15, 16, 17, 18 or 19.
• CCGs e.g., at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more CCGs
• this plurality of CCGs comprises any one, two, three, four, five, six, or seven or all of gene numbers 4 & 5, 4 to 6, 4 to 7, 4 to 8, 4 to 9, or 4 to 10 of any of Table 2, 3, 5, 6, 7, 12, 13, 14, 15, 16, 17, 18 or 19.
• the plurality of test genes comprises at least some number of CCGs (e.g., at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more CCGs) and this plurality of CCGs comprises any one, two, three, four, five, six, seven, eight, nine, 10, 1 1, 12, 13, 14, or 15 or all of gene numbers 1 & 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7, 1 to 8, 1 to 9, 1 to 10, 1 to 1 1, 1 to 12, 1 to 13, 1 to 14, or 1 to 15 of any of Table 2, 3, 5, 6, 7, 12, 13, 14, 15, 16, 17, 18 or 19.
• CCGs e.g., at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more CCGs
• this plurality of CCGs comprises any one, two, three, four, five, six, seven, eight, nine, 10, 1 1, 12, 13, 14, or 15 or all of gene numbers 1 & 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7, 1 to 8, 1
• the invention provides an method of determining a lung cancer patient's prognosis or the likelihood of the patient responding to a particular treatment comprising: (1) obtaining the measured expression levels of a plurality of genes comprising a plurality of CCGs (e.g., genes in Table 1, 2, 3, 5, 6, 7, 8, 9, 10 or 1 1; Panel A, B, C, D, E, F, G, H, J or K; or "sub-panels" of Panel F in Tables 21 to 25) in a sample from the patient; (2) obtaining a clinical score for the patient comprising (or reflecting) one or more clinical parameters relevant to the patient's lung cancer (e.g., age, gender, smoking, stage, treatment, tumor size, pleural invasion); (3) deriving a combined test value from the measured levels obtained in (1) and the clinical score obtained in (2); (4) comparing the combined test value to a combined reference value derived from measured expression levels of the plurality of genes and a clinical score comprising (or reflecting) the one or more clinical parameters in
• CCGs e
• the combined score includes CCP score and any single parameter or combination of age, gender, smoking, stage, treatment, tumor size, and pleural invasion (which single or combination of clinical parameters can be termed the "clinical score" component of the combined score).
• CCP, age and tumor size can be a continuous numeric variable.
• Tumor stage can be a numeric variable with a particular value assigned to any particular clinical stage (example shown below).
• the combined score is calculated according to the following formula:
• the clinical score is the patient's score according to a clinical nomogram for lung cancer prognosis (or for predicting response to a particular treatment).
• the combined score is calculated according to the following modified version of Formula 1 :
• CCP score is the unweighted mean of CT values for expression of the CCP genes being analyzed (e.g., any gene(s) in Table 1, 2, 3, 5, 6, 7, 8, 9, 10 or 11 ; Panel A, B, C, D, E, F, G, H, J or K; or "sub-panels" of Panel F in Tables 21 to 25), optionally normalized by the unweighted mean of the control genes so that higher values indicate higher expression (in some embodiments one unit is equivalent to a two-fold change in expression).
• the CCP score ranges from -8 to 8 or from -1.6 to 3.7.
• clinical score is represented by the numeric value assigned the patient's tumor stage as shown below:
• A, B, C and/or D is within rounding of these values (e.g., A is between 0.945 and 0.954 or between 0.325 and 0.334, B is between 0.515 and 0.524, etc.).
• A, B, C and/or D is within ⁇ 1%, ⁇ 2%, ⁇ 3%, ⁇ 4%, ⁇ 5%, ⁇ 10%, ⁇ 15%, ⁇ 20%, ⁇ 25%, ⁇ 30%, ⁇ 35%, ⁇ 40%, ⁇ 45%, ⁇ 50%, of these values (e.g., A is between 0.29 and 0.37, B is between 0.46 and 0.58, etc.). In some cases a formula may not have all of the specified coefficients (and thus not incorporate the
• A is between 0.9 and 1, 0.9 and 0.99, 0.9 and 0.95, 0.85 and 0.95, 0.86 and 0.94, 0.87 and 0.93, 0.88 and 0.92, 0.89 and 0.91, 0.85 and 0.9, 0.8 and 0.95, 0.8 and 0.9, 0.8 and 0.85, 0.75 and 0.99, 0.75 and 0.95, 0.75 and 0.9, 0.75 and 0.85, or between 0.75 and 0.8.
• B is between 0.40 and 1, 0.45 and 0.99, 0.45 and 0.95, 0.55 and 0.8, 0.55 and 0.7, 0.55 and 0.65, 0.59 and 0.63, or between 0.6 and 0.62.
• A is between 0.1 and 0.2, 0.3, 0.4, 0.5, 0.6, 0.7,
• B is between 0.1 and 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, or 20; or between 0.2 and
• A, B, and/or C is within rounding of any of these values (e.g., A is between 0.45 and 0.54, etc.).
• results of any analyses according to the invention will often be communicated to physicians, genetic counselors and/or patients (or other interested parties such as researchers) in a transmittable form that can be communicated or transmitted to any of the above parties.
• a transmittable form can vary and can be tangible or intangible.
• the results can be embodied in descriptive statements, diagrams, photographs, charts, images or any other visual forms. For example, graphs showing expression or activity level or sequence variation information for various genes can be used in explaining the results. Diagrams showing such information for additional target gene(s) are also useful in indicating some testing results.
• statements and visual forms can be recorded on a tangible medium such as papers, computer readable media such as floppy disks, compact disks, etc., or on an intangible medium, e.g., an electronic medium in the form of email or website on internet or intranet.
• results can also be recorded in a sound form and transmitted through any suitable medium, e.g., analog or digital cable lines, fiber optic cables, etc., via telephone, facsimile, wireless mobile phone, internet phone and the like.
• the information and data on a test result can be produced anywhere in the world and transmitted to a different location.
• the information and data on a test result may be generated, cast in a transmittable form as described above, and then imported into the United States.
• the present invention also encompasses a method for producing a transmittable form of information on at least one of (a) expression level or (b) activity level for at least one patient sample.
• the method comprises the steps of (1) determining at least one of (a) or (b) above according to methods of the present invention; and (2) embodying the result of the determining step in a transmittable form.
• the transmittable form is a product of such a method.
• the present invention further provides a system for determining gene expression in a tumor sample, comprising: (1) a sample analyzer for determining the expression levels of a panel of genes in a sample (e.g., a tumor sample) including at least 2, 4, 6, 8 or 10 cell-cycle genes, wherein the sample analyzer contains the sample which is from a patient having lung cancer, or mRNA molecules from the patient sample or cDNA molecules from mRNA expressed from the panel of genes; (2) a first computer program for (a) receiving gene expression data on at least 4 test genes selected from the panel of genes, (b) weighting the determined expression of each of the test genes, and (c) combining the weighted expression to provide a test value, wherein at least 20%, 50%, at least 75% or at least 90% of the test genes are cell-cycle genes (or wherein the cell-cycle genes are weighted to contribute at least 50%, 60%, 70%, 80%, 90%, 95% or 100% of the test value); and (3) a second computer program for comparing the test value to
• the amount of RNA transcribed from the panel of genes including test genes is measured in the sample.
• the amount of RNA of one or more housekeeping genes in the sample is also measured, and used to normalize or calibrate the expression of the test genes, as described above.
• the plurality of test genes includes at least 2, 3 or 4 cell-cycle genes, which constitute at least 50%, 75% or 80% of the plurality of test genes, and preferably 100% of the plurality of test genes.
• the plurality of test genes includes at least 5, 6 or 7, or at least 8 cell-cycle genes, which constitute at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80% or 90% of the plurality of test genes, and preferably 100% of the plurality of test genes.
• the plurality of test genes includes at least 8, 10, 12, 15, 20, 25 or 30 cell-cycle genes, which constitute at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80% or 90% of the plurality of test genes, and preferably 100% of the plurality of test genes.
• the sample analyzer can be any instrument useful in determining gene expression, including, e.g., a sequencing machine, a real-time PCR machine, and a microarray instrument.
• the computer-based analysis function can be implemented in any suitable language and/or browsers. For example, it may be implemented with C language and preferably using object-oriented high-level programming languages such as Visual Basic, SmallTalk, C++, and the like.
• the application can be written to suit environments such as the Microsoft WindowsTM environment including WindowsTM 98, WindowsTM 2000, WindowsTM NT, and the like.
• the application can also be written for the MacintoshTM, SUNTM, UNIX or LINUX environment.
• the functional steps can also be implemented using a universal or platform-independent programming language.
• multi-platform programming languages include, but are not limited to, hypertext markup language (HTML), JAVATM, JavaScriptTM, Flash programming language, common gateway interface/structured query language (CGI/SQL), practical extraction report language (PERL), AppleScript and other system script languages, programming language/structured query language (PL/SQL), and the like.
• JavaTM- or JavaScriptTM-enabled browsers such as HotJavaTM, MicrosoftTM ExplorerTM, or NetscapeTM can be used.
• active content web pages may include JavaTM applets or ActiveXTM controls or other active content technologies.
• the analysis function can also be embodied in computer program products and used in the systems described above or other computer- or internet-based systems. Accordingly, another aspect of the present invention relates to a computer program product comprising a computer-usable medium having computer-readable program codes or instructions embodied thereon for enabling a processor to carry out gene status analysis. These computer program instructions may be loaded onto a computer or other programmable apparatus to produce a machine, such that the instructions which execute on the computer or other programmable apparatus create means for implementing the functions or steps described above.
• These computer program instructions may also be stored in a computer-readable memory or medium that can direct a computer or other programmable apparatus to function in a particular manner, such that the instructions stored in the computer-readable memory or medium produce an article of manufacture including instruction means which implement the analysis.
• the computer program instructions may also be loaded onto a computer or other programmable apparatus to cause a series of operational steps to be performed on the computer or other programmable apparatus to produce a computer implemented process such that the instructions which execute on the computer or other programmable apparatus provide steps for implementing the functions or steps described above.
• the system comprises (1) computer program for receiving, storing, and/or retrieving a patient's CCG status data (e.g., expression level, activity level, variants) and optionally clinical parameter data (e.g., clinical stage); (2) computer program for querying this patient data; (3) computer program for concluding whether there is an increased likelihood of recurrence based on this patient data; and optionally (4) computer program for outputting/displaying this conclusion.
• this means for outputting the conclusion may comprise a computer program for informing a health care professional of the conclusion.
• Computer system [600] may include at least one input module [630] for entering patient data into the computer system [600] .
• the computer system [600] may include at least one output module [624] for indicating whether a patient has an increased or decreased likelihood of response and/or indicating suggested treatments determined by the computer system [600].
• Computer system [600] may include at least one memory module [606] in communication with the at least one input module [630] and the at least one output module
• the at least one memory module [606] may include, e.g., a removable storage drive [608], which can be in various forms, including but not limited to, a magnetic tape drive, a floppy disk drive, a VCD drive, a DVD drive, an optical disk drive, etc.
• the removable storage drive [608] may be compatible with a removable storage unit [610] such that it can read from and/or write to the removable storage unit [610].
• Removable storage unit [610] may include a computer usable storage medium having stored therein computer-readable program codes or instructions and/or computer readable data. For example, removable storage unit
• the at least one memory module [606] may also include a hard disk drive [612], which can be used to store computer readable program codes or instructions, and/or computer readable data.
• the at least one memory module [606] may further include an interface [614] and a removable storage unit [616] that is compatible with interface [614] such that software, computer readable codes or instructions can be transferred from the removable storage unit [616] into computer system [600].
• interface [614] and removable storage unit [616] pairs include, e.g., removable memory chips (e.g., EPROMs or PROMs) and sockets associated therewith, program cartridges and cartridge interface, and the like.
• Computer system [600] may also include a secondary memory module
• RAM random access memory
• Computer system [600] may include at least one processor module [602].
• the at least one processor module [602] may consist of any number of devices.
• the at least one processor module [602] may include a data processing device, such as a microprocessor or microcontroller or a central processing unit.
• the at least one processor module [602] may include another logic device such as a DMA (Direct Memory Access) processor, an integrated communication processor device, a custom VLSI (Very Large Scale Integration) device or an ASIC (Application Specific Integrated Circuit) device.
• the at least one processor module [602] may include any other type of analog or digital circuitry that is designed to perform the processing functions described herein.
• the at least one memory module [606], the at least one processor module [602], and secondary memory module [618] are all operably linked together through communication infrastructure [620], which may be a communications bus, system board, cross-bar, etc.).
• communication infrastructure may be a communications bus, system board, cross-bar, etc.
• Input interface [626] may operably connect the at least one input module [626] to the communication infrastructure [620] .
• output interface [622] may operably connect the at least one output module [624] to the communication infrastructure [620].
• the at least one input module [630] may include, for example, a keyboard, mouse, touch screen, scanner, and other input devices known in the art.
• the at least one output module [624] may include, for example, a display screen, such as a computer monitor, TV monitor, or the touch screen of the at least one input module [630] ; a printer; and audio speakers.
• Computer system [600] may also include, modems, communication ports, network cards such as Ethernet cards, and newly developed devices for accessing intranets or the internet.
• the at least one memory module [606] may be configured for storing patient data entered via the at least one input module [630] and processed via the at least one processor module [602].
• Patient data relevant to the present invention may include expression level, activity level, copy number and/or sequence information for a CCG.
• Patient data relevant to the present invention may also include clinical parameters relevant to the patient's disease (e.g., age, tumor size, node status, tumor stage). Any other patient data a physician might find useful in making treatment decisions/recommendations may also be entered into the system, including but not limited to age, gender, and race/ethnicity and lifestyle data such as diet information.
• Other possible types of patient data include symptoms currently or previously experienced, patient's history of illnesses, medications, and medical procedures.
• the at least one memory module [606] may include a computer- implemented method stored therein.
• the at least one processor module [602] may be used to execute software or computer-readable instruction codes of the computer- implemented method.
• the computer-implemented method may be configured to, based upon the patient data, indicate whether the patient has an increased likelihood of recurrence, progression or response to any particular treatment, generate a list of possible treatments, etc.
• the computer-implemented method may be configured to identify a patient as having or not having an increased likelihood of recurrence or progression. For example, the computer-implemented method may be configured to inform a physician that a particular patient has an increased likelihood of recurrence. Alternatively or additionally, the computer-implemented method may be configured to actually suggest a particular course of treatment based on the answers to/results for various queries.
• FIG.7 illustrates one embodiment of a computer-implemented method [700] of the invention that may be implemented with the computer system [600] of the invention.
• the method [700] begins with one of three queries ([710], [711]), either sequentially or substantially simultaneously. If the answer to/result for any of these queries is "Yes" [720], the method concludes [730] that the patient has an increased likelihood of recurrence or of response to a particular treatment regimen (e.g., treatment comprising chemotherapy).
• a particular treatment regimen e.g., treatment comprising chemotherapy
• the method concludes [731] that the patient does not have an increased likelihood of recurrence or of response to a particular treatment regimen (e.g., treatment comprising chemotherapy).
• the method [700] may then proceed with more queries, make a particular treatment recommendation ([740], [741]), or simply end.
• the queries When the queries are performed sequentially, they may be made in the order suggested by FIG.7 or in any other order. Whether subsequent queries are made can also be dependent on the results/answers for preceding queries.
• the method asks about clinical parameters [711] first and, if the patient has one or more clinical parameters identifying the patient as at increased likelihood of recurrence or response to a particular treatment then the method concludes such [730] or optionally confirms by querying CCG status, while if the patient has no such clinical parameters then the method proceeds to ask about CCG status [710].
• the preceding order of queries may be modified.
• an answer of "yes" to one query (e.g., [710]) prompts one or more of the remaining queries to confirm that the patient has increased risk of recurrence.
• the computer-implemented method of the invention [700] is open-ended. In other words, the apparent first step [710 and/or 711] in
• FIG.7 may actually form part of a larger process and, within this larger process, need not be the first step/query. Additional steps may also be added onto the core methods discussed above. These additional steps include, but are not limited to, informing a health care professional (or the patient itself) of the conclusion reached; combining the conclusion reached by the illustrated method [700] with other facts or conclusions to reach some additional or refined conclusion regarding the patient's diagnosis, prognosis, treatment, etc.; making a
• additional queries about additional biomarkers e.g., age, tumor size, node status, tumor stage
• additional useful patient information e.g., age at diagnosis, general patient health, etc.
• the answers to the queries may be determined by the method instituting a search of patient data for the answer.
• patient data may be searched for CCG status (e.g., CCG expression level data) and/or clinical parameters (e.g., tumor stage, nomogram score, etc.). If such a comparison has not already been performed, the method may compare these data to some reference in order to determine if the patient has an abnormal (e.g., elevated, low, negative) status. Additionally or alternatively, the method may present one or more of the queries [710, 711] to a user (e.g., a physician) of the computer system [100].
• CCG status e.g., CCG expression level data
• clinical parameters e.g., tumor stage, nomogram score, etc.
• the method may present one or more of the queries [710, 711] to a user (e.g., a physician) of the computer system [100].
• the questions [710, 711] may be presented via an output module [624].
• the user may then answer "Yes” or “No” or provide some other value (e.g., numerical or qualitative value incorporating or representing CCG status) via an input module [630].
• the method may then proceed based upon the answer received.
• the conclusions [730, 731] may be presented to a user of the computer- implemented method via an output module [624].
• the invention provides a method comprising: accessing information on a patient's CCG status stored in a computer-readable medium;
• a sample obtained from the patient shows increased expression of a plurality of test genes comprising at least 2 CCGs (e.g., a test value incorporating or representing the expression of this plurality of test genes that is weighted such that CCGs contribute at least 50% to the test value, such test value being higher than some reference value); outputting [or displaying] the quantitative or qualitative (e.g., "increased") likelihood that the patient will respond to a particular treatment regimen.
• "displaying" means communicating any information by any sensory means.
• Examples include, but are not limited to, visual displays, e.g., on a computer screen or on a sheet of paper printed at the command of the computer, and auditory displays, e.g., computer generated or recorded auditory expression of a patient's genotype.
• Computer software products of the invention typically include computer readable media having computer-executable instructions for performing the logic steps of the method of the invention.
• Suitable computer readable medium include floppy disk, CD-ROM/DVD/DVD-ROM, hard-disk drive, flash memory, ROM/RAM, magnetic tapes and etc.
• Basic computational biology methods are described in, for example, Setubal et ah, INTRODUCTION TO COMPUTATIONAL BIOLOGY METHODS (PWS Publishing Company, Boston, 1997); Salzberg et al.
• BIOINFORMATICS A PRACTICAL GUIDE FOR ANALYSIS OF GENE AND PROTEINS (Wiley & Sons, Inc., 2 ND ed., 2001); see also, U.S. Pat. No. 6,420, 108.
• the present invention may also make use of various computer program products and software for a variety of purposes, such as probe design, management of data, analysis, and instrument operation. See U.S. Pat. Nos. 5,593,839; 5,795,716; 5,733,729;
• the present invention may have embodiments that include methods for providing genetic information over networks such as the Internet as shown in U.S. Ser. Nos. 10/197,621 (U.S. Pub. No. 20030097222); 10/063,559 (U.S. Pub. No. 20020183936), 10/065,856 (U.S. Pub. No. 20030100995); 10/065,868 (U.S. Pub. No. 20030120432); 10/423,403 (U.S. Pub. No. 20040049354).
• one aspect of the present invention provides systems related to the above methods of the invention.
• the invention provides a system for determining a patient's prognosis and/or whether a patient will respond to a particular treatment regimen, comprising:
• a sample analyzer for determining the expression levels in a sample of a plurality of test genes including at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more CCGs (e.g., genes in Table 1, 2, 3, 5, 6, 7, 8, 9, 10 or 11 ; Panel A, B, C, D, E, F, G, H, J or K; or "sub-panels" of Panel F in Tables 21 to 25), wherein the sample analyzer contains the sample, RNA from the sample and expressed from the panel of genes, or DNA synthesized from said RNA;
• a second computer program for comparing the test value to one or more reference values each associated with a predetermined likelihood of recurrence or progression or a predetermined likelihood of response to a particular treatment regimen.
• the sample analyzer contains reagents for determining the expression levels in the sample of said panel of genes including at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more CCGs. In some embodiments the sample analyzer contains CCG-specific reagents as described below.
• the invention provides a system for determining gene expression in a sample (e.g., tumor sample), comprising: (1) a sample analyzer for determining the expression levels of a panel of genes in a sample including at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more CCGs, wherein the sample analyzer contains the sample which is from a patient having lung cancer, RNA from the sample and expressed from the panel of genes, or DNA synthesized from said RNA; (2) a first computer program for (a) receiving gene expression data on at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more test genes selected from the panel of genes, (b) weighting the determined expression of each of the test genes with a predefined coefficient, and (c) combining the weighted expression to provide a test value, wherein the combined weight given to said at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more CCGs is at least 40% (or 50%, 60%, 70%, 80%, 90%, 95% or 100%) of the total weight given to the expression of all of said plurality of test genes
• the system comprises a computer program for determining the patient's prognosis and/or determining (including quantifying) the patient's degree of risk of cancer recurrence or progression based at least in part on the comparison of the test value with said one or more reference values.
• the system further comprises a display module displaying the comparison between the test value and the one or more reference values, or displaying a result of the comparing step, or displaying the patient's prognosis and/or degree of risk of cancer recurrence or progression.
• the amount of RNA transcribed from the panel of genes including test genes (and/or DNA reverse transcribed therefrom) is measured in the sample.
• the amount of RNA of one or more housekeeping genes in the sample (and/or DNA reverse transcribed therefrom) is also measured, and used to normalize or calibrate the expression of the test genes, as described above.
• the plurality of test genes includes at least 2, 3 or 4 CCGs, which constitute at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80% or 90% of the plurality of test genes, and preferably 100% of the plurality of test genes.
• the plurality of test genes includes at least 5, 6 or 7, or at least 8 CCGs, which constitute at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80% or 90% of the plurality of test genes, and preferably 100% of the plurality of test genes.
• the plurality of test genes comprises at least some number of CCGs (e.g., at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more CCGs) and this plurality of CCGs comprises the top 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40 or more CCGs listed in Table 2, 3, 5, 6, 7, 12, 13, 14, 15, 16, 17, 18 or 19.
• the plurality of test genes comprises at least some number of CCGs (e.g., at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more CCGs) and this plurality of CCGs comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 of the following genes: ASPM, BIRC5, BUB1B, CCNB2, CDC2, CDC20, CDCA8, CDKN3, CENPF, DLGAP5, FOXM1, KIAA0101, KIF11, KIF2C, KIF4A, MCM10, NUSAP1, PRC1, RACGAP1, and TPX2.
• CCGs e.g., at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more CCGs
• ASPM BIRC5, BUB1B, CCNB2, CDC2, CDC20, CDCA8, CDKN3, CENPF, DLGAP5, FOXM1, KIAA0101, KIF11, KIF2C
• the plurality of test genes comprises at least some number of CCGs (e.g., at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more CCGs) and this plurality of CCGs comprises any one, two, three, four, five, six, seven, eight, nine, or ten or all of gene numbers 1 & 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7, 1 to 8, 1 to 9, or 1 to 10 of any of Table 2, 3, 5, 6, 7, 12, 13, 14, 15, 16, 17, 18 or 19.
• the plurality of test genes comprises at least some number of CCGs (e.g., at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more CCGs) and this plurality of CCGs comprises any one, two, three, four, five, six, seven, eight, nine, or ten or all of gene numbers 1 & 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7, 1 to 8, 1 to 9, or 1 to 10 of any of Table 2, 3, 5, 6,
• this plurality of CCGs comprises any one, two, three, four, five, six, seven, eight, or nine or all of gene numbers 2 & 3, 2 to 4, 2 to 5, 2 to 6, 2 to 7, 2 to 8, 2 to 9, or 2 to 10 of any of Table 2, 3, 5, 6, 7, 12, 13, 14, 15, 16, 17, 18 or 19.
• the plurality of test genes comprises at least some number of CCGs (e.g., at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more CCGs) and this plurality of CCGs comprises any one, two, three, four, five, six, seven, or eight or all of gene numbers 3 & 4, 3 to 5, 3 to 6, 3 to 7, 3 to 8, 3 to 9, or 3 to 10 of any of Table 2, 3, 5, 6, 7, 12, 13, 14, 15, 16, 17, 18 or 19.
• CCGs e.g., at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more CCGs
• this plurality of CCGs comprises any one, two, three, four, five, six, seven, or eight or all of gene numbers 3 & 4, 3 to 5, 3 to 6, 3 to 7, 3 to 8, 3 to 9, or 3 to 10 of any of Table 2, 3, 5, 6, 7, 12, 13, 14, 15, 16, 17, 18 or 19.
• the plurality of test genes comprises at least some number of CCGs (e.g., at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more CCGs) and this plurality of CCGs comprises any one, two, three, four, five, six, or seven or all of gene numbers 4 & 5, 4 to 6, 4 to 7, 4 to 8, 4 to 9, or 4 to 10 of any of Table 2, 3, 5, 6, 7, 12, 13, 14, 15, 16, 17, 18 or 19.
• CCGs e.g., at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more CCGs
• this plurality of CCGs comprises any one, two, three, four, five, six, or seven or all of gene numbers 4 & 5, 4 to 6, 4 to 7, 4 to 8, 4 to 9, or 4 to 10 of any of Table 2, 3, 5, 6, 7, 12, 13, 14, 15, 16, 17, 18 or 19.
• the plurality of test genes comprises at least some number of CCGs (e.g., at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more CCGs) and this plurality of CCGs comprises any one, two, three, four, five, six, seven, eight, nine, 10, 1 1, 12, 13, 14, or 15 or all of gene numbers 1 & 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7,
• the plurality of test genes includes at least 8,
• CCGs which constitute at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80% or 90% of the plurality of test genes, and preferably 100% of the plurality of test genes.
• the sample analyzer can be any instrument useful in determining gene expression, including, e.g., a sequencing machine (e.g., Illumina HiSeqTM, Ion Torrent PGM, ABI SOLiDTM sequencer, PacBio RS, Helicos HeliscopeTM, etc.), a real-time PCR machine (e.g., ABI 7900, Fluidigm BioMarkTM, etc.), a microarray instrument, etc.
• a sequencing machine e.g., Illumina HiSeqTM, Ion Torrent PGM, ABI SOLiDTM sequencer, PacBio RS, Helicos HeliscopeTM, etc.
• a real-time PCR machine e.g., ABI 7900, Fluidigm BioMarkTM, etc.
• microarray instrument e.g., a microarray instrument, etc.
• the present invention provides methods of treating a cancer patient comprising obtaining CCG status information (e.g., the genes in Table 1, 2, 3, 5, 6, 7, 8, 9, 10 or 11 ; Panel A, B, C, D, E, F, G, H, J or K; or "sub-panels" of Panel F in Tables 21 to 25), and recommending, prescribing or administering a treatment for the cancer patient based on the CCG status.
• CCG status information e.g., the genes in Table 1, 2, 3, 5, 6, 7, 8, 9, 10 or 11 ; Panel A, B, C, D, E, F, G, H, J or K; or "sub-panels" of Panel F in Tables 21 to 25
• the invention provides a method of treating a cancer patient comprising:
• step (2) based at least in part on the determination in step (1), recommending, prescribing or administering either
• a treatment regimen comprising chemotherapy (e.g., adjuvant chemotherapy) if the patient has increased expression of the plurality of test genes (e.g., and CCGs are weighted to contribute at least 50% to the determination of increased expression of the plurality of test genes), or
• a treatment regimen not comprising chemotherapy if the patient does not have increased expression of the plurality of test genes (e.g., and CCGs are weighted to contribute at least 50% to the determination of increased expression of the plurality of test genes).
• compositions for use in the above methods include, but are not limited to, nucleic acid probes hybridizing to a CCG, including but not limited to a CCG listed in any of Table 1, 2, 3, 5, 6, 7, 8, 9, 10 or 1 1 ; Panel A, B, C, D, E, F, G, H, J or K; or "sub-panels" of Panel F in Tables 21 to 25 (or to any nucleic acids encoded thereby or complementary thereto); nucleic acid primers and primer pairs suitable for seletively amplifying all or a portion of such a CCG or any nucleic acids encoded thereby; antibodies binding immunologically to a polypeptide encoded by such a CCG; probe sets comprising a plurality of said nucleic acid probes, nucleic acid primers, antibodies, and/or polypeptides; microarrays comprising any of these; kits comprising any of these; etc.
• the invention provides computer methods, systems, software and/or
• the invention provides a probe comprising an isolated oligonucleotide capable of selectively hybridizing to at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more of the genes in Table 1, 2, 3, 5, 6, 7, 8, 9, 10 or 1 1; Panel A, B, C, D, E, F, G, H, J or K; or "sub-panels” of Panel F in Tables 21 to 25.
• probe and "oligonucleotide” (also “oligo"), when used in the context of nucleic acids, interchangeably refer to a relatively short nucleic acid fragment or sequence.
• the invention also provides primers useful in the methods of the invention.
• Primers are probes capable, under the right conditions and with the right companion reagents, of selectively amplifying a target nucleic acid (e.g., a target gene).
• target nucleic acid e.g., a target gene
• probe is used herein to encompass “primer” since primers can generally also serve as probes.
• the probe can generally be of any suitable size/length. In some embodiments the probe has a length from about 8 to 200, 15 to 150, 15 to 100, 15 to 75, 15 to 60, or 20 to 55 bases in length. They can be labeled with detectable markers with any suitable detection marker including but not limited to, radioactive isotopes, fluorophores, biotin, enzymes (e.g., alkaline phosphatase), enzyme substrates, ligands and antibodies, etc. See Jablonski et al, NUCLEIC ACIDS RES. (1986) 14:61 15-6128; Nguyen et al, BIOTECHNIQUES (1992) 13: 1 16-123; Rigby et al, J. MOL. BIOL. (1977) 113:237-251. Indeed, probes may be modified in any conventional manner for various molecular biological applications.
• Probes according to the invention can be used in the hybridization/ amplification/ detection techniques discussed above.
• some embodiments of the invention comprise probe sets suitable for use in a microarray in detecting, amplifying and/or quantitating a plurality of CCGs.
• the probe sets have a certain proportion of their probes directed to CCGs— e.g., a probe set consisting of 10%, 20%, 30%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% probes specific for CCGs.
• the probe set comprises probes directed to at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 40, 45, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 600, 700, or 800 or more, or all, of the genes in Table 1, 2, 3, 5, 6, 7, 8, 9, 10 or 11 ; Panel A, B, C, D, E, F, G, H, J or K; or "sub-panels" of Panel F in Tables 21 to 25.
• probe sets can be incorporated into high-density arrays comprising 5,000, 10,000, 20,000, 50,000, 100,000, 200,000, 300,000, 400,000, 500,000, 600,000, 700,000, 800,000, 900,000, or 1,000,000 or more different probes.
• the probe sets comprise primers (e.g., primer pairs) for amplifying nucleic acids comprising at least a portion of one or more of the CCGs in Table 1, 2, 3, 5, 6, 7, 8, 9, 10 or 1 1; Panel A, B, C, D, E, F, G, H, J or K; or "sub-panels" of Panel F in Tables 21 to 25.
• kits for practicing the prognosis of the present invention.
• the kit may include a carrier for the various components of the kit.
• the carrier can be a container or support, in the form of, e.g., bag, box, tube, rack, and is optionally compartmentalized.
• the carrier may define an enclosed confinement for safety purposes during shipment and storage.
• the kit includes various components useful in determining the status of one or more CCGs and one or more
• the kit many include oligonucleotides specifically hybridizing under high stringency to mRNA or cDNA of the genes in Table 1, 2, 3, 5, 6, 7, 8, 9, 10 or 11 ; Panel A, B, C, D, E, F, G, H, J or K; or "sub-panels" of Panel F in Tables 21 to 25.
• oligonucleotides can be used as PCR primers in RT-PCR reactions, or hybridization probes.
• the kit comprises reagents (e.g., probes, primers, and or antibodies) for determining the expression level of a panel of genes, where said panel comprises at least 25%, 30%, 40%, 50%, 60%, 75%, 80%, 90%, 95%, 99%, or 100% CCGs (e.g., CCGs in Table 1, 2, 3, 5, 6, 7, 8, 9, 10 or 11 ; Panel A, B, C, D, E, F, G, H, J or K; or "sub-panels" of Panel F in Tables 21 to 25).
• CCGs e.g., CCGs in Table 1, 2, 3, 5, 6, 7, 8, 9, 10 or 11 ; Panel A, B, C, D, E, F, G, H, J or K; or "sub-panels" of Panel F in Tables 21 to 25.
• the kit consists of reagents (e.g., probes, primers, and or antibodies) for determining the expression level of no more than 2500 genes, wherein at least 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 150, 200, 250, or more of these genes are CCGs (e.g., CCGs in Table 1, 2, 3, 5, 6, 7, 8, 9, 10 or 1 1; Panel A, B, C, D, E, F, G, H, J or K; or "sub-panels" of Panel F in Tables 21 to 25).
• CCGs e.g., CCGs in Table 1, 2, 3, 5, 6, 7, 8, 9, 10 or 1 1
• Panel A, B, C, D, E, F, G, H, J or K or "sub-panels" of Panel F in Tables 21 to 25.
• the oligonucleotides in the detection kit can be labeled with any suitable detection marker including but not limited to, radioactive isotopes, fluorephores, biotin, enzymes (e.g., alkaline phosphatase), enzyme substrates, ligands and antibodies, etc. See Jablonski et al, Nucleic Acids Res., 14:6115-6128 (1986); Nguyen et al, Biotechniques, 13: 1 16-123 (1992); Rigby et al, J. Mol. Biol, 1 13:237-251 (1977).
• the oligonucleotides included in the kit are not labeled, and instead, one or more markers are provided in the kit so that users may label the oligonucleotides at the time of use.
• the detection kit contains one or more antibodies selectively immunoreactive with one or more proteins encoded by one or more CCGs or optionally any additional markers. Examples include antibodies that bind
• the detection kit preferably includes instructions on using the kit for practice the prognosis method of the present invention using human samples.
• the CCG score is calculated from RNA expression of 31 CCGs normalized by 15 housekeeper genes (HK). The relative numbers of CCGs and HK genes are optimized in order to minimize the variance of the CCG score.
• the CCG score is the unweighted mean of CT values for CCG expression, normalized by the unweighted mean of the HK genes so that higher values indicate higher expression.
• one unit is equivalent to a two-fold change in expression.
• the CCG scores are centered by the mean value, determined in a training set.
• the CCG score may remain stable as concentration decreased to the point of 10 failures out of a total 31 CCGs.
• samples with more than 9 missing values are not assigned a CCG score.
• samples may be obtained from an FFPE sample block.
• 5 ⁇ sections may be cut from the sample block.
• sections may be stained with haematoxylin and eosin (H&E).
• tumor areas may be marked by a pathologist.
• ⁇ sections are cut adjacent to the H&E stained sections.
• tumor areas on the unstained sections are identified by alignment with the marked areas on the H&E stain.
• tumor areas are macro-dissected manually.
• samples are deparaffinized by xylene extractions followed by washes with ethanol. In some embodiments samples are treated overnight with proteinase K.
• samples are subjected to RNA extraction.
• RNA extraction is performed using the Qiagen miRNAeasy kit.
• RNA is treated with DNASE I to remove potential genomic DNA contamination.
• RNA is converted to cDNA and synthesized cDNA serves as template for replicate pre-amplification reactions.
• samples are run on TaqmanTM low density arrays (TLDA, Applied Biosystems).
• TLDA TaqmanTM low density arrays
• the CCP score is the unweighted mean of C t values for cell cycle gene expression, normalized by the unweighted mean of the house keeper genes so that higher values indicate higher expression.
• CCP scores are centered by the mean value determined in a commercial training set.
• early stage lung adenocarcinoma samples can be used as a"training" cohort for the purpose of defining centering constants in lung tissue.
• these constants can be used to center the triplicate expression mean of CCP genes before averaging into CCP scores.
• distribution of CCP scores in the training cohort is similar to the distribution in any of the clinical sample sets.
• patient samples with early stage lung adenocarcinoma may be studied.
• pateints may be selected using staging criteria following the 6 th edition of the IASLC staging guidelines.
• other clinical data including, gender, ethnicity, smoking status, recurrence and vital status may be collected.
• survival data for the cohort includes disease-free survival (DFS, time from surgery to first recurrence or last follow-up for recurrence) and overall survival (OS, time from surgery to death or last follow-up for survival).
• DFS disease-free survival
• OS overall survival
• deaths without recurrence are censored at time of death and not included as cancer-related death events.
• a cohort may be analyzed by Cox proportional hazard analysis using disease survival as the outcome variable.
• an interaction term for adjuvant treatment and stage may be introduced to account for the known difference in treatment outcome in stage IA versus other stages.
• the test statistic for the prognostic value of the CCP score is the likelihood ratio for the full model (all clinical variable plus the CCP score) versus the reduced model (all clinical variables, no CCP score).
• a univariate analysis may show
• stage, CCP score and gender are significantly correlated with disease survival.
• the p-vaule for stage may be equal to or less than 0.05. In some embodiments the p-value for stage may be equal to or less than 0.01. In some embodiments the p-value for stage may be equal to or less than 0.00. In some embodimnets the p-value for stage may be equal to or less than 0.0001. In some embodiments the p-vaule may be equal to or less than 0.00045. In some embodiments the p-vaule for CCP score may be equal to or less than 0.05, in some embodiments the p-value for CCP score may be equal to or less than 0.01.
• the p-value for CCP score may be equal to or less than 0.0013 or less. In some embodiments the p-vaule for gender may be equal to or less than 0.05, in some embodiments the p-value for stage may be equal to or less than 0.054.
• a multivariate analysis may show that CCP score is a significant predictor of disease survival when added to a model of all clinical parameters.
• the CCP score may be equal to or less than 0.05 .
• the CCP score may be equal to or less than 0.0175.
• the Hazard Ratio may be equal to or greater than 1.52.
• the 95% confidence interval may be equal to 1.04 and 2.24.
• the lowest CCP quartile has a 5-year survival expectation of 98%, In some embodiments the highest CCP quartile has a 5-year survival rate of 60%.
• stage I and stage II patients partition across all four CCP quartiles.
• CCP score can be used to modify treatment considerations depending on risk estimates besides clinical staging criteria.
• stage IB samples may be analyzed separately.
• CCP score is a significant predictor of outcome for stage IB patients.
• the CCP score p-vaule is equal to or less than 0.05.
• the CCP score p-vaule is equal to or less than 0.02.
• CCP score may be used as a threshold for a high risk (above the mean) and low risk groups (below the mean).
• the low risk group may have a survival rate of 95% or higher.
• the high risk group may have a survival rate of 75% or lower.
• stage IB samples in the highest CCP quartile have a 5-year survival rate of 80% or higher.
• stage IB samples in the lowest CCP quartile have a 5 -year survival rate of 30% or lower.
• the CCP score not only acts as a prognostic (by identifying rapidly progressing cancers) but may also be indicative of treatment benefit (by identifying cancers that will be most susceptible to disruption of the cell cycle.).
• the test statistic is the likelihood ratio for the full model (all clinical variable, CCP score and CCP: adjuvant treatment interaction term) versus the reduced model (all clinical variables no CCP score, no interaction term).
• the interaction for CCP score and adjuvant treatment is not formally significant at the 0.05 level.
• the interaction for CCP score is equal to or less than 0.07.
• untreated patients in the highest CCP quartile have a survival rate of 30% or lower.
• untreated patients in the lowest CCP quartiel have survival rates of 70% or higher.
• patients treated with adjuvant threrapy in the highest CCP quartile have a survival rate of 70% or higher.
• a high CCP score correlates strongly with a higher likelihood of response to adjuvant chemotherapy.
• the prognostic value of CCP in terms of p-values and standardized hazard ratios from univariate, and multivariate, Cox proportional hazards models is evaluated.
• the endpoint may be death from disease within five years of surgery.
• death from disease can be defined as death following recurrence.
• patients who are lost to follow-up or died of other causes are censored from the analysis.
• univariate p-values are based on the partial likelihood ratio.
• multivariate p-values are based on the partial likelihood ratio for the change in deviance from a full model versus a reduced model.
• the full model includes all relevant covariates.
• the reduced model includes all covariates except for the covariate being evaluated, and any interaction terms involving the covariate being evaluated.
• hazard ratios are standardized to represent the increased risk associated with a one standard deviation increase in CCP score.
• CCP score may be combined with clinical variables in multivariate Cox proportional hazards models.
• clinical data for age, gender, smoking status, stage , adjuvant treatment, pleural invasion, and/or tumor size is included.
• an interaction term for stage with treatment is included.
• categorical clinical variables are coded to explain the maximum possible variability in patient outcomes.
• stage may be coded as a 4-level categorical variable (IA, IB, IIA, IIB) rather than a 2-level categorical variable (1,11).
• less significant p-values may be associated with stage.
• Cox proportional hazards models may be constructed for each of the clinical variables, consisting of the clinical variable in question, a variable designating cohort, and an interaction term.
• interaction terms may have a p-value greater than 0.05 in two-sided likelihood ratio tests.
• the appropriateness of the proportional hazards assumption may be evaluated.
• time dependence for the hazard ratio of the CCP score is not supported.
• possibility that CCP score might have a non-linear effect is evaluated.
• second- and third-order polynomials for CCP score are tested in Cox proportional hazards models but were not significant at the 5% level.
• a Cox proportional hazards models is constructed for each available clinical variable, consisting of the clinical variable in question, CCP score, and an interaction term.
• the p-value for the interaction terms is greater than 0.05.
• variables for each patient include age, gender, smoking status, stage, adjuvant treatment, tumor size, pleural invasion, cohort, and/or CCP score.
• age in years is a quantitative variable.
• gender is a binary variable (male, female).
• smoking status is a 3 -level categorical variable (never, former, current).
• pathological stage is according to the 7th edition TNM classification.
• pathological stage is a 4-level categorical variable (IA, IB, IIA, IIB).
• adjuvant treatment is a binary variable (no, yes).
• tumor size is a quantitative variable.
• tumor size is measured in centimeters.
• pleural invasion is a binary variable (no, yes).
• cohort is a 2-level categorical variable.
• CCP score is a quantitative variable.
• univariate analysis asses CCP scores ability to predict five year survival.
• the p-value is equal to or less than 0.05. In some embodiments the p-value is equal to or less than 0.01. In some embodiments the p-value is equal to or less than 0.001. In some embodiments the p-value is equal to or less than 0.0003.
• multivariate analysis assesses CCP's ability to predict five-year survival. In some embodiments the p-value is equal to or less than 0.05. In some embodiments the p- value is equal to or less than 0.01. In some embodiments the p-value is equal to or less than 0.007. In some embodiments the standardized Hazard Ratio is equal to 1.50.
• the 95% Confidence Intervals are equal to 1. 1 1 and 2.02.
• the results from multivariate analysis indicate that the CCP score is able to capture a significant amount of prognostic information independent of the many clinical variables.
• 5 -year disease survival for patients with low CCP scores is 92% or higher.
• 5-year disease survival for patients with medium CCP scores is 79% in patients or lower.
• 5-year disease survival for patients with high CCP scores is 73% or lower.
• CCP score may be used to predict survival in patients treated with adjuvant therapies.
• the technique of Zhang & Klein (Confidence bands for the difference of two survival curves under the proportional hazards model, LIFETIME DATA ANALYSIS (2001)7:243-254) may be used to evaluate the absolute difference in 5-year predicted risk of disease-related death for patients who received adjuvant treatment versus patients who did not receive adjuvant treatment over a range of observed CCP scores.
• complex contrast coding may be used to test whether the absolute difference, due to treatment, in the hazard of disease related death is greater for patients with high CCP scores than for patients with low CCP scores.
• the Zhang & Klein method may be used to test for differences in survival between two treatments (or between patients receiving treatment, and patients not receiving treatment) after adjusting for the effects of other covariates.
• estimates of absolute treatment benefit may be calculated together with point wise confidence bands, over a range of observed CCP scores.
• contrast coding may be used as to test whether the absolute decrease in the hazard of disease-related death due to adjuvant treatment is significantly greater for patients with high CCP scores than for patients with low CCP scores.
• CCP scores may be categorized as high or low using the median as the cutoff point.
• each patient may be assigned to one of four groups: high CCP with adjuvant treatment (ht), high CCP without adjuvant treatment (hu), low CCP with adjuvant treatment (It), and low CCP without adjuvant treatment (lu).
• significantly greater absolute treatment benefit is indicated for patients with high CCP scores compared to patients with low CCP scores.
• the p-value is equal to or lower than 0.05. In some embodiments the p-value is equal to or lower than 0.01. In some embodiments the p-value is equal to or lower than 0.0060.
• the association between CCP score and absolute treatment benefit maintains significance after adjusting for age, gender, smoking status, stage, tumor size, and pleural invasion status in the complex contrast model.
• the p-value is equal to or lower than 0.05. In some embodiments the p-value is equal to or lower than 0.024).
• a combined prognostic score of pathological stage (pStage) and the CCP expression score may be modeled in stage I and II patients without adjuvant treatment.
• DC values may be centered by processing site and scaled by the ratio of the standard deviations of the CCP score in qPCR and microarray data.
• the outcome measure is five year disease-specific survival.
• coefficients for the combination of CCP and pStage are derived from a bivariate Cox proportional hazards model.
• the Cox PH model may be stratified by cohort.
• cohorts are evaluated individually.
• coefficients for a final model may be derived from a combination of all cohorts.
• the final prognostic score may be scaled to represent values between 0 and 80.
• hazard ratios for CCP score and pathological stage are consistent across the various cohorts.
• CCP together with pathological stage provides the best prediction for lung cancer mortality.
• Prognostic score 20*(0.33*CCP score + 0.52*stage) + 15.
• the p-value is equal to or less than 0.05. In some embodiments the p-value is equal to or less than 0.01. In some embodiments the p-value is equal to or less than 0.001. In some embodiments the p-value is equal to or less than 0.00078.
• the combined score may differentiate 5-year lung cancer mortality risk for patients assigned the same risk based on pathological stage alone.
• pathological stage alone may provided estimates of 5-year risk of cancer- specific death.
• stage IA provides a 5-year risk of cancer-specific death estimate of 12.6% or less.
• stage IB provides a 5-year risk of cancer- specific death estimate of 22.6% or less.
• stage IIA provides a 5-year risk of cancer-specific death estimate of 38.4% or more.
• stage IIB provides a 5-year risk of cancer-specific death estimate of 60% or more.
• the prognostic score may be used to separate stage IA patients with 5-year risk estimates ranging from 6% to 24%.
• the prognostic score may be used to separate stage IB patients with 5-year risk estimates ranging from 10% to 42%. In some embodiments the prognostic score may be used to separate stage IIA patients with 5-year risk estimates ranging from 21% to 63%. In some embodiments the prognostic score may be used to separate stage IIB patients with 5-year risk estimates ranging from 32% to 75%.
• a pre-defined prognostic score is calculated for each patient.
• a PS cut-point is determined such that the percentage of stage IA patients having a PS at or below the cutpoint is close as possible to 85%.
• the association of CCP, and the PS, with 5-year lung cancer mortality is evaluated using Cox proportional hazards models, likelihood ratio tests or both.
• the Mantel-Cox logrank test is used to evaluate the difference in 5-year lung cancer mortality for patients with PS scores at or below a cut-point versus patients with scores above a cut-point.
• PS may be used to predict 5 year lung cancer specific survival.
• low and high risk may be classified by a cut-off predefined as the 85% percentile of the PS in stage IA patients.
• patients in the low PS group have a significantly more favorable 5-year survival than patients in the high PS group.
• the Log-rank p value is at least 3.8 x 10 "7 .
• risk stratification is improved by PS compared to pathological stage alone.
• patients with pathological stage 1A have an 18% risk of disease specific death within five years.
• patients with pathological stage IB have a 28% risk of disease specific death within five years.
• patients with pathological stage IIA have a 42% risk of disease specific death within five years.
• patients with pathological stage IIB have a 60% risk of disease specific death within five years.
• pathological stage is combined with CCP score resulting in the ability to assigned significantly more detailed risk to patients assigned identical risk according to pathological stage alone.
• CCP score alone is a significant prognostic marker.
• CCP score is evaluated using univariate analysis.
• the univariate p-value is at least 0.05. In some the univariate p-value is at least 0.01. In some the univariate p-value is at least 0.001. In some the univariate p-value is at least 0.0001. In some the univariate p-value is at least 0.00001. In some the univariate p-value is at least 0.000001 1. In some embodiments CCP score is evaluated using multivariate analysis. In some
• CCP score is evaluated using multivariate analysis.
• the multivariate p- value is at least 0.05. In some the multivariate p-value is at least 0.01. In some the multivariate p-value is at least 0.005.
• the prognostic value of PS is evaluated by univariate analysis. In some embodiments the p-value is at least 0.05. In some embodiments the p-value is at least 0.01. In some embodiment the p-value is at least 0.001. In some embodiments the p-value is at least 2.8 ⁇ 10 ⁇ -1 1. In some embodiments the prognostic value of PS is evaluated by bivariate analysis. In some embodiments the p-value is at least 0.05. In some embodiments the p-value is at least 0.01. In some embodiments the p-value is at least 0.093. In some embodiments the combination of pathological stage and CCP score into the Prognostic Score captures significant prognostic information that is not provided by pathological stage alone.
• the prognostic value of the PS is evaluated in IA and IB stage cancer separately using a univariate model.
• the Hazard Ratio is 1.67.
• the 95% confidence intervals are 1.27, and 2.29.
• the p-value is at least 0.05.
• the p-value is at least 0.01.
• the p-value is at least 0.001.
• the p-value is at least 0.0027.
• the prognostic value of the PS is evaluated in IA and IB stage cancer separately using a bivariate model.
• the Hazard Ratio is 1.74.
• the 95% confidence intervals are 1.16, and 2.61.
• the p-value is at least 0.05. In some embodiments the combination of pathological stage and CCP score into the Prognostic Score captures significant prognostic information that is not provided by pathological stage alone when restricted to stage IA-IB disease.
• CCP expression and pathological stage may be used to assess prognosis for post-surgical risk of death in patients diagnosed with lung carcinoids.
• CCP scores may be generated stage IA, IB, IIA, IIB, and IIIB lung carcinoid patients.
• the outcome measure is survival.
• the association of CCP with mortality is evaluated using the Cox proportional hazards model.
• the p-value in a univariate analysis is at least 0.05. In some embodiments the p-value in a univariate analysis is at least 0.01. In some embodiments the p-value in a univariate analysis is at least 0.00125. In some embodiments the p-value in a multivariate analysis is at least 0.05. In some embodiments the p- value in a multivariate analysis is at least 0.01. In some embodiments the p-value in a multivariate analysis is at least 0.0035.
• pathological stage may be used to assess prognosis for post-surgical risk of death in patients diagnosed with lung carcinoids.
• disease may be spread among two histological groups: atypical and typical.
• stage may be coded as a 4-level categorical variable.
• stages may consist of IA, IB, IIA/IIB, and IIIA/IIIB/IV.
• the association of CCP with death from disease may be evaluated using the Cox proportional hazards model.
• univariate analysis of Cox proportional hazards models may be used to evaluate the association of CCP with death from lung carcinoids.
• the p-value is at least 0.05. In some embodiments the p-value is at least 0.01. In some embodiments the p-value is at least 0.0014.
• the association of CCP with disease free survival may be evaluated using the Cox proportional hazards model. In some embodiments univariate analysis of Cox proportional hazards models may be used to evaluate the association of CCP with disease free survival. .
• the p-value is at least 0.05. In some embodiments the p-value is at least 0.01. In some embodiments the p-value is at least 0.006.
• the association of CCP and death with disease in atypical carcinoid patients may be evaluated using the Cox proportional hazards model.
• univariate analysis may be used to evaluate the association of CCP and death with disease in atypical carcinoid patients.
• CCP is a highly significant predictor of death with recurrence of disease.
• the p-value is at least 0.05. In some embodiment the p-value is at least 0.0102.

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