WO2020223233A1 - Méthodes pronostiques et thérapeutiques contre le cancer colorectal - Google Patents

Méthodes pronostiques et thérapeutiques contre le cancer colorectal Download PDF

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WO2020223233A1
WO2020223233A1 PCT/US2020/030275 US2020030275W WO2020223233A1 WO 2020223233 A1 WO2020223233 A1 WO 2020223233A1 US 2020030275 W US2020030275 W US 2020030275W WO 2020223233 A1 WO2020223233 A1 WO 2020223233A1
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avant
score
individual
gzmb
crc
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PCT/US2020/030275
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English (en)
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Thomas SANDMANN
Akshata Ramrao UDYAVAR
Anneleen Daemen
Meghna Das Thakur HARRIS
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Genentech, Inc.
F. Hoffmann-La Roche Ag
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Publication of WO2020223233A1 publication Critical patent/WO2020223233A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/118—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

  • prognostic and therapeutic methods for the treatment of colorectal cancer using a prognostic gene signature capturing stromal, proliferative, and immune functions.
  • the invention provides methods for patient selection, prognosis, and treatment.
  • CRCs Primary colorectal cancers
  • primary treatment e.g., surgical resection
  • AJCC/UICC- TNM American Joint Committee on Cancer/Union for International Cancer Control TNM classification system
  • Patients having primary CRCs classified as high risk by the AJCC/UICC-TNM generally receive chemotherapy in the adjuvant setting.
  • node positive CRC is six months of adjuvant treatment with chemotherapy. This strategy is intended to improve cure rates for patients with high-risk primary disease.
  • the present invention provides prognostic and therapeutic methods for the treatment of colorectal cancer using a prognostic gene signature capturing stromal, proliferative, and immune functions.
  • the disclosure features a method of predicting disease progression in an individual having a colorectal cancer (CRC), the method comprising determining an AVANT score from a sample from the individual, wherein an AVANT score that is above a reference AVANT score identifies the individual as one who is at high risk of CRC recurrence following surgical resection.
  • CRC colorectal cancer
  • the disclosure features a method of predicting disease recurrence in an individual who has had surgical resection of a CRC, the method comprising determining an AVANT score from a sample from the individual, wherein an AVANT score that is above a reference AVANT score indicates that the individual is at high risk of CRC recurrence.
  • the AVANT score determined from the sample is above the reference AVANT score, and the method further comprises administering to the individual an adjuvant treatment comprising a chemotherapy.
  • the AVANT score is calculated based on the expression levels of CDCA5, DTX2, E2F1, GMNN, KDM1A, ABCC9, CRYAB, DTX1, HEYL, RGS2, SPP1, TLL 1, WBSCR17, IGFBP1, KDM5D, PCSK1, REG4, SMAD3, SMAD9, TCF12, CD28, CXCL 1, and GZMB.
  • the AVANT score is calculated based on the expression levels of CDCA5, DTX2, E2F1, GMNN, KDM1A, ABCC9, CRYAB, DTX1, HEYL, RGS2, SPP1, TLL 1, WBSCR17, IGFBP1, KDM5D, PCSK1, REG4, SMAD3, SMAD9, TCF12, CD28, CXCL 1, GZMB, RPLN23, AXIN2, CDKN1B, RPS6KA 1, RHOA, SP2, SHISA5, MLH1, CENPM, CDK2, DNMT1, ANGPT1, IGFBP3, CD36, HBEGF, FOS, SERPINB13, APCDD1, SCD5, POU5F1, EPHA4, FLT1, CDK6, MAPK3, TP73, TAP1, TNF, PDCD1, FCRL5, and CXCR4.
  • the method further comprises determining a consensus molecular subtype (CMS) of the sample from the individual.
  • CMS consensus molecular subtype
  • the AVANT score determined from the sample is above the reference AVANT score and the CMS of the sample is CMS4.
  • the reference AVANT score is a score in a reference population of individuals who have had surgical resection of a CRC. In some aspects, the reference population of individuals has been administered an adjuvant treatment comprising a chemotherapy. In some aspects, the sample is obtained from the individual prior to the administration of any adjuvant treatment. In some aspects, the reference AVANT score significantly separates a first and a second subset of individuals based on responsiveness to treatment. In some aspects, responsiveness to treatment is an increase in disease- free survival (DFS), recurrence-free survival (RFS), or overall survival (OS). In some aspects, the reference AVANT score is a pre-assigned score.
  • DFS disease- free survival
  • RFS recurrence-free survival
  • OS overall survival
  • the reference AVANT score is a pre-assigned score.
  • the reference AVANT score is the 25 th percentile of AVANT scores in the reference population. In some aspects, the reference AVANT score is the 50 th percentile of AVANT scores in the reference population. In some aspects, the reference AVANT score is the 75 th percentile of AVANT scores in the reference population.
  • the disclosure features a method of predicting disease progression in an individual having a CRC, the method comprising determining an AVANT stromal score and the expression level of GZMB in a sample from the individual, wherein an AVANT stromal score that is above a reference AVANT stromal score and an expression level of GZMB that is below a reference expression level of GZMB identifies the individual as one who is at a high risk of CRC recurrence following surgical resection.
  • the disclosure features a method of predicting disease recurrence in an individual who has had surgical resection of a CRC, the method comprising determining an AVANT stromal score and the expression level of GZMB in a sample from the individual, wherein an AVANT stromal score that is above a reference AVANT stromal score and an expression level of GZMB that is below a reference expression level of GZMB indicates that the individual is at high risk of CRC recurrence.
  • the AVANT stromal score is above a reference AVANT stromal score and the expression level of GZMB is below a reference expression level of GZMB, and the method further comprises administering to the individual an adjuvant treatment comprising a chemotherapy.
  • an AVANT stromal score that is below a reference AVANT stromal score and an expression level of GZMB that is above a reference expression level of GZMB identifies the individual as one who is at low risk of CRC recurrence following surgical resection.
  • the method further comprises determining a CMS of the sample from the individual.
  • the AVANT stromal score determined from the sample is above the reference AVANT stromal score and the CMS of the sample is CMS4.
  • the AVANT stromal score is calculated based on the expression levels of ABCC9, CRYAB, DTX1, HEYL, RGS2, SPP1, TLL 1, WBSCR17, IGFBP1, KDM5D, PCSK1, REG4, SMAD3, SMAD9, and TCF12.
  • the AVANT stromal score is calculated based on the expression levels of ANGPT 1, WBSCR17, TLL 1, CRYAB, ABCC9, IGFBP3, HEYL, DTX1, CD36, SPP1, RGS2, HBEGF, FOS, SERPINB13, IGFBP1, APCDD1, SCD5, KDM5D, POU5F1, EPHA4, REG4, PCSK1, SMAD9, FLT1, TCF12, SMAD3, CDK6, and MAPK3.
  • the reference AVANT stromal score is a score in a reference population of individuals who have had surgical resection of a CRC. In some aspects, the reference population of individuals has been administered an adjuvant treatment comprising a chemotherapy. In some aspects, the sample is obtained from the individual prior to the administration of any adjuvant treatment. In some aspects, the reference AVANT stromal score significantly separates a first and a second subset of individuals based on responsiveness to treatment. In some aspects, responsiveness to treatment is an increase in DFS, RFS, or OS. In some aspects, the reference AVANT stromal score is a pre-assigned score. In some aspects, the reference AVANT stromal score is the median AVANT stromal score in the reference population.
  • the reference expression level of GZMB is a pre-assigned expression level. In some aspects, the reference expression level of GZMB is the median expression level of GZMB in the reference population.
  • the disclosure features a method of treating an individual having a CRC, the method comprising (a) determining an AVANT score from a sample from the individual, wherein the AVANT score is above a reference AVANT score; and (b) administering to the individual an adjuvant treatment comprising a chemotherapy.
  • the disclosure features a method of treating an individual having a CRC, the method comprising administering an adjuvant treatment comprising a chemotherapy to the individual, for whom an AVANT score from a sample from the individual has been determined to be above a reference AVANT score.
  • the AVANT score is calculated based on the expression levels of CDCA5, DTX2, E2F1, GMNN, KDM1A, ABCC9, CRYAB, DTX1, HEYL, RGS2, SPP1, TLL 1, WBSCR17, IGFBP1, KDM5D, PCSK1, REG4, SMAD3, SMAD9, TCF12, CD28, CXCL 1, and GZMB.
  • the AVANT score is calculated based on the expression levels of CDCA5, DTX2, E2F1, GMNN, KDM1A, ABCC9, CRYAB, DTX1, HEYL, RGS2, SPP1, TLL 1, WBSCR17, IGFBP1, KDM5D, PCSK1, REG4, SMAD3, SMAD9, TCF12, CD28, CXCL 1, GZMB, RPLN23, AXIN2, CDKN1B, RPS6KA 1, RHOA, SP2, SHISA5, MLH1, CENPM, CDK2, DNMT1, ANGPT1, IGFBP3, CD36, HBEGF, FOS, SERPINB13, APCDD1, SCD5, POU5F1, EPHA4, FLT1, CDK6, MAPK3, TP73, TAP1, TNF, PDCD1, FCRL5, and CXCR4.
  • the method further comprises determining a consensus molecular subtype (CMS) of the sample from the individual.
  • CMS consensus molecular subtype
  • the CMS of the CRC is CMS4.
  • the reference AVANT score is a score in a reference population of individuals who have had surgical resection of a CRC. In some aspects, the reference population of individuals has been administered an adjuvant treatment comprising a chemotherapy. In some aspects, the sample is obtained from the individual prior to the administration of any adjuvant treatment. In some aspects, the reference AVANT score significantly separates a first and a second subset of individuals based on responsiveness to treatment. In some aspects, responsiveness to treatment is an increase in disease- free survival (DFS), recurrence-free survival (RFS), or overall survival (OS). In some aspects, the reference AVANT score is a pre-assigned score.
  • DFS disease- free survival
  • RFS recurrence-free survival
  • OS overall survival
  • the reference AVANT score is a pre-assigned score.
  • the reference AVANT score is the 25 th percentile of AVANT scores in the reference population. In some aspects, the reference AVANT score is the 50 th percentile of AVANT scores in the reference population. In some aspects, the reference AVANT score is the 75 th percentile of AVANT scores in the reference population.
  • the disclosure features a method of treating an individual having a CRC, the method comprising (a) determining an AVANT stromal score and the expression level of GZMB in a sample from the individual, wherein the AVANT stromal score is above a reference AVANT stromal score and the expression level of GZMB is below a reference expression level of GZMB ; and (b) administering to the individual an adjuvant treatment comprising a chemotherapy.
  • the disclosure features a method of treating an individual having a CRC, the method comprising administering an adjuvant treatment comprising a chemotherapy to the individual, for whom an AVANT stromal score from a sample from the individual has been determined to be above a reference AVANT stromal score and an expression level of GZMB has been determined to be below a reference expression level of GZMB.
  • the method further comprises determining a CMS of the sample from the individual.
  • the CMS of the sample is CMS4.
  • the AVANT stromal score is calculated based on the expression levels of ABCC9, CRYAB, DTX1, HEYL, RGS2, SPP1, TLL 1, WBSCR17, IGFBP1, KDM5D, PCSK1, REG4, SMAD3, SMAD9, and TCF12. In some aspects, the AVANT stromal score is calculated based on the expression levels of ANGPT 1, WBSCR17, TLL 1, CRYAB, ABCC9, IGFBP3, HEYL, DTX1, CD36, SPP1, RGS2, HBEGF, FOS, SERPINB13, IGFBP1, APCDD1, SCD5, KDM5D, POU5F1, EPHA4, REG4,
  • the reference AVANT stromal score is a score in a reference population of individuals who have had surgical resection of a CRC. In some aspects, the reference population of individuals has been administered an adjuvant treatment comprising a chemotherapy. In some aspects, the sample is obtained from the individual prior to the administration of any adjuvant treatment. In some aspects, the reference AVANT stromal score significantly separates a first and a second subset of individuals based on responsiveness to treatment. In some aspects, responsiveness to treatment is an increase in DFS, RFS, or OS. In some aspects, the reference AVANT stromal score is a pre-assigned score. In some aspects, the reference AVANT stromal score is the median AVANT stromal score in the reference population.
  • the reference expression level of GZMB is a pre-assigned expression level. In some aspects, the reference expression level of GZMB is the median expression level of GZMB in the reference population.
  • the sample is a tissue biopsy, a whole blood sample, a buccal swab, a plasma sample, a serum sample, or a combination thereof.
  • the sample is a tissue biopsy.
  • the sample is an archival sample, a fresh sample, or a frozen sample.
  • the sample is a formalin-fixed paraffin-embedded sample.
  • the expression level is a nucleic acid expression level.
  • the nucleic acid expression level is a mRNA expression level.
  • the mRNA expression level is determined by direct digital counting of nucleic acids, RNA-seq, RT-qPCR, qPCR, multiplex qPCR or RT-qPCR, microarray analysis, or a combination thereof.
  • the digital counting of nucleic acids is by NANOSTRING ® NCOUNTER ® analysis.
  • the expression level is a protein expression level.
  • the protein expression level is determined by an immunoassay, liquid chromatography-mass spectrometry (LC-MS) technology, nephelometry, aptamer technology, or a combination thereof.
  • LC-MS liquid chromatography-mass spectrometry
  • the CRC is a stage I, stage II, or stage III CRC, according to the TNM classification system at the onset of treatment.
  • the chemotherapy comprises oxaliplatin, fluorouracil, or leucovorin. In some aspects, the chemotherapy comprises oxaliplatin, fluorouracil, and leucovorin. In some aspects, the chemotherapy consists of oxaliplatin, fluorouracil, and leucovorin. In some aspects, the chemotherapy comprises oxaliplatin and capecitabine. In some aspects, the chemotherapy consists of oxaliplatin and capecitabine. In some aspects, the adjuvant treatment further comprises bevacizumab.
  • the method further comprises administering to the individual one or more additional therapeutic agents.
  • the one or more additional therapeutic agents comprise an immunomodulatory agent.
  • the immunomodulatory agent is a PD-1 axis binding antagonist.
  • the PD-1 axis binding antagonist is a PD-L1 binding antagonist, a PD-1 binding antagonist, or a PD-L2 binding antagonist.
  • the PD-1 axis binding antagonist is a PD-L1 binding antagonist.
  • the PD-L1 binding antagonist is MPDL3280A
  • the PD-L1 binding antagonist is MPDL3280A (atezolizumab).
  • the PD-1 axis binding antagonist is a PD-1 binding antagonist.
  • the PD-1 binding antagonist is MDX-1 106 (nivolumab), MK-3475 (pembrolizumab), MEDI-0680 (AMP-514), PDR001 , REGN2810, or BGB-108.
  • the PD-1 axis binding antagonist is a PD-L2 binding antagonist.
  • the PD-L2 binding antagonist is an antibody or an immunoadhesin.
  • the individual is a human.
  • Fig. 1 A is a set of Kaplan-Meier curves for the AVANT signature in the AVANT biomarker evaluable population (BEP) dataset for disease-free survival (DFS).
  • the p-value corresponds to a log-rank test.
  • Fig. 1 B is a set of Kaplan-Meier curves for the AVANT signature in the independent validation cohort GSE39582 for recurrence-free survival (RFS).
  • the p-value corresponds to a log-rank test.
  • Fig. 1 C is a correlation plot for the genes in the AVANT signature. Blue denotes positive and red denotes negative correlation. Color intensity denotes the strength of the correlation. Signature genes were assigned to one of four clusters. Red denotes proliferative genes, light and dark green denote stromal genes, and purple denotes immune genes.
  • Fig. 1 D is a set of Kaplan-Meier curves for the proliferative genes of the AVANT signature for DFS divided at the median in the AVANT BEP dataset.
  • Fig. 1 E is a set of Kaplan-Meier curves for the immune genes of the AVANT signature for DFS divided at the median in the AVANT BEP dataset.
  • Fig. 1 F is a set of Kaplan-Meier curves for the stromal genes of the AVANT signature for DFS divided at the median in the AVANT BEP dataset.
  • Fig. 1 G is a forest plot showing RFS for the validation dataset GSE39582 showing hazard ratios and associated p-values for the AVANT signature, the OncotypeDx signature (Gray et al ., J Clin Oncol, 29(35), 461 1 -4619, 201 1 ), the T-effector signature (Mariathasan et al., Nature, 554: 544-548, 2018), the CAF signature (Isella et al., Nature Genet, 47(4), 312-319, 201 5), and the F-TBRS signature (Calon et al., Cancer Cell, 22: 571 -584, 2012) and a table showing the significance of added prognostic value (if any) provided by each published signature (OncotypeDx signature, T-effector signature, CAF signature, F- TBRS signature, and CMS subtypes (Guinney et al., Nat Med, 21 (1 1 ), 1350-1356, 201 5) when added to the
  • Fig. 2A is a set of Kaplan-Meier curves for granzyme B ( GZMB ) expression stratified by median expression in the AVANT BEP dataset for DFS.
  • Fig. 2B is a set of Kaplan-Meier curves for granzyme B (GZMB) expression stratified by median expression in the GSE39582 dataset for RFS.
  • GZMB granzyme B
  • Fig. 2C is a set of Kaplan-Meier curves for the T-effector signature stratified by median expression in the AVANT BEP dataset for DFS.
  • Fig. 2D is a set of Kaplan-Meier curves for the T-effector signature stratified by median expression in the GSE39582 dataset for RFS.
  • Fig. 2E is a forest plot showing hazard ratios (HR) and associated P-values for the T-effector signature without GZMB and for GZMB expression in the AVANT BEP dataset for DFS and a table indicating the significance of added prognostic value (if any) provided by the T-effector signature when added to GZMB (first column) and vice versa (second column).
  • HR hazard ratios
  • 2F is a forest plot showing hazard ratios (HR) and associated P-values for the T-effector signature without GZMB and for GZMB expression in the GSE39582 dataset for RFS and a table indicating the significance of added prognostic value (if any) provided by the T-effector signature when added to GZMB (first column) and vice versa (second column).
  • HR hazard ratios
  • second column a table indicating the significance of added prognostic value (if any) provided by the T-effector signature when added to GZMB (first column) and vice versa
  • Fig. 2G is a set of Kaplan-Meier curves showing the relationship between GZMB and the proliferative genes of the AVANT signature in the AVANT BEP dataset for DFS. Proliferative signature and GZMB expression were stratified by median expression. P-values correspond to a log-rank test.
  • Fig. 2H is a set of Kaplan-Meier curves showing the relationship between GZMB and the proliferative genes of the AVANT signature in the GSE39582 dataset for RFS. P-values correspond to a log-rank test.
  • Fig. 21 is a set of Kaplan-Meier curves showing the relationship between GZMB and the stromal genes of the AVANT signature in the AVANT BEP dataset for DFS. Stromal signature and GZMB expression were stratified by median expression. P-values correspond to a log-rank test.
  • Fig. 2J is a set of Kaplan-Meier curves showing the relationship between GZMB and the stromal genes of the AVANT signature in the GSE39582 dataset for RFS. P-values correspond to a log-rank test.
  • Fig. 3A is a box plot showing expression of the T-effector signature by consensus molecular subtype (CMS) in the AVANT BEP dataset.
  • CMS consensus molecular subtype
  • Fig. 3B is a box plot showing expression of the T-effector signature by CMS in the GSE39582 dataset.
  • the inset table shows p-values corresponding to a pairwise T-test.
  • Fig. 3C is a box plot showing expression of the T-effector signature by microsatellite instability (MSI) status in the AVANT BEP dataset.
  • MSS microsatellite stable or microsatellite instability low;
  • MSI- H microsatellite instability high.
  • Fig. 3D is a Pearson correlation chart showing the correlation between expression of single genes from the T-effector signature and the average expression of the other T-effector genes without the gene in question by colorectal cancer (CRC), CRC CMS 1 , CRC CMS 2, lung cancer, ovary cancer, breast cancer, head and neck cancer, and bladder cancer in The Cancer Genome Atlas (TCGA) data.
  • CRC colorectal cancer
  • CRC CMS 1 CRC CMS 2
  • TCGA Cancer Genome Atlas
  • Fig. 3E is a box plot showing GZMB expression by CMS in the AVANT BEP dataset.
  • the inset table shows p-values corresponding to a pairwise T-test.
  • Fig. 3F is a box plot showing GZMB expression by CMS in the GSE39582 dataset.
  • the inset table shows p-values corresponding to a pairwise T-test.
  • Fig. 3G is a box plot showing GZMB expression by MSI status in the AVANT BEP dataset.
  • Fig. 3H is a set of Kaplan-Meier curves for GZMB expression stratified by median in 279 CMS2 tumors with low T-effector score from the AVANT BEP dataset for DFS.
  • a T-effector score, excluding GZMB, below the mean score in the full cohort is considered low.
  • the p-value corresponds to a log-rank test.
  • Fig. 31 is a Pearson correlation chart showing the correlation between the modified T-effector signature (i.e. without granzyme A ( GZMA ) and GZMB) and GZMA or GZMB expression in AVANT patients, by CMS or MSI status.
  • Fig. 3J is a Pearson correlation chart showing the correlation between the modified T-effector signature and GZMA or GZMB expression in GSE39582 patients by CMS.
  • Fig. 4A is a t-distributed stochastic neighbor embedding (tSNE) map of the CD45+ immune cell populations in 12 procured colorectal cancer (CRC) samples. Clusters representing different cell types are denoted by distinct colors, based on marker expression. Outliers detected by density-based clustering were excluded.
  • tSNE stochastic neighbor embedding
  • Fig. 4B is a contour plot showing CD8 vs. GZMB expression in CD16+ natural killer (NK) cells. Density is indicated by color.
  • Fig. 4C is a contour plot showing CDS vs. GZMB expression in plasmacytoid dendritic cells (pDCs). Density is indicated by color.
  • Fig. 4D is a Pearson correlation chart showing the correlation between expression of single genes from the NK signature and the average expression of the other NK genes without the gene in question by colorectal cancer (CRC), CRC CMS1 , CRC CMS2, lung cancer, ovary cancer, breast cancer, head and neck cancer, and bladder cancer in TCGA data.
  • CRC colorectal cancer
  • CRC CMS1 CRC CMS1
  • CRC CMS2 CRC CMS2
  • lung cancer ovary cancer
  • breast cancer head and neck cancer
  • bladder cancer in TCGA data.
  • Fig. 4E is a box plot showing expression of the NK signature by CMS in the GSE39582 dataset.
  • the inset table shows p-values corresponding to a pairwise T-test.
  • Fig. 4F is a box plot showing expression of GZMB by CMS in CRC cell lines.
  • the inset table shows p-values corresponding to a pairwise T-test.
  • Fig. 5A is a set of Kaplan-Meier curves for the three treatment arms in the AVANT BEP dataset for overall survival (OS).
  • FOLFOX4 oxaliplatin infusion added to Fluorocil and Leucovorin ;
  • FOLFOX4+Bev FOLFOX4 plus bevacizumab
  • XELOX+Bev oxaliplatin and capecitabine plus bevacizumab.
  • the p-value corresponds to a log-rank test.
  • Fig. 5B is a set of Kaplan-Meier curves for the three treatment arms in the AVANT BEP dataset for DFS.
  • the p-value corresponds to a log-rank test.
  • Fig. 6A is a matrix showing expression of 132 CMS classifier genes (rows) in 1062 AVANT patients (columns). The predicted CMSs are denoted for each patient on the top of the chart. The row annotation denotes the subtype in which a given gene is uniquely highly expressed.
  • PAMR Prediction Analysis of Microarrays for R.
  • Fig. 6B is a matrix showing expression of 132 CMS classifier genes (rows) in TCGA CRC patients (columns).
  • the CMSs as predicted by the Guinney et al. , Nat Med, 21 (1 1 ): 1350-1362, 2015 random forest algorithm are denoted for each patient on the top of the chart.
  • Fig. 6C is a box plot showing the percentage of patients classified as having each of the CMSs in the AVANT BEP dataset. Patients that lack CMS classifier gene expression for any subtype are labeled as unclassifiable.
  • Fig. 6D is a box plot showing the percentage of patients classified as having each of the CMSs in TCGA. Patients that lack CMS classifier gene expression for any subtype are labeled as unclassifiable.
  • Fig. 6E is a matrix showing expression of 132 CMS classifier genes in the GSE39582 dataset.
  • the CMSs as predicted by the Guinney et al., Nat Med, 21 (1 1 ): 1350-1362, 2015 random forest algorithm are denoted for each patient on the top of the chart.
  • Fig. 6F is a box plot showing the percentage of patients classified as having each of the CMSs in the GSE39582 dataset.
  • Fig. 7A is a bar graph showing the percentage of patients having no mutations (WT) or at least one mutation (MUT) at an assayed position in KRAS in each CMS in the AVANT BEP dataset.
  • Fig. 7B is a bar graph showing the percentage of patients having no mutations (WT) or at least one mutation (MUT) at an assayed position in KRAS in each CMS in TCGA.
  • Fig. 7C is a bar graph showing the percentage of patients having no mutations (WT) or at least one mutation (MUT) at an assayed position in KRAS in each CMS in the GSE39582 dataset.
  • Fig. 7D is a bar graph showing the percentage of patients having no mutations (WT) or at least one mutation (MUT) at an assayed position in BRAF in each CMS in the AVANT BEP dataset.
  • Fig. 7E is a bar graph showing the percentage of patients having no mutations (WT) or at least one mutation (MUT) at an assayed position in BRAF in each CMS in TCGA.
  • Fig. 7F is a bar graph showing the percentage of patients having no mutations (WT) or at least one mutation (MUT) at an assayed position in BRAF in each CMS in the GSE39582 dataset.
  • Fig. 7G is a bar graph showing the percentage of patients having microsatellite instability status MSI-H or MSS in each CMS in the AVANT BEP dataset.
  • Fig. 7H is a bar graph showing the percentage of patients having microsatellite instability status MSI-H or MSS in each CMS in TCGA.
  • Fig. 71 is a bar graph showing the percentage of patients having right or left sidedness of the colon cancer in each CMS in the AVANT BEP dataset.
  • Fig. 8 is a matrix showing Cox-based elastic net regression results for the identification of genes prognostic for OS in AVANT using alpha values ranging from 0.1 to 0.9. Identified prognostic genes per alpha value are denoted by black tiles.
  • Fig. 9A is a box plot showing average expression of the proliferative gene cluster of the AVANT signature of Fig. 1 C by the AVANT signature quartiles in the AVANT BEP dataset.
  • the inset table shows p-values computed using the pairwise T-test for each comparison adjusted for multiplicity testing.
  • Fig. 9B is a box plot showing average expression of the immune gene cluster of the AVANT signature of Fig. 1 C by the AVANT signature quartiles in the AVANT BEP dataset.
  • Fig. 9C is a box plot showing average expression of the TGFp-enriched stromal gene cluster of the AVANT signature of Fig. 1 C by the AVANT signature quartiles in the AVANT BEP dataset.
  • Fig. 9D is a box plot showing average expression of the stromal gene cluster of the AVANT signature of Fig. 1 C by the AVANT signature quartiles in the AVANT BEP dataset.
  • Fig. 9E is a box plot showing average expression of the proliferative gene cluster of the AVANT signature of Fig. 1 C by CMS in the AVANT BEP dataset.
  • Fig. 9F is a box plot showing average expression of the immune gene cluster of the AVANT signature of Fig. 1 C by CMS in the AVANT BEP dataset.
  • Fig. 9G is a box plot showing average expression of the TGFp-enriched stromal gene cluster of the AVANT signature of Fig. 1 C by CMS in the AVANT BEP dataset
  • Fig. 9H is a box plot showing average expression of the stromal gene cluster of the AVANT signature of Fig. 1 C by CMS in the AVANT BEP dataset.
  • Fig. 91 is a matrix showing expression of the AVANT signature genes (prognostic genes; rows) in the AVANT BEP dataset (columns). Red denotes high expression and blue denotes low expression. Patients are annotated by CMS and quartile of the AVANT signature. Row annotation indicates the respective signatures from Fig. 1 C.
  • Fig. 10A is a box plot showing average expression of the proliferative gene cluster from Fig. 1 C by MSI status in the AVANT BEP dataset. P-values correspond to a T-test.
  • Fig. 10B is a box plot showing average expression of the immune gene cluster from Fig. 1 C by MSI status in the AVANT BEP dataset.
  • Fig. 10C is a box plot showing average expression of the two stromal gene clusters combined from Fig. 1 C by MSI status in the AVANT BEP dataset.
  • Fig. 10D is a set of scatter plots showing correlation between the average expression of the proliferative, immune, and stromal gene clusters of the AVANT signature in the AVANT BEP dataset, with the two stromal gene clusters combined. Pearson correlations are denoted, with font size reflecting significance.
  • Fig. 11 A is a set of Kaplan-Meier curves for the proliferative gene cluster from Fig. 1 C, stratified by median, in the GSE39582 dataset for RFS. P-values correspond to a log-rank test.
  • Fig. 11 B is a set of Kaplan-Meier curves for the immune gene cluster from Fig. 1 C, stratified by median, in the GSE39582 dataset for RFS. P-values correspond to a log-rank test.
  • Fig. 11 C is a set of Kaplan-Meier curves for the two stromal gene clusters combined from Fig.
  • Fig. 12 is a DFS forest plot showing hazard ratios and associated P-values for each individual signature in the AVANT BEP dataset and a table indicating the significance of added prognostic value (if any) provided by each published signature when added to the AVANT signature (first column) and vice versa (second column).
  • Fig. 13A is a table and a forest plot reporting multivariate analysis results for the AVANT signature in the AVANT dataset.
  • Forest plot denotes the Cox hazard ratios (HR) and p-values for the different signature quartiles after adjustment for clinical covariates.
  • HR Cox hazard ratios
  • RFS prognosis
  • covariates were age (AGE-CAT), sex, level of carcinoembryonic antigen (CEA) in blood (CEABL), Eastern Cooperative Oncology Group (ECOG) status (ECOGSTAT), and American Joint Committee on Cancer (AJCC) tumor status including lymph node status (i.e. strata).
  • Fig. 13B is a table and a forest plot reporting multivariate analysis results for the AVANT signature in the GSE39582 dataset.
  • Forest plot denotes the Cox hazard ratios (HR) and p-values for the different signature quartiles after adjustment for clinical covariates.
  • HR Cox hazard ratios
  • p-values for the different signature quartiles after adjustment for clinical covariates.
  • HR Cox hazard ratios
  • p-values for the different signature quartiles after adjustment for clinical covariates.
  • Each individual clinical covariate in each trial was tested for its effect on prognosis (DFS or RFS), and only prognostic covariates were included in the multivariate analyses. Included covariates were age (age.at.diagnosis), sex, tumor stage (tnm.stage), and lymph node status (tnm.n).
  • Fig. 14 is a set of Kaplan-Meier curves for GZMB expression stratified by median in 177 CMS2 tumors with low T-effector score from the GSE39582 dataset for RFS.
  • a T-effector score, excluding GZMB, below the mean score in the full cohort is considered low.
  • the p-value corresponds to a log-rank test.
  • Fig. 15A is a heat map showing average expression of each marker across the detected immune cell clusters from Fig. 4A. Shown are column scaled arcsinh transformed intensity values. Red denotes high expression; blue denotes low expression.
  • Fig. 15B is a bar graph showing the percentage of pooled immune cells from 12 resected stage II or stage III CRC tumors that are of a given type.
  • Fig. 15C is a Pearson correlation chart showing the correlation between expression of single genes from the pDC signature and the average expression of the other pDC genes without the gene in question by colorectal cancer (CRC), CRC CMS 1 , CRC CMS 2, lung cancer, ovary cancer, breast cancer, head and neck cancer, and bladder cancer in TCGA data.
  • CRC colorectal cancer
  • Fig. 15D is a box plot showing expression of the pDC signature by CMS in the GSE39582 dataset.
  • the inset table shows p-values corresponding to a pairwise T-test.
  • Fig. 15E is a box plot showing expression of GZMA by CMS in CRC cell lines.
  • the inset table shows p-values corresponding to a pairwise T-test.
  • Fig. 16A is a box plot showing GZMB expression by cancer type in a cohort of 671 cell lines covering 12 cancer types.
  • Fig. 16B is a box plot showing GZMA expression by cancer type in a cohort of 671 cell lines covering 12 cancer types.
  • Fig. 17A is a set of Kaplan-Meier curves for the AVANT signature in the AVANT BEP dataset for overall survival (OS).
  • the p-value corresponds to a log-rank test.
  • Fig. 17B is a set of Kaplan-Meier curves for the AVANT signature in the independent validation cohort GSE39582 for OS.
  • the p-value corresponds to a log-rank test.
  • Fig. 17C is a set of Kaplan-Meier curves for the proliferative genes of the AVANT signature for OS divided at the median in the AVANT BEP dataset.
  • Fig. 17D is a set of Kaplan-Meier curves for the immune genes of the AVANT signature for OS divided at the median in the AVANT BEP dataset.
  • Fig. 17E is a set of Kaplan-Meier curves for the stromal genes of the AVANT signature for OS divided at the median in the AVANT BEP dataset.
  • Fig. 17F is a forest plot showing OS for the validation dataset GSE39582 showing hazard ratios and associated p-values for the AVANT signature, the OncotypeDx signature, the T-effector signature, the CAF Isella et al. signature, and the F-TBRS Calon et al. signature and a table showing the significance of added prognostic value (if any) provided by each published signature (OncotypeDx signature, CAF Isella et al. signature, F-TBRS Calon et al. signature, and CMS subtypes signature) when added to the AVANT signature (first column) and the significance of added prognostic value provided by the AVANT signature when added to each of the individual published signatures (second column).
  • Fig. 18A is a set of Kaplan-Meier curves for the proliferative gene cluster from Fig. 1 C, stratified by median, in the GSE39582 dataset for OS. P-values correspond to a log-rank test.
  • Fig. 18B is a set of Kaplan-Meier curves for the immune gene cluster from Fig. 1 C, stratified by median, in the GSE39582 dataset for OS. P-values correspond to a log-rank test.
  • Fig. 18C is a set of Kaplan-Meier curves for the two stromal gene clusters combined from Fig.
  • Fig. 19A is a set of Kaplan-Meier curves for GZMB expression stratified by median expression in the AVANT BEP dataset for OS.
  • Fig. 19B is a set of Kaplan-Meier curves for GZMB expression stratified by median expression in the GSE39582 dataset for OS.
  • Fig. 19C is a set of Kaplan-Meier curves for the T-effector signature stratified by median expression in the AVANT BEP dataset for OS.
  • Fig. 19D is a set of Kaplan-Meier curves for the T-effector signature stratified by median expression in the GSE39582 dataset for OS.
  • Fig. 19E is a forest plot showing OS and associated P-values for the T-effector signature without GZMB and for GZMB expression in the AVANT BEP dataset and a table indicating the significance of added prognostic value (if any) provided by the T-effector signature when added to GZMB (first column) and vice versa (second column).
  • Fig. 19F is a forest plot showing OS and associated P-values for the T-effector signature without GZMB and for GZMB expression in the GSE39582 dataset and a table indicating the significance of added prognostic value (if any) provided by the T-effector signature when added to GZMB (first column) and vice versa (second column).
  • Fig. 19G is a set of Kaplan-Meier curves showing the relationship between GZMB and the proliferative genes of the AVANT signature in the AVANT BEP dataset for OS. Proliferative signature and GZMB expression were stratified by median expression. P-values correspond to a log-rank test.
  • Fig. 19H is a set of Kaplan-Meier curves showing the relationship between GZMB and the proliferative genes of the AVANT signature in the GSE39582 dataset for OS. Proliferative signature and GZMB expression were stratified by median expression. P-values correspond to a log-rank test.
  • Fig. 191 is a set of Kaplan-Meier curves showing the relationship between GZMB and the stromal genes of the AVANT signature in the AVANT BEP dataset for OS. Stromal signature and GZMB expression were stratified by median expression. P-values correspond to a log-rank test.
  • Fig. 19J is a set of Kaplan-Meier curves showing the relationship between GZMB and the stromal genes of the AVANT signature in the GSE39582 dataset for OS. Stromal signature and GZMB expression were stratified by median expression. P-values correspond to a log-rank test.
  • Fig. 20 is an OS forest plot showing hazard ratios and associated P-values for each individual signature in the AVANT BEP dataset and a table indicating the significance of added prognostic value (if any) provided by each published signature when added to the AVANT signature (first column) and vice versa (second column).
  • Fig. 21 A is a table and a forest plot reporting multivariate analysis results for the AVANT signature in the AVANT dataset.
  • Forest plot denotes the Cox hazard ratios (FIR) and p-values for the different signature quartiles after adjustment for clinical covariates.
  • FIR Cox hazard ratios
  • Each individual clinical covariate in each trial was tested for its effect on OS, and only prognostic covariates were included in the multivariate analyses. Included covariates were age (AGE-CAT), sex, level of carcinoembryonic antigen (CEA) in blood (CEABL), Eastern Cooperative Oncology Group (ECOG) status (ECOGSTAT), and American Joint Committee on Cancer (AJCC) tumor status including lymph node status (i.e. strata).
  • AGE-CAT age
  • CEA level of carcinoembryonic antigen
  • CEABL level of carcinoembryonic antigen
  • ECOG Eastern Cooperative Oncology Group
  • Fig. 21 B is a table and a forest plot reporting multivariate analysis results for the AVANT signature in the GSE39582 dataset.
  • Forest plot denotes the Cox hazard ratios (HR) and p-values for the different signature quartiles after adjustment for clinical covariates.
  • HR Cox hazard ratios
  • p-values for the different signature quartiles after adjustment for clinical covariates.
  • HR Cox hazard ratios
  • p-values for the different signature quartiles after adjustment for clinical covariates.
  • Each individual clinical covariate in each trial was tested for its effect on OS, and only prognostic covariates were included in the multivariate analyses. Included covariates were age (age.at.diagnosis), sex, tumor stage (tnm.stage), and lymph node status (tnm.n).
  • AVANT score refers to a numerical value that reflects an aggregated expression level for a set of genes of interest (e.g., the AVANT signature genes set forth in Table 1 or Table 2), wherein one or more genes of the set relate to proliferative biological functions, one or more genes of the set relate to stromal biological functions, and one or more genes of the set relate to immune biological functions, such that the set of genes of interest collectively reflect proliferative, stromal, and immune biological functions.
  • An AVANT score for a set of genes of interest may be determined by aggregation methods known to one skilled in the art and also disclosed herein, including, for example, by calculating the median or mean of all the expression levels of the genes of interest.
  • the expression level of each gene of interest may be normalized by using statistical methods known to one skilled in the art and also disclosed herein, including, for example, normalized to the expression level of one or more housekeeping genes, or normalized to a total library size, or normalized to the median or mean expression level value across all genes measured.
  • the normalized expression level of each gene of interest may be standardized by using statistical methods known to one skilled in the art and also disclosed herein, including, for example, by calculating the Z-score of the normalized expression level of each gene of interest.
  • each gene of interest may have an assigned weight score and the AVANT score for a set of genes of interest may be calculated by incorporating the weight score to determine the mean of all the weighted expression level of the genes of interest.
  • An AVANT score may, for example, refer to a numerical value that reflects the aggregated normalized expression level (e.g., median of the normalized expression levels, or mean of the normalized expression levels) for the AVANT signature genes set forth in Table 1 or Table 2).
  • an AVANT score may, for example, refer to a numerical value that reflects the aggregated Z-score expression level (e.g., mean of the Z-score normalized expression level, or median of the Z-score normalized expression level) for the AVANT signature genes set forth in Table 1 or Table 2.
  • An AVANT score may be detected in a sample (e.g., a blood sample (e.g., a tissue biopsy, a whole blood sample, a buccal swab, a plasma sample, a serum sample, or a combination thereof)) obtained from an individual (e.g., an individual having a CRC or having had surgical resection of a CRC).
  • a blood sample e.g., a tissue biopsy, a whole blood sample, a buccal swab, a plasma sample, a serum sample, or a combination thereof
  • an individual e.g., an individual having a CRC or having had surgical resection of a CRC.
  • the term“reference AVANT score” refers to an AVANT score against which another AVANT score is compared, e.g., to make a diagnostic, predictive, prognostic, and/or therapeutic determination.
  • the reference AVANT score may be derived from expression levels (e.g., for the AVANT signature genes set forth in Table 1 or Table 2) in a reference sample, a reference population, and/or a pre-assigned value (e.g., a cut-off value which was previously determined to significantly (e.g., statistically significantly)) separate a first subset and a second subset of individuals in the reference population based on responsiveness to treatment, e.g., overall survival (OS), disease-free survival (DFS), and/or recurrence-free survival (RFS).
  • OS overall survival
  • DFS disease-free survival
  • RFS recurrence-free survival
  • the numerical value for the reference AVANT score may vary depending on the indication (e.g., a cancer (e.g., a CRC, e.g., a stage I, stage II, stage III, or stage IV CRC), the methodology used to detect expression levels (e.g., digital counting of nucleic acids is by NANOSTRING ® NCOUNTER ® analysis), the statistical methods used to generate an AVANT score, and/or the specific combinations of genes examined (e.g., the AVANT signature genes set forth in Table 1 or Table 2).
  • a cancer e.g., a CRC, e.g., a stage I, stage II, stage III, or stage IV CRC
  • the methodology used to detect expression levels e.g., digital counting of nucleic acids is by NANOSTRING ® NCOUNTER ® analysis
  • the statistical methods used to generate an AVANT score e.g., the AVANT signature genes set forth in Table 1 or Table 2.
  • AVANT stromal score refers to a numerical value that reflects an aggregated expression level for a set of genes of interest (e.g., a set of genes comprising ABCC9, CRYAB, DTX1, HEYL, RGS2, SPP1, TLL 1, WBSCR17, IGFBP1, KDM5D, PCSK1, REG4, SMAD3, SMAD9, and TCF12, e.g., a set of genes comprising ABCC9, CRYAB, DTX1, HEYL, RGS2, SPP1, TLL 1, WBSCR17, IGFBP1, KDM5D, PCSK1, REG4, SMAD3, SMAD9, and TCF12, as well as one or more of ANGPT1, IGFBP3, CD36, HBEGF, FOS, SERPINB13, APCDD1, SCD5, POU5F1, EPHA4, FLT1, CDK6, and/or MAPK3, e.g., a set of genes comprising ANGPT1, W
  • An AVANT stromal score for a set of genes of interest may be determined by aggregation methods known to one skilled in the art and also disclosed herein, including, for example, by calculating the median or mean of all the expression levels of the genes of interest.
  • the expression level of each gene of interest may be normalized by using statistical methods known to one skilled in the art and also disclosed herein, including, for example, normalized to the expression level of one or more housekeeping genes, or normalized to a total library size, or normalized to the median or mean expression level value across all genes measured.
  • the normalized expression level of each gene of interest may be standardized by using statistical methods known to one skilled in the art and also disclosed herein, including, for example, by calculating the Z-score of the normalized expression level of each gene of interest.
  • each gene of interest may have an assigned weight score and the AVANT stromal score for a set of genes of interest may be calculated by incorporating the weight score to determine the mean of all the weighted expression level of the genes of interest.
  • An AVANT stromal score may, for example, refer to a numerical value that reflects the aggregated normalized expression level (e.g., median of the normalized expression levels, or mean of the normalized expression levels) for the set of genes comprising ABCC9, CRYAB, DTX1, HEYL, RGS2, SPP1, TLL 1, WBSCR17, IGFBP1, KDM5D, PCSK1, REG4, SMAD3, SMAD9, and TCF12, e.g., a set of genes comprising ABCC9, CRYAB, DTX1, HEYL, RGS2, SPP1, TLL 1, WBSCR17, IGFBP1, KDM5D, PCSK1, REG4, SMAD3, SMAD9, and TCF12, as well as one or more of ANGPT1, IGFBP3, CD36, HBEGF, FOS, SERPINB13, APCDD 1, SCD5, POU5F1, EPHA4, FLT1, CDK6, and/or MAPK3, e.g
  • an AVANT stromal score may, for example, refer to a numerical value that reflects the aggregated Z-score expression level (e.g., mean of the Z-score normalized expression level, or median of the Z-score normalized expression level) for the set of genes comprising ABCC9, CRYAB, DTX1, HEYL, RGS2, SPP1, TLL 1, WBSCR17, IGFBP1, KDM5D, PCSK1, REG4, SMAD3, SMAD9, and TCF12, e.g., a set of genes comprising ABCC9, CRYAB, DTX1, HEYL, RGS2, SPP1, TLL 1, WBSCR17, IGFBP1, KDM5D, PCSK1, REG4, SMAD3, SMAD9, and TCF12, as well as one or more of ANGPT1, IGFBP3, CD36, HBEGF, FOS, SERPINB13, APCDD1, SCD5, POU5F1, EPHA4, FLT1,
  • An AVANT stromal score may be detected in a sample (e.g., a blood sample (e.g., a tissue biopsy, a whole blood sample, a buccal swab, a plasma sample, a serum sample, or a combination thereof)) obtained from an individual (e.g., an individual having a CRC or having had surgical resection of a CRC).
  • a sample e.g., a blood sample (e.g., a tissue biopsy, a whole blood sample, a buccal swab, a plasma sample, a serum sample, or a combination thereof)
  • an individual e.g., an individual having a CRC or having had surgical resection of a CRC.
  • an AVANT stromal score that is above a reference AVANT stromal score when associated with an expression level of GZMB that is below a reference expression level of GZMB, identifies an individual as one who may benefit from an adjuvant treatment comprising a chemotherapy
  • an AVANT stromal score that is below a reference AVANT stromal score when associated with an expression level of GZMB that is above a reference expression level of GZMB, identifies an individual as one who is at low risk of CRC recurrence following surgical resection.
  • the term“reference AVANT stromal score” refers to an AVANT stromal score against which another AVANT stromal score is compared, e.g., to make a diagnostic, predictive, prognostic, and/or therapeutic determination.
  • the reference AVANT stromal score may be derived from expression levels (e.g., for a set of genes comprising ABCC9, CRYAB, DTX1, HEYL, RGS2, SPP1, TLL 1, WBSCR17, IGFBP1, KDM5D, PCSK1, REG4, SMAD3, SMAD9, and TCF12, e.g., a set of genes comprising ABCC9, CRYAB, DTX1, HEYL, RGS2, SPP1, TLL 1, WBSCR17, IGFBP1, KDM5D, PCSK1, REG4, SMAD3, SMAD9, and TCF12, as well as one or more of ANGPT1, IGFBP3, CD36, HBEGF, FOS, SERPINB13, APCDD1, SCD5, POU5F1, EPHA4, FLT1, CDK6, and/or MAPK3, e.g., a set of genes comprising ANGPT1, WBSCR17, TLL 1, CRYAB, ABCC
  • the numerical value for the reference AVANT stromal score may vary depending on the indication (e.g., a cancer (e.g., a CRC, e.g., a stage I, stage II, stage III, or stage IV CRC), the methodology used to detect expression levels (e.g., digital counting of nucleic acids is by NANOSTRING ® NCOUNTER ® analysis), the statistical methods used to generate an AVANT stromal score, and/or the specific combinations of genes examined (e.g., a set of genes comprising ABCC9, CRYAB, DTX1, HEYL, RGS2, SPP1, TLL 1, WBSCR17, IGFBP1, KDM5D, PCSK1, REG4, SMAD3, SMAD9, and TCF12, e.g., a set of genes comprising ABCC9, CRYAB, DTX1, HEYL, RGS2, SPP1, TLL 1, WBSCR17, IGFBP1, KDM5D, PCSK1, REG4,
  • KRAS or“GTPase KRas,” as used herein, broadly refers to any native KRAS from any mammalian source, including primates (e.g. humans) and rodents (e.g., mice and rats), unless otherwise indicated.
  • the term also encompasses naturally occurring variants of KRAS, e.g., splice variants or allelic variants.
  • the amino acid sequence of an exemplary human KRAS is shown under UniProt Accession No. P01 1 16 or in SEQ ID NO: 32.
  • KRAS status refers to the presence or absence of one or more changes at an assayed position in the nucleotide or amino acid sequence of KRAS, e.g., one or more nucleotide substitution mutations resulting in a mutation at position 12 of the KRAS amino acid sequence (e.g. a G12A, G12R, G12D, G12C, G12S, G12V, or G12D mutation) or a mutation at position 13 of the KRAS amino acid sequence (e.g., a G13D mutation), as described in Marisa et al.
  • the change in the nucleotide sequence may also be any missense or nonsense mutation that is observed in the CRC, but not in adjacent normal tissue (Stransky et al., Science, 333: 1 157-1 160, 201 1 ).
  • KRAS status is considered to be“wild-type” or“WT” if no mutations are detected at any of the assayed position, and“mutant” or“mut” if at least one mutation is detected at any of the assayed positions.
  • BRAF or“Human Serine/threonine-protein kinase B-raf,” as used herein, broadly refers to any native BRAF from any mammalian source, including primates (e.g. humans) and rodents (e.g., mice and rats), unless otherwise indicated.
  • the term also encompasses naturally occurring variants of BRAF, e.g., splice variants or allelic variants.
  • the amino acid sequence of an exemplary human BRAF is shown under UniProt Accession No. P15056 or in SEQ ID NO: 33.
  • the term“BRAF status” refers to the presence or absence of one or more changes at an assayed position in the nucleotide or amino acid sequence of BRAF.
  • the change in the nucleotide sequence may be, e.g., a c. 1799T>A nucleotide substitution mutation, e.g., a mutation resulting in a V600E change in the amino acid sequence (Marisa et al. , PLoS Med, 1 0: e1001453, 2013).
  • the change in the nucleotide sequence may also be any missense or nonsense mutation that is observed in the CRC, but not in adjacent normal tissue (Stransky et al., Science, 333: 1 157-1 160, 201 1 ).
  • BRAF status is considered to be“wild-type” or“WT” if no mutations are detected at any of the assayed position, and“mutant” or“mut” if at least one mutation is detected at any of the assayed positions.
  • adjuvant treatment or“adjuvant therapy” herein refers to treatment or therapy given after surgery (e.g., surgical resection of a CRC), where no evidence of residual disease can be detected, so as to reduce the risk of disease recurrence (e.g., recurrence of a CRC).
  • the goal of adjuvant treatment is to prevent recurrence of the cancer, and therefore to reduce the chance of cancer-related death.
  • cancer and“cancerous” refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth/proliferation. Aspects of cancer include solid tumor cancers. In some aspects, the solid tumor cancer is a colorectal cancer (CRC) or a metastatic form thereof.
  • CRC colorectal cancer
  • a CRC refers to a cancer that develops from the large intestine, e.g., the colon or rectum.
  • a CRC is characterized as consensus molecular subtype 1 (CMS1 ), CMS2, CMS3, CMS4, or unclassified.
  • CCS1 consensus molecular subtype 1
  • CMS2 CMS2, CMS3, CMS4, or unclassified.
  • a CRC is a left-sided tumor, i.e. , a tumor occurring in the distal colon (e.g., the distal third of the transverse colon, the splenic flexure the descending colon, the sigmoid colon, or the rectum).
  • a CRC is a right-sided tumor, i.e., a tumor occurring in the proximal colon (e.g., the proximal two- thirds of the transverse colon, the ascending colon, and the cecum).
  • Right-sided tumors may be associated with decreased OS.
  • the CRC is metastatic.
  • the CRC is a colon carcinoma.
  • the colon carcinoma is CMS1 , CMS2, CMS3, CMS4, or unclassified.
  • the colon carcinoma is a left-sided colon carcinoma.
  • the colon carcinoma is a right-sided colon carcinoma.
  • the stage of a CRC is assessed according to the American Joint Committee on Cancer (AJCC)/Union for International Cancer Control (UICC) TNM Classification of Malignant Tumors (TNM) classification system.
  • TNM TNM Classification of Malignant Tumors
  • cancers are designated the letter T (tumor size), N (palpable nodes), and/or M (metastases).
  • T1 , T2, T3, and T4 describe the increasing size of the primary lesion.
  • T1 , T2, T3, and T4 may additionally be classified as a or b (e.g., T4a or T4b) to provide further information about the status, e.g., local advancement, of the cancer.
  • the CRC of an individual is a stage I, stage II, or stage III CRC, e.g., a stage I, stage II, or stage III colon carcinoma.
  • an individual does not have a stage IV CRC.
  • an individual does not have a metastatic CRC.
  • the CRC of an individual in a reference population is a stage I, stage II, stage III, or stage IV CRC, e.g., a stage I, stage II, stage III, or stage IV colon carcinoma.
  • the CRC of an individual or an individual in a reference population is a stage III - N1 , stage III - N2, or high-risk stage II colon carcinoma according to the TNM classification system.
  • A“high-risk stage II colon carcinoma” e.g., high risk for recurrence of the CRC following surgical resection
  • TNM status is confirmed by histology.
  • CRC Consensus molecular subtype
  • CMS1 consensus molecular subtype 1
  • CIMP consensus molecular subtype 2
  • CCS2 consensus molecular subtype 2
  • CMS2 may be characterized by high somatic copy number alterations (SCNA) and WNT and MYC signaling activation.
  • SCNA somatic copy number alterations
  • a CRC is identified as consensus molecular subtype 3 (CMS3) (metabolic).
  • CMS3 may be characterized by mixed MSI status, low SCNA, low CIMP, KRAS mutations, and evident metabolic dysregulation.
  • a colorectal cancer is identified as consensus molecular subtype 4 (CMS4)
  • CMS4 may be characterized by high SCNA, stromal infiltration, prominent TGFp activation, angiogenesis, and worse relapse-free (recurrence-free) and overall survival.
  • a CRC has a heterogeneous expression pattern that is mixed or indeterminate relative to the CMS1 , CMS2, CMS3, and CMS4 categories.
  • “microsatellite instability status” or“MSI status” refers to a characterization of microsatellite stability in a tumor tissue of a patient.
  • the tumor tissue of a patient may be characterized as“microsatellite instability high” (“MSI-H”) or“microsatellite stable” or“microsatellite instability low” (“MSS”).
  • MSI status may be assessed, for example, by using a PCR-based approach such as the MSI Analysis System (Promega, Madison, Wl), which is comprised of 5 pseudomonomorphic mononucleotide repeats (BAT-25, BAT-26, NR-21 , NR-24, and MONO-27) to detect MSI and 2 pentanucleotide loci (PentaC and PendaD) to confirm identity between normal and tumor samples.
  • the size in bases for each microsatellite locus can be determined, e.g., by gel electrophoresis, and a tumor may be designated MSI- H if two or more mononucleotide loci vary in length compared to the germline DNA. See, e.g., Le et al. NEJM 372:2509-2520, 201 5.
  • a patient may have a low level of microsatellite instability (e.g., MSS).
  • Tumor refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues.
  • the terms“cancer,”“cancerous,”“cell proliferative disorder,”“proliferative disorder,” and“tumor” are not mutually exclusive as referred to herein.
  • Tumor cell refers to any tumor cell present in a tumor or a sample thereof. Tumor cells may be distinguished from other cells that may be present in a tumor sample, for example, stromal cells and tumor-infiltrating immune cells, using methods known in the art and/or described herein.
  • reducing or inhibiting cancer recurrence means to reduce or inhibit tumor or cancer relapse (recurrence) or tumor or cancer progression.
  • cancer recurrence and/or cancer progression include, without limitation, cancer metastasis.
  • partial response or“PR” refers to a decrease in the size of one or more tumors or lesions, or in the extent of cancer in the body, in response to treatment.
  • PR refers to at least a 30% decrease in the sum of the longest diameters (SLD) of target lesions, taking as reference the baseline SLD.
  • “stable disease” or“SD” refers to neither sufficient shrinkage of target lesions to qualify for PR, nor sufficient increase to qualify for PD, taking as reference the smallest SLD since the treatment started.
  • progressive disease or“PD” refers to at least a 20% increase in the SLD of target lesions, taking as reference the smallest SLD recorded since the treatment started or the presence of one or more new lesions.
  • the term“survival” refers to the patient remaining alive, and includes overall survival as well as progression-free survival, disease-free survival, and recurrence-free survival.
  • DFS disease-free survival
  • RFS recurrence-free survival
  • disease-free survival is defined as the time between randomization (e.g., assignment to an adjuvant treatment group) and recurrence of a colorectal cancer, new occurrence of a colorectal cancer, or death from any cause.
  • “overall survival” or“OS” refers to the percentage of individuals in a group who are likely to be alive after a particular duration of time.
  • progression-free survival refers to the length of time during and after treatment during which the disease being treated (e.g., cancer) does not get worse. Progression-free survival may include the amount of time patients have experienced a complete response or a partial response, as well as the amount of time patients have experienced stable disease.
  • extending survival is meant increasing overall survival, progression-free survival, disease- free survival, or recurrence-free survival in a treated patient relative to an untreated patient (i.e. relative to a patient not treated with the medicament).
  • hazard ratio is a statistical definition for rates of events.
  • hazard ratio is defined as representing the probability of an event (e.g., PFS or OS) in the experimental (e.g., treatment) group/arm divided by the probability of an event in the control group/arm at any specific point in time.
  • An HR with a value of 1 indicates that the relative risk of an endpoint (e.g., death) is equal in both the“treatment” and“control” groups; a value greater than 1 indicates that the risk is greater in the treatment group relative to the control group; and a value less than 1 indicates that the risk is greater in the control group relative to the treatment group.
  • “Hazard ratio” in progression-free survival analysis is a summary of the difference between two progression- free survival curves, representing the reduction in the risk of death on treatment compared to control, over a period of follow-up.
  • “Hazard ratio” in overall survival analysis is a summary of the difference between two overall survival curves, representing the reduction in the risk of death on treatment compared to control, over a period of follow-up.
  • an“effective amount” of a compound for example, a chemotherapy or a composition thereof, is at least the minimum amount required to achieve the desired therapeutic or prophylactic result, such as a measurable improvement or prevention of a particular disorder (e.g., a cell proliferative disorder, e.g., cancer, e.g., a urinary tract cancer).
  • a particular disorder e.g., a cell proliferative disorder, e.g., cancer, e.g., a urinary tract cancer.
  • An effective amount herein may vary according to factors such as the disease state, age, sex, and weight of the patient, and the ability of the antibody to elicit a desired response in the individual.
  • An effective amount is also one in which any toxic or detrimental effects of the treatment are outweighed by the therapeutically beneficial effects.
  • beneficial or desired results include results such as eliminating or reducing the risk, lessening the severity, or delaying the onset of the disease, including biochemical, histological and/or behavioral symptoms of the disease, its complications, and intermediate pathological phenotypes presenting during development of the disease.
  • beneficial or desired results include clinical results such as decreasing one or more symptoms resulting from the disease, increasing the quality of life of those suffering from the disease, decreasing the dose of other medications required to treat the disease, enhancing effect of another medication such as via targeting, delaying the progression of the disease, and/or prolonging survival.
  • an effective amount of the drug may have the effect in reducing the number of cancer cells; reducing the tumor size; inhibiting (i.e., slow to some extent or desirably stop) cancer cell infiltration into peripheral organs; inhibit (i.e., slow to some extent and desirably stop) tumor metastasis; inhibiting to some extent tumor growth ; and/or relieving to some extent one or more of the symptoms associated with the disorder.
  • An effective amount can be administered in one or more administrations.
  • composition is an amount sufficient to accomplish prophylactic or therapeutic treatment either directly or indirectly.
  • an effective amount of a drug, compound, or pharmaceutical composition may or may not be achieved in conjunction with another drug, compound, or pharmaceutical composition.
  • an“effective amount” may be considered in the context of administering one or more therapeutic agents, and a single agent may be considered to be given in an effective amount if, in conjunction with one or more other agents, a desirable result may be or is achieved.
  • pharmaceutical formulation refers to a preparation which is in such form as to permit the biological activity of an active ingredient contained therein to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered.
  • A“pharmaceutically acceptable carrier” refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, which is nontoxic to a subject.
  • a pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative.
  • treatment refers to clinical intervention in an attempt to alter the natural course of the individual being treated, and can be performed either for prophylaxis or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis.
  • a chemotherapeutic agent is used to delay development of a disease or to slow the progression of a disease.
  • anti-cancer therapy refers to a therapy useful in treating cancer.
  • anti cancer therapeutic agents include, but are not limited to, cytotoxic agents, chemotherapeutic agents, growth inhibitory agents, agents used in radiation therapy, anti-angiogenesis agents, apoptotic agents, anti-tubulin agents, and other agents to treat cancer, for example, anti-CD20 antibodies, platelet derived growth factor inhibitors (e.g., GLEEVECTM (imatinib mesylate)), a COX-2 inhibitor (e.g., celecoxib), interferons, cytokines, antagonists (e.g., neutralizing antibodies) that bind to one or more of the following targets PDGFR-b, BlyS, APRIL, BCMA receptor(s), TRAIL/ Apo2, other bioactive and organic chemical agents, and the like. Combinations thereof are also included in the invention.
  • “Chemotherapeutic agent” includes chemical compounds useful in the treatment of cancer.
  • chemotherapeutic agents include oxaliplatin (ELOXATIN®, Sanofi), 5-FU (5-fluorouracil), leucovorin, capecitabine (XELODA®); FOLFOX, an abbreviation for a treatment regimen with oxaliplatin (ELOXATINTM) combined with 5-FU and leucovorin; and XELOX, an abbreviation for a treatment regimen with oxaliplatin and capecitabine.
  • the FOLFOX treatment regimen may be, e.g., a FOLFOX4 treatment regimen.
  • Treatment with FOLFOX (e.g., FOLFOX4) or XELOX may further comprise treatment with bevacizumab (AVASTIN®, Genentech), e.g., concurrent treatment with bevacizumab.
  • chemotherapeutic agents include erlotinib (TARCEVA®, Genentech/OSI Pharm.), bortezomib (VELCADE®, Millennium Pharm.), disulfiram, epigallocatechin gallate ,
  • dynemicin including dynemicin A; bisphosphonates, such as clodronate; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN® (doxorubicin), morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino- doxorubicin and deoxydoxorubicin), epirubicin, 6-diazo-5-oxo-L-norleucine,
  • aminoglutethimide aminoglutethimide, mitotane, trilostane
  • folic acid replenisher such as frolinic acid
  • aceglatone aminoglutethimide, mitotane, trilostane
  • folic acid replenisher such as frolinic acid
  • aceglatone aminoglutethimide, mitotane, trilostane
  • folic acid replenisher such as frolinic acid
  • aceglatone aminoglutethimide, mitotane, trilostane
  • folic acid replenisher such as frolinic acid
  • aceglatone aminoglutethimide, mitotane, trilostane
  • folic acid replenisher such as frolinic acid
  • aceglatone aminoglutethimide, mitotane, trilostane
  • folic acid replenisher such as frolinic acid
  • aceglatone aminoglutethimide, mitotane, trilostane
  • aldophosphamide glycoside aminolevulinic acid
  • eniluracil amsacrine
  • bestrabucil bisantrene
  • etoglucid gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidamnol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin; sizofuran; spirogermanium; tenuazonic acid; triaziquone; 2,2’,2”-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine; mannomustine;
  • TAXOL paclitaxel
  • ABRAXANE® Cremophor-free
  • albumin-engineered nanoparticle formulations of paclitaxel American Pharmaceutical Partners, Schaumberg, III.
  • TAXOTERE® docetaxel, doxetaxel; Sanofi-Aventis
  • chloranmbucil GEMZAR® (gemcitabine); 6- thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin;
  • vinblastine etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; NAVELBINE® (vinorelbine);
  • novantrone novantrone
  • teniposide edatrexate
  • daunomycin aminopterin
  • ibandronate CPT-1 1
  • topoisomerase inhibitor RFS 2000 difluoromethylornithine (DMFO); retinoids such as retinoic acid
  • Chemotherapeutic agent also includes (i) anti-hormonal agents that act to regulate or inhibit hormone action on tumors such as anti-estrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen (including NOLVADEX®; tamoxifen citrate), raloxifene, droloxifene, iodoxyfene , 4-hydroxytamoxifen, trioxifene, keoxifene, LY1 17018, onapristone, and FARESTON® (toremifine citrate); (ii) aromatase inhibitors that inhibit the enzyme aromatase, which regulates estrogen production in the adrenal glands, such as, for example, 4(5)-imidazoles, aminoglutethimide, MEGASE® (megestrol acetate), AROMASIN® (exemestane; Pfizer), formestanie, fadrozole, RIVISOR® (vorozole), FEMARA® (
  • protein kinase inhibitors e.g., IL-12 kinase inhibitors
  • lipid kinase inhibitors e.g., antisense oligonucleotides, particularly those which inhibit expression of genes in signaling pathways implicated in aberrant cell proliferation, such as, for example, PKC-alpha, Ralf and H-Ras
  • ribozymes such as VEGF expression inhibitors (e.g., ANGIOZYME®) and HER2 expression inhibitors
  • vaccines such as gene therapy vaccines, for example,
  • Chemotherapeutic agent also includes antibodies such as bevacizumab (AVASTIN®,
  • Additional humanized monoclonal antibodies with therapeutic potential as agents in combination with the compounds of the invention include: apolizumab, aselizumab, atlizumab, bapineuzumab, bivatuzumab mertansine, cantuzumab mertansine, cedelizumab, certolizumab pegol, cidfusituzumab, cidtuzumab, daclizumab, eculizumab, efalizumab, epratuzumab, erlizumab, felvizumab, fontolizumab, gemtuzumab ozogamicin, inotuzumab ozogamicin, ipilimumab, labetuzumab, lintuzumab, matuzumab, mepolizumab, motavizumab, motovizumab, natalizumab, nimotuzumab, nolovizum
  • Chemotherapeutic agent also includes“EGFR inhibitors,” which refers to compounds that bind to or otherwise interact directly with EGFR and prevent or reduce its signaling activity, and is alternatively referred to as an“EGFR antagonist.”
  • EGFR inhibitors refers to compounds that bind to or otherwise interact directly with EGFR and prevent or reduce its signaling activity
  • Examples of such agents include antibodies and small molecules that bind to EGFR.
  • antibodies which bind to EGFR include MAb 579 (ATCC CRL HB 8506), MAb 455 (ATCC CRL HB8507), MAb 225 (ATCC CRL 8508), MAb 528 (ATCC CRL 8509) (see, US Patent No.
  • EMD7200 a humanized EGFR antibody directed against EGFR that competes with both EGF and TGF-alpha for EGFR binding (EMD/Merck); human EGFR antibody, HuMax-EGFR (GenMab); fully human antibodies known as E1 .1 , E2.4, E2.5, E6.2, E6.4, E2.1 1 , E6. 3 and E7.6. 3 and described in US 6,235,883; MDX-447 (Medarex Inc); and mAb 806 or humanized mAb 806 (Johns et al., J. Biol.
  • the anti-EGFR antibody may be conjugated with a cytotoxic agent, thus generating an immunoconjugate (see, e.g., EP659,439A2, Merck Patent GmbH).
  • EGFR antagonists include small molecules such as compounds described in US Patent Nos: 5,616,582, 5,457,105, 5,475,001 , 5,654,307, 5,679,683, 6,084,095, 6,265,410, 6,455,534, 6,521 ,620, 6,596,726, 6,713,484, 5,770,599, 6,140,332, 5,866,572, 6,399,602, 6,344,459, 6,602,863, 6,391 ,874, 6,344,455, 5,760,041 , 6,002,008, and 5,747,498, as well as the following PCT publications: W098/14451 ,
  • EGFR antagonists include OSI-774 (CP-358774, erlotinib, TARCEVA® Genentech/OSI Pharmaceuticals); PD 183805 (Cl 1033, 2- propenamide, N-[4-[(3-chloro-4-fluorophenyl)amino]-7-[3-(4-morpholinyl)propoxy]-6-quinazolinyl]-, dihydrochloride, Pfizer Inc.) ; ZD1839, gefitinib (IRESSA®) 4-(3’-Chloro-4’-fluoroanilino)-7-methoxy-6-(3- morpholinopropoxy)quinazoline, AstraZeneca); ZM 105180 ((6-amino-4-(3-methylphenyl-amino)- quinazoline, Zeneca); BIBX-1382 (N8-
  • Chemotherapeutic agents also include“tyrosine kinase inhibitors” including the EGFR-targeted drugs noted in the preceding paragraph; small molecule HER2 tyrosine kinase inhibitor such as TAK165 available from Takeda; CP-724,714, an oral selective inhibitor of the ErbB2 receptor tyrosine kinase (Pfizer and OSI); dual-HER inhibitors such as EKB-569 (available from Wyeth) which preferentially binds EGFR but inhibits both HER2 and EGFR-overexpressing cells; lapatinib (GSK572016; available from Glaxo-SmithKIine), an oral HER2 and EGFR tyrosine kinase inhibitor; PKI-166 (available from Novartis); pan-HER inhibitors such as canertinib (CI-1033; Pharmacia); Raf-1 inhibitors such as antisense agent ISIS-5132 available from ISIS Pharmaceuticals which inhibit Raf-1 signaling; non-HER targeted
  • Chemotherapeutic agents also include dexamethasone, interferons, colchicine, metoprine, cyclosporine, amphotericin, metronidazole, alemtuzumab, alitretinoin, allopurinol, amifostine, arsenic trioxide, asparaginase, BCG live, bevacuzimab, bexarotene, cladribine, clofarabine, darbepoetin alfa, denileukin, dexrazoxane, epoetin alfa, elotinib, filgrastim, histrelin acetate, ibritumomab, interferon alfa- 2a, interferon alfa-2b, lenalidomide, levamisole, mesna, methoxsalen, nandrolone, nelarabine, nofetumomab, oprelvekin
  • temozolomide temozolomide, VM-26, 6-TG, toremifene, tretinoin, ATRA, valrubicin, zoledronate, and zoledronic acid, and pharmaceutically acceptable salts thereof.
  • Chemotherapeutic agents also include hydrocortisone, hydrocortisone acetate, cortisone acetate, tixocortol pivalate, triamcinolone acetonide, triamcinolone alcohol, mometasone, amcinonide, budesonide, desonide, fluocinonide, fluocinolone acetonide, betamethasone, betamethasone sodium phosphate, dexamethasone, dexamethasone sodium phosphate, fluocortolone, hydrocortisone-1 7- butyrate, hydrocortisone-1 7-valerate, aclometasone dipropionate, betamethasone valerate,
  • TNFa tumor necrosis factor alpha
  • etanercept Enbrel
  • infliximab Resmicade
  • adalimumab Humira
  • certolizumab pegol certolizumab pegol
  • golimumab Simponi
  • interleukin 1 (IL-1 ) blockers such as anakinra (Kineret)
  • T cell costimulation blockers such as abatacept (Orencia), interleukin 6 (IL-6) blockers such as tocilizumab (ACTEMERA®); interleukin 13 (IL-13) blockers such as lebrikizumab;
  • interferon alpha (IFN) blockers such as Rontalizumab
  • beta 7 integrin blockers such as rhuMAb Beta7
  • IFN interferon alpha
  • IgE pathway blockers such as Anti-M1 prime; Secreted homotrimeric LTa3 and membrane bound heterotrimer LTa1 /p2 blockers such as anti-lymphotoxin alpha (LTa); radioactive isotopes (e.g., At 21 1 , I 131 , I 125 , Y 90 , Re 186 , Re 188 , Sm 153 , Bi 212 , P 32 , Pb 212 , and radioactive isotopes of Lu); miscellaneous
  • investigational agents such as thioplatin, PS-341 , phenylbutyrate, ET-18- OCH3, or farnesyl transferase inhibitors (L-739749, L-744832); polyphenols such as quercetin, resveratrol, piceatannol,
  • epigallocatechine gallate theaflavins, flavanols, procyanidins, betulinic acid and derivatives thereof; autophagy inhibitors such as chloroquine; delta-9-tetrahydrocannabinol (dronabinol, MARINOL®); beta- lapachone; lapachol; colchicines; betulinic acid; acetylcamptothecin, scopolectin, and 9- aminocamptothecin); podophyllotoxin; tegafur (UFTORAL®); bexarotene (TARGRETIN®);
  • autophagy inhibitors such as chloroquine; delta-9-tetrahydrocannabinol (dronabinol, MARINOL®); beta- lapachone; lapachol; colchicines; betulinic acid; acetylcamptothecin, scopolectin, and 9- aminocamptothecin); podophyllotoxin; tegafur (UF
  • bisphosphonates such as clodronate (for example, BONEFOS® or OSTAC®), etidronate (DIDROCAL®), NE-58095, zoledronic acid/zoledronate (ZOMETA®), alendronate (FOSAMAX®), pamidronate
  • AREDIA® tiludronate
  • SKELID® tiludronate
  • ACTONEL® risedronate
  • EGF-R epidermal growth factor receptor
  • vaccines such as THERATOPE® vaccine
  • perifosine COX-2 inhibitor
  • COX-2 inhibitor e.g. celecoxib or etoricoxib
  • proteosome inhibitor e.g.
  • PS341 CCI-779; tipifarnib (R1 1577); orafenib, ABT510; Bcl-2 inhibitor such as oblimersen sodium (GENASENSE®); pixantrone; farnesyltransferase inhibitors such as lonafarnib (SCH 6636, SARASARTM); and pharmaceutically acceptable salts, acids or derivatives of any of the above; as well as combinations of two or more of the above such as CHOP, an abbreviation for a combined therapy of cyclophosphamide, doxorubicin, vincristine, and prednisolone.
  • Chemotherapeutic agents also include non-steroidal anti-inflammatory drugs with analgesic, antipyretic and anti-inflammatory effects.
  • NSAIDs include non-selective inhibitors of the enzyme cyclooxygenase.
  • Specific examples of NSAIDs include aspirin, propionic acid derivatives such as ibuprofen, fenoprofen, ketoprofen, flurbiprofen, oxaprozin and naproxen, acetic acid derivatives such as indomethacin, sulindac, etodolac, diclofenac, enolic acid derivatives such as piroxicam, meloxicam, tenoxicam, droxicam, lornoxicam and isoxicam, fenamic acid derivatives such as mefenamic acid, meclofenamic acid, flufenamic acid, tolfenamic acid, and COX-2 inhibitors such as celecoxib, etoricoxib, lumirac
  • NSAIDs can be indicated for the symptomatic relief of conditions such as rheumatoid arthritis, osteoarthritis, inflammatory arthropathies, ankylosing spondylitis, psoriatic arthritis, Reiter’s syndrome, acute gout, dysmenorrhoea, metastatic bone pain, headache and migraine, postoperative pain, mild-to-moderate pain due to inflammation and tissue injury, pyrexia, ileus, and renal colic.
  • conditions such as rheumatoid arthritis, osteoarthritis, inflammatory arthropathies, ankylosing spondylitis, psoriatic arthritis, Reiter’s syndrome, acute gout, dysmenorrhoea, metastatic bone pain, headache and migraine, postoperative pain, mild-to-moderate pain due to inflammation and tissue injury, pyrexia, ileus, and renal colic.
  • the term“immunomodulatory agent” refers to a therapeutic agent that modulates (e.g., up-regulates or down-regulates) an immune response.
  • An immunomodulatory agent may be, e.g., an immune checkpoint inhibitor.
  • the term“immune checkpoint inhibitor” refers to a therapeutic agent that targets at least one immune checkpoint protein to alter the regulation of an immune response, e.g., down-modulating, inhibiting, up-modulating, or activating an immune response.
  • the term “immune checkpoint blockade” may be used to refer to a therapy comprising an immune checkpoint inhibitor.
  • Immune checkpoint proteins include, without limitation, cytotoxic T- lymphocyte antigen 4 (CTLA-4), programmed cell death 1 (PD-1 ), programmed cell death ligand 1 (PD- L1 ), programmed cell death ligand 2 (PD-L2), V-domain Ig suppressor of T cell activation (VISTA), B7-H2, B7-H3, B7-H4, B7-H6, 2B4, ICOS, HVEM, CD160, gp49B, PIR-B, KIR family receptors, TIM-1 , TIM-3, TIM-4, LAG-3, BTLA, SIRPalpha (CD47), CD48, 2B4 (CD244), B7.1 , B7.2, ILT-2, ILT-4, TIGIT, LAG-3, BTLA, IDO, 0X40, and A2aR.
  • CTL-4 cytotoxic T- lymphocyte antigen 4
  • PD-1 programmed cell death 1
  • PD- L1 programmed cell death ligand 1
  • an immune checkpoint protein may be expressed on the surface of an activated T cell.
  • Therapeutic agents that can act as immune checkpoint inhibitors useful in the methods of the present invention include, but are not limited to, therapeutic agents that target one or more of CTLA-4, PD-1 , PD-L1 , PD-L2, VISTA, B7-H2, B7-H3, B7-H4, B7-H6, 2B4, ICOS, HVEM, CD160, gp49B, PIR-B, KIR family receptors, TIM-1 , TIM-3, TIM-4, LAG- 3, BTLA, SIRPalpha (CD47), CD48, 2B4 (CD244), B7.1 , B7.2, ILT-2, ILT-4, TIGIT, LAG-3, BTLA, IDO, 0X40, and A2aR.
  • an immune checkpoint inhibitor enhances or suppresses the function of one or more targeted immune checkpoint proteins.
  • the immune checkpoint inhibitor is a PD-L1 axis binding antagonist, such as atezolizumab, as described herein.
  • PD-1 axis binding antagonist refers to a molecule that inhibits the interaction of a PD-1 axis binding partner with either one or more of its binding partner, so as to remove T cell dysfunction resulting from signaling on the PD-1 signaling axis - with a result being to restore or enhance T cell function (e.g., proliferation, cytokine production, target cell killing).
  • a PD-1 axis binding antagonist includes a PD-1 binding antagonist, a PD-L1 binding antagonist, and a PD-L2 binding antagonist.
  • PD-1 binding antagonist refers to a molecule that decreases, blocks, inhibits, abrogates or interferes with signal transduction resulting from the interaction of PD-1 with one or more of its binding partners, such as PD-L1 , PD-L2.
  • the PD-1 binding antagonist is a molecule that inhibits the binding of PD-1 to one or more of its binding partners.
  • the PD-1 binding antagonist inhibits the binding of PD-1 to PD-L1 and/or PD-L2.
  • PD-1 binding antagonists include anti-PD-1 antibodies, antigen binding fragments thereof, immunoadhesins, fusion proteins, oligopeptides and other molecules that decrease, block, inhibit, abrogate or interfere with signal transduction resulting from the interaction of PD-1 with PD-L1 and/or PD-L2.
  • a PD-1 binding antagonist reduces the negative co-stimulatory signal mediated by or through cell surface proteins expressed on T lymphocytes mediated signaling through PD-1 so as render a dysfunctional T cell less dysfunctional (e.g., enhancing effector responses to antigen recognition).
  • the PD-1 binding antagonist is an anti-PD-1 antibody.
  • a PD-1 binding antagonist is MDX-1 106 (nivolumab).
  • a PD-1 binding antagonist is MK-3475
  • a PD-1 binding antagonist is AMP-224.
  • a PD-1 binding antagonist is MED1 -0680.
  • a PD-1 binding antagonist is PDR001 (spartalizumab).
  • a PD-1 binding antagonist is REGN281 0
  • a PD-1 binding antagonist is BGB-108.
  • PD-L1 binding antagonist refers to a molecule that decreases, blocks, inhibits, abrogates or interferes with signal transduction resulting from the interaction of PD-L1 with either one or more of its binding partners, such as PD-1 and B7-1 .
  • a PD-L1 binding antagonist is a molecule that inhibits the binding of PD-L1 to its binding partners.
  • the PD-L1 binding antagonist inhibits binding of PD-L1 to PD-1 and/or B7-1 .
  • the PD-L1 binding antagonists include anti-PD-L1 antibodies, antigen binding fragments thereof, immunoadhesins, fusion proteins, oligopeptides and other molecules that decrease, block, inhibit, abrogate or interfere with signal transduction resulting from the interaction of PD-L1 with one or more of its binding partners, such as PD-1 and B7-1 .
  • a PD-L1 binding antagonist reduces the negative co-stimulatory signal mediated by or through cell surface proteins expressed on T lymphocytes mediated signaling through PD- L1 so as to render a dysfunctional T cell less dysfunctional (e.g., enhancing effector responses to antigen recognition).
  • a PD-L1 binding antagonist is an anti-PD-L1 antibody.
  • an anti-PD-L1 antibody is MPDL3280A (atezolizumab, marketed as TECENTRIQTM with a WHO Drug Information (International Nonproprietary Names for Pharmaceutical Substances),
  • an anti-PD-L1 antibody is YW243.55.S70.
  • an anti-PD-L1 antibody is MDX-1 105.
  • an anti PD-L1 antibody is MSB0015718C.
  • an anti-PD-L1 antibody is MEDI4736.
  • PD-L2 binding antagonist refers to a molecule that decreases, blocks, inhibits, abrogates or interferes with signal transduction resulting from the interaction of PD-L2 with either one or more of its binding partners, such as PD-1 .
  • a PD-L2 binding antagonist is a molecule that inhibits the binding of PD-L2 to one or more of its binding partners.
  • the PD-L2 binding antagonist inhibits binding of PD-L2 to PD-1 .
  • the PD-L2 antagonists include anti-PD-L2 antibodies, antigen binding fragments thereof, immunoadhesins, fusion proteins, oligopeptides and other molecules that decrease, block, inhibit, abrogate or interfere with signal transduction resulting from the interaction of PD-L2 with either one or more of its binding partners, such as PD-1 .
  • a PD-L2 binding antagonist reduces the negative co-stimulatory signal mediated by or through cell surface proteins expressed on T lymphocytes mediated signaling through PD-L2 so as render a dysfunctional T cell less dysfunctional (e.g., enhancing effector responses to antigen recognition).
  • a PD-L2 binding antagonist is an immunoadhesin.
  • A“subject” or an“individual” is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain aspects, the subject or individual is a human.
  • administering is meant a method of giving a dosage of a compound (e.g., a chemotherapeutic agent) to a subject.
  • a compound e.g., a chemotherapeutic agent
  • the compositions utilized in the methods herein are administered intravenously.
  • compositions utilized in the methods described herein can be administered, for example, intramuscularly, intravenously, intradermally, percutaneously, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostatically, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, intrarectally, topically, intratumorally, peritoneally, subcutaneously, subconjunctivally, intravesicularlly, mucosally, intrapericardially, intraumbilically, intraocularly, orally, topically, locally, by inhalation, by injection, by infusion, by continuous infusion, by localized perfusion bathing target cells directly, by catheter, by lavage, in cremes, or in lipid compositions.
  • the method of administration can vary depending on various factors (e.g., the compound or composition being administered and the severity of the condition, disease, or disorder being treated).
  • concurrent administration includes a dosing regimen when the administration of one or more agent(s) continues after discontinuing the administration of one or more other agent(s).
  • Reduce or inhibit is meant the ability to cause an overall decrease of 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or greater.
  • Reduce or inhibit can refer, for example, to the symptoms of the disorder being treated, the presence or size of metastases, or the size of the primary tumor.
  • the invention features prognostic gene signatures for colorectal cancer (CRC) (e.g., an AVANT gene signature and a gene signature comprising the AVANT stromal genes and Granzyme B ( GZMB )), which, when measured and quantified as a score or expression level in a sample from an individual (e.g., an AVANT score, or an AVANT stromal score and a GZMB expression level), may be used to identify individuals that are at high risk or low risk of recurrence of a CRC following surgical resection of the CRC.
  • CRC colorectal cancer
  • GZMB Granzyme B
  • prognostic gene signatures and the related scores enable improved strategies to guide diagnostic and therapeutic decisions for patients having a CRC, e.g., a stage I, stage II, or stage III CRC, or having had surgical resection of a CRC. Specifically, they may be used to identify individuals (e.g., individuals having an early stage CRC) who are at high risk for recurrence of the CRC following surgical resection and who may benefit from an adjuvant treatment comprising a chemotherapy. Similarly, the provided gene signatures and related scores may be used to identify patients who are at low risk for recurrence of a CRC following surgical resection and who are unlikely to need further treatment, e.g., an adjuvant treatment comprising a chemotherapy.
  • the invention is based, at least in part, on the discovery that measuring expression of (a) a set of genes comprising genes relating to proliferative, stromal, and immune biological functions (e.g., a set of genes comprising the AVANT gene signature) or (b) a set of genes comprising genes relating to stromal biological functions (e.g., a set of genes comprising the AVANT stromal signature) and GZMB can be used as a prognostic signature in an individual having a CRC or having had surgical resection of a CRC, e.g., for determining whether the individual is a high risk of CRC recurrence following surgical resection of a CRC.
  • the invention features a method of predicting disease progression in an individual having a colorectal cancer (CRC), the method comprising determining an AVANT score from a sample from the individual, wherein an AVANT score that is above a reference AVANT score identifies the individual as one who is at high risk of CRC recurrence following surgical resection.
  • CRC colorectal cancer
  • the invention features a method of predicting disease recurrence in an individual who has had surgical resection of a CRC, the method comprising determining an AVANT score from a sample from the individual, wherein an AVANT score that is above a reference AVANT score indicates that the individual is at high risk of CRC recurrence. ia. Determination of A VANT score
  • the invention features methods that include determining an AVANT score from a sample from an individual having a CRC or having had surgical resection of a CRC, e.g., a CRC described in Section MB herein.
  • the AVANT score is determined by measuring the expression level of a set of genes comprising the AVANT signature genes, which relate to stromal, immune, and proliferative biological functions and are set forth in Table 1 or Table 2.
  • determining the AVANT score comprises measuring the expression level of one or more genes relating to stromal biological function.
  • the gene relating to stromal biological function is WBSCR17, TLL 1, CRYAB, ABCC9, HEYL, DTX1, SPP1, RGS2, IGFBP1, KDM5D, REG4, PCSK1, SMAD9, TCF12, or SMAD3.
  • the gene relating to stromal biological function is ANGPT1, WBSCR17, TLL 1, CRYAB, ABCC9, IGFBP3, HEYL, DTX1, CD36, SPP1, RGS2, HBEGF, FOS, SERPINB13, IGFBP1, APCDD1, SCD5, KDM5D, POU5F1, EPHA4, REG4, PCSK1, SMAD9, FLT1, TCF12, SMAD3, CDK6, or MAPK3.
  • determining the AVANT score comprises measuring the expression level of one or more genes relating to immune biological function.
  • the gene relating to immune biological function is GZMB, CXCL 1, or CD28.
  • the gene relating to immune biological function is TP73, TAP1, GZMB, TNF, CXCL 1, PDCD1, FCRL5, CXCR4, or CD28.
  • determining the AVANT score comprises measuring the expression level of one or more genes relating to proliferative biological function.
  • the one or more genes relating to proliferative biological function are selected from DTX2, KDM1A, CDCA5, E2F1, or GMNN.
  • the one or more genes relating to proliferative biological function are selected from RPL23, AXIN2, CDKN1B, DTX2, RPS6KA 1, KDM1A, RHOA, SP2, SHISA5, MLH1, CDCA5, E2F1, CENPM, CDK2, GMNN, or DNMT1.
  • determining the AVANT score comprises measuring the expression level of CDCA5, DTX2, E2F1, GMNN, KDM1A, ABCC9, CRYAB, DTX1, HEYL, RGS2, SPP1, TLL 1, WBSCR17, IGFBP1, KDM5D, PCSK1, REG4, SMAD3, SMAD9, TCF12, CD28, CXCL 1, and GZMB in a sample from an individual.
  • determining the AVANT score comprises measuring the expression level of CDCA5, DTX2, E2F1, GMNN, KDM1A, ABCC9, CRYAB, DTX1, HEYL, RGS2, SPP1, TLL 1, WBSCR17, IGFBP1, KDM5D, PCSK1, REG4, SMAD3, SMAD9, TCF12, CD28, CXCL 1, GZMB, RPLN23, AXIN2, CDKN1B, RPS6KA 1, RHOA, SP2, SHISA5, MLH1, CENPM, CDK2, DNMT1, ANGPT1, IGFBP3, CD36, HBEGF, FOS, SERPINB13, APCDD1, SCD5, POU5F1, EPHA4, FLT1, CDK6, MAPK3, TP73, TAP1,
  • TNF TNF, PDCD 1, FCRL5, and CXCR4 in a sample from an individual.
  • the AVANT score may be determined from any suitable sample, e.g., a tissue biopsy, a whole blood sample, a buccal swab, a plasma sample, a serum sample, or a combination thereof, e.g., an archival sample, a fresh sample, or a frozen sample, e.g., a tissue biopsy, e.g., a formalin-fixed paraffin- embedded sample.
  • the sample is from a surgically resected CRC.
  • the sample is obtained from the individual prior to the administration of any adjuvant treatment.
  • the AVANT score is determined by measuring the expression level of a set of genes in the sample from the individual.
  • the expression level is a nucleic acid expression level, e.g., a mRNA expression level.
  • the mRNA expression level is determined by, e.g., direct digital counting of nucleic acids (e.g., digital counting of nucleic acids is by NANOSTRING ® NCOUNTER ® analysis), RNA-seq, RT-qPCR, qPCR, multiplex qPCR or RT-qPCR, microarray analysis, or a combination thereof.
  • the expression level is a protein expression level.
  • the protein expression level is determined by, e.g., an immunoassay, liquid chromatography-mass spectrometry (LC-MS) technology, nephelometry, aptamer technology, or a combination thereof.
  • LC-MS liquid chromatography-mass spectrometry
  • the AVANT score of a sample from an individual is compared to a reference AVANT score, e.g., an AVANT score in a reference population of individuals.
  • the reference population is a population of individuals who have had surgical resection of a CRC, e.g., a CRC as described in Section MB herein.
  • the reference population includes individuals having a CRC that is stage I, stage II, stage III, or stage IV, according to the TNM classification system at the onset of treatment.
  • the reference population is the population of the GSE39582 cohort (Marisa et al, PLoS Med 2013).
  • the reference population includes individuals having a histologically confirmed stage III - N1 , stage III - N2, or high-risk stage II colon carcinoma, according to the TNM classification system at the onset of treatment.
  • the reference population is the population of the AVANT clinical trial (ClinicalTrials.gov identifier NCT001 12918) (de Gramont et al., J Clin Oncol., 1 8: 2938-2947, 2000).
  • the reference population is the biomarker evaluable population (BEP) of the AVANT clinical trial.
  • the reference population of individuals has been administered an adjuvant treatment comprising a chemotherapy, e.g., a chemotherapy as described in Section NIC herein.
  • a sample e.g., a tissue biopsy, a whole blood sample, a buccal swab, a plasma sample, a serum sample, or a combination thereof, is obtained from the individual in a reference population of individuals prior to the administration of any adjuvant treatment, e.g., prior to the administration of an adjuvant treatment comprising a chemotherapy.
  • the reference AVANT score is an AVANT score that significantly separates a first subset and a second subset of individuals in the reference population based on responsiveness to treatment, e.g., overall survival (OS), disease-free survival (DFS), and/or recurrence-free survival (RFS).
  • OS overall survival
  • DFS disease-free survival
  • RFS recurrence-free survival
  • the reference AVANT score is a pre-assigned reference AVANT score. In some aspects, the reference AVANT score is defined as between the 25 th percentile and the 75 th percentile of AVANT scores in the reference population, e.g., the 25 th percentile, 26 th percentile, 27 th percentile, 28 th percentile, 29 th percentile, 30 th percentile, 31 st percentile, 32 nd percentile, 33 rd percentile, 34 th percentile, 35 th percentile, 36 th percentile, 37 th percentile, 38 th percentile, 39 th percentile, 40 th percentile, 41 st percentile, 42 nd percentile, 43 rd percentile, 44 th percentile, 45 th percentile, 46 th percentile, 47 th percentile, 48 th percentile, 49 th percentile, 50 th percentile, 51 st percentile, 52 nd percentile, 53 rd percentile, 54 th percentile, 55
  • the reference AVANT score is defined as the 25 th percentile of AVANT scores in the reference population. In some aspects, the reference AVANT score is defined as the 50 th percentile of AVANT scores in the reference population. In some aspects, the reference AVANT score is defined as the 75 th percentile of AVANT scores in the reference population. ic. Prognostic methods using A VANT score
  • the invention features a method of predicting disease progression in an individual having a CRC, the method comprising determining an AVANT score from a sample from the individual, wherein an AVANT score that is above a reference AVANT score identifies the individual as one who is at high risk of CRC recurrence following surgical resection.
  • the AVANT score of the individual is above the reference AVANT score and the individual is identified as one who is at a high risk of CRC recurrence following surgical resection.
  • the method further comprises administering to the individual an adjuvant treatment comprising a chemotherapy.
  • the AVANT score of the individual is below the reference AVANT score.
  • the individual is identified as one who is at a low risk of CRC recurrence following surgical resection.
  • the individual is not administered an adjuvant treatment comprising a chemotherapy.
  • the invention features a method of predicting disease recurrence in an individual who has had surgical resection of a CRC, the method comprising determining an AVANT score from a sample from the individual, wherein an AVANT score that is above a reference AVANT score indicates that the individual is at high risk of CRC recurrence.
  • the AVANT score of the individual is above the reference AVANT score and the individual is identified as one who is at a high risk of CRC recurrence.
  • the method further comprises administering to the individual an adjuvant treatment comprising a chemotherapy.
  • the AVANT score of the individual is below the reference AVANT score.
  • the individual is identified as one who is at a low risk of CRC recurrence.
  • the individual is not administered an adjuvant treatment comprising a chemotherapy.
  • the disclosure features a method of providing a prognosis for an individual having a CRC, the method comprising determining an AVANT score from a sample from the individual, wherein an AVANT score that is above a reference AVANT score identifies the individual as one who may have a poor prognosis.
  • the method further comprises administering to the individual an adjuvant treatment comprising a chemotherapy.
  • the invention features a method of predicting disease progression in an individual having a CRC, the method comprising determining an AVANT stromal score and the expression level of GZMB in a sample from the individual, wherein an AVANT stromal score that is above a reference AVANT stromal score and an expression level of GZMB that is below a reference expression level of GZMB identifies the individual as one who is at a high risk of CRC recurrence following surgical resection.
  • the invention features a method of predicting disease recurrence in an individual who has had surgical resection of a CRC, the method comprising determining an AVANT stromal score and the expression level of GZMB in a sample from the individual, wherein an AVANT stromal score that is above a reference AVANT stromal score and an expression level of GZMB that is below a reference expression level of GZMB indicates that the individual is at high risk of CRC recurrence. iia.
  • the invention features methods that include determining an AVANT stromal score and the expression level of GZMB from a sample from an individual having a CRC or having had surgical resection of a CRC, e.g., a CRC described in Section MB herein.
  • the AVANT stromal score is determined by measuring the expression level of one or more genes relating to stromal biological function.
  • the gene relating to stromal biological function is WBSCR17, TLL 1, CRYAB, ABCC9, HEYL, DTX1, SPP1, RGS2, IGFBP1, KDM5D, REG4, PCSK1, SMAD9, TCF12, or SMAD3.
  • the gene relating to stromal biological function is ANGPT1, WBSCR17, TLL 1, CRYAB, ABCC9, IGFBP3, HEYL, DTX1, CD36, SPP1, RGS2, HBEGF, FOS, SERPINB13, IGFBP1, APCDD1, SCD5, KDM5D, POU5F1, EPHA4, REG4, PCSK1, SMAD9, FLT1, TCF12, SMAD3, CDK6, or MAPK3.
  • determining the AVANT stromal score comprises measuring the expression level of ABCC9, CRYAB, DTX1, HEYL, RGS2, SPP1, TLL 1, WBSCR17, IGFBP1, KDM5D, PCSK1, REG4, SMAD3, SMAD9, and TCF12 in a sample from an individual.
  • determining the AVANT stromal score comprises measuring the expression level of ANGPT1, WBSCR17, TLL 1, CRYAB, ABCC9, IGFBP3, HEYL, DTX1, CD36, SPP1, RGS2, HBEGF, FOS, SERPINB13, IGFBP1, APCDD1, SCD5, KDM5D, POU5F1, EPHA4, REG4, PCSK1, SMAD9, FLT1, TCF12, SMAD3, CDK6, and MAPK3 in a sample from an individual.
  • determining the expression level of GZMB comprises measuring the expression level of GZMB.
  • the AVANT stromal score and expression level of GZMB may be determined from any suitable sample, e.g., a tissue biopsy, a whole blood sample, a buccal swab, a plasma sample, a serum sample, or a combination thereof, e.g., an archival sample, a fresh sample, or a frozen sample, e.g., a tissue biopsy, e.g., a formalin-fixed paraffin-embedded sample.
  • the sample is from a surgically resected CRC.
  • the sample is obtained from the individual prior to the administration of any adjuvant treatment.
  • the AVANT stromal score is determined by measuring the expression level of a set of genes in the sample from the individual.
  • the expression level is a nucleic acid expression level, e.g., a mRNA expression level.
  • the mRNA expression level is determined by, e.g., direct digital counting of nucleic acids (e.g., digital counting of nucleic acids is by NANOSTRING ® NCOUNTER ® analysis), RNA-seq, RT-qPCR, qPCR, multiplex qPCR or RT-qPCR, microarray analysis, or a combination thereof.
  • the expression level is a protein expression level.
  • the protein expression level is determined by, e.g., an immunoassay, liquid chromatography-mass spectrometry (LC-MS) technology, nephelometry, aptamer technology, or a combination thereof.
  • LC-MS liquid chromatography-mass spectrometry
  • the AVANT stromal score and expression level of GZMB in a sample from an individual are compared to an AVANT stromal score and expression level of GZMB in a reference population, e.g., an AVANT stromal score and expression level of GZMB as in a reference population of individuals, e.g., a reference population as described in Section 11 A (ib) herein.
  • reference expression level of GZMB alone, or the reference AVANT stromal score and reference expression level of GZMB when considered together, significantly separate a first subset and a second subset of individuals based on responsiveness to treatment, e.g., OS, DFS, and/or RFS.
  • the reference AVANT stromal score is a pre-assigned reference AVANT stromal score. In some aspects, the reference AVANT stromal score is defined as between the 25 th percentile and the 75 th percentile of AVANT stromal scores in the reference population, e.g., the 25 th percentile, 26 th percentile, 27 th percentile, 28 th percentile, 29 th percentile, 30 th percentile, 31 st percentile, 32 nd percentile, 33 rd percentile, 34 th percentile, 35 th percentile, 36 th percentile, 37 th percentile, 38 th percentile, 39 th percentile, 40 th percentile, 41 st percentile, 42 nd percentile, 43 rd percentile, 44 th percentile, 45 th percentile, 46 th percentile, 47 th percentile, 48 th percentile, 49 th percentile, 50 th percentile, 51 st percentile, 52 nd percentile, 53
  • the reference AVANT stromal score is defined as the median AVANT stromal score in the reference population.
  • the reference expression level of GZMB is a pre-assigned reference expression level of GZMB.
  • the reference expression level of GZMB is defined as between the 25 th percentile and the 75 th percentile of expression levels of GZMB in the reference population, e.g., the 25 th percentile, 26 th percentile, 27 th percentile, 28 th percentile, 29 th percentile, 30 th percentile, 31 st percentile, 32 nd percentile, 33 rd percentile, 34 th percentile, 35 th percentile, 36 th percentile, 37 th percentile, 38 th percentile, 39 th percentile, 40 th percentile, 41 st percentile, 42 nd percentile, 43 rd percentile, 44 th percentile, 45 th percentile, 46 th percentile, 47 th percentile, 48 th percentile, 49 th percentile, 50 th percentile, 51 st percentile, 52 nd percentile, 53 rd percentile
  • the reference expression level of GZMB is defined as the median expression level of GZMB in the reference population.
  • the reference AVANT stromal score is defined as the median of AVANT stromal scores in the reference population and the reference expression level of GZMB is defined as the median expression level of GZMB in the reference population. iic. Prognostic methods using A VANT stromal score and GZMB expression level
  • the invention features a method of predicting disease progression in an individual having a CRC, the method comprising determining an AVANT stromal score and the expression level of GZMB in a sample from the individual, wherein an AVANT stromal score that is above a reference AVANT stromal score and an expression level of GZMB that is below a reference expression level of GZMB identifies the individual as one who is at a high risk of CRC recurrence following surgical resection.
  • the invention features a method of predicting disease recurrence in an individual who has had surgical resection of a CRC, the method comprising determining an AVANT stromal score and the expression level of GZMB in a sample from the individual, wherein an AVANT stromal score that is above a reference AVANT stromal score and an expression level of GZMB that is below a reference expression level of GZMB indicates that the individual is at high risk of CRC recurrence.
  • the AVANT stromal score is above a reference AVANT stromal score and the expression level of GZMB is below a reference expression level of GZMB, and the method further comprises administering to the individual an adjuvant treatment comprising a chemotherapy.
  • an AVANT stromal score that is below a reference AVANT stromal score and an expression level of GZMB that is above a reference expression level of GZMB identifies the individual as one who is at low risk of CRC recurrence following surgical resection.
  • the individual is not administered an adjuvant treatment comprising a chemotherapy.
  • an AVANT stromal score that is below a reference AVANT stromal score and an expression level of GZMB that is below a reference expression level of GZMB identifies the individual as one who is at low risk of CRC recurrence following surgical resection.
  • the individual is not administered an adjuvant treatment comprising a chemotherapy.
  • the invention comprises determining (a) an AVANT score or (b) an AVANT stromal score and the expression level of GZMB for an individual, and further comprises determining one or more additional properties (e.g., consensus molecular subtype (CMS), microsatellite instability (MSI) status, KRAS status, or BRAF status) from a sample from the individual.
  • CMS consensus molecular subtype
  • MSI microsatellite instability
  • KRAS status KRAS status
  • BRAF status e.g., a sample from the individual.
  • the AVANT score or AVANT stromal score and expression level of GZMB and the one or more additional properties are determined from the same sample from the individual, e.g., a tissue biopsy, a whole blood sample, a buccal swab, a plasma sample, a serum sample, or a combination thereof, e.g., an archival sample, a fresh sample, or a frozen sample, e.g., a tissue biopsy, e.g., a formalin-fixed paraffin-embedded sample.
  • the one or more additional properties are determined from an additional sample from the individual, e.g., an additional tissue biopsy, whole blood sample, buccal swab, plasma sample, serum sample, or combination thereof.
  • the one or more additional properties are determined following surgical resection of the CRC, e.g., are determined from a surgically resected CRC tumor.
  • the sample is obtained from the individual prior to the administration of any adjuvant treatment.
  • the additional property is CMS.
  • CMS may be determined as described in Guinney et al., Nat Med, 21 (1 1 ): 1350-1362, 2015.
  • the CMS of the sample is CMS1 , CMS2, CMS3, or CMS4.
  • the CMS of the sample is CMS4.
  • the additional property is MSI status.
  • MSI status is determined to be microsatellite stable or microsatellite instability low (MSS).
  • MSI status is determined to be microsatellite instability high (MSI-H).
  • MSI status may be determined using methods known in the art, e.g., by assessing a panel of five microsatellite loci as described in Marisa et al. , PLoS Med, 10: e1001453, 2013 and Boland et al., Cancer Res, 58(22): 5248-5257, 1998.
  • KRAS status refers to the presence or absence of one or more changes in the nucleotide or amino acid sequence of KRAS, e.g., one or more nucleotide substitution mutations resulting in a mutation at position 12 of the KRAS amino acid sequence (e.g.
  • G12A, G12R, G12D, G12C, G12S, G12V, or G12D mutation or a mutation at position 13 of the KRAS amino acid sequence (e.g., a G13D mutation), as described in Marisa et al., PLoS Med, 10: e1001453, 2013, Lievre et al., J Clin Oncol, 26: 374-379, 2008, and Lievre et al., Cancer Res, 66(8): 3992-3995, 2006).
  • the change in the nucleotide sequence may also be any missense or nonsense mutation that is observed in the CRC, but not in adjacent normal tissue (Stransky et al., Science, 333: 1 157-1 160, 201 1 ).
  • KRAS status may be determined using methods known in the art, e.g., an allelic discrimination assay using TaqMan probes, as described in Lievre et al., J Clin Oncol, 26: 374- 379, 2008, or a MuTect assay, as described in Stransky et al., Science, 333: 1 157-1 160, 201 1 .
  • the additional property is BRAF status.
  • BRAF status refers to the presence or absence of one or more changes in the nucleotide or amino acid sequence of BRAF.
  • the change in the nucleotide sequence may be, e.g., a c. 1799T>A nucleotide substitution mutation, e.g., a mutation resulting in a V600E change in the amino acid sequence (Marisa et al., PLoS Med, 10:
  • the change in the nucleotide sequence may also be any missense or nonsense mutation that is observed in the CRC, but not in adjacent normal tissue (Stransky et al., Science, 333:
  • BRAF status may be determined using methods known in the art, e.g., an allelic discrimination assay using TaqMan probes, as described in Marisa et al., PLoS Med, 1 0: e1001453,
  • the individual has a colorectal cancer (CRC). In some aspects, the individual has had surgical resection of a CRC. In some aspects, the CRC of the individual is a stage I, stage II, or stage III CRC, according to the TNM classification system at the onset of treatment. In some aspects, the CRC of the individual is a stage III - N1 , stage III - N2, or high-risk stage II CRC, according to the TNM classification system at the onset of treatment, wherein a“high-risk stage II CRC” may be identified as a stage II CRC in an individual having one or more of T4 tumors; bowel obstruction or perforation;
  • the individual does not have a stage IV CRC according to the TNM classification system at the onset of treatment. In some aspects, the individual does not have a metastatic CRC. In some aspects, the CRC of the individual is characterized as consensus molecular subtype 1 (CMS1 ), CMS2, CMS3, CMS4, or unclassified, as defined in Guinney et al., Nat Med, 21 (1 1 ): 1350-1362, 2015.
  • CMS1 consensus molecular subtype 1
  • CMS2 CMS2, CMS3, CMS4, or unclassified
  • the CRC of the individual is a left-sided tumor, i.e., a tumor occurring in the distal colon (e.g., the distal third of the transverse colon, the splenic flexure the descending colon, the sigmoid colon, or the rectum) or a right sided tumor, i.e., a tumor occurring in the proximal colon (e.g., the proximal two-thirds of the transverse colon, the ascending colon, and the cecum).
  • the CRC of the individual is a colon carcinoma.
  • the colon carcinoma may be CMS1 , CMS2, CMS3, CMS4, or unclassified and may be a left sided or a right-sided colon carcinoma.
  • the colon carcinoma is a stage III - N1 , stage III - N2, or high-risk stage II colon carcinoma, according to the TNM classification system at the onset of treatment.
  • the CRC e.g., colon carcinoma
  • the CRC has been surgically resected.
  • one or more properties of the CRC of the individual e.g., CMS, are determined following surgical resection of the CRC, e.g., are determined from the surgically resected CRC tumor.
  • an individual in a reference population has a colorectal cancer (CRC).
  • the individual in a reference population has had surgical resection of a CRC.
  • the CRC of the individual in a reference population is a stage I, stage II, stage III, or stage IV CRC, according to the TNM classification system at the onset of treatment.
  • the CRC of the individual in a reference population is a stage III - N1 , stage III - N2, or high-risk stage II CRC, according to the TNM classification system at the onset of treatment, wherein a“high-risk stage II CRC” may be identified as a stage II CRC in an individual having one or more of T4 tumors; bowel obstruction or perforation ; histological signs of vascular invasion (i.e. , blood and lymphatic vessels) or perineural invasion; age less than 50 years; and sub-optimal surgery (less than 12 nodes analyzed).
  • the CRC of the individual in a reference population is characterized as consensus molecular subtype 1 (CMS1 ), CMS2, CMS3, CMS4, or unclassified, as defined in Guinney et al., Nat Med, 21 (1 1 ): 1350-1362, 2015.
  • CMS1 consensus molecular subtype 1
  • CMS2 CMS2, CMS3, CMS4, or unclassified
  • the CRC of the individual in a reference population is a left-sided tumor, i.e., a tumor occurring in the distal colon (e.g., the distal third of the transverse colon, the splenic flexure the descending colon, the sigmoid colon, or the rectum) or a right-sided tumor, i.e., a tumor occurring in the proximal colon (e.g., the proximal two-thirds of the transverse colon, the ascending colon, and the cecum).
  • the CRC of the individual in a reference population is a colon carcinoma.
  • the colon carcinoma may be CMS1 , CMS2, CMS3, CMS4, or unclassified and may be a left sided or a right-sided colon carcinoma.
  • the colon carcinoma is a stage III - N1 , stage III - N2, or high-risk stage II colon carcinoma, according to the TNM classification system at the onset of treatment.
  • the CRC e.g., colon carcinoma
  • the CRC has been surgically resected.
  • one or more properties of the CRC of the individual in a reference population, e.g., CMS are determined following surgical resection of the CRC, e.g., are determined from the surgically resected CRC tumor.
  • the invention features a method of treating an individual having a CRC or having had surgical resection of a CRC, the method comprising (a) determining an AVANT score from a sample from the individual, wherein the AVANT score is above a reference AVANT score; and (b) administering to the individual an adjuvant treatment comprising a chemotherapy.
  • the invention features a method of treating an individual having a CRC or having had surgical resection of a CRC, the method comprising administering an adjuvant treatment comprising a chemotherapy to the individual, for whom an AVANT score from a sample from the individual has been determined to be above a reference AVANT score.
  • the CRC may be a CRC as described in Section MB herein.
  • the AVANT score from a sample from the individual may be determined as described in Section IIA (ia) herein.
  • the reference AVANT score may be a reference AVANT score as described in Section IIA (ib) herein.
  • the adjuvant treatment comprising a chemotherapy may be an adjuvant treatment as described in Section MID herein.
  • the invention features a method of treating an individual having a CRC, the method comprising (a) determining an AVANT stromal score and the expression level of GZMB in a sample from the individual, wherein the AVANT stromal score is above a reference AVANT stromal score and the expression level of GZMB is below a reference expression level of GZMB ; and (b) administering to the individual an adjuvant treatment comprising a chemotherapy.
  • the invention features a method of treating an individual having a CRC, the method comprising administering an adjuvant treatment comprising a chemotherapy to the individual, for whom an AVANT stromal score from a sample from the individual has been determined to be above a reference AVANT stromal score and an expression level of GZMB has been determined to be below a reference expression level of GZMB.
  • the CRC may be a CRC as described in Section MB herein.
  • the AVANT stromal score and expression level of GZMB from a sample from the individual may be determined as described in Section IIA (iia) herein.
  • the reference AVANT stromal score and reference expression level of GZMB may be a reference AVANT stromal score and reference expression level of GZMB as described in Section IIA (lib) herein.
  • the adjuvant treatment comprising a chemotherapy may be an adjuvant treatment as described in Section MID herein.
  • an adjuvant therapy comprising a chemotherapeutic agent is used to treat or delay recurrence or progression of a colorectal cancer (CRC) in a subject in need thereof.
  • the subject is a human.
  • an individual has a colorectal cancer (CRC) or has had surgical resection of a CRC, e.g., a stage I, stage II, or stage III CRC according to the TNM classification system at the onset of treatment, e.g., a CRC as described in Section MB herein.
  • CRC colorectal cancer
  • an individual in a reference population has a colorectal cancer (CRC) or has had surgical resection of a CRC, e.g., a stage I, stage II, or stage III CRC according to the TNM classification system at the onset of treatment, e.g., a CRC as described in Section MB herein.
  • CRC colorectal cancer
  • the primary therapy comprises surgical resection of the cancer, e.g., surgical resection of a CRC.
  • the surgical resection has occurred no less than four weeks and not more than eight weeks before the onset of adjuvant treatment.
  • Adjuvant therapies comprising a chemotherapeutic agent
  • the individual is further treated with an adjuvant therapy, e.g., treated with an adjuvant therapy comprising a chemotherapeutic agent following surgical resection of a CRC.
  • an adjuvant therapy comprising a chemotherapeutic agent following surgical resection of a CRC.
  • the individual is not treated with a therapy comprising a chemotherapeutic agent preceding surgical resection of the CRC.
  • an individual is administered an adjuvant treatment comprising a
  • chemotherapeutic agent is a chemical compound useful in the treatment of cancer.
  • chemotherapeutic agents include, but are not limited to erlotinib (TARCEVA®, Genentech/OSI Pharm.), anti-hormonal agents that act to regulate or inhibit hormone action on tumors such as anti-estrogens and selective estrogen receptor modulators (SERMs), antibodies such as bevacizumab (AVASTIN®, Genentech), alemtuzumab (Campath), cetuximab (ERBITUX®, Imclone), panitumumab (VECTIBIX®, Amgen), rituximab (RITUXAN®, Genentech/Biogen pou), pertuzumab (OMNITARG®, 2C4, Genentech), or trastuzumab (HERCEPTIN®, Genentech), EGFR inhibitors (EGFR antagonists), tyrosine kinase inhibitors, and chemotherapeutic agents also include
  • the chemotherapy comprises oxaliplatin, fluorouracil, or leucovorin. In some aspects, the chemotherapy consists of oxaliplatin, fluorouracil, and leucovorin, e.g., is FOLFOX4. In some aspects, the adjuvant treatment comprises FOLFOX4 and bevacizumab. In some aspects, the adjuvant treatment comprises a first treatment comprising FOLFOX4 and bevacizumab and a second treatment comprising bevacizumab.
  • the chemotherapy comprises oxaliplatin and capecitabine, e.g., is XELOX.
  • the adjuvant treatment comprises XELOX and bevacizumab.
  • compositions utilized in the methods described herein can be administered by any suitable method, including, for example, intravenously, intramuscularly, subcutaneously, intradermally, percutaneously,
  • intravitreally e.g., by intravitreal injection
  • compositions utilized in the methods described herein can also be administered systemically or locally.
  • the method of administration can vary depending on various factors (e.g., the compound or composition being administered and the severity of the condition, disease, or disorder being treated).
  • a chemotherapeutic agent is administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally,
  • Dosing can be by any suitable route, e.g., by injections, such as intravenous or subcutaneous injections, depending in part on whether the administration is brief or chronic.
  • Various dosing schedules including but not limited to single or multiple administrations over various time-points, bolus administration, and pulse infusion are contemplated herein.
  • a chemotherapeutic agent may be formulated, dosed, and administered in a fashion consistent with good medical practice.
  • Factors for consideration in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent, the method of administration, the scheduling of
  • the immune checkpoint inhibitor need not be, but is optionally formulated with and/or administered concurrently with one or more agents currently used to prevent or treat the disorder in question, e.g., one or more of the agents provided in Section NI C herein.
  • the effective amount of such other agents depends on the amount of the chemotherapeutic agent present in the formulation, the type of disorder or treatment, and other factors discussed above. These are generally used in the same dosages and with administration routes as described herein, or about from 1 to 99% of the dosages described herein, or in any dosage and by any route that is empirically/clinically determined to be appropriate.
  • a chemotherapeutic agent described herein when used alone or in combination with one or more other additional therapeutic agents, will depend on the type of disease to be treated, the severity and course of the disease, whether the chemotherapeutic agent is administered for preventive or therapeutic purposes, previous therapy, the patient’s clinical history and response to the
  • the chemotherapeutic agent is suitably administered to the patient at one time or over a series of treatments.
  • One typical daily dosage might range from about 1 pg/kg to 100 mg/kg or more, depending on the factors mentioned above.
  • the treatment would generally be sustained until a desired suppression of disease symptoms occurs.
  • Such doses may be administered intermittently, e.g., every week or every three weeks (e.g., such that the patient receives, for example, from about two to about twenty, or e.g., about six doses of the chemotherapeutic agent).
  • An initial higher loading dose, followed by one or more lower doses may be administered.
  • other dosage regimens may be useful. The progress of this therapy is easily monitored by conventional techniques and assays.
  • the treatment comprises 24 weeks of treatment with FOLFOX4 (oxaliplatin, leucovorin calcium, and 5-Fluorouracil (5-FU)); FOLFOX4 in combination with bevacizumab; or XELOX (capecitabine) in combination with bevacizumab.
  • the treatment further comprises 24 weeks treatment with single-agent bevacizumab following the 24 week treatment with FOLFOX4 in combination with bevacizumab or XELOX in combination with bevacizumab.
  • the chemotherapeutic agent is used with one or more additional therapeutic agents, e.g., a combination therapy.
  • the composition comprising the chemotherapeutic agent further comprises the additional therapeutic agent.
  • the additional therapeutic agent is delivered in a separate composition.
  • the one or more additional therapeutic agents comprise a growth inhibitory agent, an anti-angiogenic agent, a radiation therapy, a cytotoxic agent, or a combination thereof.
  • Combination therapies as described above encompass combined administration (wherein two or more therapeutic agents are included in the same or separate formulations) and separate administration (wherein administration of a chemotherapeutic agent can occur prior to, simultaneously, and/or following, administration of the additional therapeutic agent or agents).
  • administration of a chemotherapeutic agent and administration of an additional therapeutic agent occur within about one month, or within about one, two or three weeks, or within about one, two, three, four, five, or six days, of each other.
  • the additional therapeutic agent is a growth inhibitory agent.
  • growth inhibitory agents include agents that block cell cycle progression at a place other than S phase, e.g., agents that induce G1 arrest (e.g., DNA alkylating agents such as tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin, methotrexate, 5-fluorouracil, or ara-C) or M-phase arrest (e.g., vincristine, vinblastine, taxanes (e.g., paclitaxel and docetaxel), doxorubicin, epirubicin, daunorubicin, etoposide, or bleomycin).
  • G1 arrest e.g., DNA alkylating agents such as tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin, methotrexate, 5-fluorouracil, or ara-C
  • the additional therapeutic agent is a radiation therapy.
  • Radiation therapies include the use of directed gamma rays or beta rays to induce sufficient damage to a cell so as to limit its ability to function normally or to destroy the cell altogether. Typical treatments are given as a one-time administration and typical dosages range from 10 to 200 units (Grays) per day. iii. Cytotoxic agents
  • the additional therapeutic agent is a cytotoxic agent, e.g., a substance that inhibits or prevents a cellular function and/or causes cell death or destruction.
  • Cytotoxic agents include, but are not limited to, radioactive isotopes (e.g., At 21 1 , I 131 , 1 125 , Y 90 , Re 186 , Re 188 , Sm 153 , Bi 212 , P 32 , Pb 212 , and radioactive isotopes of Lu); chemotherapeutic agents or drugs (e.g., methotrexate, adriamicin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or other intercalating agents); growth inhibitory agents; enzymes and fragments thereof such as nucleolytic enzymes; antibiotics; toxins such as small molecule toxins or enzymatic
  • the additional therapeutic agent is an immunomodulatory agent, e.g., a PD-L1 axis binding antagonist, which may be a PD-1 binding antagonist, a PD-L1 binding antagonist, or a PD-L2 binding antagonist.
  • PD-1 programmeed death 1
  • PDCD1 PDCD1
  • CD279 PD-L2 binding antagonist
  • SEB2 SLB2
  • An exemplary human PD-1 is shown in UniProtKB/Swiss-Prot Accession No. Q151 16.
  • PD-L1 (programmed death ligand 1 ) is also referred to in the art as
  • PD-L1 “programmed cell death 1 ligand 1 ,”“PDCD1 LG1 ,”“CD274,”“B7-H,” and“PDL1
  • An exemplary human PD-L1 is shown in UniProtKB/Swiss-Prot Accession No.Q9NZQ7.1 .
  • PD-L2 (programmed death ligand 2) is also referred to in the art as“programmed cell death 1 ligand 2,”“PDCD1 LG2,”“CD273,”“B7-DC,” “Btdc,” and“PDL2.”
  • An exemplary human PD-L2 is shown in UniProtKB/Swiss-Prot Accession No. Q9BQ51 .
  • PD-1 , PD-L1 , and PD-L2 are human PD-1 , PD-L1 and PD-L2.
  • the PD-1 binding antagonist is a molecule that inhibits the binding of PD-1 to its ligand binding partners.
  • the PD-1 ligand binding partners are PD-L1 and/or PD-L2.
  • a PD-L1 binding antagonist is a molecule that inhibits the binding of PD-L1 to its binding ligands.
  • PD-L1 binding partners are PD-1 and/or B7-1 .
  • the PD-L2 binding antagonist is a molecule that inhibits the binding of PD-L2 to its ligand binding partners.
  • the PD-L2 binding ligand partner is PD-1 .
  • the antagonist may be an antibody, an antigen binding fragment thereof, an immunoadhesin, a fusion protein, or oligopeptide.
  • the PD-1 binding antagonist is an anti-PD-1 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody), for example, as described below.
  • the anti-PD-1 antibody is selected from the group consisting of MDX-1 106 (nivolumab), MK-3475
  • MDX-1 106 also known as MDX- 1 106-04, ONO-4538, BMS-936558, or nivolumab, is an anti-PD-1 antibody described in W02006/121 168.
  • MK-3475 also known as pembrolizumab or lambrolizumab, is an anti-PD-1 antibody described in WO 2009/1 14335.
  • the PD-1 binding antagonist is an immunoadhesin (e.g., an immunoadhesin comprising an extracellular or PD-1 binding portion of PD-L1 or PD-L2 fused to a constant region (e.g., an Fc region of an immunoglobulin sequence).
  • the PD-1 binding antagonist is AMP-224.
  • AMP-224 also known as B7-DCIg, is a PD-L2-Fc fusion soluble receptor described in WO 2010/027827 and WO 201 1 /066342.
  • the anti-PD-1 antibody is MDX-1 106.
  • Alternative names for“MDX-1 106” include MDX-1 106-04, ONO-4538, BMS-936558, and nivolumab.
  • the anti-PD-1 antibody is nivolumab (CAS Registry Number: 946414-94-4).
  • an isolated anti-PD-1 antibody comprising a heavy chain variable region comprising the heavy chain variable region amino acid sequence from SEQ ID NO: 1 and/or a light chain variable region comprising the light chain variable region amino acid sequence from SEQ ID NO: 2.
  • an isolated anti-PD-1 antibody comprising a heavy chain and/or a light chain sequence, wherein:
  • the heavy chain sequence has at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the heavy chain sequence:
  • GNVFSCSVMHEALHNHYTQKSLSLSLGK SEQ ID NO: 1
  • the light chain sequences has at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the light chain sequence: EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTD FTLTISSLEPEDFAVYYCQQSSNWPRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYP REAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC (SEQ ID NO: 2).
  • the PD-L1 axis binding antagonist is a PD-L2 binding antagonist.
  • the PD-L2 binding antagonist is an anti-PD-L2 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody).
  • the PD-L2 binding antagonist is an immunoadhesin.
  • the PD-L1 binding antagonist is an anti-PD-L1 antibody, for example, as described below.
  • the anti-PD-L1 antibody is capable of inhibiting binding between PD- L1 and PD-1 and/or between PD-L1 and B7-1 .
  • the anti-PD-L1 antibody is a monoclonal antibody.
  • the anti-PD-L1 antibody is an antibody fragment selected from the group consisting of Fab, Fab’-SH, Fv, scFv, and (Fab’)2 fragments.
  • the anti-PD-L1 antibody is a humanized antibody.
  • the anti-PD-L1 antibody is a human antibody.
  • the anti-PD-L1 antibody is selected from the group consisting of YW243.55.S70, MPDL3280A
  • EDX-1 105 is an anti-PD-L1 described in WO 2010/077634.
  • MDX-1 105 also known as BMS- 936559, is an anti-PD-L1 antibody described in W02007/005874.
  • MEDI4736 is an anti-PD- L1 monoclonal antibody described in WO201 1 /066389 and US2013/034559.
  • anti-PD-L1 antibodies useful for the methods of this invention, and methods for making thereof are described in PCT patent application WO 2010/077634, WO 2007/005874, WO 201 1 /066389, U.S. Pat. No. 8,21 7,149, and US 2013/034559, which are incorporated herein by reference.
  • Anti-PD-L1 antibodies described in WO 2010/077634 A1 and US 8,217,149 may be used in the methods described herein.
  • the anti-PD-L1 antibody comprises a heavy chain variable region sequence of SEQ ID NO: 3 and/or a light chain variable region sequence of SEQ ID NO: 4.
  • an isolated anti-PD-L1 antibody comprising a heavy chain variable region and/or a light chain variable region sequence, wherein:
  • the heavy chain sequence has at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the heavy chain sequence:
  • the light chain sequence has at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the light chain sequence:
  • the anti-PD-L1 antibody comprises a heavy chain variable region comprising an HVR-H1 , HVR-H2 and HVR-H3 sequence, wherein:
  • HVR-H1 sequence is GFTFSXiSWIH (SEQ ID NO: 5);
  • the HVR-H2 sequence is AWIX2PYGGSX3YYADSVKG (SEQ ID NO: 6);
  • the HVR-H3 sequence is RHWPGGFDY (SEQ ID NO: 7);
  • the polypeptide further comprises variable region heavy chain framework sequences juxtaposed between the HVRs according to the formula: (FR-H1 )-(HVR-H1 )-(FR-H2)-(HVR- H2)-(FR-H3)-(HVR-H3)-(FR-H4).
  • the framework sequences are derived from human consensus framework sequences.
  • the framework sequences are VH subgroup III consensus framework.
  • at least one of the framework sequences is the following:
  • FR-H1 is EVQLVESGGGLVQPGGSLRLSCAAS (SEQ ID NO: 8)
  • FR-H2 is WVRQAPGKGLEWV (SEQ ID NO: 9)
  • FR-H3 is RFTISADTSKNTAYLQMNSLRAEDTAVYYCAR (SEQ ID NO: 10)
  • FR-H4 is WGQGTLVTVSS (SEQ ID NO: 1 1 ).
  • the heavy chain polypeptide is further combined with a variable region light chain comprising an HVR-L1 , HVR-L2 and HVR-L3, wherein:
  • HVR-L1 sequence is RASQX 4 X 5 X 6 TX7X 8 A (SEQ ID NO: 12)
  • HVR-L2 sequence is SASX9LX10S, (SEQ ID NO: 13)
  • the HVR-L3 sequence is QQX11X12X13X14PX15T (SEQ ID NO: 14) wherein: X4 IS D or V; X5 is V or I; Cb is S or N; X7 is A or F; Xs is V or L; X9 is F or T; X10 is Y or A; Xn is Y, G, F, or S; X12 IS L, Y, F or W; X13 is Y, N, A, T, G, F or l ; Xi 4 is H, V, P, T or I ; X15 is A, W, R, P or T.
  • the light chain further comprises variable region light chain framework sequences juxtaposed between the HVRs according to the formula: (FR-L1 )-(HVR-L1 )-(FR-L2)-(HVR- L2)-(FR-L3)-(HVR-L3)-(FR-L4).
  • the framework sequences are derived from human consensus framework sequences.
  • the framework sequences are VL kappa I consensus framework.
  • at least one of the framework sequence is the following:
  • FR-L1 is DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 15)
  • FR-L2 is WYQQKPGKAPKLLIY (SEQ ID NO: 16)
  • FR-L3 is GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 17)
  • FR-L4 is FGQGTKVEIKR (SEQ ID NO: 18).
  • an isolated anti-PD-L1 antibody or antigen binding fragment comprising a heavy chain and a light chain variable region sequence, wherein:
  • the heavy chain comprises an HVR-H1 , HVR-H2 and HVR-H3, wherein further:
  • the HVR-H1 sequence is GFTFSXiSWIH; (SEQ ID NO: 5)
  • HVR-H2 sequence is AWIX2PYGGSX3YYADSVKG (SEQ ID NO: 6)
  • the HVR-H3 sequence is RHWPGGFDY, and (SEQ ID NO: 7)
  • the light chain comprises an HVR-L1 , HVR-L2 and HVR-L3, wherein further:
  • the HVR-L1 sequence is RASQX 4 X 5 X 6 TX7X 8 A (SEQ ID NO: 12)
  • the HVR-L2 sequence is SASX9LX10S; and (SEQ ID NO: 13)
  • the HVR-L3 sequence is QQX11X12X13X14PX15T;
  • SEQ ID NO: 14 wherein: Xi is D or G; X2 IS S or L; X3 is T or S; X4 is D or V; Xs is V or I; Cb ⁇ e S or N; X7 IS A or F; Xe is V or L; X 9 is F or T; X10 is Y or A; Xn is Y, G, F, or S; X12 is L, Y, F or W; X13 is Y, N, A, T, G, F or I; Xi 4 is H, V, P, T or I; X15 is A, W, R, P or T.
  • the heavy chain variable region comprises one or more framework sequences juxtaposed between the HVRs as: (FR-H1 )-(HVR-H1 )-(FR-H2)-(HVR-H2)-(FR-H3)-(HVR-H3)-(FR-H4), and the light chain variable regions comprises one or more framework sequences juxtaposed between the HVRs as: (FR-L1 )-(HVR-L1 )-(FR-L2)-(HVR-L2)-(FR-L3)-(HVR-L3)-(FR-L4).
  • the framework sequences are derived from human consensus framework sequences.
  • the heavy chain framework sequences are derived from a Kabat subgroup I, II, or III sequence.
  • the heavy chain framework sequence is a VH subgroup III consensus framework.
  • one or more of the heavy chain framework sequences are set forth as SEQ ID NOs: 8, 9, 10, and 1 1 .
  • the light chain framework sequences are derived from a Kabat kappa I, II, II or IV subgroup sequence.
  • the light chain framework sequences are VL kappa I consensus framework.
  • one or more of the light chain framework sequences are set forth as SEQ ID NOs: 15, 16, 17, and 18.
  • the antibody further comprises a human or murine constant region.
  • the human constant region is selected from the group consisting of lgG1 , lgG2, lgG2, lgG3, and lgG4.
  • the human constant region is lgG1 .
  • the murine constant region is selected from the group consisting of IgG 1 , lgG2A, lgG2B, and lgG3.
  • the antibody has reduced or minimal effector function.
  • the minimal effector function results from an“effector-less Fc mutation” or aglycosylation mutation.
  • the effector-less Fc mutation is an N297A or D265A/N297A substitution in the constant region.
  • an anti-PD-L1 antibody comprising a heavy chain and a light chain variable region sequence, wherein:
  • the heavy chain further comprises an HVR-H1 , HVR-H2 and an HVR-H3 sequence
  • GFTFSDSWIH SEQ ID NO: 19
  • AWISPYGGSTYYADSVKG SEQ ID NO: 20
  • RHWPGGFDY SEQ ID NO: 21
  • the light chain further comprises an HVR-L1 , HVR-L2 and an HVR-L3 sequence having at least 85% sequence identity to RASQDVSTAVA (SEQ ID NO: 22), SASFLYS (SEQ ID NO: 23) and QQYLYHPAT (SEQ ID NO: 24), respectively.
  • sequence identity is 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.
  • the heavy chain variable region comprises one or more framework sequences juxtaposed between the HVRs as: (FR-H1 )-(HVR-H1 )-(FR-H2)-(HVR-H2)-(FR-H3)-(HVR-H3)-(FR-H4), and the light chain variable regions comprises one or more framework sequences juxtaposed between the HVRs as: (FR-L1 )-(HVR-L1 )-(FR-L2)-(HVR-L2)-(FR-L3)-(HVR-L3)-(FR-L4).
  • the framework sequences are derived from human consensus framework sequences.
  • the heavy chain framework sequences are derived from a Kabat subgroup I, II, or III sequence. In a still further aspect, the heavy chain framework sequence is a VH subgroup III consensus framework. In a still further aspect, one or more of the heavy chain framework sequences are set forth as SEQ ID NOs: 8, 9,
  • the light chain framework sequences are derived from a Kabat kappa I, II, II, or IV subgroup sequence.
  • the light chain framework sequences are VL kappa I consensus framework.
  • one or more of the light chain framework sequences are set forth as SEQ ID NOs: 15, 16, 17, and 18.
  • the heavy chain variable region comprises one or more framework sequences juxtaposed between the HVRs as: (FR-H1 )-(HVR-H1 )-(FR-H2)-(HVR-H2)-(FR-H3)-(HVR-H3)-(FR-H4), and the light chain variable regions comprises one or more framework sequences juxtaposed between the HVRs as: (FR-L1 )-(HVR-L1 )-(FR-L2)-(HVR-L2)-(FR-L3)-(HVR-L3)-(FR-L4).
  • the framework sequences are derived from human consensus framework sequences.
  • the heavy chain framework sequences are derived from a Kabat subgroup I, II, or III sequence.
  • the heavy chain framework sequence is a VH subgroup III consensus framework.
  • one or more of the heavy chain framework sequences is the following:
  • the light chain framework sequences are derived from a Kabat kappa I, II, II or IV subgroup sequence. In a still further aspect, the light chain framework sequences are VL kappa I consensus framework. In a still further aspect, one or more of the light chain framework sequences is the following:
  • the antibody further comprises a human or murine constant region.
  • the human constant region is selected from the group consisting of lgG1 , lgG2, lgG2, lgG3, and lgG4.
  • the human constant region is lgG1 .
  • the murine constant region is selected from the group consisting of IgG 1 , lgG2A, lgG2B, and lgG3.
  • the antibody has reduced or minimal effector function.
  • the minimal effector function results from an“effector-less Fc mutation” or aglycosylation.
  • the effector less Fc mutation is an N297A or D265A/N297A substitution in the constant region.
  • an anti-PD-L1 antibody comprising a heavy chain and a light chain variable region sequence, wherein: (c) the heavy chain further comprises an HVR-H1 , HVR-H2 and an HVR-H3 sequence having at least 85% sequence identity to GFTFSDSWIH (SEQ ID NO: 19), AWISPYGGSTYYADSVKG (SEQ ID NO: 20) and RHWPGGFDY (SEQ ID NO: 21 ), respectively, and/or
  • the light chain further comprises an HVR-L1 , HVR-L2 and an HVR-L3 sequence having at least 85% sequence identity to RASQDVSTAVA (SEQ ID NO: 22), SASFLYS (SEQ ID NO: 23) and QQYLYHPAT (SEQ ID NO: 24), respectively.
  • sequence identity is 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%,
  • the heavy chain variable region comprises one or more framework sequences juxtaposed between the HVRs as: (FR-H1 )-(HVR-H1 )-(FR-H2)-(HVR-H2)-(FR-H3)-(HVR-H3)-(FR-H4), and the light chain variable regions comprises one or more framework sequences juxtaposed between the HVRs as: (FR-L1 )-(HVR-L1 )-(FR-L2)-(HVR-L2)-(FR-L3)-(HVR-L3)-(FR-L4).
  • the framework sequences are derived from human consensus framework sequences.
  • the heavy chain framework sequences are derived from a Kabat subgroup I, II, or III sequence. In a still further aspect, the heavy chain framework sequence is a VH subgroup III consensus framework. In a still further aspect, one or more of the heavy chain framework sequences are set forth as SEQ ID NOs: 8, 9, 10, and WGQGTLVTVSSASTK (SEQ ID NO: 29).
  • the light chain framework sequences are derived from a Kabat kappa I, II, II or IV subgroup sequence. In a still further aspect, the light chain framework sequences are VL kappa I consensus framework. In a still further aspect, one or more of the light chain framework sequences are set forth as SEQ ID NOs: 1 5, 16, 17, and 18. In a still further specific aspect, the antibody further comprises a human or murine constant region. In a still further aspect, the human constant region is selected from the group consisting of lgG1 , lgG2, lgG2, lgG3, and lgG4. In a still further specific aspect, the human constant region is lgG1 .
  • the murine constant region is selected from the group consisting of IgG 1 , lgG2A, lgG2B, and lgG3. In a still further aspect, the murine constant region in lgG2A. In a still further specific aspect, the antibody has reduced or minimal effector function.
  • the minimal effector function results from an“effector-less Fc mutation” or aglycosylation.
  • the effector-less Fc mutation is an N297A or D265A/N297A substitution in the constant region.
  • an isolated anti-PD-L1 antibody comprising a heavy chain and a light chain variable region sequence, wherein:
  • the heavy chain sequence has at least 85% sequence identity to the heavy chain sequence:
  • the light chain sequences has at least 85% sequence identity to the light chain sequence: DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGT DFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKR (SEQ ID NO: 4).
  • an isolated anti-PD-L1 antibody comprising a heavy chain and a light chain variable region sequence, wherein the light chain variable region sequence has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 4.
  • an isolated anti-PD-L1 antibody comprising a heavy chain and a light chain variable region sequence, wherein the heavy chain variable region sequence has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 25.
  • an isolated anti-PD-L1 antibody comprising a heavy chain and a light chain variable region sequence, wherein the light chain variable region sequence has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 4 and the heavy chain variable region sequence has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 25.
  • an isolated anti-PD-L1 antibody comprising a heavy chain and a light chain sequence, wherein:
  • the heavy chain sequence has at least 85% sequence identity to the heavy chain sequence:
  • KSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 30), and/or
  • the light chain sequences has at least 85% sequence identity to the light chain sequence: DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGT DFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFY PREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSF NRGEC (SEQ ID NO: 31 ).
  • an isolated anti-PD-L1 antibody comprising a heavy chain and a light chain sequence, wherein the light chain sequence has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 31 .
  • an isolated anti-PD-L1 antibody comprising a heavy chain and a light chain sequence, wherein the heavy chain sequence has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 30.
  • an isolated anti-PD-L1 antibody comprising a heavy chain and a light chain sequence, wherein the light chain sequence has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 31 and the heavy chain sequence has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 30.
  • the isolated anti-PD-L1 antibody is aglycosylated.
  • Glycosylation of antibodies is typically either N-linked or O-linked.
  • N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue.
  • the tripeptide sequences asparagine-X-serine and asparagine-X- threonine, where X is any amino acid except proline, are the recognition sequences for enzymatic attachment of the carbohydrate moiety to the asparagine side chain.
  • O-linked glycosylation refers to the attachment of one of the sugars N-aceylgalactosamine, galactose, or xylose to a
  • glycosylation sites form an antibody is conveniently accomplished by altering the amino acid sequence such that one of the above-described tripeptide sequences (for N-linked glycosylation sites) is removed. The alteration may be made by substitution of an asparagine, serine or threonine residue within the glycosylation site another amino acid residue (e.g., glycine, alanine or a conservative substitution).
  • the isolated anti-PD-L1 antibody can bind to a human PD-L1 , for example a human PD-L1 as shown in UniProtKB/Swiss-Prot Accession No.Q9NZQ7.1 , or a variant thereof.
  • nucleic acid encoding any of the antibodies described herein.
  • the nucleic acid further comprises a vector suitable for expression of the nucleic acid encoding any of the previously described anti-PD-L1 antibodies.
  • the vector is in a host cell suitable for expression of the nucleic acid.
  • the host cell is a eukaryotic cell or a prokaryotic cell.
  • the eukaryotic cell is a mammalian cell, such as Chinese hamster ovary (CHO) cell.
  • the antibody or antigen binding fragment thereof may be made using methods known in the art, for example, by a process comprising culturing a host cell containing nucleic acid encoding any of the previously described anti-PD-L1 antibodies or antigen-binding fragments in a form suitable for expression, under conditions suitable to produce such antibody or fragment, and recovering the antibody or fragment.
  • the immune checkpoint inhibitor is an antagonist directed against a co-inhibitory molecule (e.g., a CTLA-4 antagonist (e.g., an anti-CTLA-4 antibody), a TIM-3 antagonist (e.g., an anti- TIM-3 antibody), or a LAG-3 antagonist (e.g., an anti-LAG-3 antibody)), or any combination thereof.
  • a co-inhibitory molecule e.g., a CTLA-4 antagonist (e.g., an anti-CTLA-4 antibody), a TIM-3 antagonist (e.g., an anti- TIM-3 antibody), or a LAG-3 antagonist (e.g., an anti-LAG-3 antibody)
  • the immune checkpoint inhibitor is an antagonist directed against TIGIT (e.g., an anti- TIGIT antibody).
  • TIGIT e.g., an anti- TIGIT antibody
  • anti-TIG IT antibodies are described in US Patent Application Publication No. 2018/0186875 and in International Patent Application Publication No. WO 2017/053748, which are incorporated herein by reference in their entirety.
  • Example 1 AVANT study design and sample collection
  • DFS Disease-free survival
  • FFPE paraffin-embedded
  • c fluorouracil, leucovorin, and oxaliplatin.
  • d capecitabine (Xeloda) plus oxaliplatin.
  • the raw probe intensities were corrected for background using blank (water), and then normalized using the NanostringQCPro package in R.
  • Raw counts for 1256 tumor samples were log2 transformed, normalized (common mean and standard deviation for all samples), and gene-wise expression scores were further standardized across all samples by transformation to z-scores.
  • Quality control failures were flagged based on the first principal component of normalized counts; 67 outlier samples were identified. These yielded low overall counts, indicating insufficient input material or another source of assay failure and were removed from further analysis.
  • 12 patients had 2 samples each, all of which were excluded. 1 165 samples were deemed biomarker-evaluable samples. For 102 subjects for whom gene expression data was available, no matching clinical annotations were available. The samples from these subjects were excluded from downstream analysis, leaving a final total of 1062 samples.
  • RNAseq paired-end data for colon adenocarcinoma, breast carcinoma, lung adenocarcinoma and squamous carcinoma, head and neck squamous cell carcinoma, bladder carcinoma and ovarian carcinoma were downloaded from the National Cancer Institute Genomic Data Commons and analyzed using HTSeqGenie (Goldstein et al., Cell Rep, 16(10): 2605-2617, 2016).
  • KRAS and BRAF mutation information for colon tumors was downloaded from cBioPortal for Cancer Genomics (project Colorectal Adenocarcinoma TCGA provisional) on 09/1 8/2018.
  • the GSE39582 dataset was downloaded from Gene Expression Omnibus (GEO) (GEO accession: GSE39582) (Marisa et al., PLoS Med, 10: e1001453, 2013). RNAseq data for cell lines were obtained from Klijn et al., Nat Biotechnol, 33(3): 306-312, 2015).
  • the random forest algorithm in the consensus molecular subtype (CMS) classifier R package (Guinney et al., Nat Med, 21 : 1350-1356, 2015) was used to assign CMS labels to TCGA, GSE39582 samples, and colon cell lines. Since the AVANT dataset is on a different platform, i.e. , NANOSTRING ® , we first trained a prediction analysis of microarrays (PAM) classifier on the TCGA CRC samples for the prediction of CMS, using expression of the 829 genes on the CRC
  • PAM microarrays
  • MSI Microsatellite instability
  • KRAS KRAS status
  • BRAF status was obtained by qPCR.
  • MSI-low and microsatellite stable (MSS) patients were merged into the MSS category.
  • BRAF status was assessed as the presence or absence of a c. 1799T>A nucleotide substitution mutation, e.g., a mutation resulting in a V600E mutation in the amino acid sequence of BRAF.
  • KRAS status was assessed as the presence or absence of at least one nucleotide substitution mutation resulting in a G12A, G12R, G12D, G12C, G12S, G12V, G12D, or G13D mutation in the amino acid sequence of KRAS, as described in Marisa et al ., PLoS Med, 10: e1 001453, 2013, Lievre et al ., J Clin Oncol, 26: 374-379, 2008, and Lievre et al., Cancer Res, 66(8): 3992-3995, 2006).
  • the AVANT population was comparable to other early stage populations such as GSE39582 (Marisa et al., PLoS Med, 1 0: e1001453, 2013) and The Cancer Genome Atlas (TCGA) (Cancer Genome Atlas Network, Nature, 487: 330-337, 2012) in terms of prevalence of the four CMSs (Guinney et al., Nat. Med., 21 : 1350-1356, 2015), the predominant occurrence of microsatellite instability high (MSI-H) and BRAF mutations in the CMS1 group, and the predominant occurrence of KRAS mutations in the CMS3 group (Figs. 6A-6F and 7A-7F).
  • GSE39582 Marisa et al., PLoS Med, 1 0: e1001453, 2013
  • TCGA Cancer Genome Atlas Network, Nature, 487: 330-337, 2012
  • MSI-H microsatellite instability high
  • BRAF mutations in the CMS1 group the predominant occurrence of KRA
  • the independent cohort was the GSE39582 cohort of stage l-IV colon cancer (Marisa et al, PLoS Med 2013). For validation of the signature using the independent cohort
  • the AVANT signature accurately identified a high-risk adjuvant CRC population for recurrence- free survival (RFS) and OS in the GSE39582 cohort, thus validating the gene signature (Figs. 1 B and 17B).
  • the AVANT signature was significantly more effective at predicting survival, and thus at identifying a high-risk subpopulation, than other prognostic signatures (Oncotype Dx, T-effector, CAF, F- TBRS) (Calon et al. , Cancer Cell, 22: 571 -584, 2012; Bindea et al. , Immunity, 39: 782-795, 2013) and the molecular CMSs (Guinney et al., Nat Med, 21 : 1350-1356, 2015), not only in the AVANT population, as expected (Figs. 12 and 20), but more importantly in an independent dataset not selected to be high-risk (GSE39582; Marisa et al, PLoS Med 2013) (Figs. 1 G and 17F).
  • the AVANT signature conferred significant additional prognostic value when added to each of the previously published prognostic signatures we considered.
  • Pathway signatures were calculated as the average Z-score of all the genes contained in each signature.
  • T-effector signature was modified by removing GZMB and GZMA from the signature, which we refer to as the“modified T-effector” signature.
  • Fig. 1 C Genes in the proliferative signature are provided in Fig. 1 C and are as follows: RPL23, AXIN2, CDKN1B, DTX2, RPS6KA 1, KDM1A, RHOA, SP2, SHISA5, MLH1, CDCA5, E2F1, CENPM, CDK2, GMNN, and DNMT1. Expression of the proliferative genes did not associate significantly with DFS in the AVANT dataset (Fig. 1 D). In the GSE39582 dataset, the proliferative gene set was prognostic of RFS (Fig. 1 1 A). In both the AVANT dataset and the GSE39582 dataset, the proliferative gene set was prognostic of OS (Figs. 17C and 18A).
  • stromal and TGFp stromal signatures are provided in Fig. 1 C.
  • Genes in the stromal signature are ANGPT1, WBSCR17, TLL 1, CRYAB, ABCC9, IGFBP3, HEYL, DTX1, CD36, SPP1, RGS2, HBEGF, and FOS.
  • Genes in the TGFp stromal signature are SERPINB13, IGFBP1, APCDD1, SCD5, KDM5D, POU5F1, EPHA4, REG4, PCSK1, SMAD9, FLT1, TCF12, SMAD3, CDK6, and MAPK3.
  • High expression of stromal genes was correlated with lower DFS and OS in the AVANT dataset (Figs. 1 F and 1 7E).
  • the stromal gene sets were prognostic of RFS (Fig. 1 1 C), but were not prognostic of OS (Fig. 18C).
  • Fig. 1 C Genes in the immune signature are provided in Fig. 1 C and are as follows: TP73, TAP1, GZMB, TNF, CXCL 1, PDCD1, FCRL5, CXCR4, and CD28.
  • High expression of immune genes was correlated with higher DFS in the AVANT dataset (Fig. 1 E).
  • the immune gene signature was not prognostic of OS in the AVANT dataset (Fig. 17D).
  • checkpoint inhibitors Despite the broad therapeutic benefit of checkpoint inhibitor treatment across a variety of solid tumor indications, including MSI-high CRC (Overman et al., J. Clin. Oncol., 36: 773-779, 2018), checkpoint inhibitors have demonstrated little activity in MSS CRC tumors. We were therefore surprised to find a cluster of immune-related genes that are prognostic in MSS CRC tumors (Example 4/V).
  • GZMB significantly added prognostic value to the proliferative and stromal gene sets (Figs. 2G- 2J and 1 9G-19J; likelihood ratio test for improvement of fit adding GZMB expression to the stromal set, p 4.1 e-5 or the proliferative set, p 1 .4e-5).
  • concurrent high expression of GZMB and the proliferative gene set was associated with good prognosis in AVANT and GSE38592 (Figs. 2G-2H and 19G-1 9H).
  • T-effector signature including GZMB
  • CMS1 CRC patients and MSI-H patients were highly expressed in CMS1 CRC patients and in all other cancer types considered.
  • T-effector signature genes were highly correlated in expression (Fig. 3D).
  • GZMB was additionally expressed in CMS2 patients and in a fraction of MSS patients (Figs. 3E-3G).
  • Fig. 3D In CMS2 CRC patients, GZMB expression correlated poorly with the other T-effector signature genes (Fig. 3D), suggesting that its relevance and source may not be CD8+T cells in this subset of patients.
  • tSNE stochastic neighbor embedding
  • CRC tumor samples (stage II or III, pre-treated) were procured from an external vendor (Conversant Bio) and analyzed by mass cytometry as previously described (Takahashi et al ., Cytometry A, 91 (1 ), 39-47, 2016).
  • CRC single-cell suspensions were incubated with a cisplatin- based viability dye (Fluidigm) and Human TRUSTAIN FCXTM block (Biolegend) prior to staining with a 37 parameter isotope conjugated panel of monoclonal antibodies (mABs) (see Tables 5 and 6 for clones and vendors).
  • mABs monoclonal antibodies
  • CD45+ viable singlet cells were exported into new .FCS files using FlowJo software.
  • 37 immune markers excluding pan-markers like EPCAM and CD45; markers with broad signal like CD66 and Foxp3
  • the tSNE dimensionality reduction algorithm (adjClust package) was used to obtain the tSNE map shown in Fig.
  • GZMB As a potent extracellular matrix (ECM) remodeling agent, GZMB efficiently cleaves vitronectin, fibronectin, and laminin. GZMB pretreatment of a laminin matrix significantly inhibited cell spreading of colon cancer cell line LIM1215 in vitro (Buzza et al., J. Biol. Chem., 280: 23549-23558, 2005). Thus, via disruption of integrin-dependent adhesion, GZMB has been shown to inhibit tumor cell spreading, migration, and invasion on ECM, thereby potentially inhibiting tumorigenesis. Beyond the relevance to the matrix, the impact of GZMB expression on invasion varied across a panel of CRC cell lines in the same matrix (D’Eliseo et al., J. Exp. Clin.
  • Multivariate analysis in AVANT was performed by adding the clinical covariates age, levels of Cancer Embryonic Antigen (CEA) in the blood, Eastern Cooperative Oncology Group (ECOG) status, sex, and a combination of American Joint Committee on Cancer (AJCC) tumor status (II and III) and lymph node status (N1 and N2), referred to as strata. These covariates were individually found to be prognostic in both overall survival (OS) and DFS. Multivariate analysis in GSE39582 was performed by adding the clinical covariates age, sex, tumor stage (0-4), and lymph node status. These covariates were individually found to be prognostic in both OS and recurence- free survival (RFS).

Abstract

L'invention concerne des méthodes pronostiques et thérapeutiques pour le traitement du cancer colorectal faisant appel à une signature génique pronostique capturant des fonctions stromales, prolifératives et immunitaires. En particulier, l'invention concerne des méthodes de sélection, de pronostic et de traitement de patients.
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