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WO2025034542A1 - Panel de signature génique prédisant une réponse cancéreuse à un blocage de point de contrôle immunitaire et radiothérapie - Google Patents

Panel de signature génique prédisant une réponse cancéreuse à un blocage de point de contrôle immunitaire et radiothérapie Download PDF

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WO2025034542A1
WO2025034542A1 PCT/US2024/040687 US2024040687W WO2025034542A1 WO 2025034542 A1 WO2025034542 A1 WO 2025034542A1 US 2024040687 W US2024040687 W US 2024040687W WO 2025034542 A1 WO2025034542 A1 WO 2025034542A1
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immune checkpoint
genes
cancer
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therapy
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Tim Mcgraw
Nasser ALTORKI
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Cornell University
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    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57423—Specifically defined cancers of lung
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • Immune checkpoint blockade (ICB) therapies have transformed the management of solid tumors, although these successes are tempered by the low number (-20%) of patients achieving durable response with ICB monotherapy 1 .
  • Many variables likely contribute to ICB response and resistance, including tumor type and stage, cancer cell intrinsic mechanisms (e.g., oncogene driver, antigen presentation, mutational burden, immune checkpoint expression), tumor microenvironment factors (e.g., immune landscape) and individual intrinsic factors (e.g., metabolic health, etc.) 2 .
  • SBRT stereotactic body radiation therapy
  • the primary endpoint of the trial was major pathologic response (MPR) in the resected tumor, defined as residual viable tumor of less than or equal to 10% of the tumor bed.
  • MPR was achieved in 6.66% (2 of 30 subjects) in the monotherapy anti-PD-Ll arm (Arml) and in 53.3% (16 of 30 subjects) in the dual therapy anti-PD-Ll and SBRT arm (Arm2).
  • Response in dual therapy arm remained significantly higher than in the monotherapy arm even after adjustment for baseline PD-L1 expression 3 .
  • a method of identifying the likelihood of a lung cancer in a subject to respond to an immune checkpoint and radiation combination therapy or to an immune checkpoint monotherapy comprising: a) obtaining or providing a sample comprising cancer cells from a subject having the lung cancer; b) measuring one or more characteristics selected from (1) proliferation; (2) percentage of PD-L1 + cancer cells and/or PD-L1 expression; (3) number of CD4 + Th2 and/or Thl cells; (4) expressions of glycolysis genes and/or maximum standardized uptake value (SUVmax); (5) tumor mutational burdens (TMB); (6) CD8 + PD1 + and/or CD8 + granzyme B + T cells; (7) number of Tregs (FoxP3 + CD4 + cells); and (8) expressions of MHC-II genes in the cancer cells of the subject sample; and c) comparing said one or more characteristics to a control, wherein a significant increase in the one or more characteristics selected from (l)-(7) and
  • step (b) comprises measuring characteristics ( 1 )-(8) in the cancer cells of the sample, and wherein significant increases in all characteristics of (l)-(7) and a significant decrease in characteristic (8) in the cancer cells of the subject sample relative to the control identify the lung cancer as being more likely to respond to the immune checkpoint and radiation combination therapy or to the immune checkpoint monotherapy, and wherein significant decreases in all characteristics of (l)-(7) and a significant increase in characteristic (8) in the cancer cells of the subject sample relative to the control identify the lung cancer as being less likely to respond to the immune checkpoint and radiation combination therapy or to the immune checkpoint monotherapy.
  • a method of identifying the likelihood of a lung cancer in a subject to respond to an immune checkpoint and radiation combination therapy or to an immune checkpoint monotherapy comprising: a) obtaining or providing a sample comprising cancer cells from a subject having the lung cancer; b) measuring one or more characteristics selected from (1) proliferation; (2) percentage of PD-L1 + cancer cells and/or PD-L1 expression; (3) number of CD4 + Th2 cells; (4) expressions of glycolysis genes; (5) tumor mutational burdens (TMB), and (6) expressions of MHC-II genes in the cancer cells of the subject sample; and c) comparing said one or more characteristics to a control, wherein a significant increase in the one or more characteristics selected from ( l)-(5) and/or a significant decrease in characteristic (6) in the cancer cells of the subject sample relative to the control identifies the lung cancer as being more likely to respond to the immune checkpoint and radiation combination therapy or to the immune checkpoint monotherapy, and wherein a significant decrease in
  • step (b) comprises measuring characteristics ( 1 )-(6) in the cancer cells of the sample, and wherein significant increases in all characteristics of ( l)-(5) and a significant decrease in characteristic (6) in the cancer cells of the subject sample relative to the control identify the lung cancer as being more likely to respond to the immune checkpoint and radiation combination therapy or to the immune checkpoint monotherapy, and wherein significant decreases in all characteristics of (l)-(5) and a significant increase in characteristic (6) in the cancer cells of the subject sample relative to the control identify the lung cancer as being less likely to respond to the immune checkpoint and radiation combination therapy or to the immune checkpoint monotherapy.
  • the proliferation is measured by determining expressions of proliferation-associated genes.
  • the proliferation-associated genes comprise one or more genes associated with mitotic spindle, G2M check point, DNA repair, mTOR signaling, Myc signaling, or unfolded protein response.
  • the proliferation-associated genes comprise one or more genes selected from the full gene set or the modified gene set shown in Table 3.
  • the proliferation is measured by calculating a proliferation index (PI) using the full gene set or the modified gene set shown in Table 3.
  • PI proliferation index
  • the proliferation index is calculated using the method described in Example 2.
  • the proliferation index is calculated using a method described in Venet et al. (2011) PLoS Comput Biol 7, el002240. 10.1371/journal.pcbi.1002240, the content of which is incorporated herein by reference in its entirety.
  • the proliferation is measured by determining expression of ki67 and/or ki67 + cancer cells. In some embodiments, the proliferation is measured by determining expressions of gene signatures for cell cycle phases or number of cells in S, G2 and M phases of cell cycle. In some embodiments, the expressions of gene signatures for cell cycle phases are determined as show in Example 2. In some embodiments, the expressions of gene signatures for cell cycle phases are determined using a method described in Mizuno et al. (2009). BMC Genomics 10, 137. 10.1186/1471-2164-10-137, the content of which is incorporated herein by reference in its entirety.
  • the glycolysis genes comprise one or more genes selected from the group consisting of GLUT1, HK1, HK2, GPI, PFKP, ALDOA, TPI1, GAPDH, PGK1, PGAM1, ENO1, PKM, LDHA, and MCT1.
  • the glycolysis genes comprise GLUT1, HK2, GPI, PFKP, ALDOA, TPI1, GAPDH, PGK1, PGAM1, ENO1, PKM, LDHA, and MCT1.
  • the MHC-II genes comprise one or more genes selected from the group consisting of HLA-DMA, HLA-DMB, HLA-DOA, HLA-DOB, HLA-DPA1, HLA-DPB1, HLA-DQA1, HLA-DQA2, HLA-DQB1, HLA-DQB2, HLA-DRA, HLA- DRB1, and HLA-DRB5.
  • the MHC-II genes comprise HLA-DMA, HLA-DPB1, HLA-DQB2, and HLA-DRB5.
  • a method of identifying the likelihood of a lung cancer in a subject to respond to an immune checkpoint and radiation combination therapy or to an immune checkpoint monotherapy comprising: a) obtaining or providing a sample comprising cancer cells from a subject having the lung cancer; b) measuring the copy number, amount, and/or activity of one or more genes from number 1-135 in Table 2 and/or of one or more genes from number 136-140 in Table 2 in the cancer cells of the subject sample; and c) comparing said copy number, amount, and/or activity of the one or more genes to a control, wherein a significantly increased copy number, amount, and/or activity of one or more genes from number 1-135 in Table 2, and/or a significantly decreased copy number, amount, and/or activity of one or more genes from number 136-140 in Table 2, in the cancer cells of the subject sample relative to the control identifies the lung cancer as being more likely to respond to the immune checkpoint and radiation combination therapy or to the immune checkpoint monotherapy, and where
  • step (b) comprises measuring expressions of all genes from number 1-140 in Table 2, and wherein significantly increased expressions of all genes from number 1-135 in Table 2, and significantly decreased expressions of all genes from number 136-140 in Table 2, in the cancer cells of the subject sample relative to the control identifies the lung cancer as being more likely to respond to the immune checkpoint and radiation combination therapy or to the immune checkpoint monotherapy, and wherein significantly decreased expressions of all genes from number 1-135 in Table 2, and significantly increased expressions of all genes from number 136-140 in Table 2, in the cancer cells of the subject sample relative to the control identifies the lung cancer as being less likely to respond to the immune checkpoint and radiation combination therapy or to the immune checkpoint monotherapy.
  • a method of identifying the likelihood of a lung cancer in a subject to respond to an immune checkpoint and radiation combination therapy or to an immune checkpoint monotherapy comprising: a) obtaining or providing a sample comprising cancer cells from a subject having the lung cancer; and b) measuring expressions of 140 genes in Table 2 in the cancer cells of the subject sample; wherein the lung cancer is more likely to respond to the immune checkpoint and radiation combination therapy or to the immune checkpoint monotherapy if the expressions of the 140 genes in the subject sample is clustered into a High PI group, and wherein the lung cancer is less likely to respond to the immune checkpoint and radiation combination therapy or to the immune checkpoint monotherapy if the expressions of the 140 genes in the subject sample is clustered into a Low PI group.
  • the High PI group is the High PI group shown in FIG. 2A
  • the Low PI group is the Low PI group shown in FIG. 2A
  • the High PI group is the High PI cluster of TCGA LU AD shown in FIG. 4 A
  • the Low PI group is the Low PI cluster of TCGA LU AD shown in FIG. 4A
  • the High PI group is the High PI cluster of TCGA LUSC shown in FIG. 4B
  • the Low PI group is the Low PI cluster of TCGA LUSC shown in FIG. 4B.
  • the responsiveness to the immune checkpoint and radiation combination therapy or to the immune checkpoint monotherapy is determined by achieving major pathological response (MPR).
  • MPR major pathological response
  • the lung cancer is non-small cell lung cancer (NSCLC).
  • NSCLC non-small cell lung cancer
  • the NSCLC is a NSCLC with clinical stages I-IIIA.
  • the methods described herein further comprises recommending, prescribing, or administering immune checkpoint and radiation combination therapy or to the immune checkpoint monotherapy if the lung cancer is determined to be more likely to respond to the immune checkpoint and radiation combination therapy or to the immune checkpoint monotherapy. In some embodiments, the methods described herein further comprises recommending, prescribing, or administering an anti-cancer therapy other than or in addition to the immune checkpoint and radiation combination therapy or to the immune checkpoint monotherapy if the lung cancer is determined to be less likely to respond to the immune checkpoint and radiation combination therapy or to immune checkpoint monotherapy.
  • the method is for identifying the likelihood of a lung cancer in a subject to respond to an immune checkpoint and radiation combination therapy.
  • the immune checkpoint therapy is an immune check point blockade (ICB) therapy.
  • the immune checkpoint is selected from the group consisting of CTLA-4, PD-1, VISTA, B7-H2, B7-H3, PD-L1, B7-H4, B7-H6, ICOS, HVEM, PD-L2, CD160, gp49B, PIR-B, KIR family receptors, TIM-1, TIM-3, TIM-4, LAG-3, GITR, 4-IBB, OX-40, BTLA, SIRPalpha (CD47), CD48, 2B4 (CD244), B7.1, B7.2, ILT-2, ILT-4, TIGIT, HHLA2, butyrophilins, and A2aR.
  • the immune checkpoint is PD1, PD-L1, or CTLA-4.
  • the ICB is anti-PD-Ll antibody.
  • the anti-PD-Ll antibody is Durvalumab.
  • the radiation therapy is stereotactic body radiation therapy (SBRT) therapy.
  • SBRT is a sub-ablative (clinically non-curative) dose of SBRT.
  • the sub-ablative dose is 8 Gy x 3.
  • the immune checkpoint and radiation combination therapy and/or the immune checkpoint monotherapy is a neoadjuvant therapy.
  • a method of identifying the likelihood of a solid tumor in a subject to respond to an immune checkpoint therapy comprising: a) obtaining or providing a sample comprising cancer cells from a subject having the solid tumor; b) measuring the copy number, amount, and/or activity of one or more genes from number 1-135 in Table 2 and/or of one or more genes from number 136-140 in Table 2 in the cancer cells of the subject sample; and c) comparing said copy number, amount, and/or activity of the one or more genes to a control, wherein a significantly increased copy number, amount, and/or activity of one or more genes from number 1-135 in Table 2, and/or a significantly decreased copy number, amount, and/or activity of one or more genes from number 136-140 in Table 2, in the cancer cells of the subject sample relative to the control identifies the solid tumor as being more likely to respond to the immune checkpoint therapy, and wherein a significantly decreased copy number, amount, and/or activity of one or more genes from number 1
  • step (b) comprises measuring expressions of all genes from number 1-140 in Table 2 in the cancer cells of the subject sample; and wherein significantly increased expressions of all genes from number 1-135 in Table 2, and significantly decreased expressions of all genes from number 136-140 in Table 2, in the cancer cells of the subject sample relative to the control identifies the solid tumor as being more likely to respond to the immune checkpoint therapy, and wherein significantly decreased expressions of all genes from number 1-135 in Table 2, and significantly increased expressions of all genes from number 136-140 in Table 2, in the cancer cells of the subject sample relative to the control identifies the solid tumor as being less likely to respond to the immune checkpoint therapy.
  • the solid tumor is selected from the group consisting of lung adenocarcinoma (LU AD), melanoma, breast, prostate, and pancreas cancers. In some embodiments, the solid tumor is not colon cancer or lung squamous cancer (LUSC).
  • LU AD lung adenocarcinoma
  • melanoma breast, prostate, and pancreas cancers.
  • the solid tumor is not colon cancer or lung squamous cancer (LUSC).
  • a method of identifying the likelihood of a solid tumor in a subject to respond to an immune checkpoint therapy comprising: a) obtaining or providing a sample comprising cancer cells from a subject having the lung cancer; and b) measuring expressions of 140 genes in Table 2 in the cancer cells of the subject sample; wherein the solid tumor is more likely to respond to the immune checkpoint and radiation combination therapy or to the immune checkpoint monotherapy if the expressions of the 140 genes in the subject sample is clustered into a High PI group, and wherein the solid tumor is less likely to respond to the immune checkpoint and radiation combination therapy or to the immune checkpoint monotherapy if the expressions of the 140 genes in the subject sample is clustered into a Low PI group.
  • the solid tumor is a lung adenocarcinoma (LUCD), and wherein the High PI group is the High PI cluster of TCGA LU AD shown in FIG. 4 A, and the Low PI group is the Low PI cluster of TCGA LU AD shown in FIG. 4A.
  • LUCD lung adenocarcinoma
  • the solid tumor is a melanoma
  • the High PI group is the High PI cluster of melanoma shown in FIG. 5A
  • the Low PI group is the Low PI cluster of melanoma shown in FIG. 5A.
  • the solid tumor is a breast cancer
  • the High PI group is the High PI cluster of breast cancer shown in FIG. 5B
  • the Low PI group is the Low PI cluster of breast cancer shown in FIG. 5B.
  • the solid tumor is a breast cancer
  • the High PI group is the High PI cluster of prostate cancer shown in FIG. 5C
  • the Low PI group is the Low PI cluster of prostate cancer shown in FIG. 5C.
  • the solid tumor is a breast cancer
  • the High PI group is the High PI cluster of pancreas cancer shown in FIG. 5D
  • the Low PI group is the Low PI cluster of pancreas cancer shown in FIG. 5D.
  • the responsiveness to the immune checkpoint therapy is determined by achieving increased disease-free survival (DFS) and/or overall survival (OS) than the control.
  • DFS disease-free survival
  • OS overall survival
  • the immune checkpoint therapy is an immune checkpoint monotherapy. In some embodiments, wherein the immune checkpoint therapy is an immune check point blockade (ICB) therapy.
  • the immune checkpoint is selected from the group consisting of CTLA-4, PD-1, VISTA, B7-H2, B7-H3, PD-L1, B7-H4, B7-H6, ICOS, HVEM, PD-L2, CD160, gp49B, PIR-B, KIR family receptors, TIM-1, TIM-3, TIM-4, LAG-3, GITR, 4-IBB, OX-40, BTLA, SIRPalpha (CD47), CD48, 2B4 (CD244), B7.1, B7.2, ILT-2, ILT-4, TIGIT, HHLA2, butyrophilins, and A2aR.
  • the immune checkpoint is PD1, PD-L1, or CTLA-4.
  • the ICB is anti-PD-Ll antibody.
  • the anti-PD-Ll antibody is Durvalumab.
  • the immune checkpoint therapy is a neoadjuvant therapy.
  • the expression and/or amount of a gene is determined at an mRNA level and/or a protein level. In some embodiments, the expression of a gene is determined at an mRNA level using RNA-seq.
  • control is a reference value. In some embodiments, the control is determined from a cancerous or non-cancerous sample from either the subject or a member of the same species to which the subject belongs. In some embodiments, the control is a sample that comprises cells or does not comprise cells. In some embodiments, the control sample comprises cancer cells that respond to the immune checkpoint and radiation combination therapy or to the immune checkpoint monotherapy, or that do not respond to the immune checkpoint and radiation combination therapy or to the immune checkpoint monotherapy.
  • a method of predicting survival of a subject afflicted with a solid tumor comprising: a) obtaining or providing a sample comprising cancer cells from a subject having the solid tumor; b) measuring the copy number, amount, and/or activity of one or more genes from number 1-135 in Table 2 and/or of one or more genes from number 136-140 in Table 2 in the cancer cells of the subject sample; and c) comparing said copy number, amount, and/or activity of the one or more genes to a control, wherein a significantly increased copy number, amount, and/or activity of one or more genes from number 1-135 in Table 2, and/or a significantly decreased copy number, amount, and/or activity of one or more genes from number 136-140 in Table 2, in the cancer cells of the subject sample relative to the control identifies the subject as having reduced survival, and wherein a significantly decreased copy number, amount, and/or activity of one or more genes from number 1-135 in Table 2, and/or a significantly increased copy number, amount, amount,
  • step (b) comprises measuring expressions of all 1-140 genes in Table 2 and generating a gene enrichment score, wherein a higher expression of 140-gene set score in a multivariable Cox-proportional hazards model relative to the control identifies the subject as having reduced survival, and wherein a lower expression of 140-gene set score in a multivariable Cox-proportional hazards model relative to the control identifies the subject as having increased survival.
  • the gene enrichment score adjusts for EGFR mutation status, TMB, PD-L1 and PD1 expression, tumor stage, patient’s age, and gender.
  • the solid tumor is lung adenocarcinoma (LU AD), melanoma, breast, prostate, or pancreas.
  • a method of predicting survival of a subject afflicted with a solid tumor comprising: a) obtaining or providing a sample comprising cancer cells from a subject having the solid tumor; b) measuring expressions of 140 genes in Table 2 in the cancer cells of the subject sample; wherein the subject has a reduced survival if the expressions of the 140 genes in the subject sample is clustered into a High PI group, and wherein the subject has a increased survival if the expressions of the 140 genes in the subject sample is clustered into a Low PI group.
  • the solid tumor is a lung adenocarcinoma (LUCD), and wherein the High PI group is the High PI cluster of TCGA LU AD shown in FIG. 4 A, and the Low PI group is the Low PI cluster of TCGA LU AD shown in FIG. 4A.
  • LUCD lung adenocarcinoma
  • the solid tumor is a melanoma
  • the High PI group is the High PI cluster of melanoma shown in FIG. 5A
  • the Low PI group is the Low PI cluster of melanoma shown in FIG. 5A.
  • the solid tumor is a breast cancer
  • the High PI group is the High PI cluster of breast cancer shown in FIG. 5B
  • the Low PI group is the Low PI cluster of breast cancer shown in FIG. 5B.
  • the solid tumor is a breast cancer
  • the High PI group is the High PI cluster of prostate cancer shown in FIG. 5C
  • the Low PI group is the Low PI cluster of prostate cancer shown in FIG. 5C.
  • the solid tumor is a breast cancer
  • the High PI group is the High PI cluster of pancreas cancer shown in FIG. 5D
  • the Low PI group is the Low PI cluster of pancreas cancer shown in FIG. 5D.
  • the solid tumor is not colon cancer or lung squamous cancer (LUSC).
  • the survival is disease-free survival (DFS) and/or overall survival (OS).
  • the sample is selected from the group consisting of organs, tissue, body fluids and cells.
  • the body fluid is selected from the group consisting of whole blood, serum, plasma, sputum, spinal fluid, lymph fluid, skin secretions, respiratory secrections, intestinal secretions, genitourninary tract secretions, tears, milk, buccal scrape, saliva, cerebrospinal fluid, urine, and stool.
  • the sample is whole blood, serum or plasma.
  • the subject is a mammal.
  • the mammal is a mouse or a human.
  • the mammal is a human.
  • Figures 1A-1G show pretreatment tumor characteristics of responders (MPR) to dual ICB and SBRT therapy (Arm2).
  • Figure ID Pretreatment proliferation indexes (sum z-scores) determined by the expression of 113 genes, and by a modified set of 73 genes excluding the 40 genes that are part of the 140-gene set used for clustering (Table 3).
  • Figures 2A-2H show that characteristics of proliferation and cancer cell PD-L1 expression establish two groups of pretreatment NSCLC tumors.
  • PI sum z-scores determined based on expression of 113 genes (Table 3). Mean ⁇ SEM, Unpaired, two-sided Mann- Whitney test.
  • Figure 2C Figure 2C.
  • Figures 3A-3K show that pretreatment samples clustered by proliferation genes reveal additional characteristics of the clusters.
  • Figure 3C Pretreatment expressions of MHC-II genes.
  • CD3 cells per mm 2 determined by immunofluorescence imaging analyses of five samples of each group. Mean ⁇ SEM. Unpaired, two-sided Mann-Whitney test.
  • Figure 3G CD3 + CD8 + cells per mm 2 determined by immunofluorescence imaging analyses of five samples of each group. Mean ⁇ SEM. Unpaired, two-sided Mann-Whitney test.
  • Figure 3H CD3 + CD8 + GZB + cells as a fraction of cells from immunofluorescence imaging analyses of five Low PI and four High PI tumors. One High PI sample had no CD3 + CD8 + cells. GZB, granzyme B. Mean ⁇ SEM. Unpaired, two-sided Mann-Whitney test.
  • Figure 31 The first sample was obtained from immunofluorescence imaging analyses of five Low PI and four High PI tumors.
  • GZB granzyme B.
  • CD3 + CD8 + PD1 + cells as a fraction of CD3 + CD8 + cells determined by immunofluorescence imaging analyses of five Low PI and four High PI tumors. One High PI tumor had no CD3 + CD8 + cells. Mean ⁇ SEM. Unpaired, two-sided Mann-Whitney test.
  • Figure 3J CD3 + CD4 + FoxP3 + cells (Tregs) as a fraction of CD3 + CD4 + cells determined by immunofluorescence imaging analyses of five Low PI and five High PI samples. Mean ⁇ SEM. Unpaired, two-sided Mann-Whitney test.
  • Figures 4A-4K shows MPR associated with adaptive immune responses.
  • Post treatment proliferation indexes (sum z-scores) determined by the expression of 113 genes, and by a modified gene set of 73 genes that excluding the 40 genes that are part of the 140- gene set used for clustering (Table 3).
  • Figures 5A-5I show that TCGA NSCLC tumors clustered by 140-gene signature are associated with survival in LU AD but not LUSC.
  • Figure 5C & Figure 5D DFS and OS of LUAD Low and High PI clusters.
  • Figure 5E Multivariant Cox regression DFS in LUAD using covariates listed.
  • Reference groups for EGFR mutation status is EGFR wild type, for Gender is Female, and for Tumor Stage is Stage I.
  • Figure 5F & Figure 5G DFS and OS of LUSC Low and High clusters.
  • Figure 5H Multivariant Cox regression DFS in LUSC using covariates listed. Reference groups as in panels Figure 5D and Figure 5E.
  • Figure 51 140-gene set enrichment scores.
  • Figures 6A-6J show 140-gene set associated with DFS.
  • Figures 7A-7C shows disease-free survival.
  • HR hazard ratio and CI, 95% confidence interval.
  • Figures 8A-8C shows clinical trial design and outcomes. Related to Figure 1.
  • Figure 8A Schematic of the trial design.
  • Figures 9A-9L show geatures of High and Low PI tumors.
  • Figure 9A Clustering of 16 monotherapy arm pretreatment samples by the 140 genes differentially expressed between MPR and No MPR of the dual therapy arm (Fig. ID).
  • Figure 9D Volcano plot of genes differentially expressed between low and tumor groups.
  • Figure 9E GSEA Hallmark pathways enriched in High PI group.
  • Figure 9F- Figure 9G Expressions of immune function genes. Data are scaled to the average FPKM expression in the Low PI tumor group. No significant differences (alpha 0.05) ANOVA, Kruskal-Wallis test.
  • Figure 9L Some clinical characteristics of Low and High PI tumor patient groups.
  • Figures 10A-10E show pre and post treatment features of tumors with and without MPR.
  • Figure 10C Heat map of the effect of combination therapy on expressions of the 140-gene set.
  • Figures 11A-11L show identification of MPR-signature based clusters in TCGA- LUAD. Related to Figure 5.
  • Figure HA Supervised analysis to identify robust clusters in the LU AD cohort. Left panel shows the work flow and the right panel shows the silhouette plot of the final clustering.
  • FIG. HD Fold change in expressions of MHC-I presentation genes.
  • Figure HE Fold change in gene expressions of immune modulatory genes.
  • Figure HF- Figure 11 J Enrichments calculated by xCell deconvolution of Th2 CD4 + T cells, Thl CD4 + T cells macrophages, B cells and dendritic cells.
  • Figures 12A-12J show identification of MPR-signature based clusters in TCGA- LUSC. Related to Figure 5.
  • Figure 12A Supervised analysis to identify robust clusters in the LUSC cohort. Left panel shows the workflow used, and the right panel shows the silhouette plot of the final clustering.
  • Figure 12C Fold change in gene expressions of immune modulatory genes.
  • Figure 12G Boxplot showing differences in total mutation counts between TCGA-LUSC Low and High PI clusters. Wilcoxon signed-rank test p-value.
  • Figures 13A-13E show identification of 140-gene based clusters in five TCGA solid tumors. Related to Figure 6.
  • neoadjuvant anti-PD-Ll and sub-ablative stereotactic body radiation therapy is associated with higher rates of major pathologic response compared to anti-PD-Ll alone.
  • SBRT sub-ablative stereotactic body radiation therapy
  • the present disclosure is based on the identification of a 140-gene set, enriched in genes characteristic of highly proliferating cells, associated with response to the dual therapy. Analysis of on- treatment transcriptome data indicate roles for T and B cells in response.
  • the 140-gene set is associated with disease-free survival when applied to the combined trial arms. This 140-gene set identifies a subclass of tumors in all seven Tumor Cancer Genome Atlas tumor types examined.
  • This signature of increased proliferation is accompanied by select changes in expressions of glycolysis genes and immune-related genes.
  • the changes in immune genes, indicative of a co-existing immune suppressive microenvironment include increased PD-L1 expression, enrichment of Th2 CD4 T cells and reduced expressions of MHC-II genes.
  • the 140-gene set is associated with disease-free survival independent of treatment arm, suggesting that it is predictive of response to PD-L1 ICB therapy. When this 140-gene set was applied to the NSCLC tumors in the Cancer Genome Atlas (TCGA), both lung adenocarcinoma (LU AD) and squamous cell carcinoma tumors clustered into two groups.
  • TCGA Cancer Genome Atlas
  • LU AD lung adenocarcinoma
  • squamous cell carcinoma tumors clustered into two groups.
  • 140 gene signature was identified with RNAseq data from subjects with early-stage non-small cell lung cancer in a clinical trial of AstraZeneca’s durvalumab with or without stereotactic body radiation therapy. In that data, the panel defines a new molecular subclass of lung adenocarcinoma likely respond to ICB + radiation. The panel has since been applied to melanoma, breast, prostate, pancreas, and colon cancers.
  • a method of identifying the likelihood of a lung cancer in a subject to respond to a dual immune checkpoint and radiation therapy or to an immune checkpoint monotherapy comprising: a) obtaining or providing a sample comprising cancer cells from a subject having the lung cancer; b) measuring one or more characteristics selected from (1) proliferation; (2) percentage of PD- Ll + cancer cells and/or PD-L1 expression; (3) number of Th2 CD4 + cells in the cancer cells of the subject sample; (4) expressions of glycolysis genes; (5) tumor mutational burdens (TMB), and (6) expressions of MHC-II genes; and c) comparing said one or more characteristics in a control, wherein a significant increase in the one or more characteristics selected from ( 1 )-(5) and/or a significant decrease in characteristic (6) in the cancer cells of the subject sample relative to the control identifies the lung cancer as being more likely to respond to the dual immune checkpoint and radiation therapy or to the immune checkpoint monotherapy, and wherein
  • the proliferation is measured by determining expressions of proliferation-associated genes. In some embodiments, the proliferation is measured by calculating a proliferation index (PI) using the full gene set or the modified gene set shown in Table 3. In some embodiments, the proliferation is measured by determining expression of Ki67. In some embodiments, the proliferation is measured by determining gene signatures for cell cycle phases or number of cells in S, G2 and M phases of cell cycle.
  • PI proliferation index
  • a method of identifying the likelihood of a lung cancer in a subject to respond to a dual immune checkpoint and radiation therapy or to an immune checkpoint monotherapy comprising: a) obtaining or providing a sample comprising cancer cells from a subject having the lung cancer; b) measuring the copy number, amount, and/or activity of one or more genes from number 1-135 in Table 2 and/or of one or more genes from number 136-140 in Table 2 in the cancer cells of the subject sample; and c) comparing said copy number, amount, and/or activity of the one or more genes in a control, wherein a significantly increased copy number, amount, and/or activity of one or more genes from number 1-135 in Table 2, and/or a significantly decreased copy number, amount, and/or activity of one or more genes from number 136-140 in Table 2, in the cancer cells of the subject sample relative to the control identifies the lung cancer as being more likely to respond to the dual immune checkpoint and radiation therapy or to the immune checkpoint monotherapy, and
  • the responsiveness to the dual immune checkpoint and radiation therapy or to an immune checkpoint monotherapy is determined by achieving major pathological response (MPR).
  • MPR major pathological response
  • the lung cancer is NSCLC, e.g., a NSCLC with clinical stages I-IIIA.
  • the method described herein further comprises recommending, prescribing, or administering the dual immune checkpoint and radiation therapy or the immune checkpoint monotherapy if the lung cancer is determined to be more likely to respond to the dual immune checkpoint and radiation therapy or to the immune checkpoint monotherapy. In some embodiments, the method described herein further comprises recommending, prescribing, or administering an anti-cancer therapy other than or in addition to the dual immune checkpoint and radiation therapy or to the immune checkpoint monotherapy if the lung cancer is determined to be less likely to respond to the dual immune checkpoint and radiation therapy or to the immune checkpoint monotherapy.
  • the immune checkpoint therapy is an immune check point blockade (ICB) therapy.
  • the immune checkpoint is PD1, PD-L1, or CTLA-4.
  • the ICB is antibody.
  • the anti-PD-Ll antibody is Durvalumab.
  • the radiation therapy is stereotactic body radiation therapy (SBRT) therapy.
  • a method of identifying the likelihood of a solid tumor in a subject to respond to an immune checkpoint therapy comprising: a) obtaining or providing a sample comprising cancer cells from a subject having the solid tumor; b) measuring the copy number, amount, and/or activity of one or more genes from number 1-135 in Table 2 and/or of one or more genes from number 136-140 in Table 2 in the cancer cells of the subject sample; and c) comparing said copy number, amount, and/or activity of the one or more genes in a control, wherein a significantly increased copy number, amount, and/or activity of one or more genes from number 1-135 in Table 2, and/or a significantly decreased copy number, amount, and/or activity of one or more genes from number 136-140 in Table 2, in the cancer cells of the subject sample relative to the control identifies the solid tumor as being more likely to respond to the immune checkpoint therapy, and wherein a significantly decreased copy number, amount, and/or activity of one or more genes from number 1
  • the responsiveness to the immune checkpoint therapy is determined by achieving increased disease-free survival (DFS) than the control.
  • DFS disease-free survival
  • the solid tumor is selected from the group consisting of lung, melanoma, breast, prostate, and pancreas cancers.
  • the method described herein further comprises recommending, prescribing, or administering the immune check point therapy if the lung cancer is determined to be more likely to respond to the immune check point therapy.
  • the method described herein further comprises recommending, prescribing, or administering the immune check point therapy if the lung cancer is determined to be less likely to respond to the immune check point therapy.
  • the immune checkpoint therapy is an immune check point blockade (ICB) therapy.
  • the immune checkpoint is PD1, PD-L1, or CTLA-4.
  • the ICB is anti-PD-Ll antibody.
  • the anti-PD-Ll antibody is Durvalumab.
  • control is determined from a cancerous or non-cancerous sample from either the subject or a member of the same species to which the subject belongs.
  • control is a sample that comprises cells or does not comprise cells.
  • control sample comprises cancer cells that respond to the dual immune checkpoint and radiation therapy or to the immune checkpoint monotherapy, or that do not respond to the dual immune checkpoint and radiation therapy or to the immune checkpoint monotherapy.
  • the subject is a mammal, e.g., a mouse or a human.
  • an element means one element or more than one element.
  • “About” and “approximately” shall generally mean an acceptable degree of error for the quantity measured given the nature or precision of the measurements. Typically, exemplary degrees of error are within 20%, preferably within 10%, and more preferably within 5% of a given value or range of values. Alternatively, and particularly in biological systems, the terms “about” and “approximately” may mean values that are within an order of magnitude, preferably within 5-fold and more preferably within 2-fold of a given value. Numerical quantities given herein are approximate unless stated otherwise, meaning that the term “about” or “approximately” can be inferred when not expressly stated.
  • the amount of a biomarker (e.g., one or more genes described herein such as one or more gene listed in Table 2) in a subject is “significantly” higher or lower than the normal amount of the biomarker, if the amount of the biomarker is greater or less, respectively, than the normal level by an amount greater than the standard error of the assay employed to assess amount, and preferably at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 300%, 350%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% or than that amount.
  • a biomarker e.g., one or more genes described herein such as one or more gene listed in Table 2
  • the amount of the biomarker in the subject can be considered “significantly” higher or lower than the normal amount if the amount is at least about two, and preferably at least about three, four, or five times, higher or lower, respectively, than the normal amount of the biomarker.
  • Such “significance” can also be applied to any other measured parameter described herein, such as for expression, inhibition, activity, and the like.
  • the term “assigned score” refers to the numerical value designated for each of the biomarkers after being measured in a patient sample.
  • the assigned score correlates to the absence, presence or inferred amount of the biomarker in the sample.
  • the assigned score can be generated manually (e.g., by visual inspection) or with the aid of instrumentation for image acquisition and analysis.
  • the assigned score is determined by a qualitative assessment, for example, detection of a fluorescent readout on a graded scale, or quantitative assessment.
  • an “aggregate score,” which refers to the combination of assigned scores from a plurality of measured biomarkers, is determined.
  • the aggregate score may be a summation of assigned scores.
  • combination of assigned scores may involve performing mathematical operations on the assigned scores before combining them into an aggregate score.
  • the aggregate score is also referred to herein as the “predictive score.”
  • biomarker refers to a measurable parameter of the present invention that has been determined to be predictive of (1) response and/or survival of a subject with a specific condition (e.g., lung cancer) to an agent or therapy described herein (e.g., an immune checkpoint blockade such as an anti-PD-Ll therapy), either alone or in combination with at least one other therapies (e.g., a radiation therapy such as SBRT), or (2) outcome and/or survival of a subject with a specific condition (e.g., a solid tumor such as melanoma, lung, breast, pancreas, or prostate cancer).
  • a specific condition e.g., a solid tumor such as melanoma, lung, breast, pancreas, or prostate cancer.
  • Biomarkers can include, without limitation, gene, peptide, protein, metabolite, and clinical characteristics of a subject, including those shown in the Tables (e.g., genes listed in Table 2), the Examples, the Figures, and otherwise described herein.
  • body fluid refers to fluids that are excreted or secreted from the body as well as fluids that are normally not (e.g. amniotic fluid, aqueous humor, bile, blood and blood plasma, cerebrospinal fluid, cerumen and earwax, cowper’s fluid or pre-ejaculatory fluid, chyle, chyme, stool, female ejaculate, interstitial fluid, intracellular fluid, lymph, menses, breast milk, mucus, pleural fluid, pus, saliva, sebum, semen, serum, sweat, synovial fluid, tears, urine, vaginal lubrication, vitreous humor, vomit).
  • any body fluid may be taken to detect and/or measure at least one biomarker described herein.
  • control refers to any reference standard suitable to provide a comparison to the biomarkers/products in the test sample.
  • the control comprises obtaining a “control sample” from which product or biomarker levels are detected and compared to the product or biomarker levels from the test sample.
  • a control sample may comprise any suitable sample, including but not limited to a sample from a control subject (can be stored sample or previous sample measurement) with a known outcome; normal tissue or cells isolated from a subject, a tissue or cell sample isolated from a normal subject, or a primary cells/tissues obtained from a depository.
  • control may comprise a reference standard expression product or biomarker level from any suitable source, including but not limited to housekeeping genes, an expression product level range from normal tissue (or other previously analyzed control sample), a previously determined expression product level range within a test sample from a group of patients, or a set of patients with a certain outcome or receiving a certain treatment.
  • control samples and reference standard product or biomarker levels can be used in combination as controls in the methods of the present invention.
  • the specific product or biomarker level of each patient can be assigned to a percentile level of expression, or expressed as either higher or lower than the mean or average of the reference standard expression level.
  • the control may also comprise a measured value for example, average level of expression of a particular gene in a population compared to the level of expression of a housekeeping gene in the same population.
  • administering is intended to include routes of administration which allow an agent (such as the compositions described herein) to perform its intended function.
  • routes of administration for treatment of a body which can be used include injection (subcutaneous, intravenous, parenterally, intraperitoneally, intrathecal, etc.), oral, inhalation, and transdermal routes.
  • the injection can be bolus injections or can be continuous infusion.
  • the agent can be coated with or disposed in a selected material to protect it from natural conditions which may detrimentally affect its ability to perform its intended function.
  • the agent may be administered alone, or in conjunction with a pharmaceutically acceptable carrier.
  • expression signature refers to a group of two or more coordinately expressed biomarkers.
  • the genes, proteins, metabolites, and the like making up this signature may be expressed in a specific cell lineage, stage of differentiation, or during a particular biological response.
  • the biomarkers can reflect biological aspects of the tumors in which they are expressed, such as the cell of origin of the cancer, the nature of the non-malignant cells in the biopsy, and the oncogenic mechanisms responsible for the cancer.
  • Expression data and gene expression levels can be stored on computer readable media, e.g., the computer readable medium used in conjunction with a microarray or chip reading device. Such expression data can be manipulated to generate expression signatures.
  • increased/decreased amount or “increased/decreased level” refers to increased or decreased absolute and/or relative amount and/or value of a biomarker (e.g., one or more genes described herein such as one or more gene described in Table 2) in a subject, as compared to the amount and/or value of the same biomarker in the same subject in a prior time and/or in a normal and/or control subject, or a normal/control level representative of such subjects in general.
  • a biomarker e.g., one or more genes described herein such as one or more gene described in Table 2
  • immune checkpoint refers to a group of molecules on the cell surface of CD4+ and/or CD8+ T cells that fine-tune immune responses by down-modulating or inhibiting an anti-tumor immune response.
  • Immune checkpoint proteins are well known in the art and include, without limitation, CTLA-4, PD-1, VISTA, B7-H2, B7-H3, PD-L1, B7- H4, B7-H6, 2B4, ICOS, HVEM, PD-L2, CD160, gp49B, PIR-B, KIR family receptors, TIM-
  • the term further encompasses biologically active protein fragment, as well as nucleic acids encoding full- length immune checkpoint proteins and biologically active protein fragments thereof. In some embodiment, the term further encompasses any fragment according to homology descriptions provided herein.
  • Immuno checkpoint therapy refers to the use of agents that inhibit immune checkpoint nucleic acids and/or proteins. Inhibition of one or more immune checkpoints can block or otherwise neutralize inhibitory signaling to thereby upregulate an immune response in order to more efficaciously treat cancer.
  • agents useful for inhibiting immune checkpoints include antibodies, small molecules, peptides, peptidomimetics, natural ligands, and derivatives of natural ligands, that can either bind and/or inactivate or inhibit immune checkpoint proteins, or fragments thereof; as well as RNA interference, antisense, nucleic acid aptamers, etc. that can downregulate the expression and/or activity of immune checkpoint nucleic acids, or fragments thereof.
  • Exemplary agents for upregulating an immune response include antibodies against one or more immune checkpoint proteins block the interaction between the proteins and its natural receptor(s); a non-activating form of one or more immune checkpoint proteins (e.g., a dominant negative polypeptide); small molecules or peptides that block the interaction between one or more immune checkpoint proteins and its natural receptor(s); fusion proteins (e.g. the extracellular portion of an immune checkpoint inhibition protein fused to the Fc portion of an antibody or immunoglobulin) that bind to its natural receptor(s); nucleic acid molecules that block immune checkpoint nucleic acid transcription or translation; and the like.
  • a non-activating form of one or more immune checkpoint proteins e.g., a dominant negative polypeptide
  • small molecules or peptides that block the interaction between one or more immune checkpoint proteins and its natural receptor(s)
  • fusion proteins e.g. the extracellular portion of an immune checkpoint inhibition protein fused to the Fc portion of an antibody or immunoglobulin
  • agents can directly block the interaction between the one or more immune checkpoints and its natural receptor(s) (e.g., antibodies) to prevent inhibitory signaling and upregulate an immune response.
  • agents can indirectly block the interaction between one or more immune checkpoint proteins and its natural receptor(s) to prevent inhibitory signaling and upregulate an immune response.
  • a soluble version of an immune checkpoint protein ligand such as a stabilized extracellular domain can binding to its receptor to indirectly reduce the effective concentration of the receptor to bind to an appropriate ligand.
  • anti-PD-1 antibodies e.g., Opdivo® (nivolumab) and Keytruda® (pembrolizumab)
  • anti-PD-Ll antibodies e.g., Tecentriq® (atezolizumab)
  • anti-PD-L2 antibodies e.g., Tecentriq® (atezolizumab)
  • anti-CTLA-4 antibodies either alone or in combination, are used to inhibit immune checkpoints.
  • kits is any manufacture (e.g., a package or container) comprising at least one reagent, e.g. a probe or small molecule, for specifically detecting and/or affecting the expression of a marker of the present invention.
  • the kit may be promoted, distributed, or sold as a unit for performing the methods of the present invention.
  • the kit may comprise one or more reagents necessary to express a composition useful in the methods of the present invention.
  • the kit may further comprise a reference standard.
  • One skilled in the art can envision many such controls, including, but not limited to, common molecules.
  • Reagents in the kit may be provided in individual containers or as mixtures of two or more reagents in a single container.
  • instructional materials which describe the use of the compositions within the kit can be included.
  • the “normal” level of expression and/or activity of a biomarker is the level of expression and/or activity of the biomarker in cells of a subject, e.g, a human patient, not afflicted with cancer (e.g., lung cancer).
  • an “over-expression” or “significantly higher level of expression” of a biomarker refers to an expression level in a test sample that is greater than the standard error of the assay employed to assess expression, and is preferably at least 10%, and more preferably 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 times or more higher than the expression activity or level of the biomarker in a control sample (e.g, sample from a healthy subject not having the biomarker associated disease) and preferably, the average expression level of the biomarker in several control samples.
  • a control sample e.g, sample from a healthy subject not having the biomarker associated disease
  • a “significantly lower level of expression” of a biomarker refers to an expression level in a test sample that is at least 10%, and more preferably 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 times or more lower than the expression level of the biomarker in a control sample (e.g, sample from a healthy subject not having the biomarker associated disease) and preferably, the average expression level of the biomarker in several control samples. The same determination can be made to determine overactivity or underactivity.
  • a “significantly higher level” or “significantly increased level” of a biomarker refers to an expression level, amount and/or activity level in a subject sample at one point in time that is greater than the standard error of the assay employed to assess the expression level, amount and/or activity level, and is preferably at least 10%, and more preferably 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 times or more higher than the expression level, amount or activity
  • a “significantly lower level” or “significantly decreased level” of a biomarker refers to an expression level, amount and/or activity level in a subject sample at one point in time that is at least 10%, and more preferably 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 times or more lower than the expression level, amount or activity level of the biomarker in a subject sample at another point in time.
  • prognosis includes a prediction of the probable course and outcome of cancer (e.g., solid tumor including lung cancer) in patients or the likelihood of recovery from the disease.
  • cancer e.g., solid tumor including lung cancer
  • response refers to an anti-cancer response, e.g. in the sense of reduction of tumor size or inhibiting tumor growth.
  • the terms can also refer to an improved prognosis, for example, as reflected by an increased time to recurrence, which is the period to first recurrence censoring for second primary cancer as a first event or death without evidence of recurrence, or an increased overall survival, which is the period from treatment to death from any cause.
  • a beneficial endpoint attained when exposed to a stimulus. Alternatively, a negative or detrimental symptom is minimized, mitigated or attenuated on exposure to a stimulus.
  • evaluating the likelihood that a tumor or subject will exhibit a favorable response is equivalent to evaluating the likelihood that the tumor or subject will not exhibit favorable response (ie., will exhibit a lack of response or be non-responsive).
  • the term “response to immune checkpoint therapy” or “response to therapy” relates to any response of the hyperproliferative disorder (e.g., cancer) to a therapy, such as an immune checkpoint therapy like immune checkpoint therapy, preferably to a change in tumor mass and/or volume after initiation of neoadjuvant or adjuvant chemotherapy.
  • Hyperproliferative disorder response may be assessed, for example for efficacy or in a neoadjuvant or adjuvant situation, where the size of a tumor after systemic intervention can be compared to the initial size and dimensions as measured by CT, PET, mammogram, ultrasound or palpation. Responses may also be assessed by caliper measurement or pathological examination of the tumor after biopsy or surgical resection. Response may be recorded in a quantitative fashion like percentage change in tumor volume or in a qualitative fashion like “pathological complete response” (pCR), “clinical complete remission” (cCR), “clinical partial remission” (cPR), “clinical stable disease” (cSD), “clinical progressive disease” (cPD) or other qualitative criteria.
  • pCR pathological complete response
  • cCR clinical complete remission
  • cPR clinical partial remission
  • cSD clinical stable disease
  • cPD clinical progressive disease
  • Assessment of hyperproliferative disorder response may be done early after the onset of neoadjuvant or adjuvant therapy, e.g., after a few hours, days, weeks or preferably after a few months.
  • a typical endpoint for response assessment is upon termination of neoadjuvant chemotherapy or upon surgical removal of residual tumor cells and/or the tumor bed. This is typically three months after initiation of neoadjuvant therapy.
  • clinical efficacy of the therapeutic treatments described herein may be determined by measuring the clinical benefit rate (CBR).
  • the clinical benefit rate is measured by determining the sum of the percentage of patients who are in complete remission (CR), the number of patients who are in partial remission (PR) and the number of patients having stable disease (SD) at a time point at least 6 months out from the end of therapy.
  • the CBR for a particular cancer therapeutic regimen is at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or more.
  • Additional criteria for evaluating the response to cancer therapies are related to “survival,” which includes all of the following: survival until mortality, also known as overall survival (wherein said mortality may be either irrespective of cause or tumor related); “recurrence-free survival” (wherein the term recurrence shall include both localized and distant recurrence); metastasis free survival; disease free survival (wherein the term disease shall include cancer and diseases associated therewith).
  • the length of said survival may be calculated by reference to a defined start point (e.g., time of diagnosis or start of treatment) and end point (e.g., death, recurrence or metastasis).
  • criteria for efficacy of treatment can be expanded to include response to chemotherapy, probability of survival, probability of metastasis within a given time period, and probability of tumor recurrence.
  • a particular cancer therapeutic regimen can be administered to a population of subjects and the outcome can be correlated to biomarker measurements that were determined prior to administration of any cancer therapy.
  • the outcome measurement may be pathologic response to therapy given in the neoadjuvant setting.
  • outcome measures such as overall survival and disease-free survival can be monitored over a period of time for subjects following cancer therapy for whom biomarker measurement values are known.
  • the doses administered are standard doses known in the art for cancer therapeutic agents. The period of time for which subjects are monitored can vary.
  • subjects may be monitored for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 weeks or longer, such as 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 45, 50, 55, or 60 months.
  • Biomarker measurement threshold values that correlate to outcome of a cancer therapy can be determined using well-known methods in the art, such as those described in the Examples section.
  • the response refers to major pathological response (MPR) in the resected tumor, defined as residual viable tumor of less than or equal to 10% of the tumor bed.
  • MPR major pathological response
  • survival includes all of the following: survival until mortality, also known as overall survival (wherein said mortality may be either irrespective of cause or tumor related); “recurrence-free survival” (wherein the term recurrence shall include both localized and distant recurrence); metastasis free survival; disease free survival (wherein the term disease shall include cancer and diseases associated therewith).
  • the length of said survival may be calculated by reference to a defined start point (e.g. time of diagnosis or start of treatment) and end point (e.g. death, recurrence or metastasis).
  • criteria for efficacy of treatment can be expanded to include response to chemotherapy, probability of survival, probability of metastasis within a given time period, and probability of tumor recurrence.
  • sample used for detecting or determining the presence or level of at least one biomarker is typically brain tissue, cerebrospinal fluid, whole blood, plasma, serum, saliva, urine, stool (e.g., feces), tears, and any other bodily fluid (e.g., as described above under the definition of “body fluids”), or a tissue sample (e.g., biopsy) such as a small intestine, colon sample, or surgical resection tissue.
  • the method of the present invention further comprises obtaining the sample from the individual prior to detecting or determining the presence or level of at least one biomarker in the sample.
  • the term “synergistic effect” refers to the combined effect of two or more agents described herein can be greater than the sum of the separate effects of any one of agents alone.
  • subject refers to either a human or a non-human animal. This term includes mammals such as humans, primates, livestock animals (e.g., bovines, porcines), companion animals (e.g., canines, felines) and rodents (e.g., mice, rabbits and rats).
  • livestock animals e.g., bovines, porcines
  • companion animals e.g., canines, felines
  • rodents e.g., mice, rabbits and rats.
  • Treating” a disease in a subject or “treating” a subject having a disease refers to subjecting the subject to a pharmaceutical treatment, e.g., the administration of a drug, such that at least one symptom of the disease is decreased or prevented from worsening.
  • therapeutic effect refers to a local or systemic effect in animals, particularly mammals, and more particularly humans, caused by a pharmacologically active substance.
  • the term thus means any substance intended for use in the diagnosis, cure, mitigation, treatment or prevention of disease or in the enhancement of desirable physical or mental development and conditions in an animal or human.
  • therapeutically- effective amount means that amount of such a substance that produces some desired local or systemic effect at a reasonable benefit/risk ratio applicable to any treatment.
  • a therapeutically effective amount of a compound will depend on its therapeutic index, solubility, and the like.
  • certain compounds discovered by the methods of the present invention may be administered in a sufficient amount to produce a reasonable benefit/risk ratio applicable to such treatment.
  • the subject for whom predicted likelihood of response to an immune checkpoint therapy or to an immune checkpoint and radiation combination therapy is determined is a mammal e.g., mouse, rat, primate, non-human mammal, domestic animal, such as a dog, cat, cow, horse, and the like), and is preferably a human.
  • the subject has not undergone treatment, such as chemotherapy, radiation therapy, targeted therapy, and/or immune checkpoint therapy.
  • the subject has undergone treatment, such as chemotherapy, radiation therapy, targeted therapy, and/or immune checkpoint therapy.
  • the subject has had surgery to remove cancerous or precancerous tissue.
  • the cancerous tissue has not been removed, e.g., the cancerous tissue may be located in an inoperable region of the body, such as in a tissue that is essential for life, or in a region where a surgical procedure would cause considerable risk of harm to the patient.
  • the cancer is one for which an immune checkpoint therapy (e.g., anti-PD-1 blocking antibody, anti-PD-Ll blocking antibody, CTLA-4 blocking antibody, and the like) is FDA-approved for treatment, such as those described in the Examples.
  • the cancers are solid tumors, such as lung cancer such as non-small cell lung cancer (NSCLC), breast cancer, melanoma, pancreatic cancer, and/or prostate cancer.
  • the NSCLC is an early stage (e.g., clinical stages I-IIIA) NSCLC.
  • the NSCLC is lung adenocarcinoma.
  • biomarker amount and/or activity measurement s) in a sample from a subject is compared to a predetermined control (standard) sample.
  • the sample from the subject is typically from a diseased tissue, such as cancer cells or tissues.
  • the control sample can be from the same subject or from a different subject.
  • the control sample is typically a normal, non-diseased sample.
  • the control sample can be from a diseased tissue.
  • the control sample can be a combination of samples from several different subjects.
  • the biomarker amount and/or activity measurement(s) from a subject is compared to a pre-determined level. This pre-determined level is typically obtained from normal samples.
  • a “pre-determined” biomarker amount and/or activity measurement(s) may be a biomarker amount and/or activity measurement(s) used to, by way of example only, evaluate a subject that may be selected for treatment, evaluate a response to an immune checkpoint therapy, and/or evaluate a response to a combination immune checkpoint therapy.
  • a pre-determined biomarker amount and/or activity measurement(s) may be determined in populations of patients with or without cancer.
  • the pre-determined biomarker amount and/or activity measurement s) can be a single number, equally applicable to every patient, or the pre-determined biomarker amount and/or activity measurement(s) can vary according to specific subpopulations of patients.
  • Age, weight, height, and other factors of a subject may affect the pre-determined biomarker amount and/or activity measurement(s) of the individual. Furthermore, the pre-determined biomarker amount and/or activity can be determined for each subject individually. In one embodiment, the amounts determined and/or compared in a method described herein are based on absolute measurements.
  • the amounts determined and/or compared in a method described herein are based on relative measurements, such as ratios (e.g, biomarker amount/level before a treatment vs. after a treatment, such biomarker measurements relative to a spiked or man-made control, such biomarker measurements relative to the expression of a housekeeping gene, and the like).
  • the relative analysis can be based on the ratio of pre-treatment biomarker measurement as compared to post-treatment biomarker measurement.
  • Pre-treatment biomarker measurement can be made at any time prior to initiation of anti-cancer therapy.
  • Post-treatment biomarker measurement can be made at any time after initiation of anti-cancer therapy.
  • post-treatment biomarker measurements are made 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 weeks or more after initiation of anti-cancer therapy, and even longer toward indefinitely for continued monitoring.
  • Treatment can comprise anti-cancer therapy, such as a therapeutic regimen comprising an anti-PDl monoclonal antibody (e.g., durvalumab) alone or in combination with other anti-cancer therapies, such as radiation (e.g., a sub-ablative dose of stereotactic body radiation therapy (SBRT)) described in the Examples, Figures, and Tables.
  • SBRT stereotactic body radiation therapy
  • the pre-determined biomarker amount and/or activity measurement(s) can be any suitable standard.
  • the pre-determined biomarker amount and/or activity measurement(s) can be obtained from the same or a different human for whom a patient selection is being assessed.
  • the pre-determined biomarker amount and/or activity measurement s) can be obtained from a previous assessment of the same patient. In such a manner, the progress of the selection of the patient can be monitored over time.
  • the control can be obtained from an assessment of another human or multiple humans, e.g, selected groups of humans, if the subject is a human.
  • the extent of the selection of the human for whom selection is being assessed can be compared to suitable other humans, e.g., other humans who are in a similar situation to the human of interest, such as those suffering from similar or the same condition(s) and/or of the same ethnic group.
  • the change of biomarker amount and/or activity measurement s) from the pre-determined level is about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0 fold or greater, or any range in between, inclusive.
  • Such cutoff values apply equally when the measurement is based on relative changes, such as based on the ratio of pre-treatment biomarker measurement as compared to post-treatment biomarker measurement.
  • Body fluids refer to fluids that are excreted or secreted from the body as well as fluids that are normally not (e.g., amniotic fluid, aqueous humor, bile, blood and blood plasma, cerebrospinal fluid, cerumen and earwax, cowper’s fluid or pre-ejaculatory fluid, chyle, chyme, stool, female ejaculate, interstitial fluid, intracellular fluid, lymph, menses, breast milk, mucus, pleural fluid, pus, saliva, sebum, semen, serum, sweat, synovial fluid, tears, urine, vaginal lubrication, vitreous humor, vomit).
  • the subject and/or control sample is selected from the group consisting of cells, cell lines, histological slides, paraffin embedded tissues, biopsies, whole blood, nipple aspirate, serum, plasma, buccal scrape, saliva, cerebrospinal fluid, urine, stool, and bone marrow.
  • the sample is serum, plasma, or urine. In another embodiment, the sample is serum.
  • the samples can be collected from individuals repeatedly over a longitudinal period of time (e.g., once or more on the order of days, weeks, months, annually, biannually, etc.). Obtaining numerous samples from an individual over a period of time can be used to verify results from earlier detections and/or to identify an alteration in biological pattern as a result of, for example, disease progression, drug treatment, etc. For example, subject samples can be taken and monitored every month, every two months, or combinations of one, two, or three month intervals according to the present invention.
  • biomarker amount and/or activity measurements of the subject obtained over time can be conveniently compared with each other, as well as with those of normal controls during the monitoring period, thereby providing the subject’s own values, as an internal, or personal, control for long-term monitoring.
  • Sample preparation and separation can involve any of the procedures, depending on the type of sample collected and/or analysis of biomarker measurement(s).
  • Such procedures include, by way of example only, concentration, dilution, adjustment of pH, removal of high abundance polypeptides (e.g., albumin, gamma globulin, and transferrin, etc.), addition of preservatives and calibrants, addition of protease inhibitors, addition of denaturants, desalting of samples, concentration of sample proteins, extraction and purification of lipids.
  • the sample preparation can also isolate molecules that are bound in non-covalent complexes to other protein (e.g., carrier proteins).
  • carrier proteins e.g., albumin
  • This process may isolate those molecules bound to a specific carrier protein (e.g., albumin), or use a more general process, such as the release of bound molecules from all carrier proteins via protein denaturation, for example using an acid, followed by removal of the carrier proteins.
  • Removal of undesired proteins (e.g., high abundance, uninformative, or undetectable proteins) from a sample can be achieved using high affinity reagents, high molecular weight filters, ultracentrifugation and/or electrodialysis.
  • High affinity reagents include antibodies or other reagents (e.g., aptamers) that selectively bind to high abundance proteins.
  • Sample preparation could also include ion exchange chromatography, metal ion affinity chromatography, gel filtration, hydrophobic chromatography, chromatofocusing, adsorption chromatography, isoelectric focusing and related techniques.
  • Molecular weight filters include membranes that separate molecules on the basis of size and molecular weight. Such filters may further employ reverse osmosis, nanofiltration, ultrafiltration and microfiltration.
  • Ultracentrifugation is a method for removing undesired polypeptides from a sample. Ultracentrifugation is the centrifugation of a sample at about 15,000-60,000 rpm while monitoring with an optical system the sedimentation (or lack thereof) of particles. Electrodialysis is a procedure which uses an electromembrane or semipermable membrane in a process in which ions are transported through semi-permeable membranes from one solution to another under the influence of a potential gradient.
  • the membranes used in electrodialysis may have the ability to selectively transport ions having positive or negative charge, reject ions of the opposite charge, or to allow species to migrate through a semipermable membrane based on size and charge, it renders electrodialysis useful for concentration, removal, or separation of electrolytes.
  • Separation and purification in the present invention may include any procedure known in the art, such as capillary electrophoresis (e.g., in capillary or on-chip) or chromatography (e.g., in capillary, column or on a chip).
  • Electrophoresis is a method which can be used to separate ionic molecules under the influence of an electric field. Electrophoresis can be conducted in a gel, capillary, or in a microchannel on a chip. Examples of gels used for electrophoresis include starch, acrylamide, polyethylene oxides, agarose, or combinations thereof.
  • a gel can be modified by its cross-linking, addition of detergents, or denaturants, immobilization of enzymes or antibodies (affinity electrophoresis) or substrates (zymography) and incorporation of a pH gradient.
  • capillaries used for electrophoresis include capillaries that interface with an electrospray.
  • CE Capillary electrophoresis
  • CZE capillary zone electrophoresis
  • CIEF capillary isoelectric focusing
  • cITP capillary isotachophoresis
  • CEC capillary electrochromatography
  • Capillary isotachophoresis is a technique in which the analytes move through the capillary at a constant speed but are nevertheless separated by their respective mobilities.
  • Capillary zone electrophoresis also known as free-solution CE (FSCE)
  • FSCE free-solution CE
  • CIEF Capillary isoelectric focusing
  • CEC is a hybrid technique between traditional high performance liquid chromatography (HPLC) and CE.
  • Chromatography can be based on the differential adsorption and elution of certain analytes or partitioning of analytes between mobile and stationary phases.
  • Different examples of chromatography include, but not limited to, liquid chromatography (LC), gas chromatography (GC), high performance liquid chromatography (HPLC), etc.
  • Biomarker nucleic acids and/or biomarker polypeptides can be analyzed according to the methods described herein and techniques known to the skilled artisan to identify such genetic or expression alterations useful for the present invention including, but not limited to, 1) an alteration in the level of a biomarker transcript or polypeptide, 2) a deletion or addition of one or more nucleotides from a biomarker gene, 4) a substitution of one or more nucleotides of a biomarker gene, 5) aberrant modification of a biomarker gene, such as an expression regulatory region, and the like.
  • Methods for Detection of Copy Number Methods of evaluating the copy number of a biomarker nucleic acid are well known to those of skill in the art. The presence or absence of chromosomal gain or loss can be evaluated simply by a determination of copy number of the regions or markers identified herein.
  • a biological sample is tested for the copy number changes in genomic loci containing the genomic marker.
  • the increase of copy number of at least one biomarker listed from number 1-135 in Table 2 is predictive of response to an immune checkpoint and radiation combination therapy or to an immune checkpoint monotherapy.
  • a copy number of at least 3, 4, 5, 6, 7, 8, 9, or 10 of at least one biomarker listed from number 1-135 in Table 2 is predictive of more likely responsive to an immune checkpoint and radiation combination therapy or to an immune checkpoint monotherapy.
  • the decrease of copy number of at least one biomarker listed from number 136-140 in Table 2 is predictive of less likely response to an immune checkpoint and radiation combination therapy or to an immune checkpoint monotherapy.
  • Methods of evaluating the copy number of a biomarker locus include, but are not limited to, hybridization-based assays.
  • Hybridization-based assays include, but are not limited to, traditional “direct probe” methods, such as Southern blots, in situ hybridization (e.g., FISH and FISH plus SKY) methods, and “comparative probe” methods, such as comparative genomic hybridization (CGH), e.g., cDNA-based or oligonucleotide-based CGH.
  • CGH comparative genomic hybridization
  • the methods can be used in a wide variety of formats including, but not limited to, substrate (e.g. membrane or glass) bound methods or array-based approaches.
  • evaluating the biomarker gene copy number in a sample involves a Southern Blot.
  • a Southern Blot the genomic DNA (typically fragmented and separated on an electrophoretic gel) is hybridized to a probe specific for the target region. Comparison of the intensity of the hybridization signal from the probe for the target region with control probe signal from analysis of normal genomic DNA (e.g., a non-amplified portion of the same or related cell, tissue, organ, etc.) provides an estimate of the relative copy number of the target nucleic acid.
  • a Northern blot may be utilized for evaluating the copy number of encoding nucleic acid in a sample.
  • mRNA is hybridized to a probe specific for the target region.
  • Comparison of the intensity of the hybridization signal from the probe for the target region with control probe signal from analysis of normal RNA provides an estimate of the relative copy number of the target nucleic acid.
  • RNA e.g., a non-amplified portion of the same or related cell, tissue, organ, etc.
  • other methods well known in the art to detect RNA can be used, such that higher or lower expression relative to an appropriate control (e.g., a non-amplified portion of the same or related cell tissue, organ, etc.) provides an estimate of the relative copy number of the target nucleic acid.
  • in situ hybridization comprises the following steps: (1) fixation of tissue or biological structure to be analyzed; (2) prehybridization treatment of the biological structure to increase accessibility of target DNA, and to reduce nonspecific binding; (3) hybridization of the mixture of nucleic acids to the nucleic acid in the biological structure or tissue; (4) post-hybridization washes to remove nucleic acid fragments not bound in the hybridization and (5) detection of the hybridized nucleic acid fragments.
  • the reagent used in each of these steps and the conditions for use vary depending on the particular application.
  • a nucleic acid In a typical in situ hybridization assay, cells are fixed to a solid support, typically a glass slide. If a nucleic acid is to be probed, the cells are typically denatured with heat or alkali. The cells are then contacted with a hybridization solution at a moderate temperature to permit annealing of labeled probes specific to the nucleic acid sequence encoding the protein. The targets (e.g., cells) are then typically washed at a predetermined stringency or at an increasing stringency until an appropriate signal to noise ratio is obtained. The probes are typically labeled, e.g., with radioisotopes or fluorescent reporters. In one embodiment, probes are sufficiently long so as to specifically hybridize with the target nucleic acid(s) under stringent conditions. Probes generally range in length from about 200 bases to about 1000 bases. In some applications it is necessary to block the hybridization capacity of repetitive sequences. Thus, in some embodiments, tRNA, human genomic DNA, or Cot-I DNA is used to block
  • genomic DNA is isolated from normal reference cells, as well as from test cells (e.g., tumor cells) and amplified, if necessary.
  • the two nucleic acids are differentially labeled and then hybridized in situ to metaphase chromosomes of a reference cell.
  • the repetitive sequences in both the reference and test DNAs are either removed or their hybridization capacity is reduced by some means, for example by prehybridization with appropriate blocking nucleic acids and/or including such blocking nucleic acid sequences for said repetitive sequences during said hybridization.
  • the bound, labeled DNA sequences are then rendered in a visualizable form, if necessary.
  • Chromosomal regions in the test cells which are at increased or decreased copy number can be identified by detecting regions where the ratio of signal from the two DNAs is altered. For example, those regions that have decreased in copy number in the test cells will show relatively lower signal from the test DNA than the reference compared to other regions of the genome. Regions that have been increased in copy number in the test cells will show relatively higher signal from the test DNA. Where there are chromosomal deletions or multiplications, differences in the ratio of the signals from the two labels will be detected and the ratio will provide a measure of the copy number.
  • array CGH array CGH
  • the immobilized chromosome element is replaced with a collection of solid support bound target nucleic acids on an array, allowing for a large or complete percentage of the genome to be represented in the collection of solid support bound targets.
  • Target nucleic acids may comprise cDNAs, genomic DNAs, oligonucleotides (e.g., to detect single nucleotide polymorphisms) and the like.
  • Array-based CGH may also be performed with single-color labeling (as opposed to labeling the control and the possible tumor sample with two different dyes and mixing them prior to hybridization, which will yield a ratio due to competitive hybridization of probes on the arrays).
  • amplification-based assays can be used to measure copy number.
  • the nucleic acid sequences act as a template in an amplification reaction (e.g., Polymerase Chain Reaction (PCR).
  • PCR Polymerase Chain Reaction
  • the amount of amplification product will be proportional to the amount of template in the original sample.
  • Comparison to appropriate controls, e.g. healthy tissue, provides a measure of the copy number.
  • Quantitative amplification involves simultaneously co-amplifying a known quantity of a control sequence using the same primers. This provides an internal standard that may be used to calibrate the PCR reaction.
  • Detailed protocols for quantitative PCR are provided in Innis, et al. (1990) PCR Protocols, A Guide to Methods and Applications, Academic Press, Inc. N.Y.). Measurement of DNA copy number at microsatellite loci using quantitative PCR analysis is described in Ginzonger, et al. (2000) Cancer Research 60:5405-5409.
  • the known nucleic acid sequence for the genes is sufficient to enable one of skill in the art to routinely select primers to amplify any portion of the gene.
  • Fluorogenic quantitative PCR may also be used in the methods of the present invention. In fluorogenic quantitative PCR, quantitation is based on amount of fluorescence signals, e.g., TaqMan and SYBR green.
  • ligase chain reaction (LCR) (see Wu and Wallace (1989) Genomics 4: 560, Landegren, et al. (1988) Science 241 : 1077, and Barringer et al. (1990) Gene 89: 117), transcription amplification (Kwoh, et al. (1989) Proc. Natl. Acad. Sci. USA 86: 1173), self-sustained sequence replication (Guatelli, et al. (1990) Proc. Nat. Acad. Sci. USA 87: 1874), dot PCR, and linker adapter PCR, etc.
  • LCR ligase chain reaction
  • Loss of heterozygosity (LOH) and major copy proportion (MCP) mapping (Wang, Z.C., et al. (2004) Cancer Res 64(1):64-71; Seymour, A. B., et al. (1994) Cancer Res 54, 2761-4; Hahn, S. A., et al. (1995) Cancer Res 55, 4670-5; Kimura, M., et al. (1996) Genes Chromosomes Cancer 17, 88-93; Li et al., (2008) MBC Bioinform. 9, 204-219) may also be used to identify regions of amplification or deletion.
  • LHO heterozygosity
  • MCP major copy proportion
  • Biomarker expression may be assessed by any of a wide variety of well-known methods for detecting expression of a transcribed molecule or protein.
  • Non-limiting examples of such methods include immunological methods for detection of secreted, cellsurface, cytoplasmic, or nuclear proteins, protein purification methods, protein function or activity assays, nucleic acid hybridization methods, nucleic acid reverse transcription methods, and nucleic acid amplification methods.
  • activity of a particular gene is characterized by a measure of gene transcript (e.g. mRNA), by a measure of the quantity of translated protein, or by a measure of gene product activity.
  • Marker expression can be monitored in a variety of ways, including by detecting mRNA levels, protein levels, or protein activity, any of which can be measured using standard techniques. Detection can involve quantification of the level of gene expression (e.g., genomic DNA, cDNA, mRNA, protein, or enzyme activity), or, alternatively, can be a qualitative assessment of the level of gene expression, in particular in comparison with a control level. The type of level being detected will be clear from the context.
  • detecting or determining expression levels of a biomarker and functionally similar homologs thereof, including a fragment or genetic alteration thereof (e.g., in regulatory or promoter regions thereof) comprises detecting or determining RNA levels for the marker of interest.
  • one or more cells from the subject to be tested are obtained and RNA is isolated from the cells.
  • a sample containing cancer cells are obtained from the subject.
  • RNA is obtained from a single cell.
  • a cell can be isolated from a tissue sample by laser capture microdissection (LCM).
  • LCM laser capture microdissection
  • a cell can be isolated from a tissue section, including a stained tissue section, thereby assuring that the desired cell is isolated (see, e.g., Bonner et al. (1997) Science 278: 1481; Emmert- Buck et al. (1996) Science 274:998; Fend et al. (1999) Am. J. Path. 154: 61 and Murakami et al. (2000) Kidney Int. 58: 1346).
  • Murakami et al., supra describe isolation of a cell from a previously immunostained tissue section.
  • RNA can be extracted.
  • Methods for establishing cultures of non-transformed cells, i.e., primary cell cultures, are known in the art.
  • RNA in the tissue and cells may quickly become degraded. Accordingly, in a preferred embodiment, the tissue or cells obtained from a subject is snap frozen as soon as possible.
  • RNA can be extracted from the tissue sample by a variety of methods, e.g., the guanidium thiocyanate lysis followed by CsCl centrifugation (Chirgwin et al., 1979, Biochemistry 18:5294-5299).
  • RNA from single cells can be obtained as described in methods for preparing cDNA libraries from single cells, such as those described in Dulac, C. (1998) Curr. Top. Dev. Biol. 36, 245 and Jena et al. (1996) J. Immunol. Methods 190:199. Care to avoid RNA degradation must be taken, e.g., by inclusion of a ribonuclease inhibitor.
  • RNA sample can then be enriched in particular species.
  • poly(A)+ RNA is isolated from the RNA sample.
  • such purification takes advantage of the poly-A tails on mRNA.
  • poly-T oligonucleotides may be immobilized within on a solid support to serve as affinity ligands for mRNA. Kits for this purpose are commercially available, e.g., the MessageMaker kit (Life Technologies, Grand Island, NY).
  • the RNA population is enriched in marker sequences. Enrichment can be undertaken, e.g., by primer-specific cDNA synthesis, or multiple rounds of linear amplification based on cDNA synthesis and template-directed in vitro transcription (see, e.g., Wang et al. (1989) PNAS 86, 9717; Dulac et al., supra, and Jena et al., supra).
  • RNA enriched or not in particular species or sequences
  • an “amplification process” is designed to strengthen, increase, or augment a molecule within the RNA.
  • an amplification process such as RT-PCR can be utilized to amplify the mRNA, such that a signal is detectable or detection is enhanced.
  • Such an amplification process is beneficial particularly when the biological, tissue, or tumor sample is of a small size or volume.
  • RNAscribe mRNA into cDNA followed by polymerase chain reaction RT-PCR
  • RT-AGLCR reverse transcribe mRNA into cDNA followed by symmetric gap ligase chain reaction
  • amplification methods which can be utilized herein include but are not limited to the so-called “NASBA” or “3 SR” technique described in PNAS USA 87: 1874- 1878 (1990) and also described in Nature 350 (No. 6313): 91-92 (1991); Q-beta amplification as described in published European Patent Application (EP A) No. 4544610; strand displacement amplification (as described in G. T. Walker et al., Clin. Chem. 42: 9-13 (1996) and European Patent Application No.
  • Northern analysis involves running a preparation of RNA on a denaturing agarose gel, and transferring it to a suitable support, such as activated cellulose, nitrocellulose or glass or nylon membranes. Radiolabeled cDNA or RNA is then hybridized to the preparation, washed and analyzed by autoradiography.
  • In situ hybridization visualization may also be employed, wherein a radioactively labeled antisense RNA probe is hybridized with a thin section of a biopsy sample, washed, cleaved with RNase and exposed to a sensitive emulsion for autoradiography.
  • the samples may be stained with hematoxylin to demonstrate the histological composition of the sample, and dark field imaging with a suitable light filter shows the developed emulsion.
  • Nonradioactive labels such as digoxigenin may also be used.
  • mRNA expression can be detected on a DNA array, chip or a microarray.
  • Labeled nucleic acids of a test sample obtained from a subject may be hybridized to a solid surface comprising biomarker DNA. Positive hybridization signal is obtained with the sample containing biomarker transcripts.
  • Methods of preparing DNA arrays and their use are well known in the art (see, e.g., U.S. Pat. Nos: 6,618,6796; 6,379,897; 6,664,377; 6,451,536; 548,257; U.S. 20030157485 and Schena et al. (1995) Science 20, 467- 470; Gerhold et al. (1999) Trends In Biochem. Sci.
  • Serial Analysis of Gene Expression can also be performed (See for example U.S. Patent Application 20030215858).
  • mRNA is extracted from the biological sample to be tested, reverse transcribed, and fluorescently-labeled cDNA probes are generated.
  • the microarrays capable of hybridizing to marker cDNA are then probed with the labeled cDNA probes, the slides scanned and fluorescence intensity measured. This intensity correlates with the hybridization intensity and expression levels.
  • probes that can be used in the methods described herein include cDNA, riboprobes, synthetic oligonucleotides and genomic probes.
  • the type of probe used will generally be dictated by the particular situation, such as riboprobes for in situ hybridization, and cDNA for Northern blotting, for example.
  • the probe is directed to nucleotide regions unique to the RNA.
  • the probes may be as short as is required to differentially recognize marker mRNA transcripts, and may be as short as, for example, 15 bases; however, probes of at least 17, 18, 19 or 20 or more bases can be used.
  • the primers and probes hybridize specifically under stringent conditions to a DNA fragment having the nucleotide sequence corresponding to the marker.
  • stringent conditions means hybridization will occur only if there is at least 95% identity in nucleotide sequences. In another embodiment, hybridization under “stringent conditions” occurs when there is at least 97% identity between the sequences.
  • the form of labeling of the probes may be any that is appropriate, such as the use of radioisotopes, for example, 32 P and 35 S. Labeling with radioisotopes may be achieved, whether the probe is synthesized chemically or biologically, by the use of suitably labeled bases.
  • the biological sample contains polypeptide molecules from the test subject.
  • the biological sample can contain mRNA molecules from the test subject or genomic DNA molecules from the test subject.
  • the methods further involve obtaining a control biological sample from a control subject, contacting the control sample with a compound or agent capable of detecting marker polypeptide, mRNA, genomic DNA, or fragments thereof, such that the presence of the marker polypeptide, mRNA, genomic DNA, or fragments thereof, is detected in the biological sample, and comparing the presence of the marker polypeptide, mRNA, genomic DNA, or fragments thereof, in the control sample with the presence of the marker polypeptide, mRNA, genomic DNA, or fragments thereof in the test sample.
  • the biomarker nucleic acid expression is detected by RNA sequencing (RNA-seq), a technical well-known in the art. c. Methods for Detection of Biomarker Protein Expression
  • the activity or level of a biomarker protein can be detected and/or quantified by detecting or quantifying the expressed polypeptide.
  • the polypeptide can be detected and quantified by any of a number of means well known to those of skill in the art. Aberrant levels of polypeptide expression of the polypeptides encoded by a biomarker nucleic acid and functionally similar homologs thereof, including a fragment or genetic alteration thereof (e.g., in regulatory or promoter regions thereof) are associated with the likelihood of response of a cancer to an IL3 -conjugated toxin treatment. Any method known in the art for detecting polypeptides can be used.
  • Such methods include, but are not limited to, immunodiffusion, immunoelectrophoresis, radioimmunoassay (RIA), enzyme-linked immunosorbent assays (ELISAs), immunofluorescent assays, Western blotting, binder-ligand assays, immunohistochemical techniques, agglutination, complement assays, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), hyperdiffusion chromatography, and the like (e.g., Basic and Clinical Immunology, Sites and Terr, eds., Appleton and Lange, Norwalk, Conn, pp 217-262, 1991 which is incorporated by reference).
  • binder- ligand immunoassay methods including reacting antibodies with an epitope or epitopes and competitively displacing a labeled polypeptide or derivative thereof.
  • ELISA and RIA procedures may be conducted such that a desired biomarker protein standard is labeled (with a radioisotope such as 125 I or 35 S, or an assayable enzyme, such as horseradish peroxidase or alkaline phosphatase), and, together with the unlabelled sample, brought into contact with the corresponding antibody, whereon a second antibody is used to bind the first, and radioactivity or the immobilized enzyme assayed (competitive assay).
  • a radioisotope such as 125 I or 35 S, or an assayable enzyme, such as horseradish peroxidase or alkaline phosphatase
  • biomarker protein in the sample is allowed to react with the corresponding immobilized antibody, radioisotope- or enzyme-labeled antibiomarker proteinantibody is allowed to react with the system, and radioactivity or the enzyme assayed (ELISA-sandwich assay).
  • radioactivity or the enzyme assayed ELISA-sandwich assay.
  • Other conventional methods may also be employed as suitable.
  • a “one-step” assay involves contacting antigen with immobilized antibody and, without washing, contacting the mixture with labeled antibody.
  • a “two-step” assay involves washing before contacting, the mixture with labeled antibody.
  • Other conventional methods may also be employed as suitable.
  • a method for measuring biomarker protein levels comprises the steps of: contacting a biological specimen with an antibody or variant (e.g., fragment) thereof which selectively binds the biomarker protein, and detecting whether said antibody or variant thereof is bound to said sample and thereby measuring the levels of the biomarker protein.
  • an antibody or variant e.g., fragment
  • Enzymatic and radiolabeling of biomarker protein and/or the antibodies may be effected by conventional means.
  • Such means will generally include covalent linking of the enzyme to the antigen or the antibody in question, such as by glutaraldehyde, specifically so as not to adversely affect the activity of the enzyme, by which is meant that the enzyme must still be capable of interacting with its substrate, although it is not necessary for all of the enzyme to be active, provided that enough remains active to permit the assay to be effected.
  • some techniques for binding enzyme are non-specific (such as using formaldehyde), and will only yield a proportion of active enzyme.
  • Enzymes employable for labeling are not particularly limited, but may be selected from the members of the oxidase group, for example. These catalyze production of hydrogen peroxide by reaction with their substrates, and glucose oxidase is often used for its good stability, ease of availability and cheapness, as well as the ready availability of its substrate (glucose). Activity of the oxidase may be assayed by measuring the concentration of hydrogen peroxide formed after reaction of the enzyme-labeled antibody with the substrate under controlled conditions well-known in the art.
  • biomarker protein may be detected according to a practitioner's preference based upon the present disclosure.
  • One such technique is Western blotting (Towbin et at., Proc. Nat. Acad. Sci. 76:4350 (1979)), wherein a suitably treated sample is run on an SDS-PAGE gel before being transferred to a solid support, such as a nitrocellulose filter.
  • Anti-biomarker protein antibodies (unlabeled) are then brought into contact with the support and assayed by a secondary immunological reagent, such as labeled protein A or antiimmunoglobulin (suitable labels including 125 I, horseradish peroxidase and alkaline phosphatase). Chromatographic detection may also be used.
  • Immunohistochemistry may be used to detect expression of biomarker protein, e.g., in a biopsy sample.
  • a suitable antibody is brought into contact with, for example, a thin layer of cells, washed, and then contacted with a second, labeled antibody.
  • Labeling may be by fluorescent markers, enzymes, such as peroxidase, avidin, or radiolabelling. The assay is scored visually, using microscopy.
  • Anti-biomarker protein antibodies such as intrabodies, may also be used for imaging purposes, for example, to detect the presence of biomarker protein in cells and tissues of a subject.
  • Suitable labels include radioisotopes, iodine ( 125 I, 121 I), carbon ( 14 C), sulphur ( 35 S), tritium ( 3 H), indium ( 112 In), and technetium (“mTc), fluorescent labels, such as fluorescein and rhodamine, and biotin.
  • antibodies are not detectable, as such, from outside the body, and so must be labeled, or otherwise modified, to permit detection.
  • Markers for this purpose may be any that do not substantially interfere with the antibody binding, but which allow external detection.
  • Suitable markers may include those that may be detected by X- radiography, NMR or MRI.
  • suitable markers include any radioisotope that emits detectable radiation but that is not overtly harmful to the subject, such as barium or cesium, for example.
  • Suitable markers for NMR and MRI generally include those with a detectable characteristic spin, such as deuterium, which may be incorporated into the antibody by suitable labeling of nutrients for the relevant hybridoma, for example.
  • the size of the subject, and the imaging system used, will determine the quantity of imaging moiety needed to produce diagnostic images.
  • the quantity of radioactivity injected will normally range from about 5 to 20 millicuries of technetium-99.
  • the labeled antibody or antibody fragment will then preferentially accumulate at the location of cells which contain biomarker protein. The labeled antibody or antibody fragment can then be detected using known techniques.
  • Antibodies that may be used to detect biomarker protein include any antibody, whether natural or synthetic, full length or a fragment thereof, monoclonal or polyclonal, that binds sufficiently strongly and specifically to the biomarker protein to be detected.
  • An antibody may have a Kd of at most about 10' 6 M, 10' 7 M, 10' 8 M, 10' 9 M, 10' 10 M, 10 -11 M, 10’ 12 M.
  • the phrase “specifically binds” refers to binding of, for example, an antibody to an epitope or antigen or antigenic determinant in such a manner that binding can be displaced or competed with a second preparation of identical or similar epitope, antigen or antigenic determinant.
  • An antibody may bind preferentially to the biomarker protein relative to other proteins, such as related proteins.
  • Antibodies are commercially available or may be prepared according to methods known in the art.
  • Antibodies and derivatives thereof that may be used encompass polyclonal or monoclonal antibodies, chimeric, human, humanized, primatized (CDR-grafted), veneered or single-chain antibodies as well as functional fragments, z.e., biomarker protein binding fragments, of antibodies.
  • antibody fragments capable of binding to a biomarker protein or portions thereof including, but not limited to, Fv, Fab, Fab' and F(ab') 2 fragments can be used.
  • Such fragments can be produced by enzymatic cleavage or by recombinant techniques. For example, papain or pepsin cleavage can generate Fab or F(ab') 2 fragments, respectively.
  • Fab or F(ab') 2 fragments can also be used to generate Fab or F(ab') 2 fragments.
  • Antibodies can also be produced in a variety of truncated forms using antibody genes in which one or more stop codons have been introduced upstream of the natural stop site.
  • a chimeric gene encoding a F(ab') 2 heavy chain portion can be designed to include DNA sequences encoding the CH, domain and hinge region of the heavy chain.
  • agents that specifically bind to a biomarker protein other than antibodies are used, such as peptides.
  • Peptides that specifically bind to a biomarker protein can be identified by any means known in the art. For example, specific peptide binders of a biomarker protein can be screened for using peptide phage display libraries. d. Methods for Detection of Biomarker Structural Alterations
  • biomarker nucleic acid and/or biomarker polypeptide molecule can be used to identify the presence of a structural alteration in a biomarker nucleic acid and/or biomarker polypeptide molecule in order to, for example, identify DPH1 protein that is overexpressed, overfunctional, and the like.
  • detection of the alteration involves the use of a probe/primer in a polymerase chain reaction (PCR) (see, e.g., U.S. Pat. Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR, or, alternatively, in a ligation chain reaction (LCR) (see, e.g., Landegran et al. (1988) Science 241 : 1077-1080; and Nakazawa et al. (1994) Proc. Natl. Acad. Sci.
  • PCR polymerase chain reaction
  • LCR ligation chain reaction
  • This method can include the steps of collecting a sample of cells from a subject, isolating nucleic acid (e.g., genomic, mRNA or both) from the cells of the sample, contacting the nucleic acid sample with one or more primers which specifically hybridize to a biomarker gene under conditions such that hybridization and amplification of the biomarker gene (if present) occurs, and detecting the presence or absence of an amplification product, or detecting the size of the amplification product and comparing the length to a control sample. It is anticipated that PCR and/or LCR may be desirable to use as a preliminary amplification step in conjunction with any of the techniques used for detecting mutations described herein.
  • nucleic acid e.g., genomic, mRNA or both
  • Alternative amplification methods include: self sustained sequence replication (Guatelli, J. C. et al. (1990) Proc. Natl. Acad. Sci. USA 87:1874-1878), transcriptional amplification system (Kwoh, D. Y. et al. (1989) Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-Beta Replicase (Lizardi, P. M. et al. (1988) Bio-Technology 6: 1197), or any other nucleic acid amplification method, followed by the detection of the amplified molecules using techniques well known to those of skill in the art. These detection schemes are especially useful for the detection of nucleic acid molecules if such molecules are present in very low numbers.
  • mutations in a biomarker nucleic acid from a sample cell can be identified by alterations in restriction enzyme cleavage patterns.
  • sample and control DNA is isolated, amplified (optionally), digested with one or more restriction endonucleases, and fragment length sizes are determined by gel electrophoresis and compared. Differences in fragment length sizes between sample and control DNA indicates mutations in the sample DNA.
  • sequence specific ribozymes see, for example, U.S. Pat. No. 5,498,531 can be used to score for the presence of specific mutations by development or loss of a ribozyme cleavage site.
  • biomarker nucleic acid can be identified by hybridizing a sample and control nucleic acids, e.g., DNA or RNA, to high density arrays containing hundreds or thousands of oligonucleotide probes (Cronin, M. T. et al. (1996) Hum. Mutat. 7:244-255; Kozal, M. J. et al. (1996) Nat. Med. 2:753-759).
  • biomarker genetic mutations can be identified in two dimensional arrays containing lightgenerated DNA probes as described in Cronin et al. (1996) supra.
  • a first hybridization array of probes can be used to scan through long stretches of DNA in a sample and control to identify base changes between the sequences by making linear arrays of sequential, overlapping probes. This step allows the identification of point mutations. This step is followed by a second hybridization array that allows the characterization of specific mutations by using smaller, specialized probe arrays complementary to all variants or mutations detected. Each mutation array is composed of parallel probe sets, one complementary to the wild-type gene and the other complementary to the mutant gene.
  • biomarker genetic mutations can be identified in a variety of contexts, including, for example, germline and somatic mutations.
  • any of a variety of sequencing reactions known in the art can be used to directly sequence a biomarker gene and detect mutations by comparing the sequence of the sample biomarker with the corresponding wild-type (control) sequence.
  • Examples of sequencing reactions include those based on techniques developed by Maxam and Gilbert (1977) Proc. Natl. Acad. Sci. USA 74:560 or Sanger (1977) Proc. Natl. Acad Sci. USA 74:5463. It is also contemplated that any of a variety of automated sequencing procedures can be utilized when performing the diagnostic assays (Naeve (1995) Biotechniques 19:448-53), including sequencing by mass spectrometry (see, e.g., PCT International Publication No. WO 94/16101; Cohen et al. (1996) Adv. Chromatogr. 36: 127- 162; and Griffin et al. (1993) Appl. Biochem. Biotechnol. 38: 147-159).
  • RNA/RNA or RNA/DNA heteroduplexes Other methods for detecting mutations in a biomarker gene include methods in which protection from cleavage agents is used to detect mismatched bases in RNA/RNA or RNA/DNA heteroduplexes (Myers et al. (1985) Science 230: 1242).
  • the art technique of “mismatch cleavage” starts by providing heteroduplexes formed by hybridizing (labeled) RNA or DNA containing the wild-type biomarker sequence with potentially mutant RNA or DNA obtained from a tissue sample.
  • the double-stranded duplexes are treated with an agent which cleaves single-stranded regions of the duplex such as which will exist due to base pair mismatches between the control and sample strands.
  • RNA/DNA duplexes can be treated with RNase and DNA/DNA hybrids treated with SI nuclease to enzymatically digest the mismatched regions.
  • either DNA/DNA or RNA/DNA duplexes can be treated with hydroxylamine or osmium tetroxide and with piperidine in order to digest mismatched regions. After digestion of the mismatched regions, the resulting material is then separated by size on denaturing polyacrylamide gels to determine the site of mutation. See, for example, Cotton et al. (1988) Proc. Natl. Acad. Sci. USA 85:4397 and Saleeba et al. (1992) Methods Enzymol. 217:286-295.
  • the control DNA or RNA can be labeled for detection.
  • the mismatch cleavage reaction employs one or more proteins that recognize mismatched base pairs in double-stranded DNA (so called “DNA mismatch repair” enzymes) in defined systems for detecting and mapping point mutations in biomarker cDNAs obtained from samples of cells.
  • DNA mismatch repair enzymes
  • the mutY enzyme of E. coli cleaves A at G/A mismatches and the thymidine DNA glycosylase from HeLa cells cleaves T at G/T mismatches (Hsu et al. (1994) Carcinogenesis 15: 1657-1662).
  • a probe based on a biomarker sequence e.g., a wild-type biomarker treated with a DNA mismatch repair enzyme, and the cleavage products, if any, can be detected from electrophoresis protocols or the like (e.g., U.S. Pat. No. 5,459,039.)
  • alterations in electrophoretic mobility can be used to identify mutations in biomarker genes.
  • SSCP single strand conformation polymorphism
  • Single-stranded DNA fragments of sample and control biomarker nucleic acids will be denatured and allowed to renature.
  • the secondary structure of single-stranded nucleic acids varies according to sequence, the resulting alteration in electrophoretic mobility enables the detection of even a single base change.
  • the DNA fragments may be labeled or detected with labeled probes.
  • the sensitivity of the assay may be enhanced by using RNA (rather than DNA), in which the secondary structure is more sensitive to a change in sequence.
  • the subject method utilizes heteroduplex analysis to separate double stranded heteroduplex molecules on the basis of changes in electrophoretic mobility (Keen et al. (1991) Trends Genet. 7:5).
  • the movement of mutant or wild-type fragments in polyacrylamide gels containing a gradient of denaturant is assayed using denaturing gradient gel electrophoresis (DGGE) (Myers et al. (1985) Nature 313:495).
  • DGGE denaturing gradient gel electrophoresis
  • DNA will be modified to ensure that it does not completely denature, for example by adding a GC clamp of approximately 40 bp of high-melting GC-rich DNA by PCR.
  • a temperature gradient is used in place of a denaturing gradient to identify differences in the mobility of control and sample DNA (Rosenbaum and Reissner (1987) Biophys. Chem. 265: 12753).
  • oligonucleotide primers may be prepared in which the known mutation is placed centrally and then hybridized to target DNA under conditions which permit hybridization only if a perfect match is found (Saiki et al. (1986) Nature 324: 163; Saiki et al. (1989) Proc. Natl. Acad. Set. USA 86:6230).
  • Such allele specific oligonucleotides are hybridized to PCR amplified target DNA or a number of different mutations when the oligonucleotides are attached to the hybridizing membrane and hybridized with labeled target DNA.
  • Oligonucleotides used as primers for specific amplification may carry the mutation of interest in the center of the molecule (so that amplification depends on differential hybridization) (Gibbs et al. (1989) Nucleic Acids Res. 17:2437-2448) or at the extreme 3' end of one primer where, under appropriate conditions, mismatch can prevent, or reduce polymerase extension (Prossner (1993) Tibtech 11 :238).
  • amplification may also be performed using Taq ligase for amplification (Barany (1991) Proc. Natl. Acad. Sci USA 88: 189). In such cases, ligation will occur only if there is a perfect match at the 3' end of the 5' sequence making it possible to detect the presence of a known mutation at a specific site by looking for the presence or absence of amplification.
  • immune checkpoint therapy e.g., an immune checkpoint and radiation combination therapy or an immune checkpoint monotherapy
  • immune checkpoint therapy or combinations of therapies e.g., anti-PD-1 antibodies
  • immune checkpoint therapy can be avoided once a subject is indicated as not being a likely responder to immune checkpoint therapy and an alternative treatment regimen, such as targeted and/or untargeted anti-cancer therapies can be administered.
  • Combination therapies are also contemplated and can comprise, for example, one or more chemotherapeutic agents and radiation, one or more chemotherapeutic agents and immunotherapy, or one or more chemotherapeutic agents, radiation and chemotherapy, each combination of which can be with immune checkpoint therapy.
  • targeted therapy refers to administration of agents that selectively interact with a chosen biomolecule to thereby treat cancer.
  • Immunotherapy is one form of targeted therapy that may comprise, for example, the use of cancer vaccines and/or sensitized antigen presenting cells.
  • an oncolytic virus is a virus that is able to infect and lyse cancer cells, while leaving normal cells unharmed, making them potentially useful in cancer therapy. Replication of oncolytic viruses both facilitates tumor cell destruction and also produces dose amplification at the tumor site. They may also act as vectors for anticancer genes, allowing them to be specifically delivered to the tumor site.
  • the immunotherapy can involve passive immunity for short-term protection of a host, achieved by the administration of pre-formed antibody directed against a cancer antigen or disease antigen (e.g., administration of a monoclonal antibody, optionally linked to a chemotherapeutic agent or toxin, to a tumor antigen). Immunotherapy can also focus on using the cytotoxic lymphocyte-recognized epitopes of cancer cell lines. Alternatively, antisense polynucleotides, ribozymes, RNA interference molecules, triple helix polynucleotides and the like, can be used to selectively modulate biomolecules that are linked to the initiation, progression, and/or pathology of a tumor or cancer.
  • untargeted therapy referes to administration of agents that do not selectively interact with a chosen biomolecule yet treat cancer.
  • ReRepresentative examples of untargeted therapies include, without limitation, chemotherapy, gene therapy, and radiation therapy.
  • Chemotherapy includes the administration of a chemotherapeutic agent.
  • a chemotherapeutic agent may be, but is not limited to, those selected from among the following groups of compounds: platinum compounds, cytotoxic antibiotics, antimetabolities, anti-mitotic agents, alkylating agents, arsenic compounds, DNA topoisomerase inhibitors, taxanes, nucleoside analogues, plant alkaloids, and toxins; and synthetic derivatives thereof.
  • Exemplary compounds include, but are not limited to, alkylating agents: cisplatin, treosulfan, and trofosfamide; plant alkaloids: vinblastine, paclitaxel, docetaxol; DNA topoisomerase inhibitors: teniposide, crisnatol, and mitomycin; anti-folates: methotrexate, mycophenolic acid, and hydroxyurea; pyrimidine analogs: 5-fluorouracil, doxifluridine, and cytosine arabinoside; purine analogs: mercaptopurine and thioguanine; DNA antimetabolites: 2'-deoxy-5-fluorouridine, aphidicolin glycinate, and pyrazoloimidazole; and antimitotic agents: halichondrin, colchicine, and rhizoxin.
  • alkylating agents cisplatin, treosulfan, and trofosfamide
  • compositions comprising one or more chemotherapeutic agents (e.g., FLAG, CHOP) may also be used.
  • FLAG comprises fludarabine, cytosine arabinoside (Ara-C) and G-CSF.
  • CHOP comprises cyclophosphamide, vincristine, doxorubicin, and prednisone.
  • PARP e.g., PARP-1 and/or PARP-2
  • inhibitors are well known in the art (e.g., Olaparib, ABT-888, BSI-201, BGP-15 (N-Gene Research Laboratories, Inc.); INO-lOOl (Inotek Pharmaceuticals Inc.); PJ34 (Soriano et cd.. 2001; Pacher et al., 2002b); 3 -aminobenzamide (Trevigen); 4-amino-l,8-naphthalimide; (Trevigen); 6(5H)-phenanthridinone (Trevigen); benzamide (U.S. Pat. Re.
  • the mechanism of action is generally related to the ability of PARP inhibitors to bind PARP and decrease its activity.
  • PARP catalyzes the conversion of .beta.-nicotinamide adenine dinucleotide (NAD+) into nicotinamide and poly-ADP -ribose (PAR).
  • NAD+ .beta.-nicotinamide adenine dinucleotide
  • PARPAR poly-ADP -ribose
  • Both poly (ADP-ribose) and PARP have been linked to regulation of transcription, cell proliferation, genomic stability, and carcinogenesis (Bouchard V. J. et.al. Experimental Hematology, Volume 31, Number 6, June 2003, pp. 446-454(9); Herceg Z.; Wang Z.-Q.
  • PARP1 Poly(ADP -ribose) polymerase 1
  • SSBs DNA single-strand breaks
  • DSBs DNA double-strand breaks
  • chemotherapeutic agents are illustrative, and are not intended to be limiting.
  • radiation therapy is used.
  • the radiation used in radiation therapy can be ionizing radiation.
  • Radiation therapy can also be gamma rays, X-rays, or proton beams.
  • Examples of radiation therapy include, but are not limited to, external-beam radiation therapy, interstitial implantation of radioisotopes (1-125, palladium, iridium), radioisotopes such as strontium-89, thoracic radiation therapy, intraperitoneal P-32 radiation therapy, and/or total abdominal and pelvic radiation therapy.
  • the radiation therapy can be administered as external beam radiation or teletherapy wherein the radiation is directed from a remote source.
  • the radiation treatment can also be administered as internal therapy or brachytherapy wherein a radioactive source is placed inside the body close to cancer cells or a tumor mass.
  • photodynamic therapy comprising the administration of photosensitizers, such as hematoporphyrin and its derivatives, Vertoporfin (BPD-MA), phthalocyanine, photosensitizer Pc4, demethoxy- hypocrellin A; and 2BA-2-DMHA.
  • hormone therapy is used.
  • Hormonal therapeutic treatments can comprise, for example, hormonal agonists, hormonal antagonists (e.g., flutamide, bicalutamide, tamoxifen, raloxifene, leuprolide acetate (LUPRON), LH-RH antagonists), inhibitors of hormone biosynthesis and processing, and steroids (e.g., dexamethasone, retinoids, deltoids, betamethasone, cortisol, cortisone, prednisone, dehydrotestosterone, glucocorticoids, mineralocorticoids, estrogen, testosterone, progestins), vitamin A derivatives (e.g., all-trans retinoic acid (ATRA)); vitamin D3 analogs; antigestagens (e.g., mifepristone, onapristone), or antiandrogens (e.g., cyproterone acetate).
  • hormonal antagonists e.g., flutamide, bicalu
  • hyperthermia a procedure in which body tissue is exposed to high temperatures (up to 106°F.) is used. Heat may help shrink tumors by damaging cells or depriving them of substances they need to live.
  • Hyperthermia therapy can be local, regional, and whole-body hyperthermia, using external and internal heating devices. Hyperthermia is almost always used with other forms of therapy (e.g., radiation therapy, chemotherapy, and biological therapy) to try to increase their effectiveness.
  • Local hyperthermia refers to heat that is applied to a very small area, such as a tumor. The area may be heated externally with high-frequency waves aimed at a tumor from a device outside the body.
  • sterile probes may be used, including thin, heated wires or hollow tubes filled with warm water; implanted microwave antennae; and radiofrequency electrodes.
  • regional hyperthermia an organ or a limb is heated. Magnets and devices that produce high energy are placed over the region to be heated.
  • perfusion some of the patient's blood is removed, heated, and then pumped (perfused) into the region that is to be heated internally.
  • Whole-body heating is used to treat metastatic cancer that has spread throughout the body. It can be accomplished using warm-water blankets, hot wax, inductive coils (like those in electric blankets), or thermal chambers (similar to large incubators). Hyperthermia does not cause any marked increase in radiation side effects or complications. Heat applied directly to the skin, however, can cause discomfort or even significant local pain in about half the patients treated. It can also cause blisters, which generally heal rapidly.
  • photodynamic therapy also called PDT, photoradiation therapy, phototherapy, or photochemotherapy
  • PDT photoradiation therapy
  • phototherapy phototherapy
  • photochemotherapy is used for the treatment of some types of cancer. It is based on the discovery that certain chemicals known as photosensitizing agents can kill one-celled organisms when the organisms are exposed to a particular type of light.
  • PDT destroys cancer cells through the use of a fixed-frequency laser light in combination with a photosensitizing agent.
  • the photosensitizing agent is injected into the bloodstream and absorbed by cells all over the body. The agent remains in cancer cells for a longer time than it does in normal cells.
  • the photosensitizing agent absorbs the light and produces an active form of oxygen that destroys the treated cancer cells.
  • the laser light used in PDT can be directed through a fiber-optic (a very thin glass strand).
  • the fiber-optic is placed close to the cancer to deliver the proper amount of light.
  • the fiber-optic can be directed through a bronchoscope into the lungs for the treatment of lung cancer or through an endoscope into the esophagus for the treatment of esophageal cancer.
  • PDT is mainly used to treat tumors on or just under the skin or on the lining of internal organs.
  • Photodynamic therapy makes the skin and eyes sensitive to light for 6 weeks or more after treatment. Patients are advised to avoid direct sunlight and bright indoor light for at least 6 weeks. If patients must go outdoors, they need to wear protective clothing, including sunglasses.
  • Other temporary side effects of PDT are related to the treatment of specific areas and can include coughing, trouble swallowing, abdominal pain, and painful breathing or shortness of breath. In December 1995, the U.S.
  • FDA Food and Drug Administration
  • porfimer sodium or Photofrin®
  • Photofrin® a photosensitizing agent
  • the FDA approved porfimer sodium for the treatment of early nonsmall cell lung cancer in patients for whom the usual treatments for lung cancer are not appropriate.
  • the National Cancer Institute and other institutions are supporting clinical trials (research studies) to evaluate the use of photodynamic therapy for several types of cancer, including cancers of the bladder, brain, larynx, and oral cavity.
  • laser therapy is used to harness high-intensity light to destroy cancer cells.
  • This technique is often used to relieve symptoms of cancer such as bleeding or obstruction, especially when the cancer cannot be cured by other treatments. It may also be used to treat cancer by shrinking or destroying tumors.
  • the term “laser” stands for light amplification by stimulated emission of radiation. Ordinary light, such as that from a light bulb, has many wavelengths and spreads in all directions. Laser light, on the other hand, has a specific wavelength and is focused in a narrow beam. This type of high-intensity light contains a lot of energy. Lasers are very powerful and may be used to cut through steel or to shape diamonds.
  • CO2 laser Carbon dioxide
  • This type of laser can remove thin layers from the skin's surface without penetrating the deeper layers. This technique is particularly useful in treating tumors that have not spread deep into the skin and certain precancerous conditions.
  • the CO2 laser is also able to cut the skin. The laser is used in this way to remove skin cancers.
  • Neodymium:yttrium-aluminum-gamet (Nd: YAG) laser Light from this laser can penetrate deeper into tissue than light from the other types of lasers, and it can cause blood to clot quickly. It can be carried through optical fibers to less accessible parts of the body. This type of laser is sometimes used to treat throat cancers.
  • Argon laser This laser can pass through only superficial layers of tissue and is therefore useful in dermatology and in eye surgery. It also is used with light-sensitive dyes to treat tumors in a procedure known as photodynamic therapy (PDT). Lasers have several advantages over standard surgical tools, including: Lasers are more precise than scalpels. Tissue near an incision is protected, since there is little contact with surrounding skin or other tissue.
  • Lasers sterilizes the surgery site, thus reducing the risk of infection. Less operating time may be needed because the precision of the laser allows for a smaller incision. Healing time is often shortened; since laser heat seals blood vessels, there is less bleeding, swelling, or scarring. Laser surgery may be less complicated. For example, with fiber optics, laser light can be directed to parts of the body without making a large incision. More procedures may be done on an outpatient basis. Lasers can be used in two ways to treat cancer: by shrinking or destroying a tumor with heat, or by activating a chemical— known as a photosensitizing agent— that destroys cancer cells.
  • a chemical known as a photosensitizing agent
  • a photosensitizing agent is retained in cancer cells and can be stimulated by light to cause a reaction that kills cancer cells.
  • CO2 and Nd: YAG lasers are used to shrink or destroy tumors. They may be used with endoscopes, tubes that allow physicians to see into certain areas of the body, such as the bladder. The light from some lasers can be transmitted through a flexible endoscope fitted with fiber optics. This allows physicians to see and work in parts of the body that could not otherwise be reached except by surgery and therefore allows very precise aiming of the laser beam. Lasers also may be used with low-power microscopes, giving the doctor a clear view of the site being treated.
  • Lasers Used with other instruments, laser systems can produce a cutting area as small as 200 microns in diameter— less than the width of a very fine thread.
  • Lasers are used to treat many types of cancer.
  • Laser surgery is a standard treatment for certain stages of glottis (vocal cord), cervical, skin, lung, vaginal, vulvar, and penile cancers.
  • laser surgery is also used to help relieve symptoms caused by cancer (palliative care).
  • lasers may be used to shrink or destroy a tumor that is blocking a patient's trachea (windpipe), making it easier to breathe. It is also sometimes used for palliation in colorectal and anal cancer.
  • LITT Laser-induced interstitial thermotherapy
  • hyperthermia a cancer treatment
  • heat may help shrink tumors by damaging cells or depriving them of substances they need to live.
  • lasers are directed to interstitial areas (areas between organs) in the body. The laser light then raises the temperature of the tumor, which damages or destroys cancer cells.
  • the duration and/or dose of treatment with anti-immune checkpoint therapies may vary according to the particular anti-immune checkpoint agent or combination thereof.
  • An appropriate treatment time for a particular cancer therapeutic agent will be appreciated by the skilled artisan.
  • the present invention contemplates the continued assessment of optimal treatment schedules for each cancer therapeutic agent, where the phenotype of the cancer of the subject as determined by the methods of the present invention is a factor in determining optimal treatment doses and schedules.
  • any means for the introduction of a polynucleotide into mammals, human or nonhuman, or cells thereof may be adapted to the practice of this invention for the delivery of the various constructs of the present invention into the intended recipient.
  • the DNA constructs are delivered to cells by transfection, z.e., by delivery of “naked” DNA or in a complex with a colloidal dispersion system.
  • a colloidal system includes macromolecule complexes, nanocapsules, microspheres, beads, and lipid- based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes.
  • the preferred colloidal system of this invention is a lipid-complexed or liposome-formulated DNA.
  • a plasmid containing a transgene bearing the desired DNA constructs may first be experimentally optimized for expression (e.g., inclusion of an intron in the 5' untranslated region and elimination of unnecessary sequences (Feigner, et al., Ann NY Acad Sci 126-139, 1995).
  • Formulation of DNA, e.g. with various lipid or liposome materials may then be effected using known methods and materials and delivered to the recipient mammal.
  • the targeting of liposomes can be classified based on anatomical and mechanistic factors.
  • Anatomical classification is based on the level of selectivity, for example, organspecific, cell-specific, and organelle-specific.
  • Mechanistic targeting can be distinguished based upon whether it is passive or active. Passive targeting utilizes the natural tendency of liposomes to distribute to cells of the reticulo-endothelial system (RES) in organs, which contain sinusoidal capillaries.
  • RES reticulo-endothelial system
  • Active targeting involves alteration of the liposome by coupling the liposome to a specific ligand such as a monoclonal antibody, sugar, glycolipid, or protein, or by changing the composition or size of the liposome in order to achieve targeting to organs and cell types other than the naturally occurring sites of localization.
  • a specific ligand such as a monoclonal antibody, sugar, glycolipid, or protein
  • the surface of the targeted delivery system may be modified in a variety of ways.
  • lipid groups can be incorporated into the lipid bilayer of the liposome in order to maintain the targeting ligand in stable association with the liposomal bilayer.
  • Various linking groups can be used for joining the lipid chains to the targeting ligand. Naked DNA or DNA associated with a delivery vehicle, e.g., liposomes, can be administered to several sites in a subject (see below).
  • Nucleic acids can be delivered in any desired vector. These include viral or non-viral vectors, including adenovirus vectors, adeno-associated virus vectors, retrovirus vectors, lentivirus vectors, and plasmid vectors. Exemplary types of viruses include HSV (herpes simplex virus), AAV (adeno associated virus), HIV (human immunodeficiency virus), BIV (bovine immunodeficiency virus), and MLV (murine leukemia virus). Nucleic acids can be administered in any desired format that provides sufficiently efficient delivery levels, including in virus particles, in liposomes, in nanoparticles, and complexed to polymers.
  • viral or non-viral vectors including adenovirus vectors, adeno-associated virus vectors, retrovirus vectors, lentivirus vectors, and plasmid vectors. Exemplary types of viruses include HSV (herpes simplex virus), AAV (adeno associated virus), HIV (human immunodeficiency virus), BIV (bovine
  • the nucleic acids encoding a protein or nucleic acid of interest may be in a plasmid or viral vector, or other vector as is known in the art. Such vectors are well known and any can be selected for a particular application.
  • the gene delivery vehicle comprises a promoter and a demethylase coding sequence.
  • Preferred promoters are tissue-specific promoters and promoters which are activated by cellular proliferation, such as the thymidine kinase and thymidylate synthase promoters.
  • promoters which are activatable by infection with a virus such as the a- and P-interferon promoters, and promoters which are activatable by a hormone, such as estrogen.
  • promoters which can be used include the Moloney virus LTR, the CMV promoter, and the mouse albumin promoter.
  • a promoter may be constitutive or inducible.
  • naked polynucleotide molecules are used as gene delivery vehicles, as described in WO 90/11092 and U.S. Patent 5,580,859.
  • gene delivery vehicles can be either growth factor DNA or RNA and, in certain embodiments, are linked to killed adenovirus. Curiel et al., Hum. Gene. Ther. 3: 147-154, 1992.
  • Other vehicles which can optionally be used include DNA-ligand (Wu et al., J. Biol. Chem. 264: 16985-16987, 1989), lipid-DNA combinations (Feigner et al., Proc. Natl. Acad. Sci.
  • a gene delivery vehicle can optionally comprise viral sequences such as a viral origin of replication or packaging signal. These viral sequences can be selected from viruses such as astrovirus, coronavirus, orthomyxovirus, papovavirus, paramyxovirus, parvovirus, picornavirus, poxvirus, retrovirus, togavirus or adenovirus.
  • the growth factor gene delivery vehicle is a recombinant retroviral vector.
  • Recombinant retroviruses and various uses thereof have been described in numerous references including, for example, Mann et al., Cell 33: 153, 1983, Cane and Mulligan, Proc. Nat'l. Acad. Sci. USA 81 :6349, 1984, Miller et al., Human Gene Therapy 1 :5-14, 1990, U.S. Patent Nos. 4,405,712, 4,861,719, and 4,980,289, and PCT Application Nos. WO 89/02,468, WO 89/05,349, and WO 90/02,806.
  • Numerous retroviral gene delivery vehicles can be utilized in the present invention, including for example those described in EP 0,415,731; WO 90/07936; WO 94/03622; WO 93/25698; WO 93/25234; U.S. Patent No. 5,219,740; WO 9311230; WO 9310218; Vile and Hart, Cancer Res. 53:3860-3864, 1993; Vile and Hart, Cancer Res. 53:962-967, 1993; Ram et al., Cancer Res. 53:83-88, 1993; Takamiya et al., J. Neurosci. Res. 33:493-503, 1992; Baba et al., J. Neurosurg. 79:729-735, 1993 (U.S. Patent No. 4,777,127, GB 2,200,651, EP 0,345,242 and W091/02805).
  • Herpes virus e.g., Herpes Simplex Virus (U.S. Patent No. 5,631,236 by Woo et al., issued May 20, 1997 and WO 00/08191 by Neurovex), vaccinia virus (Ridgeway (1988) Ridgeway, “Mammalian expression vectors,” In: Rodriguez R L, Denhardt D T, ed.
  • Vectors A survey of molecular cloning vectors and their uses.
  • RNA viruses include an alphavirus, a poxivirus, an arena virus, a vaccinia virus, a polio virus, and the like. They offer several attractive features for various mammalian cells (Friedmann (1989) Science, 244: 1275-1281; Ridgeway, 1988, supra; Baichwal and Sugden, 1986, supra; Coupar et al., 1988; Horwich et al. (1990) J. Virol., 64:642-650).
  • target DNA in the genome can be manipulated using well- known methods in the art.
  • the target DNA in the genome can be manipulated by deletion, insertion, and/or mutation are retroviral insertion, artificial chromosome techniques, gene insertion, random insertion with tissue specific promoters, gene targeting, transposable elements and/or any other method for introducing foreign DNA or producing modified DNA/modified nuclear DNA.
  • Other modification techniques include deleting DNA sequences from a genome and/or altering nuclear DNA sequences. Nuclear DNA sequences, for example, may be altered by site-directed mutagenesis.
  • biomarker polypeptides, and fragments thereof can be administered to subjects.
  • fusion proteins can be constructed and administered which have enhanced biological properties.
  • biomarker polypeptides, and fragment thereof can be modified according to well-known pharmacological methods in the art (e.g., pegylation, glycosylation, oligomerization, etc.) in order to further enhance desirable biological activities, such as increased bioavailability and decreased proteolytic degradation.
  • the response to a therapy relates to any response of the cancer, e.g., a tumor, to the therapy, preferably to a change in tumor mass and/or volume after initiation of neoadjuvant or adjuvant chemotherapy.
  • Tumor response may be assessed in a neoadjuvant or adjuvant situation where the size of a tumor after systemic intervention can be compared to the initial size and dimensions as measured by CT, PET, mammogram, ultrasound or palpation and the cellularity of a tumor can be estimated histologically and compared to the cellularity of a tumor biopsy taken before initiation of treatment.
  • Response may also be assessed by caliper measurement or pathological examination of the tumor after biopsy or surgical resection.
  • Response may be recorded in a quantitative fashion like percentage change in tumor volume or cellularity or using a semi-quantitative scoring system such as residual cancer burden (Symmans et aL, J. Clin. Oncol. (2007) 25:4414-4422) or Miller-Payne score (Ogston et al., (2003) Breast (Edinburgh, Scotland) 12:320-327) in a qualitative fashion like “pathological complete response” (pCR), “clinical complete remission” (cCR), “clinical partial remission” (cPR), “clinical stable disease” (cSD), “clinical progressive disease” (cPD) or other qualitative criteria.
  • pathological complete response pCR
  • cCR clinical complete remission
  • cPR clinical partial remission
  • cSD clinical stable disease
  • cPD clinical progressive disease
  • Assessment of tumor response may be performed early after the onset of neoadjuvant or adjuvant therapy, e.g., after a few hours, days, weeks or preferably after a few months.
  • a typical endpoint for response assessment is upon termination of neoadjuvant chemotherapy or upon surgical removal of residual tumor cells and/or the tumor bed.
  • clinical efficacy of the therapeutic treatments described herein may be determined by measuring the clinical benefit rate (CBR).
  • CBR clinical benefit rate
  • the clinical benefit rate is measured by determining the sum of the percentage of patients who are in complete remission (CR), the number of patients who are in partial remission (PR) and the number of patients having stable disease (SD) at a time point at least 6 months out from the end of therapy.
  • the CBR for a particular anti-immune checkpoint therapeutic regimen is at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or more.
  • Additional criteria for evaluating the response to anti-immune checkpoint therapies are related to “survival,” which includes all of the following: survival until mortality, also known as overall survival (wherein said mortality may be either irrespective of cause or tumor related); “recurrence-free survival” (wherein the term recurrence shall include both localized and distant recurrence); metastasis free survival; disease free survival (wherein the term disease shall include cancer and diseases associated therewith).
  • the length of said survival may be calculated by reference to a defined start point (e.g., time of diagnosis or start of treatment) and end point (e.g., death, recurrence or metastasis).
  • criteria for efficacy of treatment can be expanded to include response to chemotherapy, probability of survival, probability of metastasis within a given time period, and probability of tumor recurrence.
  • a particular anti- immune checkpoint therapeutic regimen can be administered to a population of subjects and the outcome can be correlated to biomarker measurements that were determined prior to administration of any immune checkpoint therapy.
  • the outcome measurement may be pathologic response to therapy given in the neoadjuvant setting.
  • outcome measures such as overall survival and disease-free survival can be monitored over a period of time for subjects following immune checkpoint therapy for whom biomarker measurement values are known.
  • the same doses of anti-immune checkpoint agents are administered to each subject.
  • the doses administered are standard doses known in the art for anti-immune checkpoint agents. The period of time for which subjects are monitored can vary.
  • Biomarker measurement threshold values that correlate to outcome of an immune checkpoint therapy can be determined using methods such as those described in the Examples section.
  • the methods described herein can be used in a variety of diagnostic, prognostic, and therapeutic applications.
  • any method described herein such as a diagnostic method, prognostic method, therapeutic method, or combination thereof, all steps of the method can be performed by a single actor or, alternatively, by more than one actor.
  • diagnosis can be performed directly by the actor providing therapeutic treatment.
  • a person providing a therapeutic agent can request that a diagnostic assay be performed.
  • the diagnostician and/or the therapeutic interventionist can interpret the diagnostic assay results to determine a therapeutic strategy.
  • such alternative processes can apply to other assays, such as prognostic assays.
  • compositions described herein can also be used in a variety of diagnostic, prognostic, and therapeutic applications regarding biomarkers described herein, such as those listed in Table 2. Moreover, any method of diagnosis, prognosis, prevention, and the like described herein can be applied to a therapy or test agent of interest, such as immune checkpoint therapies, radiation therapies, anti-cancer therapies, and the like. a. Screening Methods
  • One aspect of the present invention relates to screening assays, including non-cell based assays.
  • the assays provide a method for identifying whether a cancer is likely to respond to immune checkpoint therapy and/or whether an agent can inhibit the growth of or kill a cancer cell that is unlikely to respond to immune checkpoint therapy.
  • the present invention relates to assays for screening test agents which modulates the amount of at least one biomarker listed in Table 2.
  • a method for identifying such an agent entails determining the ability of the agent to modulate, e.g., reduce the amount of the at least one biomarker listed from number 1-135 in Table 2, and/or increase the amount of the at least one biomarker listed from number 136-140 in Table 2.
  • an assay is a cell-free or cell-based assay, comprising contacting at least one biomarker listed in Table 2, with a test agent, and determining the ability of the test agent to modulate the amount of the biomarker (e.g., reduce the amount of the at least one biomarker listed from number 1-135 in Table 2, and/or increase the amount of the at least one biomarker listed from number 136-140 in Table), such as by measuring direct binding of substrates or by measuring indirect parameters as described below.
  • biomarker molecules can be coupled with a radioisotope or enzymatic label such that binding can be determined by detecting the labeled protein or molecule in a complex.
  • the targets can be labeled with 125 1, 35 S, 14 C, or 3 H, either directly or indirectly, and the radioisotope detected by direct counting of radioemmission or by scintillation counting.
  • the targets can be enzymatically labeled with, for example, horseradish peroxidase, alkaline phosphatase, or luciferase, and the enzymatic label detected by determination of conversion of an appropriate substrate to product.
  • Determining the interaction between biomarker and substrate can also be accomplished using standard binding or enzymatic analysis assays.
  • Binding of a test agent to a target can be accomplished in any vessel suitable for containing the reactants.
  • vessels include microtiter plates, test tubes, and micro-centrifuge tubes.
  • Immobilized forms of the antibodies of the present invention can also include antibodies bound to a solid phase like a porous, microporous (with an average pore diameter less than about one micron) or macroporous (with an average pore diameter of more than about 10 microns) material, such as a membrane, cellulose, nitrocellulose, or glass fibers; a bead, such as that made of agarose or polyacrylamide or latex; or a surface of a dish, plate, or well, such as one made of polystyrene.
  • a solid phase like a porous, microporous (with an average pore diameter less than about one micron) or macroporous (with an average pore diameter of more than about 10 microns) material, such as a membrane, cellulose, nitrocellulose, or glass fibers
  • determining the ability of the agent to modulate the amount of the biomarker can be accomplished by determining the ability of the test agent to modulate the activity of a polypeptide or other product that functions downstream or upstream of its position within the signaling pathway (e.g., feedback loops).
  • feedback loops are well-known in the art (see, for example, Chen and Guillemin (2009) Ini. J. Tryptophan Res. 2: 1-19).
  • the present invention further pertains to novel agents identified by the abovedescribed screening assays. Accordingly, it is within the scope of this invention to further use an agent identified as described herein in an appropriate animal model.
  • an agent identified as described herein can be used in an animal model to determine the efficacy, toxicity, or side effects of treatment with such an agent.
  • an antibody identified as described herein can be used in an animal model to determine the mechanism of action of such an agent.
  • the present invention also pertains to the field of predictive medicine in which diagnostic assays, prognostic assays, and monitoring clinical trials are used for prognostic (predictive) purposes to thereby treat an individual prophylactically.
  • diagnostic assays for determining the amount and/or activity level of a biomarker listed in Table 2 in the context of a biological sample (e.g., blood, serum, cells, or tissue) to thereby determine whether an individual afflicted with a cancer (e.g., a lung cancer, a breast cancer, a prostate cancer, a melanoma, or a pancreatic cancer) is likely to respond to immune checkpoint therapy, whether in an original or recurrent cancer.
  • a biological sample e.g., blood, serum, cells, or tissue
  • a cancer e.g., a lung cancer, a breast cancer, a prostate cancer, a melanoma, or a pancreatic cancer
  • Another aspect of the present invention pertains to monitoring the influence of agents (e.g., drugs, compounds, and small nucleic acid-based molecules) on the expression or activity of a biomarker listed in Table 2.
  • agents e.g., drugs, compounds, and small nucleic acid-based molecules
  • the methods of the present invention implement a computer program and computer system.
  • a computer program can be used to perform the algorithms described herein.
  • a computer system can also store and manipulate data generated by the methods of the present invention which comprises a plurality of biomarker signal changes/profiles which can be used by a computer system in implementing the methods of this invention.
  • a computer system receives biomarker expression data; (ii) stores the data; and (iii) compares the data in any number of ways described herein (e.g., analysis relative to appropriate controls) to determine the state of informative biomarkers from cancerous or pre-cancerous tissue.
  • a computer system (i) compares the determined expression biomarker level to a threshold value; and (ii) outputs an indication of whether said biomarker level is significantly modulated (e.g., above or below) the threshold value, or a phenotype based on said indication.
  • such computer systems are also considered part of the present invention.
  • Numerous types of computer systems can be used to implement the analytic methods of this invention according to knowledge possessed by a skilled artisan in the bioinformatics and/or computer arts.
  • Several software components can be loaded into memory during operation of such a computer system.
  • the software components can comprise both software components that are standard in the art and components that are special to the present invention (e.g., dCHIP software described in Lin et al. (2004) Bioinformatics 20, 1233-1240; radial basis machine learning algorithms (RBM) known in the art).
  • dCHIP software described in Lin et al. (2004) Bioinformatics 20, 1233-1240
  • RBM radial basis machine learning algorithms
  • the methods of the present invention can also be programmed or modeled in mathematical software packages that allow symbolic entry of equations and high-level specification of processing, including specific algorithms to be used, thereby freeing a user of the need to procedurally program individual equations and algorithms.
  • Such packages include, e.g., Matlab from Mathworks (Natick, Mass.), Mathematica from Wolfram Research (Champaign, Ill.) or S-Plus from MathSoft (Seattle, Wash.).
  • the computer comprises a database for storage of biomarker data.
  • biomarker production profiles of a sample derived from the non-cancerous tissue of a subject and/or profiles generated from populationbased distributions of informative loci of interest in relevant populations of the same species can be stored and later compared to that of a sample derived from the cancerous tissue of the subject or tissue suspected of being cancerous of the subject.
  • the present invention provides, in part, methods, systems, and code for accurately classifying whether a biological sample is associated with a cancer that is likely to respond to immune checkpoint therapy (e.g., an immune checkpoint and radiation combination therapy or to an immune checkpoint monotherapy).
  • immune checkpoint therapy e.g., an immune checkpoint and radiation combination therapy or to an immune checkpoint monotherapy.
  • the present invention is useful for classifying a sample (e.g., from a subject) as associated with or at risk for responding to or not responding to immune checkpoint therapy using a statistical algorithm and/or empirical data (e.g., the amount or activity of a biomarker listed in Table 2).
  • An exemplary method for detecting the amount or activity of a biomarker listed in Table 2, and thus useful for classifying whether a sample is likely or unlikely to respond to immune checkpoint therapy involves obtaining a biological sample from a test subject and contacting the biological sample with an agent, such as a protein-binding agent like an antibody or antigen-binding fragment thereof, or a nucleic acid-binding agent like an oligonucleotide, capable of detecting the amount or activity of the biomarker in the biological sample.
  • an agent such as a protein-binding agent like an antibody or antigen-binding fragment thereof, or a nucleic acid-binding agent like an oligonucleotide, capable of detecting the amount or activity of the biomarker in the biological sample.
  • the statistical algorithm is a single learning statistical classifier system.
  • a single learning statistical classifier system can be used to classify a sample as a based upon a prediction or probability value and the presence or level of the biomarker.
  • a single learning statistical classifier system typically classifies the sample as, for example, a likely immune checkpoint therapy responder or progressor sample with a sensitivity, specificity, positive predictive value, negative predictive value, and/or overall accuracy of at least about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.
  • learning statistical classifier systems include a machine learning algorithmic technique capable of adapting to complex data sets (e.g., panel of markers of interest) and making decisions based upon such data sets.
  • a single learning statistical classifier system such as a classification tree (e.g., random forest) is used.
  • a combination of 2, 3, 4, 5, 6, 7, 8, 9, 10, or more learning statistical classifier systems are used, preferably in tandem.
  • Examples of learning statistical classifier systems include, but are not limited to, those using inductive learning (e.g., decision/classification trees such as random forests, classification and regression trees (C&RT), boosted trees, etc.), Probably Approximately Correct (PAC) learning, connect!
  • inductive learning e.g., decision/classification trees such as random forests, classification and regression trees (C&RT), boosted trees, etc.
  • PAC Probably Approximately Correct
  • onist learning e.g., neural networks (NN), artificial neural networks (ANN), neuro fuzzy networks (NFN), network structures, perceptrons such as multi-layer perceptrons, multi-layer feed-forward networks, applications of neural networks, Bayesian learning in belief networks, etc.
  • reinforcement learning e.g., passive learning in a known environment such as naive learning, adaptive dynamic learning, and temporal difference learning, passive learning in an unknown environment, active learning in an unknown environment, learning action-value functions, applications of reinforcement learning, etc.
  • genetic algorithms and evolutionary programming e.g., neural networks (NN), artificial neural networks (ANN), neuro fuzzy networks (NFN), network structures, perceptrons such as multi-layer perceptrons, multi-layer feed-forward networks, applications of neural networks, Bayesian learning in belief networks, etc.
  • reinforcement learning e.g., passive learning in a known environment such as naive learning, adaptive dynamic learning, and temporal difference learning, passive learning in an unknown environment, active learning in an unknown environment, learning action-value functions, applications of reinforcement
  • the method of the present invention further comprises sending the sample classification results to a clinician, e.g., an oncologist.
  • a clinician e.g., an oncologist.
  • the diagnosis of a subject is followed by administering to the individual a therapeutically effective amount of a defined treatment based upon the diagnosis.
  • the methods further involve obtaining a control biological sample (e.g., biological sample from a subject who does not have a cancer or whose cancer is susceptible to immune checkpoint therapy), a biological sample from the subject during remission, or a biological sample from the subject during treatment for developing a cancer progressing despite immune checkpoint therapy.
  • a control biological sample e.g., biological sample from a subject who does not have a cancer or whose cancer is susceptible to immune checkpoint therapy
  • a biological sample from the subject during remission e.g., a biological sample from the subject during remission
  • a biological sample from the subject during treatment for developing a cancer progressing despite immune checkpoint therapy e.g., prognostic Assays
  • the prognostic assays can be utilized to predict: (1) outcome (e.g., survival) of a subject having a cancer (e.g., a lung cancer, a breast cancer, a prostate cancer, a melanoma, or a pancreatic cancer), or (2) outcome (e.g., effects or survival) of a subject having a cancer (e.g., a lung cancer, a breast cancer, a prostate cancer, a melanoma, or a pancreatic cancer) undergoing an immunotherapy (e.g., an immune checkpoint and radiation combination therapy or to an immune checkpoint monotherapy).
  • an immunotherapy e.g., an immune checkpoint and radiation combination therapy or to an immune checkpoint monotherapy.
  • the prognostic assays described herein can be used to determine whether a subject can be administered an agent (e.g., an immune checkpoint and radiation combination therapy or to an immune checkpoint monotherapy) to treat a disease or disorder associated with the aberrant biomarker expression or activity.
  • an agent e.g., an immune checkpoint and radiation combination therapy or to an immune checkpoint monotherapy
  • compositions described herein can be used in a variety of in vitro and in vivo therapeutic applications using the formulations and/or combinations described herein.
  • anti-immune checkpoint agents (alone or in combination with radiation therapy such as a sub-ablative dose of SBRT) can be used to treat cancers determined to be responsive thereto.
  • antibodies that block the interaction between PD-L1, PD- L2, and/or CTLA-4 and their receptors e.g., PD-L1 binding to PD-1, PD-L2 binding to PD- 1, and the like
  • kits for detecting and/or modulating biomarkers described herein may also include instructional materials disclosing or describing the use of the kit in a method of the disclosed invention as provided herein.
  • a kit may also include additional components to facilitate the particular application for which the kit is designed.
  • a kit may additionally contain means of detecting the label (e.g., enzyme substrates for enzymatic labels, filter sets to detect fluorescent labels, appropriate secondary labels such as a sheep anti-mouse-HRP, etc. and reagents necessary for controls (e.g., control biological samples or standards).
  • a kit may additionally include buffers and other reagents recognized for use in a method of the disclosed invention. Non-limiting examples include agents to reduce non-specific binding, such as a carrier protein or a detergent.
  • Embodiment 1 A method of identifying the likelihood of a lung cancer in a subject to respond to a dual immune checkpoint and radiation therapy or to an immune checkpoint monotherapy, the method comprising: a) obtaining or providing a sample comprising cancer cells from a subject having the lung cancer; b) measuring one or more characteristics selected from (1) proliferation; (2) percentage of PD-L1 + cancer cells and/or PD-L1 expression; (3) number of CD4 + Th2 cells in the cancer cells of the subject sample; (4) expressions of glycolysis genes; (5) tumor mutational burdens (TMB), and (6) expressions of MHC-II genes; and c) comparing said one or more characteristics in a control, wherein a significant increase in the one or more characteristics selected from ( l)-(5) and/or a significant decrease in characteristic (6) in the cancer cells of the subject sample relative to the control identifies the lung cancer as being more likely to respond to the dual immune checkpoint and radiation therapy or to the immune checkpoint monotherapy, and wherein a significant decrease in the one
  • Embodiment 2 The method of embodiment 1, wherein the proliferation is measured by determining expressions of proliferation-associated genes.
  • Embodiment 3 The method of embodiment 1 or 2, wherein the proliferation is measured by calculating a proliferation index (PI) using the full gene set or the modified gene set shown in Table 3.
  • PI proliferation index
  • Embodiment 4 The method of any one of embodiments 1-3, wherein the proliferation is measured by determining expression of Ki67.
  • Embodiment 5 The method of embodiment 1 or 2, wherein the proliferation is measured by determining gene signatures for cell cycle phases or number of cells in S, G2 and M phases of cell cycle.
  • Embodiment 6 A method of identifying the likelihood of a lung cancer in a subject to respond to a dual immune checkpoint and radiation therapy or to an immune checkpoint monotherapy, the method comprising: a) obtaining or providing a sample comprising cancer cells from a subject having the lung cancer; b) measuring the copy number, amount, and/or activity of one or more genes from number 1-135 in Table 2 and/or of one or more genes from number 136-140 in Table 2 in the cancer cells of the subject sample; and c) comparing said copy number, amount, and/or activity of the one or more genes in a control, wherein a significantly increased copy number, amount, and/or activity of one or more genes from number 1-135 in Table 2, and/or a significantly decreased copy number, amount, and/or activity of one or more genes from number 136-140 in Table 2, in the cancer cells of the subject sample relative to the control identifies the lung cancer as being more likely to respond to the dual immune checkpoint and radiation therapy or to the immune checkpoint monotherapy, and wherein
  • Embodiment 7 The method of any one of embodiments 1-6, wherein the responsiveness to the dual immune checkpoint and radiation therapy or to an the immune checkpoint monotherapy is determined by achieving major pathological response (MPR).
  • MPR major pathological response
  • Embodiment 8 The method of any one of embodiments 1-7, wherein the lung cancer is NSCLC, e.g., a NSCLC with clinical stages I-IIIA.
  • NSCLC e.g., a NSCLC with clinical stages I-IIIA.
  • Embodiment 9 The method of any one of embodiments 1-8, further comprising recommending, prescribing, or administering dual immune checkpoint and radiation therapy or to the immune checkpoint monotherapy if the lung cancer is determined to be more likely to respond to the dual immune checkpoint and radiation therapy or to the immune checkpoint monotherapy.
  • Embodiment 10 The method of any one of embodiments 1-9, further comprising recommending, prescribing, or administering an anti-cancer therapy other than or in addition to the dual immune checkpoint and radiation therapy or to the immune checkpoint monotherapy if the lung cancer is determined to be less likely to respond to the dual immune checkpoint and radiation therapy or to immune checkpoint monotherapy.
  • Embodiment 11 The method of any one of embodiments 1-10, wherein the immune checkpoint therapy is an immune check point blockade (ICB) therapy.
  • Embodiment 12 The method of embodiment 11, wherein the immune checkpoint is PD1, PD-L1, or CTLA-4.
  • Embodiment 13 The method of embodiment 11 or 12, wherein the ICB is anti-PD-Ll antibody.
  • Embodiment 14 The method of embodiment 13, wherein the anti-PD-Ll antibody is Durvalumab.
  • Embodiment 15 The method of any one of embodiments 1-14, wherein the radiation therapy is stereotactic body radiation therapy (SBRT) therapy.
  • SBRT stereotactic body radiation therapy
  • Embodiment 16 A method of identifying the likelihood of a solid tumor in a subject to respond to an immune checkpoint therapy, the method comprising: a) obtaining or providing a sample comprising cancer cells from a subject having the solid tumor; b) measuring the copy number, amount, and/or activity of one or more genes from number 1- 135 in Table 2 and/or of one or more genes from number 136-140 in Table 2 in the cancer cells of the subject sample; and c) comparing said copy number, amount, and/or activity of the one or more genes in a control, wherein a significantly increased copy number, amount, and/or activity of one or more genes from number 1-135 in Table 2, and/or a significantly decreased copy number, amount, and/or activity of one or more genes from number 136-140 in Table 2, in the cancer cells of the subject sample relative to the control identifies the solid tumor as being more likely to respond to the immune checkpoint therapy, and wherein a significantly decreased copy number, amount, and/or activity of one or more genes from number 1-135 in
  • Embodiment 17 The method of embodiment 16, wherein the responsiveness to the immune checkpoint therapy is determined by achieving increased disease-free survival (DFS) than the control.
  • DFS disease-free survival
  • Embodiment 18 The method of embodiment 16 or 17, wherein the solid tumor is selected from the group consisting of lung, melanoma, breast, prostate, and pancreas cancers.
  • Embodiment 19 The method of any one of embodiments 16-18, further comprising recommending, prescribing, or administering the immune check point therapy if the lung cancer is determined to be more likely to respond to the immune check point therapy.
  • Embodiment 20 The method of any one of embodiments 16-19, further comprising recommending, prescribing, or administering the immune check point therapy if the lung cancer is determined to be less likely to respond to the immune check point therapy.
  • Embodiment 21 The method of any one of embodiments 16-20, wherein the immune checkpoint therapy is an immune check point blockade (ICB) therapy.
  • IICB immune check point blockade
  • Embodiment 22 The method of any one of embodiments 16-21, wherein the immune checkpoint is PD1, PD-L1, or CTLA-4.
  • Embodiment 23 The method of any one of embodiments 16-22, wherein the ICB is anti -PD -LI antibody.
  • Embodiment 24 The method of embodiment 23, wherein the anti-PD-Ll antibody is Durvalumab.
  • Embodiment 25 The method of any one of embodiments 1-24, wherein the control is determined from a cancerous or non-cancerous sample from either the subject or a member of the same species to which the subject belongs.
  • Embodiment 26 The method of any one of embodiments 1-25, wherein the control is a sample that comprises cells or does not comprise cells.
  • Embodiment 27 The method of embodiment 26, wherein the control sample comprises cancer cells that respond to the dual immune checkpoint and radiation therapy or to the immune checkpoint monotherapy, or that do not respond to the dual immune checkpoint and radiation therapy or to the immune checkpoint monotherapy.
  • Embodiment 28 The method of any one of embodiments 1-27, wherein the subject is a mammal.
  • Embodiment 29 The method of embodiment 28, wherein the mammal is a mouse or a human.
  • Biopsy tissue samples and tissue from resected tumors for this study were obtained from patients participating in a clinical trial approved by the New York- Presbyterian-Weill Cornell Medicine institutional review board and patient consent was obtained for biospecimen collection and analysis 3 . Participant profile information is provided in Table 1. Table 1. Study cohort. Related to Figure 1.
  • PD-L1 expression The immunohistochemistry for PD-L1 was performed with the Ventana PD-L1 (SP263) FDA approved platform that uses clone SP263 on a Ventana Benchmark instrument (Ventana Medical Systems Inc, Arlington AZ) 60 . All tumor samples were analyzed using tumor proportion score of the percentage of tumor cells showing any intensity of membranous staining, with a minimum of 100 cells needed for analysis.
  • Tumor cellularity A Hematoxylin and eosin stain slide was obtained after unstained sections were cut for molecular studies and the tissue dimensions compared to the prior unstained slides. Tumor containing areas were assessed for the relative area of tumor cells from non-tumor cells using the method of Dudley et al 61 with the modification of using 10% intervals. These tumor rich areas were marked on the unstained slides for manual microdissection of material slated for nucleic acid extraction.
  • RNA-seq RNA was extracted from FFPE samples with RNeasy FFPE kit from Qiagen, Part# 73504. Tissue from tumor adjacent lung were used as controls. NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (NEB #E7760, New England BioLabs, Ipswich, MA) was used for sample library preparation according to the manufacturer’s instruction. PCR enrichment of adaptor ligated cDNAs were pooled and hybridized with probes from Twist Exome Kit #2 (Twist Bioscience, San Francisco, CA). After post capture PCR amplification and purification, the quality of final libraries was checked and loaded to Illumina NovaSeq6000 for sequencing at PE2xl00 cycles. The raw sequencing reads in BCL format were processed through bcl2fastq 2.19 (Illumina) for FASTQ conversion and demultiplexing.
  • RNA sequencing (RNA-seq) data processed using the established Genomic Data Commons (GDC) guidelines as gene counts and FPKMs (Fragments Per Kilobase of transcript per Million mapped reads) were obtained from the GDC Data Portal (https://portal.gdc.cancer.gov). These data used the human reference genome GRCh38 (https://gdc.cancer.gov/about-data/gdc-data-harmonization). The corresponding clinical traits data (including Overall survival OS and Progression free survival PFS) was obtained from a previous publication 66 . Mutation data processed according to the Multi Center Mutation Calling in Multiple Cancers (MC3) 67 pipeline was also downloaded from the GDC data portal.
  • GDC Genomic Data Commons
  • 140-gene set cluster analysis in TCGA The cluster analysis to identify Low and High PI sample groups within each TCGA cancer type were performed using ConsensusClusterPlus 68 supervised using the 140-gene signature. Prior to clustering, signature genes with zero variance across all samples were excluded and the sample were restricted to primary tumors. The data was median centered and clustered using pam, k- means, and hierarchal clustering methods (with 1,000 random selections, 80% re-sampling). Samples classified into the same cluster by all three methods were retained. A silhouette analysis was performed using the cluster package in R, and samples identified as being misclassified in this analysis were removed from further analysis. These steps led to the identification of two robust clusters of samples with Low and High PI characteristics.
  • DEGs Differentially expressed genes
  • DESeq2 69 with apeglm shrinkage
  • Significant pathways associate with the ranked DEGs were identified using the MSigDB 70 pathway database in / .scv/ R package (FDR q ⁇ 0.25).
  • Cell type deconvolution was done in xCell 11 using FPKM expression profiles.
  • the data was visualized as heatmaps with ComplexHeatmap in R.
  • Survival analysis was performed using the Cox Proportional-Hazards (Cox-PH) models and results were visualized as Kaplan-Meier curves or forest plots.
  • Immunofluorescence imaging of a subset of pretherapy biopsy samples was performed using the Neogenomics platform as previously described 71,72 . Briefly, formalin-fixed tissue slides were baked at 65°C for 1 hour. Slides were deparaffinized with xylene, rehydrated by decreasing ethanol concentration washes, and then processed for antigen retrieval. A two-step antigen retrieval was adopted to allow antibodies with different antigen retrieval conditions to be used together on the same samples 73 .
  • MultiOmyx image analytics The acquired images from sequential rounds were registered using DAPI images acquired in the first round of staining via a rigid registration algorithm for each region of interest. The parameters of transformation were then applied to the subsequent rounds, which ensured that the pixel coordinates across all the imaging rounds corresponded to the same physical locations on the tissue. Classification and co-expression analysis were performed in multiple stages. First, a nuclear segmentation algorithm was applied on the DAPI image to delineate and identify individual cells. Location information and expression of all the markers were computed for every cell identified. Then, morphologic image analysis and shape detection were performed using proprietary algorithms (Neogenomics Laboratories; https://neogenomics.com/pharma-services/lab- services/multiomyx).
  • Example 2 Response to combination ICB and SBRT therapy was associated with a gene expression profile of increased proliferation.
  • the gene expression profile of tumors achieving MPR showed significant upregulation of 135 genes and downregulation of 5 genes (false discover rate (FDR) ⁇ 0.05, Benjamini -Hochberg (BH) correction) (Fig. lC, Fig.8C, Table 2).
  • PI Proliferation index
  • GSEA Gene set enrichment analysis revealed significant upregulation (FDR ⁇ 0.05) in the MPR tumor group of pathways associated with proliferation, including although not limited to mitotic spindle, G2M check point, DNA repair, mTOR and Myc signaling and unfolded protein response (Fig. IF).
  • metabolic and signaling pathways related to rapid proliferation are also enriched in the MPR group, although not achieving significance at FDR ⁇ 0.05.
  • GSEA also identified significant (FDR ⁇ 0.05) upregulation of Hallmark cell proliferation pathways and Glycolysis gene sets (Fig.9E). Supporting an increased glycolytic activity of the High PI tumors, pretreatment SUVmax (diagnostic fluorodeoxyglucose- positron emission tomography), a surrogate for glycolytic metabolism, was higher in the High PI tumor group (Fig.3B). These data suggest a hyper-glycolytic phenotype as an additional characteristic of the High PI tumors; thereby, associating pretreatment glycolytic metabolism with response to the dual therapy.
  • TMB tumor mutational burdens
  • Example 3 Tumors with MPR are characterized by post-treatment enrichments of genes involved in B cell biology and tertiary lymphoid structures.
  • Example 4 MPR in dual therapy arm is associated with post-treatment anti-tumor immune response.
  • Immune related biological pathways dominated the pathways enriched among the genes upregulated in the MPR group (Fig.4H).
  • pathways of tissue repair e.g., migration and signaling are enriched among the upregulated genes.
  • the pretreatment 140-gene expression signature associated with MPR was reversed post-treatment, whereas there were no pre- to post-treatment differences in the expressions of the 140-genes for the Arm2 tumors that did not achieve MPR (Fig.10C).
  • expressions of MHC-II genes were increased and expressions of glycolytic pathway genes decreased post-treatment relative to pre-treatment for the tumors that achieved MPR but unchanged in Arm2 tumors that did not achieve MPR (Fig.10D, E).
  • the upregulated genes 206 are in the analyses of the non-matched samples and of the down regulated genes, 333 are in the analyses of the non-matched samples.
  • RNA-seq Deconvolution of the post-treatment RNA-seq data revealed an increase in CD8 + T cells, dendritic cells, and Ml and M2 macrophages, all changes supporting increased antitumor immunity in the responding tumors (Fig.4I-K, Table 12).
  • Table 12 xCell deconvolution of combination therapy arm (Arm2) RNAseq of tumors that achieved MPR, contrasting pre to post-treatment.
  • Arm2 combination therapy arm
  • Example 5 The 140-gene set identifies two molecular subclasses of NSCLC tumors.
  • the 140-gene set classified pretreatment tumors from the trial into two molecular subtypes (Fig.2). To validate these results, we separately clustered the TCGA NSCLC adenocarcinoma and squamous samples by expressions of these 140 genes. Of the 455 primary TCGA lung adenocarcinoma (LU AD) samples, 407 (89.4%) were clustered into two subclasses by the 140-gene set (Fig.5 A, Fig.11 A). There were 196 LU AD samples in the Low PI group (43.1% of TCGA LUADs) and 211 in the High PI group (46.4% of TCGA LUADs).
  • LU AD primary TCGA lung adenocarcinoma
  • the LU AD High PI group was also characterized by reductions in MHC-II genes and increases in glycolysis genes, similar to the differences between our clinical trial clusters (Fig.11B-C). Since both MHC-II genes and glycolysis genes were not a part of the 140-gene set used to cluster the tumors, we concluded that the observed patterns of expression for these genes was a distinctive feature of the LU AD High PI group. There were no differences between LU AD Low and High PI groups in expressions of MHC-I genes nor did the gene expression profile support robust differences in antigen processing and presentation (Fig.1 ID). There were, however, increases in the expressions of some immune modulatory genes, prominently PD-L1, PD1, LAG3 and IDO1 (Fig. HE).
  • Example 6 Association of the 140-gene set with survival.
  • TMB tumor stage
  • III stages referenced to stage I was also associated with increased risk.
  • Example 7 High PI subclasses in the solid tumors.
  • Example 8 Association of proliferation signature with disease-free survival.
  • the 140-gene set was derived by contrasting gene expression profiles of dual therapy tumors with or without MPR (measured within a few weeks of the therapy at the time of resection).
  • the 140-gene set was established based on MPR within the dual treatment arm, these data suggest an association with DFS that is independent of treatment arm, in which case the 140-gene set might be predictive of improved DFS to anti-PD-Ll -based therapy.
  • TMB has been positively associated with cell cycle 21 and a positive correlation between proliferation and TMB has been reported in breast cancer and melanoma 22 ' 24 .
  • Three, in this subclass features of the tumor microenvironment are biased towards immune suppression, including increased cancer cell PD-L1 expression, reduced MHC-II expression and increased abundance of Tregs.
  • MHC-II expression is highest in the professional antigen-presenting cell types of DCs, macrophages and B cells, and therefore the reduction in tumor MHC-II expression might reflect a mechanism for the High PI tumors to evade immune surveillance 25,26 .
  • Increased CD4 Th2 cells is also a characteristic of this subclass. These cells, mediators of type 2 immunity, are best described for their roles in response to helminths and in wound healing, although there is emerging evidence for a role of these cells in cancer 27 .
  • Example 9 Radiation enhanced of ICB therapy.
  • Radiation has a number of possible immunomodulatory effects that can synergize with ICB, including the cancer-cell intrinsic effects of increased release of tumor antigens, induction of damage-associated molecular patterns, induction of immunologic cell death that promotes release of Type I interferon, as well as effects on the tumor stromal components such as activation of antigen presenting cells and the recruitment of T cells into the tumor bed 31 ' 37 .
  • MPR as a metric of response, our data suggest that the effects of radiation that augment response to anti-PD-Ll therapy are linked to the proliferative status of the cells.
  • Example 10 The 140-gene set is associated with response to and survival following anti- PD-L1 therapy.
  • the 140-gene set was discovered by exploring gene expression programs associated with SBRT enhancement of PD-L1 ICB based on pathology response, a metric of early antitumor response because it is determined within a few weeks (15.5 days median) of the neoadjuvant therapy.
  • the 140-gene set was also associated with improved DFS, which might have been expected based on the recently documented association between MPR and improved DFS in NSCLC 49 .
  • improvement in DFS in patients with the High PI was independent of MPR because it was also associated with improved DFS in patients in monotherapy arm (Arml), of which only one had MPR 3 .
  • the constellation of biological features identifying a subclass tumors as more proliferative is potentially predictive of MPR after anti-PD-Ll plus SBRT dual therapy as well as an improved DFS in response to anti-PD-Ll monotherapy.
  • the monotherapy anti-PD- Ll might be particularly effective in shifting the balance towards anti -turn or immunity in High PI tumors, inducing systemic immune response sufficient to reduce distal disease and improved survival even in the absence of MPR 50,51 .
  • Example 11 The 140-gene set identifies a tumor subclass in five other solid tumor types examined.
  • PI index used here has been negatively associated with survival in 7 of the 19 TCGA cancers examined, including LUAD 53 .
  • One contribution of our study is that we propose High PI as one characteristic of a molecular tumor subclass that is broadly associated with worse survival in untreated individuals and potentially associated with improved survival after anti- PD-L1 therapy.
  • Example 12 Biomarkers of ICB response.
  • Proliferation has also been associated with response to ICB.
  • cell cycle and DNA repair genes were associated with improved response to anti-PD-Ll in locally advanced or metastatic NSCLC and in metastatic urothelial carcinoma 54 .
  • tumors were segregated into tertiles of proliferation based on the expressions of 10 genes 55 .
  • Patients in the middle proliferation tertile had improved survival to ICB when compared to those in the pooled lowest and highest tertials 55 .
  • Our study links molecular determinants of highly proliferative tumors to response and survival after PD- L1 blockade.
  • biomarkers may be specific to stage and the specifics of ICB therapy 56 .
  • a recent study identified aneuploidy as a biomarker of response to combination ablative radiotherapy and ICB (ipilimumab plus nivolumab) in metastatic NSCLC 57 . Proliferation was not reported as a biomarker of response in that trial. This discordance is likely due to differences in tumor stage and or the specifics of the therapy. We did not have sufficient exome sequencing data to assess aneuploidy in our cohort.
  • Neoadjuvant durvalumab with or without stereotactic body radiotherapy in patients with early-stage non-smallcell lung cancer a single-centre, randomised phase 2 trial. Lancet Oncol 22, 824-835. 10.1016/S1470-2045(21)00149-2.
  • B cells and tertiary lymphoid structures promote immunotherapy response. Nature 577, 549-555. 10.1038/s41586- 019-1922-8.
  • Neoadjuvant durvalumab for resectable non-small-cell lung cancer results from a multicenter study (IFCT-1601 IONESCO). J Immunother Cancer 10. 10.1136/jitc-2022-005636. Janopaul-Naylor, J.R., Shen, Y., Qian, D.C., and Buchwald, Z.S. (2021).
  • RNA sequencing-based cell proliferation analysis across 19 cancers identifies a subset of proliferation-informative cancers with a common survival signature.
  • Oncotarget S 38668-38681. 10.18632/oncotarget.16961.
  • Banchereau, R. Leng, N., Zill, O., Sokol, E., Liu, G., Pavlick, D., Maund, S., Liu, L.F., Kadel, E., 3rd, Baldwin, N., et al. (2021).
  • PANTHER version 14 more genomes, a new PANTHER GO-slim and improvements in enrichment analysis tools.

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Abstract

La présente invention concerne l'identification de panels de signatures géniques et de caractéristiques tumorales pour prédire des tumeurs solides (par exemple, le cancer du poumon) en réponse à un blocage de point de contrôle immunitaire et/ou à une radiothérapie, et des procédés d'utilisation associés.
PCT/US2024/040687 2023-08-04 2024-08-02 Panel de signature génique prédisant une réponse cancéreuse à un blocage de point de contrôle immunitaire et radiothérapie WO2025034542A1 (fr)

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