WO2023172913A2 - Marqueurs protéogénomiques de résistance et de réponse à une chimiothérapie contre un cancer du sein triple négatif - Google Patents

Marqueurs protéogénomiques de résistance et de réponse à une chimiothérapie contre un cancer du sein triple négatif Download PDF

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WO2023172913A2
WO2023172913A2 PCT/US2023/063863 US2023063863W WO2023172913A2 WO 2023172913 A2 WO2023172913 A2 WO 2023172913A2 US 2023063863 W US2023063863 W US 2023063863W WO 2023172913 A2 WO2023172913 A2 WO 2023172913A2
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lig1
protein
individual
cancer
sections
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WO2023172913A3 (fr
Inventor
Meenakshi ANURAG
Matthew J. Ellis
Bing Zhang
Eric JAEHNIG
Anh M. TRAN-HUYNH
Shankha SATPATHY
Steven Carr
Karsten KRUG
Michael A. GILLETTE
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Baylor College of Medicine
General Hospital Corp
Broad Institute Inc
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Baylor College of Medicine
General Hospital Corp
Broad Institute Inc
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • This disclosure relates at least to the fields of medicine, oncology, and proteogenomics.
  • TNBC triple negative
  • HER2 human epidermal growth factor receptor 2
  • ER estrogen receptor
  • PR progesterone receptor
  • Salvage therapy with adjuvant capecitabine has modest efficacy in this setting [4].
  • the programmed cell death receptor (PDl)-targ eting antibody pembrolizumab is also approved for neoadjuvant/adjuvant TNBC treatment [5], but outcome improvements are unfortunately not accurately predicted by PD-Ll-based immunohistochemistry (IHC) [6].
  • IHC immunohistochemistry
  • Embodiments herein concern methods of detecting genetic alterations and/or deletions.
  • methods concern methods of detecting deletion and/or amplification of one or more genes on cytoband 19ql3.31-33 or 8q21.3 respectively in an individual.
  • the genes may comprise DNA ligase 1 (LIG1), POLDI and XRCC1 on cytoband 19ql3.31-33 and RIPK2, RMDN1, CPNE3, DECR1, and OTUB6B on cytoband 8q21.3, in specific embodiments.
  • a sample from the individual identifies gene deletion and/or low mRNA expression and/or low protein level of LIG1
  • the individual is administered a therapeutically effective amount of a therapy that is not a platinum-based drug.
  • Particular embodiments provide a method of determining a treatment therapy for an individual with cancer, comprising the step of administering a therapy other than a platinum-based drug to an individual having gene deletion and/or low mRNA expression and/or low protein level of DNA ligase 1 (LIG1).
  • the method comprises the step of combining into multiple vessels a plurality of sections from different regions of the biological sample, said biological sample optionally being a biopsy.
  • the combining step may provide a mixture of cells and/or tissues from different regions of the biopsy for the plurality of sections.
  • the method comprises the step of isolating DNA from the plurality of sections in a first vessel, isolating RNA from the plurality of sections in a second vessel, and isolating protein from the plurality of sections in a third vessel.
  • the method comprises the step of analyzing the isolated DNA, analyzing the isolated RNA, and/or analyzing the isolated protein to determine the status of at least one marker present in cells in the biopsy, wherein the marker comprises LTG1 .
  • the protein may be denatured, in certain embodiments.
  • the method comprises the step of sectioning the biopsy. In some embodiments the sectioning and combining step are performed concurrently or separately. In some embodiments, a plurality of sections from a fourth vessel are used for sample quality analysis. In some embodiments, the plurality of sections for at least one of the 1st, 2nd, and/or 3rd vessels comprises nonadjacent sections from the different regions. In some embodiments, analyzing the isolated protein further comprises determining the phosphorylation status of LIG1. The phosphorylation status of any cancer marker may be determined by mass spectrometry, in specific embodiments.
  • analyzing the isolated DNA, analyzing the isolated RNA, and analyzing the isolated protein determines the status of LIG1, POLDI and/or XRCC1 in the biopsy.
  • the method further comprises the step of administering to the individual one or more therapies, wherein the therapies do not comprise a platinum-based drug.
  • DNA and protein are analyzed from the same vessel.
  • deletion occurs in the gene (DNA) but may also manifest as lower expression in RNA and/or protein.
  • the platinum-based drug may comprise carboplatin.
  • the individual has, or is suspected of having, a cancer.
  • Certain embodiments concern methods of determining the susceptibility of an individual having, or suspected of having, a cancer to a cancer treatment.
  • a sample from an individual is subjected to any method encompassed herein.
  • an individual is administered or not administered carboplatin.
  • an individual has or is suspected of having breast cancer, such as triple negative breast cancer. The individual may or may not have a family history of triple negative breast cancer.
  • Certain embodiments concern a method of treating an individual having, or suspected of having, a cancer using a biological sample from the individual comprising the steps of: combining into multiple vessels a plurality of sections from different regions of the biological sample, said biological sample optionally being a biopsy; isolating DNA from the plurality of sections in a first vessel, isolating RNA from the plurality of sections in a second vessel, and isolating protein from the plurality of sections in a third vessel; analyzing the isolated DNA, analyzing the isolated RNA, and/or analyzing the isolated protein to determine the status of at least one marker present in cells in the biopsy, wherein the marker comprises LIG1; determining the LIG1 status in any one, two, or all three vessels having sections from different regions of the biological sample; and administering or not administering a particular therapeutic.
  • the sections are staggered so that the different vessels are as similar as possible even though they are collected from different sections; one embodiment is to minimize regional differences between the different analytes (DNA, protein, RNA) collected in each vessel by alternating the sections added to each.
  • an individual is not administered a platinum-based drug, inhibitor of EGFR (including tyrosine kinase inhibitors such as erlotinib and/or gefitinib and including monoclonal antibodies such as cetuximab and/or necitumumab) or PI3K inhibitor (such as copanlisib, duvelisib and/or idelalisib) when LIG1 is reduced or deleted in one or more of DNA, RNA, or protein.
  • EGFR including tyrosine kinase inhibitors such as erlotinib and/or gefitinib and including monoclonal antibodies such as cetuximab and/or necitumumab
  • PI3K inhibitor such as copanlisib, duvelisib and/or idelalisib
  • an individual is administered one or more CDK2 inhibitors (such as Flavopiridol and/or TG02); one or more Chk2 inhibitors (4,4'- diacetyldiphenylurea-bis(guanylhydrazone) (NSC 109555); PV1019 (NSC 744039) [7-nitro-lH- indole-2-carboxylic acid ⁇ 4-[l-(guanidinohydrazone)-ethyl]-phenyl ⁇ -amide]; and/or PHI-101); and/or one or more PARP inhibitors (such as olaparib, rucaparib, and/or niraparib) when LIG1 is reduced or deleted in one or more of DNA, RNA, or protein.
  • an individual is administered a platinum-based drug when the LIG1 status is determined to not be deleted in the sample.
  • an individual is not administered a platinum-based drug when the LIG1 status is determined to be deleted
  • LIG1 RNA, protein, and/or DNA are depleted or reduced in level from a sample, the individual should not be administered a platinum-based drug, EGFR inhibitor, or PI3K inhibitor, and/or the individual should be administered a CDK2 inhibitor, Chk2 inhibitor, and/or PARP inhibitor.
  • Certain embodiments concern a method of determining an individual’s susceptibility to a platinum-based drug comprising the steps of analyzing genomic DNA from the sample at a locus comprising LIG1 and/or measuring expression of LIG1 mRNA and/or protein in the sample.
  • FIGS. 1A-1F show an overview of TNBC patient samples.
  • FIG. 1A REMARK diagram showing pre- and on-treatment sample accrual schema from triple negative breast cancer patients enrolled in two clinical trials (NCT02547987 (BCM), NCT02124902 (WashU)) and treated with carboplatin and docetaxel in the neoadjuvant setting. * samples with ⁇ 45% tumor content were later excluded from analysis based on evidence from data QC.
  • FIG. IB Overview of available omics datasets from 59 patients (22 tumors with pCR and 37 tumors without pCR). Pathogenic BRCA1/2 and PALB2 mutation status, residual cancer burden (RCB) and patient race are indicated via color-coded annotation tracks.
  • FIG. 1C Venn-diagram showing overlap of gene IDs detected across multiple analytes and omics data profiled.
  • FIG. ID Hallmark metabolism pathways (PMID:26771021) are acutely induced by chemotherapy exclusively at the protein level.
  • MSigDB Hallmark metabolism pathways (PMID:26771021) are elevated in baseline non-pCR tumors at the protein level while immune and cell cycle pathways are elevated in baseline pCR tumors at both RNA and protein levels.
  • Scatter plot shows the signed -loglO fdr values from Gene Set Enrichment Analysis (GSEA; PMID: 16199517) using ranked lists of signed (by direction of change) -loglO p-values from Wilcoxon rank sum tests comparing RNA (x-axis) and protein (y- axis) levels in non-pCR tumors to pCR tumors. (FIG.
  • FIGS. 2A-2F show proteogenomic features associated with pCR in TNBC tumors.
  • FIG. 2A Proteogenomic features associated with the immune microenvironment are elevated in pCR tumors relative to non-pCR tumors.
  • Heatmap shows protein-based Hallmark single sample GSEA (ssGSEA) scores (PMID:26771021; PMID: 19847166), protein-based immune modulator scores (PMID: 31433971; PMID: 33212010), RNA-based immune profiles (PMID: 24113773; PMID: 2582280; PMID: 29141660), and proteogenomic features for immune checkpoint genes that are targets of FDA-approved inhibitors.
  • ssGSEA protein-based Hallmark single sample GSEA
  • FIG. 2D-2E Scatter plots showing correlation between PDL1 IHC levels with PDL1 protein (FIG. 2D) and phosphoprotein levels (FIG. 2E).
  • FIG. 2F While cell cycle features are elevated in pCR tumors, a subset of non-pCR tumors have elevated levels of CDK4 target sites and Rb levels.
  • Heatmap shows RNA- and protein-based multi-gene proliferation scores (MGPS; PMID: 28045625; PMID: 12058064), single sample PTM-SEA scores for CDK4 and CDK2 target sites (PMID: 30563849), ssGSEA scores for E2F targets (PMID:26771021; PMID: 19847166), and other proteogenomic features for genes regulating the Gl/S transition of the cell cycle (see pathway on right). Box outlines samples from non-pCR tumors with high CDK4 activity and Rb phosphorylation. Asterisks indicate p ⁇ 0.05 by Wilcoxon rank sum test comparing non- pCR to pCR tumors.
  • FIGS. 3A-3C show proteogenomic features associated with lack of pCR and that are altered upon treatment in TNBC tumors.
  • FIG. 3A Heatmap showing single-sample GSEA NES for metabolic Hallmark pathways that are significantly higher in non-pCR cases, arranged by RCBO (pCR) and RCBT/TT/TTT (non-pCR). Shown are enrichment scores for 4 pathways as assessed at the level of mRNA (yellow), protein (blue) and phospho-protcin (red). Wilcoxon rank sum test was used to compare scores for non-pCR Vs pCR scores, * p ⁇ 0.05.
  • FIG. 3B Membership of differentially regulated proteins to pathways highlighted in A.
  • Proteins (rows) belonging to a given pathway are shown in light green.
  • the differential expression at protein and mRNA levels for each gene along with mRNA-protein correlation scores are shown as signed -log 10 p- value (signedp).
  • FIG. 3C A multi-omics metabolic gene signature derived for genes that are correlated at mRNA and protein level was further investigated in patients treated with carboplatin and paclitaxel in the BrighTNess clinical trial. The mean mRNA expression score for this signature was significantly higher in higher RCB tumors.
  • FIGS. 4A-4E show discovery of DNA repair and replication components enriched in non-pCR TNBC tumors.
  • FIG. 4A Workflow depicts the strategy that was used to identify enriched or depleted chromosomal cytobands using mRNA and protein lists ranked by signed - loglO p-value derived from differential expression for non-pCR vs. pCR samples.
  • FIG. 4B Plot showing significantly enriched or depleted cytobands obtained by running differential mRNA and protein ranked lists through GSEA.
  • FIG. 4C Venn-diagram showing differential (non-pCR vs. pCR) mRNA and proteins located on cytoband 19ql3.3.
  • HR Forest plot showing hazard ratios (HR) and p-values for metastasis free survival associated with LIG1, POLDI, XRCC1 and ERCC2.
  • HR is based on categorizing samples using median expression cutoff for each gene in the Hatzis dataset (PMID: 21558518).
  • FIG. 5 shows proteogenomic features associated with LIG1.
  • Heatmap showing copy number, mRNA and protein levels of LIG1, which are significantly (Wilcoxon rank sum test) lower (blue) in non-pCR tumors.
  • T-test and wilcoxon rank sum test were used to compare LIG1 loss cases to LIG1 intact (WT/Gain) cases.
  • FIGS. 6A-6D show LIG1 association with advanced TNBC disease in preclinical models.
  • FIG. 6A Proteogenomic status of LIG1, POLDI and XRCC1 in three PDX models derived from longitudinal biopsies from the same TNBC patient prior to any treatment (WHIM68), at the time of surgery after completing 5 months of neoadjuvant carboplatin and docetaxel (WHIM74), and from a liver metastasis one year after treatment initiation (WHIM75).
  • WHIM68 with highest LIG1 protein levels, was most sensitive to carboplatin while WHIM74 and 75, which displayed progressive LIG1 loss at the copy number, mRNA, and protein levels, were insensitive to carboplatin treatment.
  • P-values derived from a general linear model within each PDX were computed using estimated mean log2 fold changes in tumor volume at Day 28 vs Day 0 for each treatment arm.
  • FIG. 6C Boxplots showing LIG1 mRNA levels in TNBCs PDXs categorized into complete response (CR) and non-CR groups. After 4 of carboplatin weeks treatment, CR was defined as PDXs with non-palpable tumors, and non-CR was defined as PDXs with residual tumors with measurable dimensions.
  • FIG. 6D Association between LIG1 copy number loss and treatment response in patient-derived xenograft organoids (PDXOs) obtained from the BCaPE database (PMID 27641504). Carboplatin and docetaxel are highlighted in red.
  • FIGS. 7A-7E show pan-cancer analysis of LIG1 loss.
  • FIG. 7B Boxplot showing higher fraction genome altered (FGA) in tumors with LIG1 copy number loss tumors (shown in teal) relative to tumors that were LIG1 wild-type or displayed LTG1 gain (shown in orange).
  • FGA fraction genome altered
  • FIG. 7C Violin plot showing significantly lower (Wilcoxon rank sum test) COSMIC Signature 3 scores in LIG1 loss tumors (shown in teal).
  • FIG. 7D Forest plot showing impact of LIG1 copy number loss on PFS by cancer type along with LIG1 WT/gain/loss frequency, HR and corresponding p-value.
  • FIG. 7E Boxplot showing significantly higher (Wilcoxon rank sum test) FGA (representing chromosomal instability) in tumors that had LIG1 copy number loss versus tumors with either wild-type LIG1 or with LIG1 copy number gain. Shown are the only 5 cancers (HNSCC, UCEC, COAD, PRAD and KIRP) that displayed significant association between LIG1 loss and adverse PFS.
  • FIGS. 8A-8G show cohort characteristics.
  • FIG. 8A Proportion plot showing distribution of samples obtained from baseline tumors grouped by PAM50 (Basal, HER2, LumA, LumB), TNBC subtyping (BLl-basal like 1, BL2-basal like 2, IM- immunomodulatory, LAR- luminal androgen receptor, M-mesenchymal, MSL-mesenchymal stem like, UNS -unspecified tumors) and Race (AA-african american, C-caucasian, Others).
  • the left panel shows all samples, and the right panel shows samples segregated into two groups based on pCR. P-values were obtained using Fisher’s exact test.
  • FIG. 8A Proportion plot showing distribution of samples obtained from baseline tumors grouped by PAM50 (Basal, HER2, LumA, LumB), TNBC subtyping (BLl-basal like 1, BL2-basal like 2, IM- immunomodulatory, LAR- luminal androgen receptor, M-mesenchymal, M
  • FIG. 8B Correlation between average tumor content as assessed by immunohistochemistry (IHC) and mRNA- protein correlation for all baseline samples for which there were paired mRNA and protein data. Samples indicated by teal points were excluded from this study due to poor tumor content and poor mRNA-protein correlation (tumor content ⁇ 45%).
  • FIG. 8C Pairwise correlation analysis depicting correlations across (green) and within (red) TMT-plexes at the protein (left) and phosphopeptide level (right). A common reference sample from the prospective BRCA study (PMID: 33212010) was measured in each TMT plex as part of this study and showed consistently high correlations across all 8 TMT plexes. Pairwise within-plex correlations of tumor samples are shown in red. (FIG.
  • FIG. 8D Gene-wise mRNA-protein correlations ordered from most negative to most positive by Spearman rank correlation coefficient are shown in the top panel. Signed loglO p-values were used as input for GSEA using the KEGG pathway database. Top pathways enriched for genes that had positive correlation between mRNA and protein are shown in the middle panel while top pathways enriched for genes with lower correlation are shown in the bottom panel.
  • FIG. 8E Gene function prediction performance quantified by AUROC shows co-expression networks based on proteomic profiles outperformed RNA-based networks for predicting KEGG pathway membership. Dotted lines indicate 10% increase or decrease in prediction performance.
  • Genotoxic stress sites sites induced by nocodazole, UV, and ionizing radiation
  • ATM and CDK1/2 target sites arc acutely induced by chemotherapy.
  • FIG. 8G Heatmap showing distribution of COSMIC mutational signatures in the baseline (pre-treatment) samples. Signatures 6 and 10, which are associated with MMR and POLE mutations respectively, were higher in RCB II/III categories (ANOVA test, p ⁇ 0.05).
  • FIGS. 9A-9D show PDL1 IHC, distribution of Rb phosphorylation, and association of Rb protein and drug sensitivity in cell lines.
  • FIGS. 9A-9B Representative images for patient samples with high (A) and low (B) PDL1 IHC staining.
  • FIG. 9C The distribution of Rb phosphorylation levels in non-pCR tumors (brown) is overlapping with but also shifted towards higher levels than in pCR tumors (green). Density plots for each group are shown. (FIG.
  • platinum compounds such as cisplatin and carboplatin, as well as an alkylating agent that induces DNA damage, temozolomide, were among the most positively correlated pairs (higher Rb protein in TNBC cell lines with increasing resistance to those drugs) while response to palbociclib was negatively correlated (higher Rb protein in TNBC cell lines with decreasing resistance to palbociclib).
  • FIG. 10 shows metabolic multi-omic signature and results from PTM-SEA.
  • the multi- omics metabolic gene signature identified in this study was further investigated in patients treated with carboplatin and paclitaxel in the BrighTNess clinical trial. The mean mRNA expression score for this signature was significantly higher in residual disease. P-value was calculated using the Wilcoxon rank sum test.
  • FIGS. 11A-11D show proteogenomic prioritization of candidates driving chemoresistance in TNBC.
  • FIG. 11A Chromosome-wise differences in the SCNA landscape in non- pCR and pCR samples are shown as the top two tracks. Amplification and deletion events are indicated by red and blue respectively. The bottom panels show differential mRNAs and proteins up- and down -regulated in non-pCR compared to pCR cases arranged based on their chromosomal location to align with the SCNA tracks.
  • FIG. 11B Vcnn-diagram showing differential (non-pCR vs. pCR) mRNA and proteins located on cytoband 8q21.3.
  • FIG. 11A Chromosome-wise differences in the SCNA landscape in non- pCR and pCR samples are shown as the top two tracks. Amplification and deletion events are indicated by red and blue respectively. The bottom panels show differential mRNAs and proteins up- and down -regulated in non-pCR compared to pCR cases arranged
  • FIG. 11C Pircos (proteogenomic circos) plots for chromosome 19 showing signed -loglO p-values for mRNA and protein based on Wilcoxon rank-sum tests comparing non-pCR to pCR cases and frequency of amplifications and deletions (CNV) in non-pCR and pCR. Genes annotated in blue on the outermost track represent those to be low at the CNV, mRNA, and protein level from (C).
  • FIG. 11D Kaplan-Meier (KM) curve depicting metastasis free survival of patients with TNBC breast cancer from the Hatzis dataset. Tumors are categorized into high and low LIG1 based on mean RNA expression cutoff.
  • FIGS. 12A-12E show features associated with LIG1 genomic status.
  • FIG. 12C Boxplot showing immune stimulatory scores across LIG1 copy number and chemotherapy response categories. P-values are calculated using the Wilcoxon ranksum test.
  • FIG. 12D LIG1 loss tumors have higher CDK1/2 activity than non-loss tumors.
  • FIG. 12E Heatmap showing ssPTMSEA scores for significantly differential kinases and pathways shown in D. LIG1 single copy loss and RCB class are shown as additional rows.
  • FIGS. 13A-13D show LIG1 association with advanced TNBC disease in preclinical models and independent cohorts.
  • FIG. 13A Proportion plot showing distribution of LIG1 copy number alterations in TCGA-Breast (primary disease) and INSERM (metastatic disease) cohorts. P-value was obtained using Fisher’s exact test.
  • FIG. 13B Quantification by densitometry using the same western blots for LIG1, POLDI and XRCC1, along with GAPDH as a control, in WHIM 68, 74 and 75 as shown in FIG. 6A.
  • FIG. 13C Tumor volumes in PDX models treated with 4 weekly cycles of vehicle or 20 mg/kg docetaxel. P-values derived from a general linear model within each PDX were computed using estimated mean log2 fold changes in tumor volume at Day 28 vs Day 0 for each treatment arm.
  • FIG. 13D Boxplots showing LIG1 mRNA levels in TNBCs PDXs categorized into complete response (CR) and non- CR groups. After 4 weeks of docetaxel treatment, CR was defined as PDXs with non-palpablc tumors and non-CR defined for PDXs with residual tumors with measurable dimensions. Wilcoxon rank sum test was used to compare the two groups.
  • FIGS. 14A-14B show LIG1 loss across TCGA Pan-Cancer cohort.
  • FIG. 14B Chromosomal Instability (CIN) scores in LIG1 WT/gain and loss in 3 CPTAC cancer cohorts.
  • HSCC Head and Neck Squamous Cell Carcinoma (PMID: 33417831) and UCEC: Endometrial Carcinoma (PMID: 32059776), and COAD: Colon Cancer PMID: 31031003).
  • FIG. 15 demonstrates that L1G1 loss tumors have higher CDK2 activity than non-loss tumors.
  • Volcano plot shows results from Post-Translational Modification-Set Enrichment Analysis using the signed -log 10 p-values from Wilcoxon rank sum tests comparing phosphosite levels in LIG1 loss with LIG1 WT or gain cases.
  • FIGS. 16A-16B Reanalyzed data from three independent CRISPR (clustered regularly interspersed palindromic repeats) screens to identify genes and pathways that mediate cellular resistance to Olaparib, a clinically approved PARP inhibitor (Zimmerman et al, 2018). The Venn-diagram shows that LIG1 is one of 13 genes that showed increased sensitivity to PARP inhibitors across all the three models when suppressed.
  • FIGS. 17A-17C boxplot showing LIG1 estimates (by FISH) by manual counting of 2000 cells from the three PDX models from 2 slides. Evidence suggests higher LIG1- loss heterogeneity in primary (WHIM68,74) as compared to the metastatic site (WHIM75).
  • FIG. 17B Dot plots showing a higher % of LIG1 -haploid cells in non-CR than in CR patients (FIG. 17B) with BRCA1/2 mutated cases (FIG. 17C) without BRCA1/2 mutated cases. 200 cells were manually counted for each of the 50 TNBC PDXs. DETAILED DESCRIPTION
  • A, B, and/or C includes: A alone, B alone, C alone, a combination of A and B, a combination of A and C, a combination of B and C, or a combination of A, B, and C.
  • A, B, and/or C includes: A alone, B alone, C alone, a combination of A and B, a combination of A and C, a combination of B and C, or a combination of A, B, and C.
  • “and/or” operates as an inclusive or.
  • the term “cancer,” as used herein, may be used to describe a solid tumor, metastatic cancer, or non-metastatic cancer.
  • the cancer may originate in the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, duodenum, small intestine, large intestine, colon, rectum, anus, gum, head, kidney, liver, lung, nasopharynx, neck, ovary, pancreas, prostate, skin, stomach, testis, tongue, or uterus.
  • the cancer is recurrent cancer.
  • the cancer is Stage I cancer.
  • the cancer is Stage II cancer.
  • the cancer is Stage III cancer.
  • the cancer is Stage IV cancer.
  • compositions and methods for their use can “comprise,” “consist essentially of,” or “consist oT’ any of the ingredients or steps disclosed throughout the specification.
  • Compositions and methods “consisting essentially of’ any of the ingredients or steps disclosed limits the scope of the claim to the specified materials or steps which do not materially affect the basic and novel characteristic of the claimed invention.
  • a “protein” or “polypeptide” refers to a molecule comprising at least five amino acid residues.
  • wild-type refers to the endogenous version of a molecule that occurs naturally in an organism.
  • the genetic alteration may be in one or more cells in an individual.
  • the genetic alteration may directly or indirectly cause or contribute to oncogenicity of the cells.
  • the genetic alteration may directly or indirectly cause or contribute to drug resistance in the cells.
  • the genetic alteration directly or indirectly causes or contributes to resistance to a platinum-based drug.
  • the platinum-based drug may comprise carboplatin, cisplatin, oxaliplatin, nedaplatin, heptaplatin, and/or lobaplatin, as examples.
  • the drug may be a platinum pro-drug and/ or metabolites such as satraplatin and/or JM518, JM559, JM383, JM118.
  • the genetic alteration comprises one or more alterations to genomic DNA in the cell, one or more alterations to expression of at least one gene or regulatory molecule, and/or one or more alterations to a biomolecule in the cell, including a protein and/or nucleic acid.
  • the genetic alteration comprises alteration of a genomic locus.
  • the genomic locus may comprise the 8q21 .3 and/or 19q 13.31 -33 cytobands.
  • the genomic locus may comprise the LIG1, PPP5C, BCL3, and/or NOS IP genes.
  • the deletion may be a deletion in all or part of a genomic locus that comprises the 8q21.3 and/or 19ql3.31-33 cytobands.
  • the 19ql3.31-33 genomic locus may comprise the LIG1, POLDI, XRCC1, PPP5C, BCL3, and/or NOSIP genes.
  • the 8q21.3 genomic locus may comprise the RIPK2, RMDN1 , CPNE3, DECR1, and/or OTUB6B genes.
  • the deletion (19ql3.3) or amplification (8q21.2) may cause or contribute to drug resistance in the cells.
  • the deletion directly or indirectly causes or contributes to resistance to a platinum-based drug.
  • the platinum-based drug may comprise carboplatin, cisplatin, oxaliplatin, nedaplatin, heptaplatin, and/or lobaplatin.
  • a genetic alteration and/or deletion to LIG1 in a cell and/or tumor results in drug resistance in said cell and/or tumor.
  • Ligl loss is a marker of lack of response to certain therapies, such as platinum-based drugs.
  • the drug resistance may comprise a complete or partial lack of response to the drug.
  • the genetic alteration and/or deletion may cause the cell and/or tumor not to respond to the drug in a manner that a cell and/or tumor not having the genetic alteration and/or deletion may have.
  • a cytotoxic drug may have less of an effect on a cell and/or tumor having a LIG1 alteration and/or deletion compared to a cell and/or tumor not having the alteration and/or deletion.
  • the drug may comprise a platinum-based drug.
  • Embodiments herein refer to LIG1, LIG1, DNA Ligase 1, which may comprise the gene, its expressed mRNA, and/or protein.
  • references to one may comprise any of the biomolecules.
  • a reference to alteration of LIG1 may refer to an alteration of the gene in genomic DNA and/or an alteration to the expressed mRNA and/or alteration to the expression of protein.
  • Detection of the genetic alteration and/or deletion may be achieved through any method known in the art, including any method disclosed herein. In some embodiments, detection occurs through one or a combination of the proteogenomic methods disclosed herein. The proteogemoic methods are discussed in PCT Application No. PCT/US2020/045962, which is incorporated by reference herein in its entirety.
  • compositions, systems, kits, and methods described herein may be useful for detecting one or more genetic alterations.
  • One skilled in the art would understand that the methods disclosed herein may be used for determining genetic alterations that cause or contribute to oncogenicity and/or drug resistance or the like.
  • Certain embodiments concern assays useful for diagnosing and/or treating cancer.
  • the cancer may specifically be of the following histological type, though it is not limited to these: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; basal-like 1 triple-negative breast cancer; basal-like 2 triple-negative breast cancer; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposis coli; solid carcinoma; carcinoid tumor
  • Particular embodiments concern the methods of detecting a genetic signature in an individual.
  • the genetic signature may identify a treatment regimen for an individual and/or may identify whether an individual will have poor metastasis-free survival.
  • one or more genes from cytoband 19ql3.31-33 are analyzed to either determine an appropriate treatment and/or determine whether or not the individual will have poor metastasis-free survivial.
  • the individual when a sample from an individual identifies gene deletion and/or low mRNA expression and/or low protein level of L1G1; POLDI; and/or XRCC1, the individual is administered a therapeutically effective amount of a particular therapy, including of a therapy that is not a platinum-based drug.
  • the individual when a sample from an individual identifies gene deletion and/or low mRNA expression and/or low protein level of LIG1 ; POLDI ; and/or XRCC1, the individual will have poor metastasis-free survival. In either case, the individual may be administered a therapeutically effective amount of one or more therapies that are not a platinumbased drug.
  • a genetic signature may encompass the genetic DNA and/or its expression into mRNA and/or protein.
  • the method for detecting the genetic signature may include selective oligonucleotide probes, arrays, allele- specific hybridization, molecular beacons, restriction fragment length polymorphism analysis, enzymatic chain reaction, flap endonuclease analysis, primer extension, 5’-nuclease analysis, oligonucleotide ligation assay, single strand conformation polymorphism analysis, temperature gradient gel electrophoresis, denaturing high performance liquid chromatography, high-resolution melting, DNA mismatch binding protein analysis, surveyor nuclease assay, sequencing, RNASeq, BEAMing, or a combination thereof, for example.
  • the method for detecting the genetic signature may include fluorescent in situ hybridization, comparative genomic hybridization, arrays, polymerase chain reaction, sequencing, or a combination thereof, for example.
  • the detection of the genetic signature may involve using a particular method to detect one feature of the genetic signature and additionally use the same method or a different method to detect a different feature of the genetic signature. Multiple different methods independently or in combination may be used to detect the same feature or a plurality of features.
  • the present disclosure concerns methods that facilitate analysis of biological samples for accurate treatment or prognosis for an individual.
  • an individual that has cancer or is suspected of having cancer is subject to methods of the disclosure to provide a correct assessment to allow for selection of one or more suitable treatments.
  • the methods greatly reduce the risk of inaccurate treatment regimens at least in part because they employ proteogenomics that encompass analysis of DNA, RNA, and protein as part of the evaluation for the individual.
  • the particular methods of the disclosure reduce the level of required tissue and include uniform distribution of sample parts (such as sections) each for the DNA analysis, RNA analysis, and protein analysis.
  • the DNA analysis, RNA analysis, and protein analysis may or may not occur in parallel, although in particular cases the different analyses occur generally concomitantly.
  • mass spectrometry may be utilized to analyze the proteome and/or phosphoproteome from the biological sample.
  • analysis of the proteome and/or phosphoproteome utilizes a scale of tissue on the order of micrograms.
  • Certain embodiments of the disclosure concern at least one biological sample of any kind, such as a biopsy of any kind, taken from an individual, including any individual encompassed herein.
  • the individual may have cancer, may be suspected of having cancer, or may be at increased risk for having cancer compared to the general population (for example, a personal or family history, a smoker, the elderly, exposure to the sun or environmental conditions, a combination thereof, and so forth).
  • the individual may be a research subject, including any mammal that is part of a research study.
  • a biopsy may be obtained as part of a routine preventative practice or as part of a directed concern or suspected indication for the onset of cancer.
  • the biopsy may be taken from the breast, for example from a mass in the breast. Alternatively nipple aspirate may provide the biological sample.
  • the one or more biological samples may be taken from the individual at any time, such as before, after, and/or simultaneously with diagnosis of a cancer, or such as before, after, and/or simultaneously with the administration of one or more therapies. At least one of the biological samples taken from the individual may be taken from a tumor or other cancer cells present in the individual. A tumor may or may not be benign or suspected of being benign. In some embodiments, at least one of the biological samples taken from the individual may be taken from non-cancerous tissue or other biological material in the individual.
  • the biological sample may be taken from the individual using any method known in the art, including a(n) needle biopsy (including core-needle biopsy), guided biopsy, aspiration biopsy, surgical biopsy, core biopsy, open biopsy, punch biopsy, sentinel lymph node biopsy, shave biopsy, endoscopic biopsy, or a combination thereof.
  • the biological sample is taken from the individual by a core-needle biopsy, including a core-needle biopsy using a 14 gauge, 15 gauge, 16 gauge, 17 gauge, 18 gauge, 19 gauge, 20 gauge, 21 gauge, or 22 gauge needle.
  • the biological sample may be from tissue, bone, blood, serum, plasma, urine, stool, sputum, saliva, semen, vaginal fluids, mucus, fat, or a combination thereof.
  • a biological sample comprises a biopsy that is taken from a mass in a breast of an individual.
  • the biological sample may be prepared, processed, stored, handled, and/or fixed using any method known in the art.
  • the sample may or may not be stored prior to processing.
  • at least one biological sample is embedded in optimal cutting temperature (OCT) medium.
  • OCT optimal cutting temperature
  • at least one biological sample is stored in cryogenic storage, such as at a temperature lower than -80°C, lower than -70°C, lower than -60°C, lower than -50°C, lower than -40°C, lower than -30°C, or lower than -20°C.
  • at least one biological sample is prepared and/or sectioned using a microtome, such as a crytostat.
  • the sectioning is done at a temperature lower than -30°C, lower than -20°C, or lower than -10°C. In some embodiments, the sectioning is done at a temperature between -30°C to - 10°C, or between -23°C to -15°C. In some embodiments, the biological sample is sectioned at a thickness between 3 microns and 100 microns, or between 4 microns and 100 microns, or between 5 microns and 100 microns, or between 3 microns and 50 microns, or between 4 microns and 50 microns, or between 5 microns and 50 microns. However, the biological sample may be sectioned to any thickness that is suitable for practicing the methods of the disclosure. In some embodiments, one or more sections from the biological samples are placed into one or more vessels suitable for comprising the sections.
  • Certain embodiments of the present disclosure concern methods of generating DNA, RNA, and/or protein from at least one biological sample, such as a biopsy.
  • the DNA, RNA, and/or protein are isolated in individual vessels, including vessels comprising one or more sections of the biological sample(s).
  • the DNA, RNA, and/or protein isolated into individual vessels may have originated from different regions of the biological sample, such that the isolated DNA, RNA, and/or protein in an individual vessel comprises DNA, RNA, and/or protein originating from different regions of the biological sample.
  • at least one biological sample contained in a vessel is used for the preparation of the section(s) for microscopic analysis.
  • some sections of the biological sample are combined with other sections of the biological sample.
  • the combining of some sections with other sections may comprise combining sections of one region of the biological sample with at least one other region of the biological sample.
  • non-adjacent sections from the biological sample are combined, including non-adjacent sections from different regions of the biological sample.
  • At least three sections are generated from a biopsy, followed by adding any one of the three sections to a first vessel, adding any one of the two remaining sections to a second vessel, and adding the remaining section to a third vessel.
  • the process may be repeated indefinitely. The processes may be repeated until a sufficient number of sections, from the biological samples, for practice of the disclosure are generated and placed into vessels. A sufficient number may be the number required to produce sufficient RNA, DNA, and/or protein for analysis.
  • four sections are generated followed by, in any order: adding any one of the four sections to a first vessel, adding any one of the four sections not in the first vessel to a second vessel, adding any one of the four sections not in the first or second vessel to a third vessel, and preparing any one of the four sections not in the first, second, or third vessel for microscopic analysis.
  • the process may be repeated indefinitely. The processes may be repeated until a sufficient number of sections, from the biological samples, for practice of the disclosure are generated and placed into vessels and/or prepared for microscopic analysis. A sufficient number may be the number required to produce sufficient RNA, DNA, and/or protein for analysis.
  • a sufficient number of sections for practice of the disclosure will produce approximately between 10 pg to 45 pg of isolated protein. In some embodiments, a sufficient number of sections for practice of the disclosure will produce approximately 10 pg, 15 pg, 20 pg, 25 pg, 30 pg, 35 pg, 40 pg, 45 pg, or more than 45 pg of isolated protein. In some embodiments, a sufficient number of sections for practice of the disclosure will produce approximately between 0.1 pg to 1 pg of isolated DNA.
  • a sufficient number of sections for practice of the disclosure will produce approximately 0.1 pg, 0.2 pg, 0.3 pg, 0.4 pg, 0.5 pg, 0.6 pg, 0.7 pg, 0.8 pg, 0.9 pg, 1.0 pg, or more than 1.0 pg of isolated DNA. In some embodiments, a sufficient number of sections for practice of the disclosure will produce approximately between 0.1 pg to 1 pg of isolated RNA.
  • a sufficient number of sections for practice of the disclosure will produce approximately 0.1 pg, 0.2 pg, 0.3 pg, 0.4 pg, 0.5 pg, 0.6 pg, 0.7 pg, 0.8 pg, 0.9 pg, 1.0 pg, or more than 1.0 pg of isolated RNA.
  • DNA, RNA, and/or protein may be isolated using any method known in the art.
  • the DNA may be isolated from one or more sections of at least one biological sample by digesting the section(s) with a proteinase and an RNase, then purifying the DNA, such as by ethanol precipitation and/or a column purification system.
  • the RNA may be isolated from one or more sections of at least one biological sample by incubating the section(s) with an RNA extraction reagent, such as TRIzol reagent.
  • the TRIzol reagent incubated sections may be sonicated and the organic layer may be extracted using an organic solvent, such as chloroform.
  • the resulting RNA may be dissolved in a suitable solution (including water) and further purified, such as by ethanol precipitation and/or a column purification system.
  • protein including native and/or denatured protein, may be isolated from one or more sections of at least one biological sample such as by, optionally precipitating the sections with ethanol, followed by incubation with a suitable lysis buffer.
  • the DNA, RNA, and/or protein isolated are subjected to quality control analysis.
  • RNA, and/or Protein C. Biological Sample Analysis of DNA, RNA, and/or Protein
  • Certain embodiments of the present disclosure concern methods for analyzing biological samples taken from an individual, including any individual encompassed herein.
  • DNA, RNA, and/or protein isolated from one or more sections of at least one biological sample is analyzed.
  • Analyzing DNA, RNA, and/or protein may comprise, for example, any PCR technique (such as allele- specific PCR, qPCR, RT-qPCR, multiplex PCR, and/or digital PCR), the use of restriction enzymes (such as for restriction fragment length polymorphism analysis or the like), any sequencing technique (such as Sanger sequencing, next generation sequencing, high throughput sequencing, deep sequencing, nanopore sequencing, exome sequencing, and/or single cell sequencing), Northern blotting, Western blotting, Southern blotting, flow cytometry, mass spectrometry, NMR spectroscopy, electrophoresis, or a combination thereof. Additionally, DNA may also be isolated from blood samples from the same individual to predict if a genomic alteration is somatic or germline.
  • any PCR technique such as allele- specific PCR, qPCR, RT-qPCR, multiplex PCR, and/or digital PCR
  • restriction enzymes such as for restriction fragment length polymorphism analysis or the like
  • any sequencing technique such as Sanger sequencing,
  • DNA may be analyzed by sequencing.
  • the DNA may be prepared for sequencing by any method known in the art, such as library preparation, hybrid capture, sample quality control, product-utilized ligation-based library preparation, or a combination thereof.
  • the DNA may be prepared for any sequencing technique, including whole exome sequencing.
  • a unique genetic readout for each sample may be generated by genotyping one or more highly polymorphic SNPs.
  • sequencing such as 76 base pair, paired-end sequencing, may be performed to cover approximately 70%, 75%, 80%, 85%, 90%, 95%, 99%, or greater percentage of targets at more than 20x, 25x, 30x, 35x, 40x, 45x, 50x, or greater than 50x coverage.
  • mutations, SNPS, INDELS, copy number alterations (somatic and/or germline), or other genetic differences may be identified from the sequencing, such as whole exome sequencing, using at least one bioinformatics tool, including Strelka2, Mutect2, CARNAC, Pindel , GISTIC,, any R package (including CopywriteR) and/or Annovar.
  • bioinformatics tool including Strelka2, Mutect2, CARNAC, Pindel , GISTIC, any R package (including CopywriteR) and/or Annovar.
  • RNA may be analyzed by sequencing.
  • the RNA may be prepared for sequencing by any method known in the art, such as poly-A selection, cDNA synthesis, stranded or nonstranded library preparation, or a combination thereof.
  • the RNA may be prepared for any type of RNA sequencing technique, including stranded specific RNA sequencing. In some embodiments, sequencing may be performed to generate approximately 10M, 15M, 20M, 25M, 30M, 35M, 40M or more reads, including paired reads.
  • the sequencing may be performed at a read length of approximately 50 bp, 55 bp, 60 bp, 65 bp, 70 bp, 75 bp, 80 bp, 85 bp, 90 bp, 95 bp, 100 bp, 105 bp, 110 bp, or longer.
  • raw sequencing data may be converted to estimated read counts (RSEM), fragments per kilobase of transcript per million mapped reads (FPKM), and/or reads per kilobase of transcript per million mapped reads (RPKM).
  • RSEM estimated read counts
  • FPKM fragments per kilobase of transcript per million mapped reads
  • RPKM reads per kilobase of transcript per million mapped reads
  • one or more bioinformatics tools may be used to infer stroma content, immune infiltration, and/or tumor immune cell profiles, such as by using upper quartile normalized FPKM data.
  • protein from samples is analyzed, including denatured protein.
  • the protein may be analyzed by mass spectrometry.
  • the protein may be prepared for mass spectrometry using any method known in the art.
  • the protein is digested to produce peptides that are then analyzed.
  • Protein, including any isolated protein encompassed herein may be treated with DTT followed by iodoacetamide.
  • the protein may be incubated with at least one peptidase, including an endopeptidase, proteinase, protease, or any enzyme that cleaves proteins.
  • protein is incubated with the endopeptidase, LysC and/or trypsin.
  • the protein may be incubated with one or more protein-cleaving enzymes at any ratio, including a ratio of pg of enzyme to pg protein at approximately 1:1000, 1:100, 1:90, 1:80, 1:70, 1:60, 1:50, 1:40, 1:30, 1:20, 1:10, 1:1, or any range between.
  • the cleaved proteins may be purified, such as by column purification.
  • purified peptides may be snap-frozen and/or dried, such as dried under vacuum.
  • the purified peptides may be fractionated, such as by reverse phase chromatography or basic reverse phase chromatography. Fractions may be combined for practice of the methods of the disclosure.
  • one or more fractions, including the combined fractions are subject to enrichment based on one or more post- translational modifications, such as phosphopeptide enrichment, including phospho-enrichment by affinity chromatography and/or binding, ion exchange chromatography, chemical derivatization, immunoprecipitation, co-precipitation, or a combination thereof.
  • the entirety or a portion of one or more fractions, including the combined fractions and/or phospho -enriched fractions may be subject to mass spectrometry.
  • the raw mass spectrometry data may be processed and normalized using at least one relevant bioinformatics tool.
  • the protein is analyzed with mass spectrometry instead of antibody-based analysis.
  • methods involve obtaining a sample from a subject.
  • the methods of obtaining provided herein may include methods of biopsy such as fine needle aspiration, core needle biopsy, vacuum assisted biopsy, incisional biopsy, excisional biopsy, punch biopsy, shave biopsy or skin biopsy.
  • the sample is obtained from a biopsy from esophageal tissue by any of the biopsy methods previously mentioned.
  • the sample may be obtained from any of the tissues provided herein that include but are not limited to non-cancerous or cancerous tissue and non-cancerous or cancerous tissue from the serum, gall bladder, mucosal, skin, heart, lung, breast, pancreas, blood, liver, muscle, kidney, smooth muscle, bladder, colon, intestine, brain, prostate, esophagus, or thyroid tissue.
  • the sample may be obtained from any other source including but not limited to blood, sweat, hair follicle, buccal tissue, tears, menses, feces, or saliva.
  • any medical professional such as a doctor, nurse or medical technician may obtain a biological sample for testing.
  • the biological sample can be obtained without the assistance of a medical professional.
  • a sample may include but is not limited to, tissue, cells, or biological material from cells or derived from cells of a subject.
  • the biological sample may be a heterogeneous or homogeneous population of cells or tissues.
  • the biological sample may be obtained using any method known to the art that can provide a sample suitable for the analytical methods described herein.
  • the sample may be obtained by non-invasive methods including but not limited to: scraping of the skin or cervix, swabbing of the cheek, saliva collection, urine collection, feces collection, collection of menses, tears, or semen.
  • the sample may be obtained by methods known in the art.
  • the samples are obtained by biopsy.
  • the sample is obtained by swabbing, endoscopy, scraping, phlebotomy, or any other methods known in the art.
  • the sample may be obtained, stored, or transported using components of a kit of the present methods.
  • multiple samples such as multiple esophageal samples may be obtained for diagnosis by the methods described herein.
  • multiple samples such as one or more samples from one tissue type (for example esophagus) and one or more samples from another specimen (for example scrum) may be obtained for diagnosis by the methods.
  • multiple samples such as one or more samples from one tissue type (e.g.
  • samples from another specimen may be obtained at the same or different times.
  • Samples may be obtained at different times are stored and/or analyzed by different methods. For example, a sample may be obtained and analyzed by routine staining methods or any other cytological analysis methods.
  • the biological sample may be obtained by a physician, nurse, or other medical professional such as a medical technician, endocrinologist, cytologist, phlebotomist, radiologist, or a pulmonologist.
  • the medical professional may indicate the appropriate test or assay to perform on the sample.
  • a molecular profiling business may consult on which assays or tests are most appropriately indicated.
  • the patient or subject may obtain a biological sample for testing without the assistance of a medical professional, such as obtaining a whole blood sample, a urine sample, a fecal sample, a buccal sample, or a saliva sample.
  • the sample is obtained by an invasive procedure including but not limited to: biopsy, needle aspiration, endoscopy, or phlebotomy.
  • the method of needle aspiration may further include fine needle aspiration, core needle biopsy, vacuum assisted biopsy, or large core biopsy.
  • multiple samples may be obtained by the methods herein to ensure a sufficient amount of biological material.
  • the sample is a fine needle aspirate of a esophageal or a suspected esophageal tumor or neoplasm.
  • the fine needle aspirate sampling procedure may be guided by the use of an ultrasound, X-ray, or other imaging device.
  • the molecular profiling business may obtain the biological sample from a subject directly, from a medical professional, from a third party, or from a kit provided by a molecular profiling business or a third party.
  • the biological sample may be obtained by the molecular profiling business after the subject, a medical professional, or a third party acquires and sends the biological sample to the molecular profiling business.
  • the molecular profiling business may provide suitable containers, and excipients for storage and transport of the biological sample to the molecular profiling business.
  • a medical professional need not be involved in the initial diagnosis or sample acquisition.
  • An individual may alternatively obtain a sample through the use of an over the counter (OTC) kit.
  • OTC kit may contain a means for obtaining said sample as described herein, a means for storing said sample for inspection, and instructions for proper use of the kit.
  • molecular profiling services are included in the price for purchase of the kit. In other cases, the molecular profiling services are billed separately.
  • a sample suitable for use by the molecular profiling business may be any material containing tissues, cells, nucleic acids, genes, gene fragments, expression products, gene expression products, or gene expression product fragments of an individual to be tested. Methods for determining sample suitability and/or adequacy are provided.
  • the subject may be referred to a specialist such as an oncologist, surgeon, or endocrinologist.
  • the specialist may likewise obtain a biological sample for testing or refer the individual to a testing center or laboratory for submission of the biological sample.
  • the medical professional may refer the subject to a testing center or laboratory for submission of the biological sample.
  • the subject may provide the sample.
  • a molecular profiling business may obtain the sample.
  • Particular embodiments of the disclosure concern methods of detecting a SNP in an individual.
  • One may employ any of the known general methods for detecting SNPs for detecting the particular SNP in this disclosure, for example.
  • Such methods include, but are not limited to, selective oligonucleotide probes, arrays, allele- specific hybridization, molecular beacons, restriction fragment length polymorphism analysis, enzymatic chain reaction, flap endonuclease analysis, primer extension, 5’-nuclease analysis, oligonucleotide ligation assay, single strand conformation polymorphism analysis, temperature gradient gel electrophoresis, denaturing high performance liquid chromatography, high-resolution melting, DNA mismatch binding protein analysis, surveyor nuclease assay, sequencing, or a combination thereof.
  • the method used to detect the SNP comprises sequencing nucleic acid material from the individual and/or using selective oligonucleotide probes.
  • Sequencing the nucleic acid material from the individual may involve obtaining the nucleic acid material from the individual in the form of genomic DNA, complementary DNA that is reverse transcribed from RNA, or RNA, for example. Any standard sequencing technique may be employed, including Sanger sequencing, chain extension sequencing, Maxam-Gilbert sequencing, shotgun sequencing, bridge PCR sequencing, high-throughput methods for sequencing, next generation sequencing, RNA sequencing, or a combination thereof.
  • Any standard sequencing technique may be employed, including Sanger sequencing, chain extension sequencing, Maxam-Gilbert sequencing, shotgun sequencing, bridge PCR sequencing, high-throughput methods for sequencing, next generation sequencing, RNA sequencing, or a combination thereof.
  • After sequencing the nucleic acid from the individual one may utilize any data processing software or technique to determine which particular nucleotide is present in the individual at the particular SNP.
  • the nucleotide at the particular SNP is detected by selective oligonucleotide probes.
  • the probes may be used on nucleic acid material from the individual, including genomic DNA, complementary DNA that is reverse transcribed from RNA, or RNA, for example.
  • Selective oligonucleotide probes preferentially bind to a complementary strand based on the particular nucleotide present at the SNP.
  • one selective oligonucleotide probe binds to a complementary strand that has an A nucleotide at the SNP on the coding strand but not a G nucleotide at the SNP on the coding strand
  • a different selective oligonucleotide probe binds to a complementary strand that has a G nucleotide at the SNP on the coding strand but not an A nucleotide at the SNP on the coding strand.
  • Similar methods could be used to design a probe that selectively binds to the coding strand that has a C or a T nucleotide, but not both, at the SNP.
  • any method to determine binding of one selective oligonucleotide probe over another selective oligonucleotide probe could be used to determine the nucleotide present at the SNP.
  • One method for detecting SNPs using oligonucleotide probes comprises the steps of analyzing the quality and measuring quantity of the nucleic acid material by a spectrophotometer and/or a gel electrophoresis assay; processing the nucleic acid material into a reaction mixture with at least one selective oligonucleotide probe, PCR primers, and a mixture with components needed to perform a quantitative PCR (qPCR), which could comprise a polymerase, deoxynucleotides, and a suitable buffer for the reaction; and cycling the processed reaction mixture while monitoring the reaction.
  • qPCR quantitative PCR
  • the polymerase used for the qPCR will encounter the selective oligonucleotide probe binding to the strand being amplified and, using endonuclease activity, degrade the selective oligonucleotide probe. The detection of the degraded probe determines if the probe was binding to the amplified strand.
  • Another method for determining binding of the selective oligonucleotide probe to a particular nucleotide comprises using the selective oligonucleotide probe as a PCR primer, wherein the selective oligonucleotide probe binds preferentially to a particular nucleotide at the SNP position.
  • the probe is generally designed so the 3’ end of the probe pairs with the SNP.
  • the probe will be extended during the amplification step of the PCR. For example, if there is a T nucleotide at the 3’ position of the probe and there is an A nucleotide at the SNP position, the probe will bind to the SNP and be extended during the amplification step of the PCR. However, if the same probe is used (with a T at the 3’ end) and there is a G nucleotide at the SNP position, the probe will not fully bind and will not be extended during the amplification step of the PCR.
  • the SNP position is not at the terminal end of the PCR primer, but rather located within the PCR primer.
  • the PCR primer should be of sufficient length and homology in that the PCR primer can selectively bind to one variant, for example the SNP having an A nucleotide, but not bind to another variant, for example the SNP having a G nucleotide.
  • the PCR primer may also be designed to selectively bind particularly to the SNP having a G nucleotide but not bind to a variant with an A, C, or T nucleotide.
  • PCR primers could be designed to bind to the SNP having a C or a T nucleotide, but not both, which then does not bind to a variant with a G, A, or T nucleotide or G, A, or C nucleotide respectively.
  • the PCR primer is at least or no more than 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,3 5, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, or more nucleotides in length with 100% homology to the template sequence, with the potential exception of non-homology the SNP location.
  • the SNP can be determined to have the A nucleotide and not the G nucleotide.
  • a germline or somatic SNP in the LIG1 gene of an individual predicts susceptibility or resistance to a chemotherapy, including a platinum-based chemotherapy.
  • CNV Copy Number Variation Detection
  • the CNV is detected using an array, wherein the array is capable of detecting CNVs on the entire X chromosome and/or all targets of miR-362.
  • Array platforms such as those from Agilent, Illumina, or Affymetrix may be used, or custom arrays could be designed.
  • One example of how an array may be used includes methods that comprise one or more of the steps of isolating nucleic acid material in a suitable manner from an individual suspected of having the CNV and, at least in some cases from an individual or reference genome that does not have the CNV; processing the nucleic acid material by fragmentation, labelling the nucleic acid with, for example, fluorescent labels, and purifying the fragmented and labeled nucleic acid material; hybridizing the nucleic acid material to the array for a sufficient time, such as for at least 24 hours; washing the array after hybridization; scanning the array using an array scanner; and analyzing the array using suitable software.
  • the software may be used to compare the nucleic acid material from the individual suspected of having the CNV to the nucleic acid material of an individual who is known not to have the CNV or a reference genome.
  • PCR primers can be employed to amplify nucleic acid at or near the CNV wherein an individual with a CNV will result in measurable higher levels of PCR product when compared to a PCR product from a reference genome.
  • the detection of PCR product amounts could be measured by quantitative PCR (qPCR) or could be measured by gel electrophoresis, as examples.
  • Quantification using gel electrophoresis comprises subjecting the resulting PCR product, along with nucleic acid standards of known size, to an electrical current on an agarose gel and measuring the size and intensity of the resulting band.
  • the size of the resulting band can be compared to the known standards to determine the size of the resulting band.
  • the amplification of the copy number (CN) will result in a band that has a larger size than a band that is amplified, using the same primers as were used to detect the CNV, from a reference genome or an individual that does not have the CNV being detected.
  • the resulting band from the CN amplification may be nearly double, double, or more than double the resulting band from the reference genome or the resulting band from an individual that does not have the CNV being detected.
  • the deletion of the CN will result in a band that has a smaller size than a band that is deleted, using the same primers as were used to detect the CNV, from a reference genome or an individual that does not have the CNV being detected.
  • the resulting band from the CN deletion may be nearly half, half, or less than half the resulting band from the reference genome or the resulting band from an individual that does not have the CNV being detected.
  • the CNV can be detected using nucleic acid sequencing. Sequencing techniques that could be used include, but are not limited to, whole genome sequencing, whole exome sequencing, and/or targeted sequencing.
  • a germline or a somatic CNV in the LIG1 gene of an individual predicts susceptibility or resistance to a chemotherapy, including a platinum-based chemotherapy.
  • DNA may be analyzed by sequencing.
  • the DNA may be prepared for sequencing by any method known in the art, such as library preparation, hybrid capture, sample quality control, product-utilized ligation-based library preparation, or a combination thereof.
  • the DNA may be prepared for any sequencing technique.
  • a unique genetic readout for each sample may be generated by genotyping one or more highly polymorphic SNPs.
  • sequencing such as 76 base pair, paired- end sequencing, may be performed to cover approximately 70%, 75%, 80%, 85%, 90%, 95%, 99%, or greater percentage of targets at more than 20x, 25x, 30x, 35x, 40x, 45x, 50x, or greater than 50x coverage.
  • mutations, SNPS, INDELS, copy number alterations (somatic and/or germline), or other genetic differences may be identified from the sequencing using at least one bioinformatics tool, including Strelka2, Mutect2, CARNAC, Pindel , GISTIC, any R package (including CopywriteR) and/or Annovar.
  • bioinformatics tool including Strelka2, Mutect2, CARNAC, Pindel , GISTIC, any R package (including CopywriteR) and/or Annovar.
  • RNA may be analyzed by sequencing.
  • the RNA may be prepared for sequencing by any method known in the art, such as poly-A selection, cDNA synthesis, stranded or nonstranded library preparation, or a combination thereof.
  • the RNA may be prepared for any type of RNA sequencing technique, including stranded specific RNA sequencing. In some embodiments, sequencing may be performed to generate approximately 10M, 15M, 20M, 25M, 30M, 35M, 40M or more reads, including paired reads.
  • the sequencing may be performed at a read length of approximately 50 bp, 55 bp, 60 bp, 65 bp, 70 bp, 75 bp, 80 bp, 85 bp, 90 bp, 95 bp, 100 bp, 105 bp, 110 bp, or longer.
  • raw sequencing data may be converted to estimated read counts (RSEM), fragments per kilobase of transcript per million mapped reads (FPKM), and/or reads per kilobase of transcript per million mapped reads (RPKM).
  • RSEM estimated read counts
  • FPKM fragments per kilobase of transcript per million mapped reads
  • RPKM reads per kilobase of transcript per million mapped reads
  • one or more bioinformatics tools may be used to infer stroma content, immune infiltration, and/or tumor immune cell profiles, such as by using upper quartile normalized RSEM data.
  • protein may be analyzed by mass spectrometry.
  • the protein may be prepared for mass spectrometry using any method known in the art. Protein, including any isolated protein encompassed herein, may be treated with DTT or TCEP followed by iodoacetamide.
  • the protein may be incubated with at least one peptidase, including an endopeptidase, proteinase, protease, or any enzyme that cleaves proteins. In some embodiments, protein is incubated with the endopeptidase, LysC and/or trypsin.
  • the protein may be incubated with one or more protein cleaving enzymes at any ratio, including a ratio of pg of enzyme to pg protein at approximately 1:1000, 1:100, 1:90, 1:80, 1:70, 1:60, 1:50, 1:40, 1:30, 1:20, 1:10, 1:1, or any range between.
  • the cleaved proteins may be purified, such as by column purification.
  • purified peptides may be snap-frozen and/or dried, such as dried under vacuum.
  • the purified peptides may be fractionated, such as by reverse phase chromatography or basic reverse phase chromatography. Fractions may be combined for practice of the methods of the disclosure.
  • one or more fractions, including the combined fractions are subject to phosphopeptide enrichment, including phosphoenrichment by affinity chromatography and/or binding, ion exchange chromatography, chemical derivatization, immunoprecipitation, co-precipitation, or a combination thereof.
  • the entirety or a portion of one or more fractions, including the combined fractions and/or phospho-enriched fractions may be subject to mass spectrometry.
  • the raw mass spectrometry data may be processed and normalized using at least one relevant bioinformatics tool.
  • kits, and/or systems can be utilized to detect the SNP and/or the CNV related to the genetic signature for diagnosing an individual (the detection either individually or in combination).
  • the reagents can be combined into at least one of the established formats for kits and/or systems as known in the art.
  • kits and “systems” refer to embodiments such as combinations of at least one SNP detection reagent, for example at least one selective oligonucleotide probe, and at least one CNV detection reagent, for example at least one PCR primer.
  • kits could also contain other reagents, chemicals, buffers, enzymes, packages, containers, electronic hardware components, etc.
  • the kits/systems could also contain packaged sets of PCR primers, oligonucleotides, arrays, beads, or other detection reagents. Any number of probes could be implemented for a detection array.
  • the detection reagents and/or the kits/systems are paired with chemiluminescent or fluorescent detection reagents.
  • Particular embodiments of kits/systems include the use of electronic hardware components, such as DNA chips or arrays, or microfluidic systems, for example.
  • the kit also comprises one or more therapeutic or prophylactic interventions in the event the individual is determined to be in need of.
  • the kit may comprise one or both of a composition for detecting a polymorphism and a composition for detecting a CNV.
  • the polymorphism detected is polymorphism rs4567 as represented by position 101 of SEQ ID NO:1.
  • the CNV detected is represented by SEQ ID NO:2.
  • the composition in the kit for detecting the polymorphism may be selected from the group consisting of oligonucleotide, one or more primers suitable for amplifying the polymorphism, one or more sequencing reagents, and a combination thereof.
  • the composition in the kit for detecting the CNV may be selected from the group consisting of one or more primers suitable for amplifying the polymorphism, one or more sequencing reagents, one or more arrays, and a combination thereof.
  • the therapy provided herein may comprise administration of one or a combination of therapeutic agents, such as a first cancer therapy and a second cancer therapy.
  • the therapies may be administered in any suitable manner known in the art.
  • the first and second cancer treatment may be administered sequentially (at different times) or concurrently (at the same time).
  • the first and second cancer treatments are administered in a separate composition.
  • the first and second cancer treatments arc in the same composition.
  • the first therapy and the second therapy are administered substantially simultaneously. In some embodiments, the first therapy and the second therapy are administered sequentially. In some embodiments, the first therapy, the second therapy, and a third therapy are administered sequentially. In some embodiments, the first therapy is administered before administering the second therapy. In some embodiments, the first therapy is administered after administering the second therapy.
  • Embodiments of the disclosure relate to compositions and methods comprising therapeutic compositions.
  • the different therapies may be administered in one composition or in more than one composition, such as 2 compositions, 3 compositions, or 4 compositions.
  • Various combinations of the agents may be employed.
  • the cancer therapy is administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intraventricularly, or intranasally.
  • the antibiotic is administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intraventricularly, or intranasally.
  • the appropriate dosage may be determined based on the type of disease to be treated, severity and course of the disease, the clinical condition of the individual, the individual's clinical history and response to the treatment, and the discretion of the attending physician.
  • the treatments may include various “unit doses.”
  • Unit dose is defined as containing a predetermined-quantity of the therapeutic composition.
  • the quantity to be administered, and the particular route and formulation, is within the skill of determination of those in the clinical arts.
  • a unit dose need not be administered as a single injection but may comprise continuous infusion over a set period of time.
  • a unit dose comprises a single administrable dose.
  • the quantity to be administered depends on the treatment effect desired.
  • An effective dose is understood to refer to an amount necessary to achieve a particular effect. In the practice in certain embodiments, it is contemplated that doses in the range from 10 mg/kg to 200 mg/kg can affect the protective capability of these agents.
  • doses include doses of about 0.1 , 0.5, 1 , 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, and 200, 300, 400, 500, 1000 pg/kg, mg/kg, pg/day, or mg/day or any range derivable therein.
  • doses can be administered at multiple times during a day, and/or on multiple days, weeks, or months.
  • the effective dose of the pharmaceutical composition is one which can provide a blood level of about 1 pM to 150 pM.
  • the effective dose provides a blood level of about 4 pM to 100 pM.; or about 1 pM to 100 pM; or about 1 pM to 50 pM; or about 1 pM to 40 pM; or about 1 pM to 30 pM; or about 1 pM to 20 pM; or about 1 pM to 10 pM; or about 10 pM to 150 pM; or about 10 pM to 100 pM; or about 10 pM to 50 pM; or about 25 pM to 150 pM; or about 25 pM to 100 pM; or about 25 pM to 50 pM; or about 50 pM to 150 pM; or about 50 pM to 100 pM (or any range derivable therein).
  • the dose can provide the following blood level of the agent
  • the therapeutic agent that is administered to a subject is metabolized in the body to a metabolized therapeutic agent, in which case the blood levels may refer to the amount of that agent.
  • the blood levels discussed herein may refer to the unmetabolized therapeutic agent.
  • Precise amounts of the therapeutic composition also depend on the judgment of the practitioner and are peculiar to each individual. Factors affecting dose include physical and clinical state of the patient, the route of administration, the intended goal of treatment (alleviation of symptoms versus cure) and the potency, stability and toxicity of the particular therapeutic substance or other therapies a subject may be undergoing.
  • dosage units of pg/kg or mg/kg of body weight can be converted and expressed in comparable concentration units of pg/ml or mM (blood levels), such as 4 pM to 100 pM. It is also understood that uptake is species and organ/tissue dependent. The applicable conversion factors and physiological assumptions to be made concerning uptake and concentration measurement are well-known and would permit those of skill in the art to convert one concentration measurement to another and make reasonable comparisons and conclusions regarding the doses, efficacies and results described herein.
  • administrations of the composition e.g., 2, 3, 4, 5, 6 or more administrations.
  • the administrations can be at 1, 2, 3, 4, 5, 6, 7, 8, to 5, 6, 7, 8, 9, 10, 11, or 12 week intervals, including all ranges there between.
  • phrases “pharmaceutically acceptable” or “pharmacologically acceptable” refer to molecular entities and compositions that do not produce an adverse, allergic, or other untoward reaction when administered to an animal or human.
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, anti-bacterial and anti-fungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredients, its use in immunogenic and therapeutic compositions is contemplated. Supplementary active ingredients, such as other anti-infective agents and vaccines, can also be incorporated into the compositions.
  • the active compounds can be formulated for parenteral administration, e.g., formulated for injection via the intravenous, intramuscular, subcutaneous, or intraperitoneal routes.
  • parenteral administration e.g., formulated for injection via the intravenous, intramuscular, subcutaneous, or intraperitoneal routes.
  • such compositions can be prepared as either liquid solutions or suspensions; solid forms suitable for use to prepare solutions or suspensions upon the addition of a liquid prior to injection can also be prepared; and, the preparations can also be emulsified.
  • the pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including, for example, aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
  • the form must be sterile and must be fluid to the extent that it may be easily injected. It also should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.
  • a pharmaceutical composition can include a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.
  • a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.
  • the proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion, and by the use of surfactants.
  • the prevention of the action of microorganisms can be brought about by various anti-bacterial and anti-fungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars or sodium chloride.
  • Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various other ingredients enumerated above, as required, followed by filtered sterilization or an equivalent procedure.
  • dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum-drying and freeze-drying techniques, which yield a powder of the active ingredient, plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • compositions will typically be via any common route. This includes, but is not limited to oral, or intravenous administration. Alternatively, administration may be by orthotopic, intradermal, subcutaneous, intramuscular, intraperitoneal, or intranasal administration. Such compositions would normally be administered as pharmaceutically acceptable compositions that include physiologically acceptable carriers, buffers or other excipients.
  • solutions Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically or prophylactically effective.
  • the formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above.
  • the method further comprises administering a cancer therapy to the patient.
  • the cancer therapy may be chosen based on the assay measurements, including any or all of the proteogenomic methods, alone or in combination with a clinical risk score calculated for the patient.
  • the cancer therapy comprises a local cancer therapy.
  • the cancer therapy excludes a systemic cancer therapy.
  • the cancer therapy excludes a local therapy.
  • the cancer therapy comprises a local cancer therapy without the administration of a system cancer therapy.
  • the cancer therapy does not comprise a platinum-based drug, such as does not comprise carboplatin, cisplatin, oxaliplatin, nedaplatin, heptaplatin, and/or lobaplatin.
  • the cancer therapy comprises an immunotherapy, which may be an immune checkpoint therapy. Any of these cancer therapies may also be excluded. Combinations of these therapies may also be administered.
  • Curative surgery includes resection in which all or part of cancerous tissue is physically removed, excised, and/or destroyed and may be used in conjunction with other therapies, such as the treatment of the present embodiments, chemotherapy, radiotherapy, hormonal therapy, gene therapy, immunotherapy, and/or alternative therapies.
  • Tumor resection refers to physical removal of at least part of a tumor.
  • treatment by surgery includes laser surgery, cryosurgery, electrosurgery, and microscopically-controlled surgery (Mohs’ surgery).
  • a cavity may be formed in the body.
  • Treatment may be accomplished by perfusion, direct injection, or local application of the area with an additional anti-cancer therapy. Such treatment may be repeated, for example, every 1, 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, 4, and 5 weeks or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. These treatments may be of varying dosages as well.
  • wild-type versions of a protein or polypeptide or a modified protein are assayed.
  • a “modified protein” or “modified polypeptide” or a “variant” refers to a protein or polypeptide whose chemical structure, particularly its amino acid sequence, is altered with respect to the wild-type protein or polypeptide.
  • a modified/variant protein or polypeptide has at least one modified activity or function (recognizing that proteins or polypeptides may have multiple activities or functions). It is specifically contemplated that a modified/variant protein or polypeptide may be altered with respect to one activity or function yet retain a wild-type activity or function in other respects, such as immunogenicity.
  • a protein is specifically mentioned herein, it is in general a reference to a native (wild-type) or recombinant protein.
  • the protein may be isolated directly from the organism of which it is native, produced by recombinant DNA/exogenous expression methods, or produced by solid-phase peptide synthesis (SPPS) or other in vitro methods.
  • SPPS solid-phase peptide synthesis
  • recombinant may be used in conjunction with a polypeptide or the name of a specific polypeptide, and this generally refers to a polypeptide produced from a nucleic acid molecule that has been manipulated in vitro or that is a replication product of such a molecule.
  • the size of a protein or polypeptide may comprise, but is not limited to, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210
  • polypeptides may be mutated by truncation, rendering them shorter than their corresponding wild-type form, also, they might be altered by fusing or conjugating a heterologous protein or polypeptide sequence with a particular function (e.g., for targeting or localization, for enhanced immunogenicity, for purification purposes, etc.).
  • domain refers to any distinct functional or structural unit of a protein or polypeptide, and generally refers to a sequence of amino acids with a structure or function recognizable by one skilled in the art.
  • polypeptides, proteins, or polynucleotides encoding such polypeptides or proteins of the disclosure may include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, or 50 (or any derivable range therein) or more variant amino acids or nucleic acid substitutions or be at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% (or any
  • nucleotide as well as the protein, polypeptide, and peptide sequences for various genes have been previously disclosed, and may be found in the recognized computerized databases.
  • Two commonly used databases are the National Center for Biotechnology Information’s Genbank and GenPept databases (on the World Wide Web at ncbi.nlm.nih.gov/) and The Universal Protein Resource (UniProt; on the World Wide Web at uniprot.org).
  • Genbank and GenPept databases on the World Wide Web at ncbi.nlm.nih.gov/
  • the Universal Protein Resource UniProt; on the World Wide Web at uniprot.org.
  • the coding regions for these genes may be assayed and/or amplified and/or expressed using the techniques disclosed herein or as would be known to those of ordinary skill in the art.
  • Amino acid sequence assayed by the methods of the disclosure can be substitutional, insertional, or deletion variants.
  • a variation in a polypeptide of the disclosure may affect 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, or more non-contiguous or contiguous amino acids of the protein or polypeptide, as compared to wild-type.
  • a variant can comprise an amino acid sequence that is at least 50%, 60%, 70%, 80%, or 90%, including all values and ranges there between, identical to any sequence provided or referenced herein.
  • a variant can include 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more substitute amino acids.
  • Deletion variants typically lack one or more residues of the native or wild type protein. Individual residues can be deleted or a number of contiguous amino acids can be deleted. A stop codon may be introduced (by substitution or insertion) into an encoding nucleic acid sequence to generate a truncated protein.
  • Insertional mutants typically involve the addition of amino acid residues at a nonterminal point in the polypeptide. This may include the insertion of one or more amino acid residues. Terminal additions may also be generated and can include fusion proteins which are multimers or concatemers of one or more peptides or polypeptides described or referenced herein.
  • Substitutional variants typically contain the exchange of one amino acid for another at one or more sites within the protein or polypeptide, and may be designed to modulate one or more properties of the polypeptide, with or without the loss of other functions or properties. Substitutions may be conservative, that is, one amino acid is replaced with one of similar chemical properties.
  • Constant amino acid substitutions may involve exchange of a member of one amino acid class with another member of the same class.
  • Conservative substitutions are well known in the art and include, for example, the changes of: alanine to serine; arginine to lysine; asparagine to glutamine or histidine; aspartate to glutamate; cysteine to serine; glutamine to asparagine; glutamate to aspartate; glycine to proline; histidine to asparagine or glutamine; isoleucine to leucine or valine; leucine to valine or isoleucine; lysine to arginine; methionine to leucine or isoleucine; phenylalanine to tyrosine, leucine or methionine; serine to threonine; threonine to serine; tryptophan to tyrosine; tyrosine to tryptophan or phenylalanine; and valine to iso
  • amino acid substitutions may encompass non-naturally occurring amino acid residues, which are typically incorporated by chemical peptide synthesis rather than by synthesis in biological systems. These include peptidomimetics or other reversed or inverted forms of amino acid moieties.
  • substitutions may be “non-conservative”, such that a function or activity of the polypeptide is affected. Non-conservative changes typically involve substituting an amino acid residue with one that is chemically dissimilar, such as a polar or charged amino acid for a nonpolar or uncharged amino acid, and vice versa. Non-conservative substitutions may involve the exchange of a member of one of the amino acid classes for a member from another class.
  • Example 1 LIG1 deletion predicts chemotherapy resistance, chromosomal instability, and poor prognosis in triple negative breast cancer
  • Proteogenomic analysis of neoadjuvant chemotherapy resistance in triple negative breast cancer reveals a complex landscape of response associates, including a 19ql3.31-33 somatic deletion affecting lagging- strand DNA synthesis genes that associates with lack of pathological complete response, carboplatin-selective chemotherapy resistance, poor prognosis and chromosomal instability in multiple cancer types.
  • TNBC Sporadic Triple Negative Breast Cancer
  • BC breast cancers
  • Microscaled proteogenomics (PMID: 31988290) is a discovery technique that integrates DNA and RNA sequencing with mass spectrometry based proteomics. MPG was applied to snap-frozen TNBC clinical trial core needle biopsies obtained before treatment with carboplatin and docetaxel (PMID: 34173924, BCM NCT02547987; WashU: NCT02124902). Proteogenomic profiling included whole exome sequencing (WES), RNA-seq, and tandem mass tag-based proteomics and phosphoprotcomics. Clinical endpoints were pathological complete response (pCR) and residual cancer burden (RCB). Standard non-parametric statistical tests were employed to identify proteogenomic analytes associated with these endpoints.
  • WES whole exome sequencing
  • RNA-seq RNA-seq
  • tandem mass tag-based proteomics and phosphoprotcomics
  • Clinical endpoints were pathological complete response (pCR) and residual cancer burden (RCB). Standard non-parametric statistical tests were
  • CNA recurrent copy number aberrations
  • COSMIC mutational signature 3 homologous recombination defect signature
  • LIG1 deletion is associated with chromosomal instability in TNBC and specifically occurs in tumors without evidence for defects in homologous recombination.
  • the role of LIG1 deletion as a negative predictive marker for carboplatin, and a discussion of how LIG1 deletion causes chromosomal instability and tumorigenesis (PMID: 12124343) is encompassed herein.
  • Microscaled proteogenomics was deployed to probe the molecular basis for differential response to neoadjuvant carboplatin & taxotere combination chemotherapy for triple negative breast cancer (TNBC).
  • TNBC triple negative breast cancer
  • Proteomic analyses of pretreatment biopsies uniquely revealed that metabolic pathways including oxidative phosphorylation, fatty acid metabolism and glycolysis were resistance associated.
  • Proteogenomic analyses of somatic copy number aberrations identified a resistance-associated 8q21.3 gain and 19ql3.31-33 deletion where LIG1, POLDI and XRCC1 are located.
  • LIG1 DNA ligase I involved in lagging strand synthesis gene deletion and/or low mRNA expression were associated with lack of pathological complete response and poor prognosis in TNBC, as well as selective carboplatin-resistance in TNBC patient- derived xenograft models. Low expression or LIG1 loss was also associated with higher chromosomal instability index (CIN) and poor prognosis in other cancer types, demonstrating that deletion of lagging strand synthesis components has broad clinical significance.
  • CIN chromosomal instability index
  • OCT-embedded snap-frozen core needle biopsies were accrued from consented patients with TNBC (70% Caucasian, 27% African American, and 3% other ethnicities). Samples from 59 patients taken before treatment had >25% tumor content. For 16 patients, an additional sample was obtained 48 to 72 hours after initiating chemotherapy. All patients received six cycles of neoadjuvant carboplatin and docetaxel combination chemotherapy (NCT02547987, NCT02124902). A REMARK diagram provides information on sample flow into different analytical pipelines (FIG.1A).
  • RNA-based PAM50 [9] and TNBCtype [10] intrinsic subtyping of baseline samples revealed “Basal-like” to be the most prevalent subtype (75%) although all Lehman TNBC subtypes were represented (FIG.8A). Sample-level mRNA and protein correlations deteriorated in samples with an average tumor content (TC) below 45% (FIG.8B), and these samples were excluded from further bioinformatics analyses. As previously reported [7], TMT11 plexes were linked using a common reference which served as a common denominator for calculating protein and phosphositc ratios.
  • FIG.8C As a measure of excellent data quality across plcxcs, common references across multiple plexes showed very strong correlations (FIG.8C). For each qualified sample, DNA, RNA and protein level information were available for an average of 10,500 genes (FIG.1C). Overall, phosphoproteomic analysis quantified -27,000 phosphorylation sites in -5,000 distinct phosphoproteins (FIG.1C). Median mRNA and protein correlation per gene in qualified samples was 0.37, similar to previous CPTAC proteogenomic studies [11] (FIG.8D). Genes with significant negative RNA-protein correlations were enriched in KEGG pathways involved in cellular respiration and amino-acid and lipid metabolism.
  • genes with both higher mRNA and protein expression in pCR cases showed enrichment for immune (interferon alpha and gamma response) and cell cycle (G2M checkpoint and E2F and MYC target) pathways (FIG.1E).
  • Enrichment analysis of differential phosphorylation sites (PTM-SEA [20]) showed elevated phosphoproteome-driven signatures for treatment with inhibitors targeting microtubule depolymerization or with DNA damage-inducing agents such as etoposide, hydroxyurea and ionizing radiation in pCR tumors (FIG. IF).
  • Elevated MARK2 target sites were enriched in non- pCR tumors (FIG.1F), corroborating prior evidence for higher MARK2 levels in cisplatin resistance in other cancer types [21, 22]. Consistent with elevated cell cycle pathways observed for pCR samples in the RNA and protein data, CDK1 , 2, and 7 and CDC7 target phosphosites were also significantly higher in pCR. A notable exception was CDK4, for which target sites were significantly higher in non-pCR.
  • FIG.2A A heatmap detailing baseline proteogenomic features associated with the immune microenvironment is provided (FIG.2A). Protein-derived immune stimulatory scores, previously found to be well correlated with immune infiltration [11], as well as PD-L1 RNA, protein, and phosphorylation levels were significantly higher in pCR tumors (Fig. 2B). Both PD-L1 protein and phosphoprotein levels significantly correlated with PD-L1 IHC levels (Fig. 2D-E). Representative IHC images for high and low PD-L1 staining are shown in FIG.9A and B, respectively.
  • CTN chromosomal instability
  • RNA- and protein-based multi-gene proliferation scores MGPS
  • ssGSEA single-sample GSEA
  • PTM-Signature Enrichment Analysis PTM-SEA
  • ssGSEA scores for differential metabolic pathways including oxidative phosphorylation, adipogenesis, fatty acid metabolism, and glycolysis were significantly higher in pre-treatment tumors that did not achieve pCR (RCBO) (FIG.3A).
  • RCBO pCR
  • metabolic pathway enrichment was specific to proteomic data, which may account for the lack of these associations in prior mRNA-based analyses.
  • Metabolism-related proteins significantly associated with resistance included many mitochondrial proteins such as those directly involved in the tricarboxylic acid (TCA) cycle (ACO2, FH, MDH2, SUCLG1, SUCLG2, PDP1, DLAT), the electron transport chain (SDHC, UQCR10), fatty acid metabolism (CRAT, ACADS, ACAT1, DECR1, ECHS1, HADHB), and amino acid catabolism (ALDH6A1, HMGCL, DBT, BCKDHB). While the majority of metabolic genes contributing to these pathway scores showed association with non-pCR only at the protein level, a subset of metabolic genes from the differential Hallmark pathways (FIG.3B).
  • RNA expression of this subset of 29 genes was tested against pCR status in an independent cohort of patients who received combination treatment with carboplatin and docetaxel in the BrighTNESS TNBC trial [26].
  • the somatic mutation landscape of TNBC is dominated by chromosomal instability (CIN), which produces recurrent copy number aberrations [27].
  • CIN chromosomal instability
  • the overall copy number landscape in this dataset was typical for TNBCs, and alignment of the differential expression results for RNA and protein by position in the genome demonstrated chromosomal regions where the differential expression of multiple genes is determined by CNA (FIG. 12A).
  • GSEA was utilized to statistically evaluate relationships between cytoband location and upregulated or downregulated gene expression (FIG.4A). Individual genes were subsequently examined to identify examples that were up- or down- regulated with respect to pCR status.
  • RIPK2 which mediates metastasis in patients with advanced breast cancer [27], also located on 8q21.3, was significantly higher in non-pCR tumors, but only at the protein level.
  • Hallmark pathway GSEA analysis of the genes on cytoband 19ql3.31-33 showed enrichment in the DNA damage repair (DDR) pathway at the mRNA level, with LTG1 , XRCC1 , POLDI and ERCC1 comprising the leading-edge genes (FIG.4C).
  • LIG1 showed the strongest association with treatment response at the protein level, followed by POLDI (FIG.4C).
  • RNA expression levels for LIG1 and XRCC1 were also significantly associated with poor metastasis-free survival in the TNBC subset of another chemotherapy-treated TNBC patient cohort (Hatzis dataset [28]) (FIG.4E).
  • LIG1 was the most consistently associated with chemotherapy resistance and poor metastasis-free survival in multiple independent datasets (FIG.4D, 4E, 11D).
  • IM immune stimulatory
  • the IL33 pathway was significantly upregulated in tumors without LIG1 loss, further indicating a less active immune environment associated with LIG1 loss (FIG.12D-E). Tumors with LIG1 loss also had significantly higher protein-based proliferation scores (pMGPS, FIG.5) as well as upregulation of CDK1/2 activity (FIG.12D) by Single-sample PTM-SEA [20], indicating increased cell cycle activity.
  • Example 8 LIG1 copy number loss is associated with poor progression-free survival and CIN across multiple cancer types
  • proteogenomic analysis provides at least five new perspectives.
  • integrated proteogenomic information provides extensive information on the immune microenvironment that could be used as alternatives to PDL1 IHC.
  • oxidative phosphorylation and fatty acid metabolism were strongly associated with chemotherapy resistance but uniquely at the proteomic level, indicating models to predict chemotherapy response could be strengthened by the inclusion of proteomic data.
  • G2M checkpoint components, E2F regulation and MYC target pathways provide an additional rich resource of biomarkers for predictive model-building.
  • proteomics can help prioritize genomic observations in tumors with large numbers of chromosomal aberrations such as TNBC demonstrated by the identification of LIG1 as chemotherapy resistance marker.
  • proteomic analyses of preclinical models suggest that LIG1 loss is a selective biomarker for carboplatin resistance.
  • the use of carboplatin in TNBC remains controversial and could be potentially avoided in LIGl-depleted tumors. Further investigation of LIG1/XRCC1/POLD1 expression and 19ql3.31-33 deletion status in randomized trials of carboplatin should therefore be pursued in an effort to reduce carboplatin use.
  • LIG1 encodes an ATP-dependent DNA ligase that seals DNA nicks during replication, recombination, and a variety of DNA damage responses [30].
  • ligase family it was first discovered as the main enzyme responsible for ligating Okazaki fragments together during lagging-strand synthesis at the replication fork during S-phase [31-33].
  • LIG1 also ligates single-stranded or double- stranded DNA breaks in various DNA damage repair pathways including long-patch base-excision repair, nucleotide-excision repair, and alternative non-homologous end-joining repair [34, 35].
  • a phenotype for LIG1 deficiency in humans was first identified in an immunodeficient patient with homozygous germline hypomorphic LIG1 alleles causing impaired Okazaki fragment ligation [36]. Insufficient LIG1 activity results in the accumulation of replication intermediates that cause single- stranded (SSB) and double-stranded breaks (DSB) [37, 38], ultimately leading to reduced genome integrity. In transgenic systems hypomorphic LIG1 alleles are associated with high susceptibility to cancer formation [39].
  • LIG1 hypomorphic alleles can be questioned because single copy loss is most likely observed, with the remaining LIG1 allele intact, in sporadic TNBC and other cancers, raising the question of whether single copy loss produces sufficient functional deficiency to generate a phenotype.
  • One obvious possibility is that co-deletion of POLDI and XRCC1 produce a compound deficiency phenotype since all three genes serve lagging strand synthesis.
  • XRCC1 is particularly noteworthy because LIG3/XRCC1 provides a backup pathway for LIG1 during DNA repair and lagging strand synthesis but is known to be less efficient than LIG1 [40].
  • LIG1 loss was found to be orthogonal to HRD signatures. Consequently, LIG1 cells may still be paradoxically proficient at double strand break repair.
  • the PDX study hints at this, as the model derived from the baseline study had a BRCA2 frameshift mutation and no LIG1 loss, but the BRCA2 mutation was undetectable in the both the PDX from treatment-resistant residual disease at surgery and the PDX from a liver metastasis, both of had gained a 19ql3.3/LIGl hemizygous deletion. It remains unclear why LIG1 loss is so strongly associated with chromosomal instability across many cancer types. We have not demonstrated cause and effect in this paper, but clearly cells that enter mitosis with unrepaired strand gaps are at risk for chromosomal breakage and illicit chromosomal fusion events, likely driving aneuploidy.
  • CDK1 and CDK2 are targets in LIG1 depleted tumors, in some embodiments. Because targeting CDK1 in cancer patients may lead to toxicity in normal proliferating cells [9], one can focus on CDK2 inhibition. Similarly, preliminary data also indicates elevated levels of Chk2 targets in LIG1 depleted TNBCs, and one can characterize Chk2 inhibitors in this setting.
  • RNA, DNA, and proteomic profiling were also profiled.
  • pre-treatment WHIM68 — LIG1 high
  • WHIM74 - LIG1 heterogenous at surgery after chemotherapy
  • WHIM75 - LIG1 low liver metastatic site
  • NCT02547987 BCM
  • NCT02124902 WashU
  • Eligible patients for the two clinical trials included pre or post-menopausal women at least 18 years old, with clinical stages II/III ER negative and HER2 negative (0 or 1 + by IHC or FISH negative) invasive breast cancer.
  • the study was approved by the TRB at both participating sites WashU and BCM and followed the Declaration of Helsinki and Good Clinical Practice guidelines.
  • the protocol and informed consent documents were approved by WUSM and BCM.
  • PDL 1 staining was performed using the PD-L 1 IHC 22C3 pharmDx kit per regulated protocol on the Dako Autolink-48 platform (SK006; Agilent). Pathology slide scoring was performed using established professional guidelines for TNBC, when appropriate. All immunohistochemistry results were evaluated against positive and negative tissue controls.
  • RNA-Seq data Transciptome data was generated for 60 samples in this study. For this, strand-specific, poly-A+ RNA-seq libraries for sequencing on the Illumina platform were prepared as previously described (PMID: 25360585). Briefly, poly-A+ mRNA was extracted from 1 p.g total RNA, followed by fragmentation and first strand cDNA synthesis. The resultant cDNA was end- repaired, A-tailed and ligated with Illumina Dual barcode adapters. Eibraries were sequenced on NovaSeq 6000 instruments using the S4 reagent kit (300 cycles) to generate 2xl50bp paired-end reads. Between 59.96 and 112.62M total reads were generated for these 60 samples. The average strand-specificity and rRNA rate was 97.04% and 1.79% respectively. The transcripts for 22868 to 27856 genes were detected in these samples.
  • Somatic and copy number variant calling Somatic variants were called using paired tumor and blood normal from WES data.
  • Tools used for somatic variant calling are Strelka2, Mutect2, CARNAC, and Pindel (v 0.2.5b9). Variants reported by these tools were filtered using GATK VariantFiltration (v 3.8.0) with parameters window 35, cluster 3, FS > 30.0, and QD ⁇ 2.0.
  • GATK VariantFiltration v 3.8.0
  • a 10X coverage cutoff was applied for both tumor and normal sequence depth.
  • Somatic SNVs and indels were filtered somatic SNVs and indels by a minimal variant allele frequency (VAF) of 0.02. Then annovar (v 04.16.2018) was used to annotate remaining variants. Somatic mutations were called comparing tumor DNA against matched blood normal DNA using WES data. Similarly, germline mutations were called by comparing normal WES against the reference genome. Hgl9.UCSC.add_miR.140312.refgene was used to map the copy number information to genes. COSMIC mutational signature scores for every sample were estimated using dcconstructSigs [43].
  • bam files were processed by the CopywriteR package [44] to derive log2 tumor-to-normal copy number ratios, and the circular binary segmentation (CBS) algorithm [45] implemented in the CopywriteR package was used for the copy number segmentation, with the default parameters.
  • Chromosomal instability for each chromosome in each sample was inferred from the segmentation data using a weighted-sum approach in which the absolute values of the log2 ratios of all segments within a chromosome were weighted by the segment length and summed up [13].
  • the genome- wide chromosome instability index (CIN) was derived by adding up the instability scores for all 22 autosomes in each sample.
  • GISTIC2[46] was used to retrieve gene-level copy number values and call significant copy number alterations in the cohort.
  • a threshold of +/-0.3 was applied to log2 copy number ratio to identify gene-wise gain or loss of copy number, respectively.
  • Each gene of every sample was assigned a thresholded copy number level that reflects the magnitude of its deletion or amplification. These are integer values ranging from -2 to 2, where 0 means no amplification or deletion of magnitude greater than the threshold parameters described above.
  • Amplifications are represented by positive numbers: 1 means amplification above the amplification threshold; 2 means amplification larger than the arm level amplifications observed in the sample.
  • Deletions are represented by negative numbers: -1 means deletion beyond the threshold; -2 means deletions greater than the minimum arm-level copy number observed in the sample.
  • GISTIC value +/- 2 exceed the high-level thresholds for amplifications/deep deletions, and those with +/- 1 exceed the low-level thresholds but not the high-level thresholds.
  • the low-level thresholds are just the 'ampthresh' and 'delthresh' noise threshold input values to GISTIC (typically 0.1 or 0.3) and are the same for every thresholds.
  • Proteomic sample preparation Samples were prepared for proteomic analysis as described in a previous microscaled proteogenomic study [7]. Protein lysates in 8 M urea were reduced with 1 mM DTT for 45 min, then alkylated with iodoacetamide (IAA) for 45 min protected from light. Before digestion, urea was diluted to a final concentration of 2 M with 50 mM Tris- HC1 pH 8.5. Protein lysates were then treated with endopeptidase LysC (Promega) at a 1:40 enzyme mass to BCA-estimated protein mass ratio, followed by overnight treatment with trypsin (Promcga) at a 1:30 ratio.
  • endopeptidase LysC Promega
  • TMT labeling A total of 30 ug peptides in 100 uL 50 mM HEPES, pH 8.5, were labeled with 240 ug TMT reagent for an 8:1 TMT:peptide ratio and incubated at 25C for 1 hour. Before quenching excess TMT, labeling was assessed by stage-tip desalting 1 uL per sample and running 0.5 ug peptides on a 30 min gradient. Mixing ratios were assessed by pooling and stage-tip desalting 2 uL from each sample, and running 0.5 ug peptides on a 110 min gradient.
  • TMT site labeling was required to be over 97%, and mixing ratios were balanced to be within +/- 15% when compared to the common references in each 11-plex (see Experimental design for proteomics and phospho-proteomics).
  • Excess TMT reagent was quenched by incubating with 5 uL 5% hydroxylamine (Sigma) for 15 min. Samples within each plex were combined according to the ratios determined by the mixing controls to achieve equal sample representation within each plex.
  • the combined peptides were desalted on a 100 mg tC18 Sep-Pak (Waters), eluted with 50% acetonitrile/0.1% FA, and dried in a vacuum centrifuge.
  • paired pre- and post-treatment tumor samples from a patient were grouped within the same 11-plex.
  • protein and phosphopeptide ratios were obtained between prospective BRCA common reference and the core common reference, and the results arc shown in FIG.8C.
  • Phosphopeptides were enriched using Fe3+ immobilized metal affinity chromatography (IMAC) as previously described [7].
  • IMAC iron-based metal affinity chromatography
  • Ni-NTA (Qiagen) beads were washed three times with HPLC grade water followed by incubation with 100 mM EDTA (Sigma) to strip nickel from the beads. Beads were washed three more times with HPLC grade water and incubated in 10 mM FeC13 (Sigma) for 30 min.
  • Fe3+-loaded beads were resuspended in a 1:1:1 solution of methanol, acetonitrile, and 0.01% acetic acid in water.
  • Dried peptides were resuspended to a final volume of 500 uL with 50% acetonitrile and 0.1% trifluoroacetic acid (TFA) followed by 100% acetonitrile and 0.1 % TFA for a final concentration of 80% acetonitrile.
  • TFA trifluoroacetic acid
  • Each reconstituted fraction was added to 20 uL of 50% bead slurry and incubated while rotating end-over-end at room temperature for 30 min. Beads were then spun down and supernatant removed. The beads were transferred to a conditioned C18 stage- tip in 200 uL of 80% acetonitrile and 0.1% TFA.
  • Phosphopeptides were eluted from the beads with 500 mM potassium phosphate, pH 7, onto the Cl 8, washed with 1% formic acid, and eluted into an autosampler vial with 50% acetonitrile and 0.1% FA.
  • Proteome and phosphoproteome data acquisition was performed with a Proxeon nLC- 1200 coupled to Thermo Lumos instrumentation.
  • proteomic analysis peptides were run on a 110 min gradient with 86 min of effective gradient (6 to 30% buffer B containing 90% ACN and 0.1%FA).
  • phosphoproteomics analysis two injections were run per fraction: a first injection over a 90 min gradient with 70 min of effective gradient (6 to 30% buffer B containing 90% ACN and 0.1% FA), and a second injection over a 140 min gradient with 120 min of effective gradient (6 to 35% buffer B containing 90% ACN and 0.1% FA).
  • MS2 injection time was increased to 250 s, and the MS2 AGC decreased to 5e4.
  • a cycle time of 2 s was used for all methods.
  • Carb amidomethylation of cysteines was set as a fixed modification, and N-terminal protein acetylation, oxidation of methionine (Met-ox), de-amidation of asparagine, hydroxylation of proline, TMT over-labeling of serine, threonine, and tyrosine, and cyclization of peptide N-terminal glutamine and carbamidomethylated cysteine to pyroglutamic acid (pyroGlu) and pyro-carbamidomethyl cysteine were set as variable modifications.
  • N-terminal protein acetylation, oxidation of methionine (Met-ox), de-amidation of asparagine, hydroxylation of proline, TMT over-labeling of serine, threonine, and tyrosine and cyclization of peptide N-terminal glutamine and carbamidomethylated cysteine to pyroglutamic acid (pyroGlu) and pyro-carba
  • phosphorylation of serine, threonine, and tyrosine were allowed as additional variable modifications, while de-amidation of asparagine and hydroxylation of proline were disabled. Trypsin Allow P was specified as the proteolytic enzyme with up to 4 missed cleavage sites allowed.
  • the allowed precursor mass shift range was -18 to 326 Da.
  • the range was -18 to 272 Da, to allow for up to 3 phosphorylations and 2 Met-ox per peptide.
  • Precursor and product mass tolerances were set to ⁇ 20 ppm.
  • peptide FDR limits were set to 0.8% for charge states 2-4 and 0.4% for charge states 5-6, and for PDX analysis peptide FDR limits were set to 0.6% for 2-4 and 0.3% for 5-6, employing a target-decoy approach using reversed protein sequences (PMID: 20013364).
  • SGS subgroup- specific
  • reporter ion signals were corrected for isotopic impurities. Relative abundances of proteins and phosphosites were selected as the median of TMT reporter ion intensity ratios from all PSMs matching to the protein or phosphosite. PSMs were excluded if they lacked a TMT label, had a precursor ion purity ⁇ 50%, or had a negative delta forward -reverse score. To normalize across 11-plex experiments, TMT intensities were divided by the common reference for each protein and phosphosite. Log2 TMT ratios were further normalized by median centering and median absolute deviation scaling. Proteins and phosphosites quantified in fewer than 30% of samples (i.e., missing in > 70% of samples) were removed from the respective datasets.
  • cryopulverized PDX tumor tissues were lysed and digested as described above. 50ug peptides were dissolved in 200ul 50 mM HEPES, pH 8.5 and labeled with 400ug of TMT reagent. TMT sample generation, basic reverse fractionation and protcomic analysis was performed identical to that of clinical core biopsies. Raw files were searched against the human and mouse (PDX samples) UniProt protein databases complemented with 553 smallopen reading frames (smORFs) and common contaminants (Human and mouse: UniProt.human.mouse.20171228.RIsnrNF.553smORFs.264contams.fasta) using Spectrum Mill .
  • smORFs smallopen reading frames
  • common contaminants Human and mouse: UniProt.human.mouse.20171228.RIsnrNF.553smORFs.264contams.fasta
  • WebGestaltR (PMID: 31114916) and PTM-SEA[20] were used to identify MSigDB Hallmark pathways (gene level data) and PTM signature sets (phosphosite level data), respectively, that show enrichment in pCR or non-pCR tumors by applying the GSEA/PTM-SEA algorithms to signed (by direction of change) log 10 p-values from the differential expression analysis.
  • ssGSEA R package [47, 48] was applied to data from three “omes” and scores for Hallmark pathways were obtained for individual samples). Normalized enrichment scores (NES) were utilized for visualization purposes.
  • the Wilcoxon signed rank test in R was used for paired differential analysis of on-treatment to baseline measurements for RNA, protein, phosphosite, and phosphoprotein data for 14 patients with matched on-treatment and baseline biopsies (only 13 had matched RNA data).
  • GSEA using WebGestaltR (PMID: 31114916) and PTM-SEA were applied to signed loglO transformed p-values from this analysis.
  • PTM-SEA was also applied to phosphosite log2 TMT ratios for each baseline sample to obtain single sample kinase activity scores (normalized enrichment scores for kinase target PTM sets).
  • RNA-based multi-gene proliferation scores were calculated as described previously [11, 49] by averaging the gene-centered log2 RSEM data for all genes previously characterized as cycle-regulated [50] in each sample.
  • Protein-based MGPS were generated for each sample by averaging log2 TMT ratios for all proteins that showed significant correlation with the RNA-bascd MGPS (Pearson correlation, p ⁇ 0.01 after Bcnjamini-Hochbcrg fdr correction).
  • Immune profile and microenvironment scores were inferred from the FPKM version of the RNA- seq data using ESTIMATE (R package; PMID: 24113773), Cibersort (webtool (https://cibersort.stanford.edu/) run in absolute mode; PMID: 25822800), and xCell (webtool (https://xcell.ucsf.edu/); PMID: 29141660).
  • Protein-based immune modulator scores were calculated as described previously [11] by averaging log2 TMT ratios for expert curated sets of immune modulators belonging to three categories: immune stimulatory, immune inhibitory, and human leukocyte antigen (HLA) [51].
  • genomics and transcriptomics data has been deposited in the dbGAP database under the accession code phs002505.vl. All genomics and proteomic raw data associated with this study will be available via dbGAP and CPTAC portal (https://proteomics.cancer.gov/data-portal) respectively upon publication.
  • MARK2 enhances cisplatin resistance via PI3K/AKT/NF- kappaB signaling pathway in osteosarcoma cells.
  • O'Meara T Safonov A, Casadevall D, et al. Immune microenvironment of triple-negative breast cancer in African-American and Caucasian women.
  • Iorio F Knijnenburg TA, Vis DJ, et al. A Landscape of Pharmacogenomic Interactions in Cancer. Cell 2016;166(3):740-754.
  • Picco G Chen ED, Alonso LG, et al.
  • DNA ligase I deficiency leads to replication-dependent DNA damage and impacts cell morphology without blocking cell cycle progression. Mol Cell Biol 2009;29(8):2032-41. Harrison C, Ketchen AM, Redhead NJ, et al. Replication failure, genome instability, and increased cancer susceptibility in mice with a point mutation in the DNA ligase I gene. Cancer Res 2002;62(14):4065- 74. Bentley D, Selfridge J, Millar JK, et al. DNA ligase I is required for fetal liver erythropoiesis but is not essential for mammalian cell viability. Nat Genet 1996; 13(4):489- 91. Le Chalony C, Hoffschir F, Gauthier LR, et al.

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Abstract

La protéogénomique microscopique a été déployée pour sonder la base moléculaire d'une réponse différentielle à une chimiothérapie d'association de carboplatine et de taxotère néoadjuvants contre un cancer du sein triple négatif (TNBC). Des analyses protéomiques de biopsies de prétraitement ont révélé de manière unique que des voies métaboliques comprenant la phosphorylation oxydative, le métabolisme des acides gras et la glycolyse ont été associées à la résistance. Les protéomiques aussi bien que les transcriptomiques ont révélé que la sensibilité a été marquée par élévation de la réparation de l'ADN, des cibles E2F, du point de contrôle G2M, de la réponse interféron-gamma et des composants de point de contrôle immunitaire. Des analyses protéogénomiques d'aberrations de nombre de copies somatiques ont identifié une délétion de 19q13.31-33 associée à une résistance dans laquelle LIG1, POLD1 et XRCC1 se situent. Dans des ensembles de données orthogonaux, LIG1 (ADN ligase I impliquée dans la synthèse de brin de retard) une délétion de gène et/ou une faible expression d'ARNm ont été associées à un manque de réponse complète pathologique et un mauvais pronostic dans le TNBC, ainsi qu'une résistance à la carboplatine sélective dans des modèles de xénogreffe dérivés de patients TNBC. Une faible expression ou une faible perte de LIG1 a également été associée à un indice d'instabilité chromosomique supérieur (CIN) et à un mauvais pronostic dans d'autres types de cancer, ce qui démontre que la délétion de composants de synthèse de brin de retard revêt d'une grande importance clinique.
PCT/US2023/063863 2022-03-07 2023-03-07 Marqueurs protéogénomiques de résistance et de réponse à une chimiothérapie contre un cancer du sein triple négatif Ceased WO2023172913A2 (fr)

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