Colorectal cancer is the third most common cancer worldwide, with 1.2 million patients diagnosed annually. In late-stage colorectal cancer, the most commonly used targeted therapies are the ...monoclonal antibodies cetuximab and panitumumab, which prevent epidermal growth factor receptor (EGFR) activation. Recent studies have identified alterations in KRAS and other genes as likely mechanisms of primary and secondary resistance to anti-EGFR antibody therapy. Despite these efforts, additional mechanisms of resistance to EGFR blockade are thought to be present in colorectal cancer and little is known about determinants of sensitivity to this therapy. To examine the effect of somatic genetic changes in colorectal cancer on response to anti-EGFR antibody therapy, here we perform complete exome sequence and copy number analyses of 129 patient-derived tumour grafts and targeted genomic analyses of 55 patient tumours, all of which were KRAS wild-type. We analysed the response of tumours to anti-EGFR antibody blockade in tumour graft models and in clinical settings and functionally linked therapeutic responses to mutational data. In addition to previously identified genes, we detected mutations in ERBB2, EGFR, FGFR1, PDGFRA, and MAP2K1 as potential mechanisms of primary resistance to this therapy. Novel alterations in the ectodomain of EGFR were identified in patients with acquired resistance to EGFR blockade. Amplifications and sequence changes in the tyrosine kinase receptor adaptor gene IRS2 were identified in tumours with increased sensitivity to anti-EGFR therapy. Therapeutic resistance to EGFR blockade could be overcome in tumour graft models through combinatorial therapies targeting actionable genes. These analyses provide a systematic approach to evaluating response to targeted therapies in human cancer, highlight new mechanisms of responsiveness to anti-EGFR therapies, and delineate new avenues for intervention in managing colorectal cancer.
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DOBA, IJS, IZUM, KILJ, KISLJ, NUK, PILJ, PNG, SAZU, SBMB, SIK, UILJ, UKNU, UL, UM, UPUK
EGF receptor (EGFR)-targeted monoclonal antibodies are effective in a subset of metastatic colorectal cancers. Inevitably, all patients develop resistance, which occurs through emergence of KRAS ...mutations in approximately 50% of the cases. We show that amplification of the MET proto-oncogene is associated with acquired resistance in tumors that do not develop KRAS mutations during anti-EGFR therapy. Amplification of the MET locus was present in circulating tumor DNA before relapse was clinically evident. Functional studies show that MET activation confers resistance to anti-EGFR therapy both in vitro and in vivo. Notably, in patient-derived colorectal cancer xenografts, MET amplification correlated with resistance to EGFR blockade, which could be overcome by MET kinase inhibitors. These results highlight the role of MET in mediating primary and secondary resistance to anti-EGFR therapies in colorectal cancer and encourage the use of MET inhibitors in patients displaying resistance as a result of MET amplification.
Neurofibromatosis 1 is a hereditary syndrome characterized by the development of numerous benign neurofibromas, a small subset of which progress to malignant peripheral nerve sheath tumors (MPNSTs). ...To better understand the genetic basis for MPNSTs, we performed genome-wide or targeted sequencing on 50 cases. Sixteen MPNSTs but none of the neurofibromas tested were found to have somatic mutations in SUZ12, implicating it as having a central role in malignant transformation.
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DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
Variability in the accuracy of somatic mutation detection may affect the discovery of alterations and the therapeutic management of cancer patients. To address this issue, we developed a somatic ...mutation discovery approach based on machine learning that outperformed existing methods in identifying experimentally validated tumor alterations (sensitivity of 97% versus 90 to 99%; positive predictive value of 98% versus 34 to 92%). Analysis of paired tumor-normal exome data from 1368 TCGA (The Cancer Genome Atlas) samples using this method revealed concordance for 74% of mutation calls but also identified likely false-positive and false-negative changes in TCGA data, including in clinically actionable genes. Determination of high-quality somatic mutation calls improved tumor mutation load-based predictions of clinical outcome for melanoma and lung cancer patients previously treated with immune checkpoint inhibitors. Integration of high-quality machine learning mutation detection in clinical next-generation sequencing (NGS) analyses increased the accuracy of test results compared to other clinical sequencing analyses. These analyses provide an approach for improved identification of tumor-specific mutations and have important implications for research and clinical management of cancer patients.
Circulating cell-free tumor DNA (ctDNA) can serve as a real-time biomarker of tumor burden and provide unique insights into the evolving molecular landscape of cancers under the selective pressure of ...immunotherapy. Tracking the landscape of genomic alterations detected in ctDNA may reveal the clonal architecture of the metastatic cascade and thus improve our understanding of the molecular wiring of therapeutic responses. While liquid biopsies may provide a rapid and accurate evaluation of tumor burden dynamics during immunotherapy, the complexity of antitumor immune responses is not fully captured through single-feature ctDNA analyses. This underscores a need for integrative studies modeling the tumor and the immune compartment to understand the kinetics of tumor clearance in association with the quality of antitumor immune responses. Clinical applications of ctDNA testing in patients treated with immune checkpoint inhibitors have shown both predictive and prognostic value through the detection of genomic biomarkers, such as tumor mutational burden and microsatellite instability, as well as allowing for real-time monitoring of circulating tumor burden and the assessment of early on-therapy responses. These efforts highlight the emerging role of liquid biopsies in selecting patients for cancer immunotherapy, monitoring therapeutic efficacy, determining the optimal duration of treatment and ultimately guiding treatment selection and sequencing. The clinical translation of liquid biopsies is propelled by the increasing number of ctDNA-directed interventional clinical trials in the immuno-oncology space, signifying a critical step towards implementation of liquid biopsies in precision immuno-oncology.
Adenoid cystic carcinomas (ACC) of the salivary glands are challenging to understand, treat, and cure. To better understand the genetic alterations underlying the pathogenesis of these tumors, we ...performed comprehensive genome analyses of 25 fresh-frozen tumors, including whole-genome sequencing and expression and pathway analyses. In addition to the well-described MYB-NFIB fusion that was found in 11 tumors (44%), we observed five different rearrangements involving the NFIB transcription factor gene in seven tumors (28%). Taken together, NFIB translocations occurred in 15 of 25 samples (60%, 95% CI, 41%-77%). In addition, mRNA expression analysis of 17 tumors revealed overexpression of NFIB in ACC tumors compared with normal tissues (P = 0.002). There was no difference in NFIB mRNA expression in tumors with NFIB fusions compared with those without. We also report somatic mutations of genes involved in the axonal guidance and Rho family signaling pathways. Finally, we confirm previously described alterations in genes related to chromatin regulation and Notch signaling. Our findings suggest a separate role for NFIB in ACC oncogenesis and highlight important signaling pathways for future functional characterization and potential therapeutic targeting.
Pancreatic ductal adenocarcinoma (PDA) is a lethal cancer with complex genomes and dense fibrotic stroma. This study was designed to identify clinically relevant somatic aberrations in pancreatic ...cancer genomes of patients with primary and metastatic disease enrolled and treated in two clinical trials.
Tumour nuclei were flow sorted prior to whole genome copy number variant (CNV) analysis. Targeted or whole exome sequencing was performed on most samples. We profiled biopsies from 68 patients enrolled in two Stand Up to Cancer (SU2C)-sponsored clinical trials. These included 38 resected chemoradiation naïve tumours (SU2C 20206-003) and metastases from 30 patients who progressed on prior therapies (SU2C 20206-001). Patient outcomes including progression-free survival (PFS) and overall survival (OS) were observed.
We defined: (a) CDKN2A homozygous deletions that included the adjacent MTAP gene, only its' 3' region, or excluded MTAP; (b) SMAD4 homozygous deletions that included ME2; (c) a pancreas-specific MYC super-enhancer region; (d) DNA repair-deficient genomes; and (e) copy number aberrations present in PDA patients with long-term (⩾ 40 months) and short-term (⩽ 12 months) survival after surgical resection.
We provide a clinically relevant framework for genomic drivers of PDA and for advancing novel treatments.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
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Background: Tumor mutational burden (TMB) is a predictive biomarker of response to immune checkpoint inhibitors across multiple cancers. In Phase 1 of the Friends of Cancer ...Research Harmonization Project, we demonstrated a robust correlation between TMB estimated using targeted next-generation sequencing (NGS) gene panels and whole exome sequencing (WES) applied to MC3-TCGA data. These findings demonstrated variability in TMB estimates across different panels. Phase 2 evaluates sustainable TMB reference standard materials for TMB alignment to assess this variability. The goal of this effort is to establish best practices for estimating TMB in order to improve consistency across panels, for the sake of optimizing clinical application and facilitating integration of datasets generated from multiple assays. Methods: Fifteen laboratories with targeted panels at different stages of development participated. We identified a set of reference standards consisting of 10 well-characterized human-derived lung and breast tumor-normal matched cell lines. WES was performed using a uniform bioinformatics pipeline agreed upon by all team members (WES-TMB). Each laboratory used their own sequencing and bioinformatics pipelines (tumor-only and tumor-normal) to estimate TMB according to genes represented in their respective panels (panel-TMB). The association between WES-TMB and each panel-TMB was investigated using regression analyses. Bias (relative to WES-TMB) and variability in TMB estimates across panels were rigorously assessed. All analyses were blinded. Results: The set of reference standards spanned a clinically meaningful TMB range (4.3 to 31.4 mut/Mb). Preliminary data from 12 laboratories shows a good correlation between panel-TMB and WES-TMB in this empirical analysis. Across panels, regression R
2
values range 0.77-0.96 with slopes ranging 0.60-1.26. Calibration analyses that seek to minimize variability of TMB estimates across panels using the established set of reference standards are ongoing, as well as investigating cancer type dependence on the relationship between panel-TMB vs. WES-TMB, which will be available at the time of presentation. Conclusions: Preliminary findings demonstrate feasibility of using sustainable reference control cell lines to standardize and align estimation of TMB across different targeted NGS assays. Future studies aim to validate reference standard material as a reliable alignment tool by using formalin-fixed paraffin-embedded human tumor samples.
Abstract
Cancer is a genetic disease resulting from the accumulation of genetic alterations which initiate and promote uncontrolled cellular growth and invasion. These genetic alterations are the ...targets of approved therapies that preferentially inhibit tumor cells while limiting damage to normal cells. Liquid biopsies of circulating tumor DNA (ctDNA) offer a considerable advantage for analyses of cancer patients as they allow for the detection of alterations in multiple metastatic lesions throughout the course of therapy. However, the detection of tumor specific genomic alterations in ctDNA has largely centered on point mutations. The identification of tumor-derived structural changes including gene fusions and amplifications has been challenging. Approaches that analyze allelic imbalance are often not sufficiently specific to accurately define such events for solid tumor malignancies that shed low levels of DNA. Since many clinical responses to targeted therapies have been those directed at gene products from amplified loci, as well as those involved in activating translocations, we developed digital genomic approaches to detect such changes in a highly specific manner. These include amplification of the MET and ERBB2 tyrosine kinases as well as translocation of the ALK and ROS1 tyrosine kinases.
Development of the METDetect assay to capture and sequence the genomic regions corresponding to the MET gene, including coding and non-coding loci as well as control regions yielded an average of 33Gb of sequence data at a coverage of 3,125-fold per analyzed base when applied to 205 plasma samples from a variety of cancer patients, including gastric, lung and others. Analytical validation analyses showed that amplification-associated rearrangements resulting from focal amplification of the MET gene were readily detected to a level of 0.10% mutant DNA molecules while these were not present in tumors or cell-free DNA without such alterations. MET amplification was also evaluated in the tissue of patients with MET amplification through either FISH or next-generation sequence approaches, and ranged from 2.2-fold to 30-fold. A concordance of >90% was achieved when MET amplification in plasma DNA was compared to MET amplification status in matched tumor tissue. No MET amplification associated rearrangements or amplified copy number results were observed in plasma samples from 19 patients whose tumors did not harbor MET amplification. Extension of these noninvasive approaches to clinically actionable 63 gene and 9 gene panels demonstrated that ERBB2 amplification, as well as ALK and ROS1 translocation were detectable with a lower limit of detection of 0.20% and 0.10% in the plasma of patients with breast and lung cancers, respectively. Taken together, these data indicate that structural alterations, including amplifications and translocations, can be readily identified using noninvasive approaches in the circulation of cancer patients.
Citation Format: Mark Sausen. Comprehensive detection of structural alterations in the circulation of cancer patients. abstract. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr CN02-02.
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