Abstract 3614: Evaluation of a commercial targeted NGS panel for tumor mutation burden assessment in FFPE tissue
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Abstract Checkpoint inhibitors have been approved for the treatment of solid tumor and hematological malignancies. While significant responses have been observed in a subset of patients, outcomes are variable and there is a need to identify additional predictive biomarkers beyond PD-L1 levels as measured by IHC. Tumor mutation burden (TMB) has been correlated with response to checkpoint inhibitors and is emerging as a key biomarker for predicting checkpoint inhibitor response. So far, the methods used to assess tumor mutation burden have included exome sequencing and multiple laboratory-developed targeted NGS panels (e.g., FoundationOne and MSK-IMPACT). In order to fully determine the value of TMB as a predictive biomarker for immunotherapy, a standardized panel, workflow and data analysis pipeline for TMB assessment are needed. In this study we evaluated the performance of a commercially available targeted NGS panel and workflow for TMB analysis. A set of 30 FFPE tumor samples including colon, renal, gastric, endometrial, and lung tissues was analyzed. DNA and RNA were extracted using the RecoverAll Total Nucleic Acid Isolation Kit. DNA quantity and quality were assessed using Qubit and qPCR, respectively. Samples were analyzed with the ThermoFisher Oncomine™ Mutation Load Research Assay (TML), which evaluates tumor mutation load (mutations/Mb) by interrogating 409 cancer-related genes, spanning ~1.7 megabases of the genome. TMB was measured by counting somatic single-base substitutions per Mb at ≥10% allele frequency in single, non-matched, tumor DNA samples. The impact of DNA quality on the TMB score was evaluated. Deamination errors (i.e. G>A and C>T) in poor quality FFPE samples was found to cause the overestimation of TMB. Therefore, a delta Ct cutoff was established to qualify DNA samples for TMB analysis. 12 of the 30 samples were also analyzed using a comparator NGS panel covering ~1.25 megabases. The correlation of TMB results between the two panels was 0.87. Overall, TMB was lowest in RCC (9-17/Mb) compared to NSCLC and CRC (16-37/Mb). MSI status was determined using the Promega MSI Analysis System v1.2. A correlation was observed between TMB and MSI status in a subset of samples. Reproducibility of the assay was also evaluated. To identify clinically relevant mutations and genetic alteration associated with high mutation burden, the Oncomine Comprehensive assay v3 (OCAv3) was also used to analyze the sample set. Mutations in genes involved in several DNA repair pathways were found to correlate with TMB. This study demonstrated the feasibility of utilizing a commercial targeted NGS panel and data analysis pipeline for TMB evaluation in clinical FFPE tumor samples. Standardization of TMB analysis will enable the clinical validation of TMB as a predictive biomarker for therapy selection. Citation Format: Peng Fang, Zhenyu Yan, Quyen Vu, David Smith, Chad Galderisi, Cynthia S. Spittle, Jin Li. Evaluation of a commercial targeted NGS panel for tumor mutation burden assessment in FFPE tissue [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3614.Keywords:
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Processes that cause or contribute to cancer, such as aging, exposure to carcinogens, or DNA damage repair deficiency (DDRd), create predictable and traceable nucleotide alterations in one's genetic code (termed "mutational signatures"). Large studies have previously identified various such mutational signatures across cancers that can be attributed to the specific causative processes. To gain further insight into the processes in glioma development, the authors analyzed mutational signatures in adult diffuse gliomas (DGs).
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We describe a patient with both gastric adenocarcinoma and metastatic squamous cell carcinoma (SCC) of unknown primary site. The possibility of a single malignant clonal process as opposed to differing primaries was supported by the finding of both histologies exhibiting high microsatellite instability. Despite evidence of tumor microsatellite instability, the patient's disease process did not respond to immune checkpoint inhibition. Our pursuit of whole-exome sequencing and comparing the single-nucleotide variant profiles of both tumors supported a single clonal process with the development of significant intratumoral heterogeneity. High intratumoral heterogeneity has posed a challenge to precision medicine approaches, but we also provide a review of the literature of this phenomenon mediating resistance to immunotherapy strategies.
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Abstract BACKGROUND: Multiple gastric cancer (MGC) is one special type of gastric cancer with more than two different tumors at various locations of stomach. However, the clonal relationship and carcinogenesis of MGC are still remain unclear. Therefore, we investigated the clonal relationship and role of germline mutations playing in the carcinogenesis of MGC. METHODS: We collected 16 multiple gastric cancer patients who underwent subtotal and total gastrectomy at Peking Cancer Hospital form January 2016 to December 2017. Thirty-three tumor samples and sixteen normal gastric tissue or blood samples were obtained for experiment. We also conducted analysis for 208 gastric cancer and 49 esophagogastric junction cancer (GC-EGJ) tumors from TCGA. DNA extraction from our samples was conducted for whole exome sequencing. We analyzed nonsynonymous mutations and somatic copy number variations (CNVs) based on exome data. Driver mutations, germline and cancer predisposing genes, significantly mutated genes (SMGs) were also analyzed. RESULTS: Tumor mutation burden (TMB) was not statically significant within database and our data in GCs-EGJ groups (P=0.0591). And the TMB level was also similar between two databases in GCs groups (P=0.3113). The mutation spectrum and mutation signatures also showed uniform distributions in GCs and GCs-EGJ groups within our data and TCGA database. Among sixteen patients, four were identified as monoclonal, in which 11, 10, 26 and 6 somatic mutations were shared within different tumors of P7, P8, P9 and P16 respectively. However, no common mutation between different tumors of same patient was found from the other 12 patients in spite of thousands of somatic mutations in P2. After identifying predisposing genes, we found that germline MSH2 and NCOR2 mutations were significantly dominant in 8/12 and 10/12 of genetic MGCs patients. And all patients were identified MSH2 mutations in cancer samples of those same patients. Interestingly, the NCOR2 mutations were not detected significantly with non-frameshift INDELs in only three patients. Taking genetic MGCs as a whole, we identified that TP53 were significantly mutated in 14 of 25 tumor samples. And MSH2 mutations were found in all patients. The correlation analysis between clinical information and mutation signatures showed that the signature B was associated with the level of CA72.4. CONCLUSIONS: Whole exome sequencing analyses are suggestive of monoclonal and polyclonal origin of MGC, which may promote the classification MGC into genetic and metastatic MGC. For genetic MGC patients, germline MSH2 mutations may contribute to the carcinogenesis of them, may thus giving rise to the consideration of more radical surgery and PD-1/PD-L1 therapy. Citation Format: Anqiang Wang, Zhongwu Li, Xin Ji, Tao Fu, Xiaojiang Wu, Ji Zhang, Zhaode Bu, Jiafu Ji. Multiple gastric cancer and predisposing genes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1417.
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Abstract Gastric cancer contributes to a significant health care burden, being the third most common cause of cancer related deaths worldwide. In the US, gastric cancer incidence and mortality in the Hispanic population is more than double compared to Non-Hispanic whites (NHW). However, a majority of the large-scale gastric cancer genome characterization studies that have been carried so far have been largely limited to non-Hispanic (NH) populations, predominantly Non-Hispanic Whites (NHW). This “mono-ethnic” approach is likely to miss driver mutations that are unique or common in Hispanics but rare in NHWs and can lead to future genomic-driven cancer health disparities. Identification of driver mutations is important to understand the molecular basis of tumorigenesis and to develop better preventive and therapeutic strategies in minority groups like Hispanics. Therefore, the purpose of this study was to characterize gastric cancer somatic changes in the understudied Hispanic population. Somatic mutations were identified using whole exome sequencing (WES) of 36 tumor-normal pairs from Hispanic gastric cancer patients. Microsatellite stability tasting was also performed on all the samples and samples were divided into 27 microsatellite stable (MSS) and 9 microsatellite instable (MSI) cases. Interestingly, preliminary analysis of exome sequencing data delineates differences in mutational profile in our study population compared to publish studies in NH. For example, we found that the frequency of TP53, the most commonly mutated gastric cancer driver in NHs, had a significantly lower frequency in our Hispanic sample (33% in MSS tumors and 11% in MSI tumors compared to 50% in MSS and 35% in MSI tumors reported in TCGA dataset). Similar differences were also found in other frequently mutated gastric cancer genes, which suggest that the genetic pathways leading to gastric cancer in Hispanics are likely to be different to the ones involved in NHW tumors. In summary, our sequencing study of Hispanic gastric cancer cases have identified important differences with other populations, are likely to identify novel cancer drivers in this tumor type and would help bridge the gap in heath care disparities Citation Format: Rodrigo Prieto-Sanchez, Ruta Madhusudan Sahasrabudhe, Paul Lott, Mabel Bohorquez, Jhon Jairo Suarez, Gilbert Mateus, Javier Torres, Magdalena Echeverry, Luis Carvajal-Carmona. Somatic mutation profile of gastric cancer cases from the Hispanic population. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4620. doi:10.1158/1538-7445.AM2015-4620
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Background Colorectal cancer (CRC) is with approximately 1 million cases the third most common cancer worldwide. Extensive research is ongoing to decipher the underlying genetic patterns with the hope to improve early cancer diagnosis and treatment. In this direction, the recent progress in next generation sequencing technologies has revolutionized the field of cancer genomics. However, one caveat of these studies remains the large amount of genetic variations identified and their interpretation. Methodology/Principal Findings Here we present the first work on whole exome NGS of primary colon cancers. We performed 454 whole exome pyrosequencing of tumor as well as adjacent not affected normal colonic tissue from microsatellite stable (MSS) and microsatellite instable (MSI) colon cancer patients and identified more than 50,000 small nucleotide variations for each tissue. According to predictions based on MSS and MSI pathomechanisms we identified eight times more somatic non-synonymous variations in MSI cancers than in MSS and we were able to reproduce the result in four additional CRCs. Our bioinformatics filtering approach narrowed down the rate of most significant mutations to 359 for MSI and 45 for MSS CRCs with predicted altered protein functions. In both CRCs, MSI and MSS, we found somatic mutations in the intracellular kinase domain of bone morphogenetic protein receptor 1A, BMPR1A, a gene where so far germline mutations are associated with juvenile polyposis syndrome, and show that the mutations functionally impair the protein function. Conclusions/Significance We conclude that with deep sequencing of tumor exomes one may be able to predict the microsatellite status of CRC and in addition identify potentially clinically relevant mutations.
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Several studies have evaluated the relationship between tumor mutational burden (TMB) and outcomes of immune checkpoint inhibitors. In the phase II KEYNOTE-158 study of pembrolizumab monotherapy for previously treated recurrent or metastatic cancer, high TMB as assessed by the FoundationOne CDx was associated with an improved objective response rate (ORR).We retrospectively assessed the relationship between TMB and efficacy in participants with previously treated advanced solid tumors enrolled in 12 trials that evaluated pembrolizumab monotherapy, including 3 randomized trials that compared pembrolizumab with chemotherapy. TMB was assessed in formalin-fixed, paraffin-embedded pretreatment tumor samples by whole-exome sequencing. High TMB was defined as ≥175 mutations/exome. Microsatellite instability (MSI) phenotype was based on whole-exome sequencing results. Programmed death ligand 1 (PD-L1) expression was assessed by immunohistochemistry. The primary end point was ORR assessed per RECIST V.1.1 by independent central review. Other end points included progression-free survival (PFS) assessed per RECIST V.1.1 by independent central review and overall survival (OS).Of the 2234 participants in the analysis, 1772 received pembrolizumab monotherapy and 462 received chemotherapy. Among the pembrolizumab-treated participants, ORR was 31.4% (95% CI 27.1 to 36.0) in the 433 participants with TMB ≥175 mutations/exome and 9.5% (95% CI 8.0 to 11.2) in the 1339 participants with TMB <175 mutations/exome. The association of TMB with ORR was observed regardless of PD-L1 expression and not driven by specific tumor types or participants with very high TMB or high MSI. In the 3 randomized controlled trials, TMB was associated with ORR (p≤0.016), PFS (p≤0.005), and OS (p≤0.029) of pembrolizumab but not of chemotherapy (p≥0.340, p≥0.643, and p≥0.174, respectively), and pembrolizumab improved efficacy versus chemotherapy in participants with TMB ≥175 mutations/exome.TMB ≥175 mutations/exome is associated with clinically meaningful improvement in the efficacy of pembrolizumab monotherapy and improved outcomes for pembrolizumab versus chemotherapy across a wide range of previously treated advanced solid tumor types. These data suggest TMB has broad clinical utility irrespective of tumor type, PD-L1 expression, or MSI status and support its use as a predictive biomarker for pembrolizumab monotherapy in participants with previously treated advanced solid tumors.
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Microsatellite instability (MSI) testing and tumor mutational burden (TMB) are genomic biomarkers used to identify patients who are likely to benefit from immune checkpoint inhibitors. Pembrolizumab was recently approved by the Food and Drug Administration for use in TMB-high (TMB-H) tumors, regardless of histology, based on KEYNOTE-158. The primary objective of this retrospective study was real-world applicability and use of immunotherapy in TMB/MSI-high patients to lend credence to and refine this biomarker.Charts of patients with advanced solid tumors who had MSI/TMB status determined by next generation sequencing (NGS) (FoundationOne CDx) were reviewed. Demographics, diagnosis, treatment history, and overall response rate (ORR) were abstracted. Progression-free survival (PFS) was determined from Kaplan-Meier curves. PFS1 (chemotherapy PFS) and PFS2 (immunotherapy PFS) were determined for patients who received immunotherapy after progressing on chemotherapy. The median PFS2/PFS1 ratio was recorded.MSI-high or TMB-H [≥20 mutations per megabase (mut/MB)] was detected in 157 adults with a total of 27 distinct tumor histologies. Median turnaround time for NGS was 73 days. ORR for most recent chemotherapy was 34.4%. ORR for immunotherapy was 55.9%. Median PFS for patients who received chemotherapy versus immunotherapy was 6.75 months (95% confidence interval, 3.9-10.9 months) and 24.2 months (95% confidence interval, 9.6 months to not reached), respectively (P = 0.042). Median PFS2/PFS1 ratio was 4.7 in favor of immunotherapy.This real-world study reinforces the use of TMB as a predictive biomarker. Barriers exist to the timely implementation of NGS-based biomarkers and more data are needed to raise awareness about the clinical utility of TMB. Clinicians should consider treating TMB-H patients with immunotherapy regardless of their histology.
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Microsatellite instability (MSI), resulting from a defective mismatch repair system, occurs in approximately 15% of sporadic colorectal cancers (CRC). Since MSI is associated with a poor response to 5-fluorouracile based chemotherapy and is a positive predictive marker of immunotherapy, it is routine practice to evaluate the MSI status of resected tumors in CRC patients. MSIsensor is a novel computational tool for determining MSI status using Next Generation Sequencing. However, it is not widely used in the clinic and has not been independently validated in exome data from CRC. To facilitate clinical implementation of computational determination of MSI status, we compared MSIsensor to current gold standard methods for MSI testing.MSI status was determined for 130 CRC patients (UICC stage I-IV) using immunohistochemistry, PCR based microsatellite stability testing and by applying MSIsensor to exome sequenced tumors and paired germline DNA. Furthermore, we investigated correlation between MSI status, mutational load and mutational signatures.Eighteen out of 130 (13.8%) patients were microsatellite instable. We found a 100% agreement between MSIsensor and gold standard methods for MSI testing. All MSI tumors were hypermutated. In addition, two microsatellite stable (MSS) tumors were hypermutated, which was explained by a dominant POLE signature and pathogenic POLE mutations (p.Pro286Arg and p.Ser459Phe).MSIsensor is a robust tool, which can be used to determine MSI status of tumor samples from exome sequenced CRC patients.
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Immunotherapy has recently shown important clinical successes in a substantial number of oncology indications. Additionally, the tumor somatic mutation load has been shown to associate with response to these therapeutic agents, and specific mutational signatures are hypothesized to improve this association, including signatures related to pathogen insults. We sought to study in silico the validity of these observations and how they relate to each other. We first addressed the question whether somatic mutations typically involved in cancer may increase, in a statistically meaningful manner, the similarity between common pathogens and the human exome. Our study shows that common mutagenic processes like those resulting from exposure to ultraviolet light (in melanoma) or smoking (in lung cancer) increase, in the upper range of biologically plausible frequencies, the similarity between cancer exomes and pathogen DNA at a scale of 12 to 16 nucleotide sequences (corresponding to peptides of 4 – 5 amino acids). Second, we investigated whether this increased similarity is due to the specific mutation distribution of the considered mutagenic processes or whether uniformly random mutations at equal rate would trigger the same effect. Our results show that, depending on the combination of pathogen and mutagenic process, these effects need not be distinguishable. Third, we studied the impact of mutation rate and showed that increasing mutation rate generally results in an increased similarity between the cancer exome and pathogen DNA, again at a scale of 4 – 5 amino acids. Finally, we investigated whether the considered mutational processes result in amino-acid changes with functional relevance that are more likely to be immunogenic. We showed that functional tolerance to mutagenic processes across species generally suggests more resilience to mutagenic processes that are due to exposure to elements of nature than to mutagenic processes that are due to exposure to cancer-causing artificial substances. These results support the idea that recognition of pathogen sequences as well as differential functional tolerance to mutagenic processes may play an important role in the immune recognition process involved in tumor infiltration by lymphocytes.
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Abstract Background: Gastric cancer is increasing in incidence among young white populations in the US. Gastric cancer in young patients is notable for its enrichment of diffuse histology as well as female predominance and aggressive clinical course, whereas gastric cancer is generally more frequent in males. Thus, clinicopathological characteristics of diffuse-type gastric cancer differ according to patient age, but the molecular mechanisms for early-onset gastric cancer's unique clinicopathological characteristics have not been elucidated. While the incidence of intestinal-type gastric cancer is decreasing worldwide, that of diffuse-type gastric cancer remained constant. Nonetheless, relatively small numbers of diffuse-type gastric cancers have been represented in whole exome sequencing studies such as TCGA project. We therefore investigated germline and somatic mutation profiles of diffuse-type gastric cancers with regards to patient age. Methods: We conducted whole exome and targeted sequencing and SNP6.0 array analyses of resected tumor tissue and blood samples collected from young (45 years old or younger) Korean patients with diffuse-type gastric cancer, and compared the genomic data with those from older Korean patients with diffuse-type gastric cancer. Genomic data of Korean diffuse-type gastric cancers were also compared with TCGA diffuse-type gastric cancers, 70% of which were from Caucasians. Results: Among 84 young Korean patients with DGC, 7 patients (8.3%) harbored germline mutations in TP53, CDH1, ATM, RAD51D, or PALB2. Three tumors with strong mutation signatures for inherited DNA repair defects harbored germline mutations in either RAD51D or PALB2, with loss-of-heterozygosity in the tumors. The most significant somatic mutations in young patients with diffuse gastric cancer were CDH1, TP53, ARID1A, KRAS, PIK3CA, ERBB3, TGFBR1, FBXW7, RHOA, and MAP2K1. Within the Korean population and within populations of other ethnicities, mutations in CDH1 (42.2%) or TGFBR1 (7.3%) were more frequent in younger patients than in older patients (17.4% and 0.9%, respectively). In contrast, the RHOA mutation (9.2%) was less frequent in younger patients than in older patients (19.1%). CDH1 alterations, but not RHOA mutations, were associated with poor prognosis (hazard ratio, 3.4 (95% CI, 1.5-7.7)), which may be associated with the aggressive clinical course of diffuse-type gastric cancers in young patients. Conclusions: Our largest-ever genomic analysis of diffuse-type gastric cancer reveals young age-specific mutation profiles (supported by National Cancer Center Grant 1710809 and Multi-omic Research Program). Citation Format: Hark K. Kim, Soo Y. Cho. Genomic correlates of patient age in diffuse-type gastric cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3421.
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