Tumor Mutation Profiles Research Articles

BRAF mutations may identify a clinically distinct subset of glioblastoma

Glioblastoma (GBM) is the most common primary malignant brain tumor in adults. Prior studies examining the mutational landscape of GBM revealed recurrent alterations in genes that regulate the same growth control pathways. To this regard, ~ 40% of GBM harbor EGFR alterations, whereas BRAF variants are rare. Existing data suggests that gain-of-function mutations in these genes are mutually exclusive. This study was designed to explore the clinical, pathological, and molecular differences between EGFR- and BRAF-mutated GBM. We reviewed retrospective clinical data from 89 GBM patients referred for molecular testing between November 2012 and December 2015. Differences in tumor mutational profile, location, histology, and survival outcomes were compared in patients with EGFR- versus BRAF-mutated tumors, and microarray data from The Cancer Genome Atlas was used to assess differential gene expression between the groups. Individuals with BRAF-mutant tumors were typically younger and survived longer relative to those with EGFR-mutant tumors, even in the absence of targeted treatments. BRAF-mutant tumors lacked distinct histomorphology but exhibited unique localization in the brain, typically arising adjacent to the lateral ventricles. Compared to EGFR- and IDH1-mutant tumors, BRAF-mutant tumors showed increased expression of genes related to a trophoblast-like phenotype, specifically HLA-G and pregnancy specific glycoproteins, that have been implicated in invasion and immune evasion. Taken together, these observations suggest a distinct clinical presentation, brain location, and gene expression profile for BRAF-mutant tumors. Pending further study, this may prove useful in the stratification and management of GBM.

FP04.02 RATIONALE-307: Updated Biomarker Analysis of Phase 3 Study of Tislelizumab Plus Chemo vs Chemo Alone For 1L Advanced Sq-NSCLC

In the RATIONALE-307 trial (NCT03594747), tislelizumab plus platinum-based chemotherapy significantly improved clinical outcomes vs chemotherapy alone in treatment-naïve advanced squamous non-small cell lung cancer (sq-NSCLC). Previously, we showed superior clinical efficacy of tislelizumab plus chemotherapy vs chemotherapy alone regardless of PD-L1 expression (J Clin Oncol 38:2020[suppl; Abstr 9554]) and blood tumor mutational burden (Ann Oncol 2020;31[4):S754–S840). Here we report the updated biomarker analysis of PD-L1 expression, tissue tumor mutational burden (tTMB) and gene expression profiling (GEP) in baseline tumor samples.

Sensitive detection of tumor mutations from blood and its application to immunotherapy prognosis

Cell-free DNA (cfDNA) is attractive for many applications, including detecting cancer, identifying the tissue of origin, and monitoring. A fundamental task underlying these applications is SNV calling from cfDNA, which is hindered by the very low tumor content. Thus sensitive and accurate detection of low-frequency mutations (<5%) remains challenging for existing SNV callers. Here we present cfSNV, a method incorporating multi-layer error suppression and hierarchical mutation calling, to address this challenge. Furthermore, by leveraging cfDNA’s comprehensive coverage of tumor clonal landscape, cfSNV can profile mutations in subclones. In both simulated and real patient data, cfSNV outperforms existing tools in sensitivity while maintaining high precision. cfSNV enhances the clinical utilities of cfDNA by improving mutation detection performance in medium-depth sequencing data, therefore making Whole-Exome Sequencing a viable option. As an example, we demonstrate that the tumor mutation profile from cfDNA WES data can provide an effective biomarker to predict immunotherapy outcomes.

Comparison of the Predictive Power of a Combination versus Individual Biomarker Testing in Non-Small Cell Lung Cancer Patients Treated with Immune Checkpoint Inhibitors.

PurposeSince tumor mutational burden (TMB) and gene expression profiling (GEP) have complementary effects, they may have improved predictive power when used in combination. Here, we investigated the ability of TMB and GEP to predict the immunotherapy response in patients with non–small cell lung cancer (NSCLC) and assessed if this combination can improve predictive power compared to that when used individually.Materials and MethodsThis retrospective cohort study included 30 patients with NSCLC who received immune checkpoint inhibitors (ICI) therapy at the Seoul National University Bundang Hospital. programmed cell death-ligand-1 (PD-L1) protein expression was assessed using immunohistochemistry, and TMB was measured by targeted deep sequencing. Gene expression was determined using NanoString nCounter analysis for the PanCancer IO360 panel, and enrichment analysis were performed.ResultsEleven patients (36.7%) showed a durable clinical benefit (DCB), whereas 19 (63.3%) showed no durable benefit (NDB). TMB and enrichment scores (ES) showed significant differences between the DCB and NDB groups (p=0.044 and p=0.017, respectively); however, no significant correlations were observed among TMB, ES, and PD-L1. ES was the best single biomarker for predicting DCB (area under the curve [AUC], 0.794), followed by TMB (AUC, 0.679) and PD-L1 (AUC, 0.622). TMB and ES showed the highest AUC (0.837) among other combinations (AUC [TMB and PD-L1], 0.777; AUC [PD-L1 and ES], 0.763) and was similar to that of all biomarkers used together (0.832).ConclusionThe combination of TMB and ES may be an effective predictive tool to identify patients with NSCLC patients who would possibly benefit from ICI therapies.

Abstract 471: Reference materials for tumor mutational burden transcriptome profiling

Abstract Immune Checkpoint Inhibitor (ICI) therapies continue to revolutionize treatment of advanced malignancies. However, identification of individuals who respond to ICI treatment remains challenging. Along with programmed death ligand 1(PD-L1 aka CD274) expression and microsatellite instability, tumor mutational burden (TMB), defined as the number of nonsynonymous mutations per megabase of genome, has emerged as a promising biomarker for differentiating responders from non-responders, especially in non-small cell lung cancer. Although promising, TMB is not a perfect biomarker for ICI efficacy with some individuals responding conversely to expectations. The imperfection of TMB as a prognostic biomarker may result from it being a proxy measurement of potential neoantigens. Indeed, all mutations that contribute to TMB will not be expressed as neoantigens and not all neoantigens will illicit immune responses. In refining ICI response prediction, tumor abundance of nonsynonymous mutations has aided in neoantigen prediction. In order to begin development of quality reference materials for neoantigen prediction, we have characterized the transcriptomes of four tumor cell lines that were previously used to formulate TMB reference materials. Sequencing libraries were prepared with the TruSeq Stranded Total RNA with Illumina Ribo-Zero Plus rRNA Depletion Kit and Invitrogen Collibri Stranded RNA Library Prep Kit from Illumina with Collibri H/M/R rRNA Depletion Kit. The resultant transcriptome libraries were sequenced on an Illumina NextSeq2000 platform and relative transcript counts were determined. Broad ranges of expression were observed among transcripts containing nonsynonymous mutations in each of the tumor cell lines tested. These datasets will serve as benchmarks for others attempting to combine TMB and tumor abundance in predicting neoantigen potential. Citation Format: Matthew G. Butler, Yves Konigshofer, Jessica Dickens, Bharathi Anekella. Reference materials for tumor mutational burden transcriptome profiling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 471.

Abstract 2734: Impact of the mutation profile and versican status on lymphocyte infiltration in early age onset colorectal cancer

Abstract Background: The incidence of colorectal cancer (CRC) in those of pre-screening age continues to rise. A deeper understanding of the differences in tumor biology in early age onset (EAO) cancers is needed. Differences in the tumor microenvironment have been understudied in this setting. We previously have identified versican (VCAN), a large matrix proteoglycan, as an important factor in lymphocyte exclusion in CRC. Here we explore the impact of the mutation profile and VCAN status on the infiltration of CD8+ and CD4+ lymphocytes in EAO CRC. Methods: Cancer tissues from 153 patients with CRC were stained via immunohistochemistry for CD4, CD8, and VCAN. CD4 and CD8 stains were quantified by number of positive-staining tumor-infiltrating lymphocytes per high powered field (TILs/HPF). VCAN stains were quantified on an intensity scale from 0-3+. 121 of these patients had tissue that was sequenced using the Qiagen Comprehensive Cancer targeted sequencing panel, and variations were called using Strelka. Patients were split by age into early-onset (EAO; age at diagnosis &amp;lt;50) and later-onset (LAO; age at diagnosis 50 or later). Results: Mutations in BRAF and APC were significantly more common in LAO cancers (p&amp;lt;0.05 for both). TP53 mutations were correlated with significantly lower CD4+ and CD8+ TILs/HPF in the LAO cohort (12.6 CD4+ TILs/HPF for TP53-wild type [WT] vs 3.5 for TP53-mutant, p&amp;lt;0.001; 10.0 CD8+ TILs/HPF for WT vs 3.2 for mutant, p&amp;lt;0.01), but trended opposite, though not statistically significant, in the EAO group (2.4 vs 5.9 CD4+ TILs/HPF; 3.0 vs 5.9 CD8+ TILs/HPF). PIK3CA mutations were correlated with an increase in CD8+ TILs in the LAO cohort (5.3 vs 11.9 CD8+ TILs/HPF; p&amp;lt;0.05) but a decrease in CD4+ TILs in the EAO cohort (4.9 vs 0.8 CD4+ TILs/HPF; p&amp;lt;0.05). TP53-WT tumors are disproportionately low in VCAN in the LAO cohort (p&amp;lt;0.01). Differences in VCAN accumulation could account for the changes in CD8+ and CD4+ T lymphocyte infiltration across the age cohorts and TP53 mutation status. Conclusions: The impacts of TP53 and PIK3CA mutations on immune infiltration in CRC differ in EAO compared to later onset cancers, indicating that the processes controlling immune infiltration vary between age cohorts. Such differences in immune infiltration may be linked to changes in microenvironmental factors such as VCAN. These data warrant further investigation into the relationship between tumor microenvironment and mutation profiles in EAO CRC. Citation Format: Katherine A. Johnson, Philip B. Emmerich, Anna L. Lippert, Cheri A. Pasch, Linda Clipson, Wei Zhang, Kristina A. Matkowskyj, Dustin A. Deming. Impact of the mutation profile and versican status on lymphocyte infiltration in early age onset colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2734.

Abstract 620: Interrogating the molecular profile of lung cancer tumors in Puerto Rican Hispanics

Abstract Background: In the United States, lung cancer accounted for approximately one-quarter of all cancer deaths; meanwhile, it was reported as the second cause of death in Puerto Rico (PR) for 2018. Worldwide, the development of new technologies such as next-generation sequencing (NGS) has allowed describing the profile of tumors and identifying mutation drivers in oncologic patients. The information on these is limited for Puerto Rican Hispanics (PRH). Methods: Using retrospective data from CARIS Life Sciences from 2015 to 2020, we were able to estimate the prevalence of somatic mutations of 119 PRH with lung cancer. In addition, we compared them with the mutational profiles reported for lung cancer from the TCGA Pan-Lung data available in the cBioPortal for Cancer Genomics. Frequencies and percentages were calculated to describe the data. Results: The results showed that the 10 most mutated genes for lung cancer tumors in PRH were TP53 (53.7%, n=67), EGFR (20.6%, n=73), KRAS (20.6%, n=73), T.M.L. (14.1%, n=64), APC (11.0, n=73), PTEN (10.3%, n=68), ARID1A (13.6%, n=44), PIK3CA (6.9%, n=73), STK11 (6.9%, n=72), and BRAF (5.5%, n=73). Additionally, only one positive mutation was found in MET (1.4%, n=72), and no mutations were identified in ALK and RET. When comparing to the top 10 tumor mutational profiles from the TCGA study, only BRAF was observed with a mutational frequency of 67.7% (n=1,444). Conclusion: Understanding the common molecular pathways that affect the PRH population is necessary to develop new therapeutic modalities. Moreover, this preliminary review of oncogenic drivers in PRH with lung cancer will inform clinicians, scientists, and the health community about actionable mutations to guide research, treatment, and health policy efforts. Citation Format: Hilmaris Centeno-Girona, Ingrid M. Montes-Rodríguez, Camila Rivera-Lynch, Marievelisse Soto-Salgado, Noridza Rivera, Marcia Cruz-Correa. Interrogating the molecular profile of lung cancer tumors in Puerto Rican Hispanics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 620.

Abstract 612: Interrogating the molecular profile of colorectal cancer tumors in Puerto Rican Hispanics

Abstract Background: Colorectal cancer (CRC) is the second and third most common diagnosed cancer in males and females, respectively, and is the second leading cause of cancer-related deaths in Puerto Rico (PR). This differs from USA, were CRC is the third most commonly diagnosed cancer and the third leading cause of cancer-related deaths for men and woman, respectively. CRC is a heterogeneous disease and the genetic characteristics of the tumors are considered to evaluate prognostic outcome and selection of targeted therapies. Currently, techniques such as next-generation sequencing (NGS) are being used for tumor profiling, in order to classify and identify driver genetic mutations in CRC patients. Even though there have been numerous studies that have use genetic profiling to classify and to identify driver genetic mutations for CRC, comprehensive genetic profiling of cancer patients in the Hispanic population of PR has not been performed. Methods: We retrospectively evaluated the mutational profile of CRC tumors from 218 Puerto Rican Hispanics (PRH) that underwent NGS testing from 2015 to 2020. The data was provided by CARIS Life Sciences that uses a 592 gene panel in order to detect mutations, indels and copy number variants (CNV). We estimated the prevalence of somatic mutations of PRH CRC tumors and compared them with the mutational profiles reported for Colorectal Adenocarcinoma from the TCGA Pan-Cancer Clinical Data available in the cBioPortal for Cancer Genomics. Descriptive statistics were performed to characterize the database. Results: For our population, 7.8% were diagnosed with CRC before 50 years of age (early-onset CRC) and 92.2% were diagnosed after 50 years of age. These analyses showed that the most commonly mutated genes for CRC tumors in PRH are APC (82%), TP53 (76%), KRAS (56%), PIK3CA (18%), SMAD4 (13%), AMER1 (10%), FBXW7 (9%), BRAF (5%), RNF43 (4%), and MUTYH (4%). Several genes are shared among the top ten most mutated genes when comparing the CRC tumor mutational profiles from PRH with that reported in the CRC tumors from TCGA study, including APC, TP53, KRAS, and PIK3CA, having mutational frequencies of 73%, 59%, 41%, and 28%, respectively. The most common CNVs for PRH CRC tumors were CDX2 (20%), CDKN1B (7%), and HNRNPA2B1 (5%) genes. These CNVs have also been reported in the CRC TCGA Pan-Cancer study with frequencies of 6.4%, 0.3% and 0.3 % for CDX2, CDKN1B, and HNRNPA2B1 genes, respectively. Conclusion: This study is the first to reports the mutational profile of CRC tumors from Puerto Rican Hispanics. CRC tumors from PRH and the TCGA study, which has only 0.8% of Hispanic/Latino patients, share mutated genes and CNV, although with different mutational frequencies. Understanding the most common carcinogenic molecular pathways that affect PRH with CRC is crucial to guide research efforts in the discovery of new therapeutic modalities. Citation Format: Ingrid M. Montes-Rodriguez, Hilmaris Centeno-Girona, Camila Rivera-Lynch, Marievelisse Soto-Salgado, Noridza Rivera, Marcia Cruz-Correa. Interrogating the molecular profile of colorectal cancer tumors in Puerto Rican Hispanics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 612.

The Impact of EBV upon the Tumor Microenvironment and Mutational Profile of Primary CNS Lymphoma in PTLD

Primary CNS Lymphoma (PCNSL) with diffuse large B-cell lymphoma (DLBCL) histology occurring after organ transplantation (EBV+ PCNSL-PTLD) is characterized by extremely poor outcome, almost universal EBV-positivity and late presentation relative to systemic PTLD (Fink, AJT 2012; Evens, AJT 2013). However, as the incidence is low and biopsy material limited, characterization of its immunobiology is minimal. Furthermore, there is no comparative data with EBV+ DLBCL occurring as systemic PTLD (EBV+ syDLBCL-PTLD), or with EBV-ve DLBCL in the non-PTLD setting occurring either as PCNSL (EBV-ve PCSNL) or systemically (EBV-ve syDLBCL).Here, we outline results of a detailed comparison of the genetic landscape and tumor microenvironment (TME) between PCSNL and systemic DLBCL (syDLBCL), stratified by EBV and PTLD status. A combination of targeted sequencing (involving NFk-B, immune response, cell cycle and epigenetic genes), CNV analysis, nanoString gene expression (for macrophage, immune checkpoint and effector molecules), and in-vitro assays was employed. 191 adult patients with DLBCL (13 EBV+ PCNSL-PTLD; 27 EBV-ve PCNSL; 11 EBV+ syDLBCL-PTLD; and 140 EBV-ve syDLBCL) were included, with 16 non-malignant lymph nodes as controls.EBV+ PCNSL-PTLD was typically viral latency III. In EBV-ve PCNSL, in broad agreement with previous studies (Gonzalez-Aguilar, CCR 2012; Chapuy, Blood 2015; Fukumura, Acta Neuropath 2016; Nakamura, Neuropath Appl Neurobiol 2016), common nonsynonymous mutations in known cancer drivers were PIM1 (76%), MYD88 (72%), CD79B (68%), TBL1XR1 (48%), KMT2D (44%), TOX (28%), PRDM 1 (24%), EP300, CREBBP1 and CIITA (all 20%). Interestingly, the mutation rate in EBV-ve PCNSL was considerably higher than in EBV+ PCNSL-PTLD, and was also higher than EBV-ve syDLBCL and EBV+ syDLBCL-PTLD.In line with previous reports, there was high occurrence of CN loss at the HLA-class I/II loci (Riemersma, Blood 2000) in EBV-ve PCNSL. However, this was relatively infrequent in EBV+ PCNSL-PTLD and EBV+ syDLBCL-PTLD, and intermediate in EBV-ve syDLBCL. To investigate the TME gene expression profile, a selection of immune effector and checkpoint genes were quantified in combined PCNSL cases and compared to syDLBCL i.e. irrespective of viral status. CD4 and CD8 levels were similar in PCNSL vs. sysDLBCL, whereas the NK cell marker CD56 was 9-fold higher in PCNSL (p<0.0001). A key feature was that the immune activation marker CD137 was 2-fold higher in syDLBCL than PCNSL (p<0.0001), indicative of reduced immune effector function in PCNSL. Consistent with this, there was a 6-fold increase in the M2 immunosuppressive tumor-associated macrophage marker CD163 in PCNSL vs. syDLBCL (p<0.0001). Furthermore, CD4 and CD8 correlated with CD163, suggesting an adaptive immune response in which the malignant B-cell up-regulates M2 macrophages to evade immune surveillance. To test this, healthy monocytes were co-incubated with a PCNSL cell-line. This resulted in the elevated expression of CD163+, PD-L1+ and PD-L2+ M2 polarized monocyte/macrophages in PCNSL relative to a syDLBCL control cell-line (all ≥7-fold, p<0.0001), confirming the differential impact of PCNSL to upregulate immunosuppressive monocyte/macrophages.Next, differences between the TME in EBV-ve PCNSL and EBV+ PCNSL-PTLD were examined. PD-1 levels were similar. However, consistent with our previous findings in EBV+/-ve Hodgkin Lymphoma, levels of CD163 (Jones, CCR 2012) and LAG3 (Gandhi, Blood 2006) genes were both 4-fold higher in EBV+ PCNSL-PTLD biopsies than EBV-ve PCNSL (both p≤0.005). Furthermore, both PD-L1 and PD-L2 were 7-fold and 3-fold higher in EBV+ PCNSL-PTLD than EBV-ve PCNSL respectively (both p<0.005). These data implicate EBV as a major determinant of the TME within the CNS.Combined, these results indicate that ‘immune privilege’ occurring in the context of lymphoma within the CNS is more accurately described as an adaptive immune response in which the malignant B-cell actively utilizes a variety of mechanisms to evade immune surveillance (Fig 1). For EBV+ PCNSL-PTLD this involves up-regulation of PD-L1+/PD-L2+ M2 monocyte/macrophages and LAG3, whereas with EBV-ve PCNSL the emphasis is on genetically mediated immune evasion including loss of HLA-I/II loci. The findings will help guide the rational design of novel immunotherapeutic strategies for EBV+ PCNSL-PTLD. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Population-Scale Identification of Clonal Hematopoiesis Derived Mutations in Solid Tumor Mutation Profiling

▪Background: Clonal hematopoiesis (CH) is the commonly observed age-related acquisition of somatic mutations in hematopoietic lineage leading to clonal expansion of mutated cells. We recently showed 25% of advanced solid tumor patients harbor CH related mutations in their blood. As tumor tissue is typically infiltrated by blood cells, we hypothesized that sequencing tumors without the utilization of matched blood in a paired analysis could erroneously attribute CH mutations to be of tumor-specific origin. In review of patient clinical records, we encountered two patients (one endometrial adenocarcinoma and one mucosal melanoma) with DNMT3A R882H mutations reported by an external tumor-only NGS assay. In each case, paired tumor/blood sequencing subsequently confirmed the hematopoietic origin. Here, we show the prevalence of CH derived mutations in the solid tumors by analyzing a cohort of patients where tumor/normal sequencing is performed using MSK-IMPACT.Methods: We analyzed deep coverage, targeted next-generation sequencing data from January 2014 - May 2017 of paired blood and tumor samples from 15,432 patients with advanced solid malignancies using the MSK-IMPACT platform. MSK-IMPACT detects point mutations and indels in coding exons of currently 468 genes with older versions targeting 341 and 410 genes. We called mutations in blood DNA and identified those associated with CH using a custom filtering schema: First, germline mutations were eliminated based on variant allele fraction (VAF) > 0.35, except for mutations reported in COSMIC on at least 10 occasions. Second, we required mutations to be supported by at least 8 reads and present at twice the variant allele fraction (VAF) or greater in blood cells compared to the tumor. Third, we retained mutations that were detected with a VAF greater than 0.02 for a selected panel of leukemia genes and 0.05 for other genes. For cases where at least one mutation exceeded these thresholds, we reduced the VAF threshold in blood to 0.01 to detect subsequent events for leukemia genes and 0.03 for non-leukemia genes.Results: We identified 4,044 (26.2%) patients with at least one CH derived mutation in their blood corresponding to 6,588 presumptive somatic, non-silent mutations in 350 genes. Out of these, we identified 963 CH mutations from 808 patients (5.2%) that could have been classified as tumor-derived mutations in the absence of paired blood sequencing given a tumor VAF > 0.02. The number of mutations per patient ranged from 1-7 with a median VAF of 0.04 and 0.17 in paired tumor and blood samples, respectively. 34.5% of these variants are present in the COSMIC database with occurrence counts greater than 5 and include well-characterized driver alterations such as DNMT3A R882H, NRAS G12D, and IDH2 R140Q, which could readily be mistaken for somatic mutations in the corresponding solid tumor.Conclusion: With the potential for admixed leukocytes in biopsy specimens, these data demonstrate that failure to employ a paired blood sample in solid tumor sequencing can lead to the misclassification of CH mutations as somatic in the tumor, with clear diagnostic, prognostic, and therapeutic implications. DisclosuresCoombs:H3 Biomedicine: Honoraria; Pharmacyclics: Honoraria. Arcila:Raindance Tecnologies: Honoraria; Archer: Honoraria; Invivoscribe: Honoraria. Levine:Roche: Research Funding; Qiagen: Equity Ownership; Celgene: Research Funding; Celgene: Research Funding; Roche: Research Funding; Qiagen: Equity Ownership.

Enhanced specificity of clinical high-sensitivity tumor mutation profiling in cell-free DNA via paired normal sequencing using MSK-ACCESS

Circulating cell-free DNA from blood plasma of cancer patients can be used to non-invasively interrogate somatic tumor alterations. Here we develop MSK-ACCESS (Memorial Sloan Kettering - Analysis of Circulating cfDNA to Examine Somatic Status), an NGS assay for detection of very low frequency somatic alterations in 129 genes. Analytical validation demonstrated 92% sensitivity in de-novo mutation calling down to 0.5% allele frequency and 99% for a priori mutation profiling. To evaluate the performance of MSK-ACCESS, we report results from 681 prospective blood samples that underwent clinical analysis to guide patient management. Somatic alterations are detected in 73% of the samples, 56% of which have clinically actionable alterations. The utilization of matched normal sequencing allows retention of somatic alterations while removing over 10,000 germline and clonal hematopoiesis variants. Our experience illustrates the importance of analyzing matched normal samples when interpreting cfDNA results and highlights the importance of cfDNA as a genomic profiling source for cancer patients.

Therapeutic Implications of Germline Testing in Patients With Advanced Cancers.

Tumor mutational profiling is increasingly performed in patients with advanced cancer. We determined the extent to which germline mutation profiling guides therapy selection in patients with advanced cancer. Patients with cancer undergoing tumor genomic profiling were prospectively consented for germline cancer predisposition gene analysis (2015-2019). In patients harboring germline likely pathogenic or pathogenic (LP/P) alterations, therapeutic actionability was classified using a precision oncology knowledge base. Patients with metastatic or recurrent cancer receiving germline genotype-directed therapy were determined. Among 11,947 patients across > 50 malignancies, 17% (n = 2,037) harbored a germline LP/P variant. By oncology knowledge base classification, 9% (n = 1042) had an LP/P variant in a gene with therapeutic implications (4% level 1; 4% level 3B; < 1% level 4). BRCA1/2 variants accounted for 42% of therapeutically actionable findings, followed by CHEK2 (13%), ATM (12%), mismatch repair genes (11%), and PALB2 (5%). When limited to the 9,079 patients with metastatic or recurrent cancer, 8% (n = 710) harbored level 1 or 3B genetic findings and 3.2% (n = 289) received germline genotype-directed therapy. Germline genotype-directed therapy was received by 61% and 18% of metastatic cancer patients with level 1 and level 3B findings, respectively, and by 54% of BRCA1/2, 75% of mismatch repair, 43% of PALB2, 35% of RAD51C/D, 24% of BRIP1, and 19% of ATM carriers. Of BRCA1/2 patients receiving a poly(ADP-ribose) polymerase inhibitor, 45% (84 of 188) had tumors other than breast or ovarian cancer, wherein the drug, at time of delivery, was delivered in an investigational setting. In a pan-cancer analysis, 8% of patients with advanced cancer harbored a germline variant with therapeutic actionability with 40% of these patients receiving germline genotype-directed treatment. Germline sequence analysis is additive to tumor sequence analysis for therapy selection and should be considered for all patients with advanced cancer.

Increased Radiosensitivity of Solid Tumors Harboring ATM and BRCA1/2 Mutations.

PurposePreclinical data indicate that response to radiotherapy (RT) depends on DNA damage repair. In this study, we investigated the role of mutations in genes related to DNA damage repair in treatment outcome after RT.Materials and MethodsPatients with solid tumor who participated in next generation sequencing panel screening using biopsied tumor tissue between October 2013 and February 2019 were reviewed and 97 patients that received RT were included in this study. Best response to RT and the cumulative local recurrence rate (LRR) were compared according to absence or presence of missense, nonsense, and frameshift mutations in ATM and/or BRCA1/2.ResultsOf the 97 patients, five patients harbored mutation only in ATM, 22 in only BRCA1/2, and six in both ATM and BRCA1/2 (ATMmtBRCAmt). Propensity score matching was performed to select the control group without mutations (ATMwtBRCAwt, n=33). In total, 90 RT-treated target lesions were evaluated in 66 patients. Highest objective response rate of 80% was observed in ATMmtBRCAmt lesions (p=0.007), which was mostly durable. Furthermore, the cumulative 1-year LRR was the lowest in ATMmtBRCAmt lesions and the highest in ATMwtBRCAwt lesions (0% vs. 47.9%, p=0.008). RT-associated toxicities were observed in 10 treatments with no significant difference among the subgroups (p=0.680).ConclusionTumors with ATM and BRCA1/2 mutations exhibited superior tumor response and local control after RT compared to tumors without these mutations. The results are hypothesis generating and suggest the need for integrating the tumor mutation profile of DNA repair genes during treatment planning.

Germline testing in community oncology patients with somatic BRCA1/2 mutations.

1523 Background: The primary purpose of tumor mutation profiling (TMP) is to molecularly characterize tumors to identify targeted treatments and improve outcomes, but it may also uncover germline mutations with implications for patients and families. In September 2016, the National Comprehensive Cancer Network (NCCN) added “BRCA1/2 mutation detected by TMP” as a criterion for germline BRCA1/2 testing. This study aims to assess rates of germline testing, with and without genetic consultations, for individuals with a somatic BRCA1/2 finding in community oncology centers. Methods: Retrospective data was abstracted from an internal database of results from four TMP laboratories. Individuals with a somatic BRCA1/2 pathogenic/likely pathogenic (P/LP) variant reported from January 1, 2017 to December 31, 2019 were included. Clinical data was obtained from electronic medical records. The project was approved by the Texas Oncology Privacy Board. Results: 221 patients had a P/LP somatic BRCA1/2 result on TMP, 138 of which were BRCA1/2 spectrum tumors (breast, ovary, pancreas, prostate). 144/221 patients (65.2%) had BRCA1/2 germline testing. 133/221 (60.2%) met NCCN guidelines for germline BRCA1/2 testing independent of their somatic results; they were statistically more likely to undergo germline testing than patients who did not otherwise meet germline BRCA1/2 testing criteria (p=2.3e-16) (Table). 70/144 (48.6%) had a germline P/LP BRCA1/2 mutation identified. At locations with genetic providers versus those without, there was a significant difference in rates of genetic consultation (p=0.02) but not in the rate of germline testing (p=0.3). This indicates patients were more likely to receive pre- and post-test counseling at clinics offering in-house genetic consultations. Conclusions: Most individuals who had a P/LP somatic BRCA1/2 mutation identified on TMP underwent germline genetic testing. As 60.2% of our cohort qualified independently for germline testing, somatic BRCA1/2results may not have been the driving force behind germline testing. However, individuals who had a non- BRCA1/2-spectrum tumor were significantly less likely to have the recommended confirmatory germline testing. Quality improvement initiatives can focus on improving rates of counseling and germline testing for patients with somatic BRCA1/2 mutations, regardless of tumor type.[Table: see text]

Association of PIK3CA, PTCH1, FGF6, and NRAS mutations with venous thromboembolism in advanced lung and gastrointestinal cancer.

e15087 Background: Venous thromboembolism (VTE) in patients with gastrointestinal (GI) and non‐small cell lung cancer (NSCLC) is common and increases morbidity and mortality. In advanced GI and NSCLC, molecular subtyping has increased use of next-generation sequencing (NGS). The association between tumor mutation profile and VTE risk remains undetermined. Methods: We conducted a retrospective cohort study of consecutive GI/NSCLC patients from 2014-2019 with NGS and follow‐up at Brown Cancer Center. The NGS platform detected substitutions, indels, copy number alterations and select rearrangements in 324 genes. Patients with thrombophilia, anticoagulation use or &gt;1 malignancy were excluded. VTE was defined as deep vein thrombosis, pulmonary embolism or visceral vein thrombosis within 6 months prior to diagnosis or any time after. For statistical analysis SAS 9.5 was used with significance at alpha=0.05. Multinomial logistic regression was performed, in which the log odds of VTE was modeled as a linear combination of the genes. Odds ratios and 95% confidence intervals for VTE were generated. Results: A total of 364 patients were reviewed; after exclusions 326 patients were included comprising Stage III/IV NSCLC (58%), metastatic colorectal (33%) and other metastatic GI cancers - gastric, duodenal, esophageal, pancreatic and cholangiocarcinoma (9%). Approximately half (53%) were males with mean age of 59.1 yrs and 76.4% current/former smokers. There was a low level of microsatellite instability (0.9%). VTE occurred in 75 patients (23%) during a mean follow‐up of 24.5 months. Only 1 patient had surgery within 90 days prior to VTE. Of 248 genes mutated in the VTE group, 9 were more prevalent compared to those without VTE (Table). Statistical analysis showed PIK3CA, PTCH1, FGF6, and NRAS mutations significantly increased odds of VTE, with PIK3CA the most prevalent of these (Table). Conclusions: Patients with PIK3CA, PTCH1, FGF6 and NRAS mutations had significantly higher VTE risk. These tumor mutations and associated pathways may provide novel insight into risk stratification, prevention and targeted treatment of VTE in cancer. [Table: see text]

Overall survival (OS) with palbociclib (PAL) + fulvestrant (FUL) in women with hormone receptor–positive (HR+), human epidermal growth factor receptor 2–negative (HER2–) advanced breast cancer (ABC): Updated analyses from PALOMA-3.

1000 Background: In PALOMA-3, a randomized, double-blind, placebo-controlled, phase 3 study, PAL+FUL significantly prolonged progression-free survival (PFS) compared with placebo (PBO) + FUL (1-sided P&lt;0.0001). The final protocol-specified OS analysis, which was conducted with a median follow-up of 44.8 months (mo), showed improved OS with PAL+FUL vs PBO+FUL (median OS, 34.9 vs 28.0 mo; hazard ratio, 0.814 [95% CI, 0.644–1.029]; 1-sided P=0.0429). Here, we report the results from an OS analysis with a longer median follow-up of 73.3 mo. Methods: A total of 521 patients (pts) with HR+/HER2– ABC who had progressed on prior endocrine therapy were randomized 2:1 to PAL (125 mg/d orally, 3/1 week schedule) + FUL (500 mg intramuscular injection) or PBO+FUL. Investigator-assessed PFS was the primary endpoint; OS was a key secondary endpoint. An ad hoc OS analysis was performed when 393 events (75% of the total population) were observed. Circulating tumor DNA (ctDNA) analysis was conducted among pts who consented for this study. Results: Improvement in OS continues to be observed with longer follow-up, with a hazard ratio of 0.806 (95% CI, 0.654–0.994; 1-sided nominal P=0.0221). The 5-year OS rate was 23.3% (95% CI, 18.7–28.2) with PAL+FUL and 16.8% (95% CI, 11.2–23.3) with PBO+FUL. Favorable OS with PAL+FUL vs PBO+FUL was observed in most subgroups except among pts who were endocrine resistant or had prior chemotherapy for ABC. No new safety signals were identified. Eighteen pts remain on study treatment, including 15 (4.3%) on PAL+FUL and 3 (1.7%) on PBO+FUL. A post-study cyclin-dependent kinase 4/6 inhibitor was received by 20 pts (7.5%) in the PAL+FUL arm and 32 pts (22.2%) in the PBO+FUL arm. ctDNA analyses of tumor mutation profiles (ie, ESR1, PIK3CA, RB1) at the end of treatment and their effect on OS will also be presented. Conclusions: The clinically meaningful improvement in OS with PAL+FUL was maintained with &gt;6 years of median follow-up in pts with HR+/HER2– ABC who had progressed on prior endocrine treatment. Pfizer (NCT01942135) Clinical trial information: NCT01942135 .[Table: see text]

Associations of mutation profiles with clinical outcomes among metastatic colorectal cancer patients at the United States-Mexico border.

e15519 Background: The incidence of CRC among Hispanics living in the USA varies according to their country of origin, supports the idea that differences in ancestry may contribute to the differences in the incidence of CRC. Moreover, Hispanics-Latino(HL) patients usually present with a more advanced stage and have a higher mortality rate compared to other ethnic cohorts. Although it is unknown, the incidence of CRC has been substantially increasing among younger Hispanics. We sought to characterize the tumor mutation profile of mCRC patients and associate with clinical outcomes among Hispanic population. Methods: We retrospectively collected the data from next generation sequencing of 49 patients with metastatic CRC (treated at TTUHSC from 2012 to 2020. We identified the most frequent alterations in our study sample and associated with the clinical outcomes. Association analyses were performed using chi square test, unpaired t-test, log rank test and the Cox proportional hazards regressions. Results: Of 49 patients with mCRC, the average age of patients at the time of diagnosis was 57 years with 98% Hispanic, and 32(65%) male. Most of the patients had stage IV disease (98%) and left side CRC (31, 67%). In our study cohort, the most commonly mutated genes identified as APC (37/11, 77.08%), TP53 (29/19, 60.42%), KRAS (23/25, 47.92%), NOTCH 12/36 (25.00%), BRCA 1&amp;2 11/37 (22.92%) and FLT1 11/37 (22.92%). None of the identified mutations were found to be associated with overall survival. However, the presence of the FLT1 mutation was associated with a reduced risk of progression to additional chemotherapy (P=0.031). Compared to the left side CRC, the BRCA mutation appeared slightly higher on the right side of the CRC (p=0.057). In compare to data from larger national and international colon cancer databases ( COSMIC and METABRICS); HL patients showed a significantly higher proportion of frequent mutations. Compared to other ethnic cohorts, HL patients were relatively younger (57 vs. 63 years) and the male to female ratio was observed to be remarkably higher as well (1.77:1 vs. 1.32:1). In our study, the combination of mutations (TP53, APC, and KRAS) was associated with worse clinical outcomes. Our study demonstrated that worse clinical outcomes were correlated with. Conclusions: Our study is the first to characterize the most common genetic alterations among HL patients with mCRC. The most frequent identified mutations including TP53, APC, KRAS, NOTCH, and BRCA were even higher in HL cohort than the national and international databases ( COSMIC and METABRICS). Our data support the motion that molecular drivers of colon cancer might be different in HL patients.

BRAF and NRAS mutation status and response to checkpoint inhibition in advanced melanoma.

9558 Background: The ability to analyze tumor mutation profiles has altered the oncology treatment landscape over the past decades. However, little is known about the effect of specific gene mutations on the response to immune checkpoint inhibitors (ICIs) in patients with advanced melanoma. Methods: All unresectable stage IIIc and IV patients with BRAF V600, NRAS mutations and BRAF and NRAS wild-type patients treated with anti-PD-1 or ipilimumab-nivolumab between 2012 and 2020 were included from the Dutch Melanoma Treatment Registry, a nationwide population-based registry. Outcomes were objective response rate (ORR), progression-free survival (PFS), and overall survival (OS). A Cox model was used to analyze the association of possible prognostic factors with PFS and OS. Results: In total 1358 first-line patients treated with anti-PD-1 and 524 treated with ipilimumab-nivolumab were included. Median follow-up was 25.6 months for anti-PD-1 treated patients and 16.3 months for ipilimumab-nivolumab treated patients. The highest ORR, in first-line, to anti-PD-1 was in patients who were BRAF and NRAS wildtype (50.2%), compared to BRAF V600 (43.8%) and NRAS mutated patients (49.8%). ORR to ipilimumab-nivolumab was highest in NRAS mutated patients (44.9%), while ORR was 39.5% for BRAF mutated patients and 40.3% for wild-type patients. Median PFS in the anti-PD-1 treatment regimen was significantly higher (p = 0.049) for double wild-type patients (16.7 months) patients than for BRAF mutated patients (9.9 months) and NRAS mutated patients (11.3 months). PFS was not significantly different (p = 0.11) in the ipilimumab-nivolumab treatment cohort, with a median PFS of 6.5 months for the wild-type group, 10.8 months for the BRAF group, and 9.1 months for the NRAS group. In the anti-PD-1 treated patients, median OS was significantly higher (p &lt; 0.001) for BRAF mutated patients (32.8 months) compared to NRAS (21.0 months) and wild-type patients (23.0 months). For ipilimumab-nivolumab treated patients, median OS was also significantly higher (p &lt; 0.001) for BRAF mutated patients (36.5 months) than for NRAS mutated patients (11.8 months) and wild-type patients (16.1 months). After adjustment for potential confounders, the presence of a BRAF mutation remained associated with lower PFS in the anti-PD-1 treatment cohort and better OS in both treatment cohorts. Higher age, higher ECOG score, elevated LDH levels, liver metastases and brain metastases were associated with worse survival. Conclusions: PFS in first-line PD-1 was significantly higher for double wild-type patients than for BRAF mutant and NRAS mutant patients. PFS in ipilimumab-nivolumab treated patients did not significantly differ between BRAF mutant, NRAS mutant and double wild-type patients. OS was significantly higher for BRAF mutant patients in both treatment strata, which is probably the result of the subsequent BRAF/MEK-inhibition treatment option in this group.

AGAPP: efficacy of first-line cisplatin, 5-fluorouracil with afatinib in inoperable gastric and gastroesophageal junction carcinomas. A Hellenic Cooperative Oncology Group study

Purpose Gastric cancer is the fifth most common neoplasm worldwide with high rates of mortality. Afatinib, a low molecular, irreversible potent inhibitor of ErbB trans-membrane receptor family, has shown promising results according to preclinical and phase I clinical trial data when combined with chemotherapy. We aimed at evaluating the safety and efficacy of the combination of cisplatin, 5FU with afatinib in molecularly unselected patients with advanced gastric cancer. Methods Patients with locally advanced or metastatic gastric/gastroesophageal junction adenocarcinoma received first line combination therapy of cisplatin, 5FU and afatinib every 21 days, followed by afatinib maintenance monotherapy. The primary endpoint was the Objective Response Rate (ORR); secondary endpoints included Overall Survival (OS), Progression Free Survival (PFS) and the safety profile. Unplanned exploratory analysis of HER2 and tumor mutational profile was performed. Results Among 55 patients (ITT population) enrolled, 19 (34.5%) achieved an objective tumor response; stable disease was observed in 16 patients (29.1%) and progressive disease in 10 patients (18.2%). The ORR in the per protocol population (PP) was 42.9%. Within a median follow-up of 56 months, the median PFS and OS in the ITT population was 5.0 and 8.7 months, respectively. Seven of the 47 HER2 informative cases carried HER2 positive tumors while TP53, BRCA2 and SMAD4 were the most frequently mutated genes. The most common toxicities were neutrophil count and white blood cell decrease occurring in 56.4% of patients, followed by anemia (50.9%), hyperglycemia (40%), and diarrhea (38.2%). Conclusions The combination of cisplatin/5FU with afatinib did not surpass the benchmarks of efficacy of the contemporary therapeutic regimens that are being applied for the treatment of patients with advanced gastric cancer. However, the observed efficacy and the improved safety profile support that our administration schedule may be further investigated to overcome toxicity problems when integrating afatinib to cytotoxic chemotherapy. Clinical trial registration NCT01743365

Intrahepatic Cholangiocarcinoma with Lymph Node Metastasis: Treatment-Related Outcomes and the Role of Tumor Genomics in Patient Selection.

Lymph node metastasis (LNM) drastically reduces survival after resection of intrahepatic cholangiocarcinoma (IHC). Optimal treatment is ill defined, and it is unclear whether tumor mutational profiling can support treatment decisions. Patients with liver-limited IHC with or without LNM treated with resection (N = 237), hepatic arterial infusion chemotherapy (HAIC; N = 196), or systemic chemotherapy alone (SYS; N = 140) at our institution between 2000 and 2018 were included. Genomic sequencing was analyzed to determine whether genetic alterations could stratify outcomes for patients with LNM. For node-negative patients, resection was associated with the longest median overall survival [OS, 59.9 months; 95% confidence interval (CI), 47.2-74.31], followed by HAIC (24.9 months; 95% CI, 20.3-29.6), and SYS (13.7 months; 95% CI, 8.9-15.9; P < 0.001). There was no difference in survival for node-positive patients treated with resection (median OS, 19.7 months; 95% CI, 12.1-27.2) or HAIC (18.1 months; 95% CI, 14.1-26.6; P = 0.560); however, survival in both groups was greater than SYS (11.2 months; 95% CI, 14.1-26.6; P = 0.024). Node-positive patients with at least one high-risk genetic alteration (TP53 mutation, KRAS mutation, CDKN2A/B deletion) had worse survival compared to wild-type patients (median OS, 12.1 months; 95% CI, 5.7-21.5; P = 0.002), regardless of treatment. Conversely, there was no difference in survival for node-positive patients with IDH1/2 mutations compared to wild-type patients. There was no difference in OS for patients with node-positive IHC treated by resection versus HAIC, and both treatments had better survival than SYS alone. The presence of high-risk genetic alterations provides valuable prognostic information that may help guide treatment.