Tumor Next-generation Sequencing Research Articles

Patients' and Oncologists' Knowledge and Expectations Regarding Tumor Multigene Next-Generation Sequencing: A Narrative Review.

Tumor multigene next-generation sequencing (NGS) is increasingly being offered to cancer patients to guide clinical management and determine eligibility for clinical trials. We undertook a review of studies examining the knowledge and attitudes of patients and oncologists regarding the primary results and potential secondary findings of such testing. A search was conducted through the MEDLINE database using the following keywords: "neoplasms" and "molecular sequencing / genome sequencing / tumor profiling / NGS / whole exome sequencing" and "patient / oncologist" and "knowledge / attitudes / satisfaction / experience / evaluation / perspective / practice / preference." Articles meeting the inclusion criteria and additional relevant articles from their references were selected. From 1,142 publications identified by the search and 9 from references, 21 publications were included in the final review. Patients generally had positive attitudes toward tumor NGS despite relatively little knowledge of test-related genetics concepts, but their expectations often exceeded the reality of low clinical utility. Patients with higher education and greater genetics knowledge had more realistic expectations and a more altruistic view of the role of NGS. Attitudes toward disclosure of secondary findings were highly variable. Oncologists had poor to moderate genomic literacy; they communicated challenges with tempering patient expectations and deciding what information to disclose. Patients considering undergoing tumor NGS should be provided with easily understandable resources explaining the procedure, goals, and probable outcomes, whenever possible based on evidence-based guidelines. Continuing medical education programs on this topic for oncology health care professionals should strive to improve their genomic literacy and instruct them on how to optimally present this information to their patients. Oncologists are increasingly offering tumor multigene testing to patients with advanced cancers to guide more "personalized" treatment and/or determine eligibility for clinical trials. However, patients often have inadequate understanding and unrealistic expectations. Oncologists must ensure that they themselves have sufficient knowledge of the benefits and limitations of testing and must provide their patients with appropriate educational resources. Prior to testing, patients should be told the likelihood of finding a mutation in their specific tumor type for which a targeted treatment or clinical trial is available. Patients also need clear information about the possibility and implications of secondary findings.

Learnings From Precision Clinical Trial Matching for Oncology Patients Who Received NGS Testing.

PURPOSETumor next-generation sequencing reports typically generate trial recommendations for patients based on their diagnosis and genomic profile. However, these require additional refinement and prescreening, which can add to physician burden. We wanted to use human prescreening efforts to efficiently refine these trial options and also elucidate the high-value parameters that have a major impact on efficient trial matching.METHODSClinical trial recommendations were generated based on diagnosis and biomarker criteria using an informatics platform and were further refined by manual prescreening. The refined results were then compared with the initial trial recommendations and the reasons for false-positive matches were evaluated.RESULTSManual prescreening significantly reduced the number of false positives from the informatics generated trial recommendations, as expected. We found that trial-specific criteria, especially recruiting status for individual trial arms, were a high value parameter and led to the largest number of automated false-positive matches.CONCLUSIONReflex clinical trial matching approaches that refine trial recommendations based on the clinical details as well as trial-specific criteria have the potential to help alleviate physician burden for selecting the most appropriate trial for their patient. Investing in publicly available resources that capture the recruiting status of a trial at the cohort or arm level would, therefore, allow us to make meaningful contributions to increase the clinical trial enrollments by eliminating false positives.

Phase II Trial of the Combination of Temsirolimus and Sorafenib in Advanced Hepatocellular Carcinoma with Tumor Mutation Profiling

Background: The mammalian target of rapamycin (mTOR) pathway is upregulated in nearly half of hepatocellular carcinoma (HCC) tumors and is associated with poor prognosis. In preclinical models of HCC, the combination of mTOR pathway inhibition with the multikinase inhibitor sorafenib improves treatment efficacy. A prior phase I study of the allosteric mTOR inhibitor temsirolimus combined with sorafenib demonstrated acceptable safety at the recommended phase II dose. Methods: We conducted a single-arm, multicenter phase II trial of the combination of temsirolimus 10 mg intravenously weekly plus sorafenib 200 mg b.i.d. The primary endpoint was time to progression (TTP) with efficacy target of median TTP of at least 6 months; secondary endpoints included overall survival (OS), objective response rate, safety, and alpha-fetoprotein (AFP) tumor marker response. Next-generation tumor sequencing was performed as an exploratory endpoint. Results: Twenty-nine patients were enrolled, including 48% with hepatitis C virus infection and 28% with hepatitis B virus; 86% had Barcelona clinic liver cancer stage C disease. Among 28 patients evaluable for efficacy, the median TTP was 3.7 (95% confidence interval [CI]: 2.2, 5.3) months, with 14% of patients achieving TTP of at least 6 months. The median OS was 8.8 (95% CI: 6.8, 14.8) months. There were no complete or partial responses; 75% of patients had stable disease as best response. AFP decline by at least 50% was associated with prolonged TTP and OS. Serious adverse events occurred in 21%; the most common treatment-related adverse events of CTCAE grade 3 or higher were hypophosphatemia (36%), thrombocytopenia (14%), and rash (11%). There were no grade 5 events attributed to sorafenib or temsirolimus. Tumor next-generation sequencing (NGS) was performed in a subgroup of 24 patients with adequate tumor samples. Tumor mTOR pathway mutations were identified in 42%. There was no association between tumor mutation profile and OS or TTP. Conclusions: The combination of temsirolimus and sorafenib demonstrated acceptable safety but did not achieve the target threshold for efficacy in this phase II study. Tumor NGS including the presence of mTOR pathway mutations was not associated with treatment response in an exploratory subgroup analysis.

Plasma biopsy by Tag-sequencing: an acceptable alternative to tumor tissue profiling in non-small-cell lung cancer.

Tag-sequencing is amodified next-generation sequencing (NGS) approach wherein targeted regions are tagged with unique molecular identifiers enabling error-free detection ofrare genomic alterations. We aimed to perform this high- fidelity sequencing to identify actionable variants from theplasma oflung cancer patients. Targeted sequencing was performed from plasma-derived cell-free nucleic acid in twenty-one advanced, treatment naïve, non-small-cell lung cancer (NSCLC) patients. Clinically significant genetic alterations were compared with matched tumor NGS profile for each patient (patient-level), and separately for each alteration (variant-level). Cross-platform validation was done for EGFR and KRAS mutations (real-time PCR) and ALK1 rearrangement (immunohistochemistry). Forty-seven alterations (26 in plasma and 21 in tumor tissue) were detected in 19/21 tested cases. Overall-concordance between thetwo assays was 94.87% (κ of0.71, 95% CI: 0.54-0.89). Patient-level and genic-concordance was 57.1% (12/21 cases) and 67.86%, respectively. Almost perfect agreement was reached for detecting actionable EGFR mutations and ALK1 rearrangement (κ of0.89 and κ of1, respectively), which was confirmed by single-gene testing. Substantial agreement between theassays makes Tag-sequencing aviable option for identifying multibiomarkers from theplasma ofadvanced NSCLC patients in special circumstances where tissue has depleted/tumor is inaccessible/high risk ofbiopsy due to existing comorbidities.

Research Related Tumour Biopsies in Early-Phase Trials with Simultaneous Molecular Characterisation – a Single Unit Experience

Early-phase cancer clinical trials are becoming increasingly accessible for patients with advanced cancer who have exhausted standard treatment options and later phase trial options. Many of these trials mandate research tissue biopsies. Research biopsies have been perceived as ethically fraught due to the perception of potential coercion of vulnerable human subjects. We performed an audit of two years of practice to assess the safety of ultrasound (US)-guided research biopsies, and to look at the yield of a simultaneous tumour next-generation sequencing (NGS) and immunohistochemistry (IHC) molecular characterisation programme. We show that in our institution, US-guided research biopsies were safe, produced adequate tumour content and in a selected subset who underwent in-house NGS sequencing, showed a high rate of actionable mutations with 30% having a Tier 1 variant. Nevertheless, these research biopsies may only provide direct benefit for a minority of patients and we conclude with a reflection on the importance of obtaining truly informed consent.

Usefulness and Applicability of Next Generation Sequencing in Neuroendocrine Neoplasms

Background: Neuroendocrine neoplasms (NENs) are rare tumors that can arise anywhere in the body and treatment options are limited due to their rarity. Knowledge of their mutational status might allow for tumor agnostic treatments, suggest a familial component or aid in enrollment in clinical trials, especially in the metastatic setting. Objective: We aimed to evaluate the clinical relevance of results from next generation sequencing (NGS) in NEN patients and determine their applicability to patient management. Patients and methods: Eligible NEN patients on an institutional, IRB approved protocol, who had NGS as standard of care and were treated in the past 24 months, were included. Tumors were categorized by location and histologic grade. We explored the actual and theoretical eligibility for tumor agnostic treatments and enrollment in clinical trials as available on clinicaltrials.gov. Results: Between August 2017 and July 2019 a total of 107 patients were eligible. Globally 102 clinical trials included patients with NEN and specific mutations. NGS detected one (1%) case of MSI high and one (1) TRK fusion positive tumor, eligible for checkpoint inhibitor and TRK inhibitor therapy respectively. Moreover, tumor NGS identified 16 (15%) cases of MEN1, 1 (1%) of RET, 2 (2%) of NF1 and 3 (2.8%) of MUTYH, 2 (2%) TSC or TSC2, BRCA in 1 (1%). These patients were appropriately referred to genetic counseling. About 51.5% of patients would in theory be eligible for an investigational treatment based on NGS and global clinical trial availability. Fifty two of 107 patients (48.5%) would not have been eligible for a clinical trial with reasons varying between no mutations (24%), sample failure (8.4%) or nonactionable mutations (15.9%). Conclusion: NGS can point to clinical trial eligibility and guide genetic counseling and should probably be considered as a standard approach in the evaluation of new metastatic NEN patients.

Intestinal metaplasia of the urinary tract harbors potentially oncogenic genetic variants.

In the urinary tract, there is an uncertain relationship between intestinal metaplasia (IM), primary adenocarcinoma, and urothelial carcinoma. Although IM is usually found adjacent to concurrent urothelial carcinoma or adenocarcinoma, small retrospective series have shown that most bladder biopsies with only IM do not subsequently develop cancer. However, IM with dysplasia does seem to be associated with a higher risk of concurrent malignancy or progressing to cancer. Since the molecular landscape of these lesions has remained largely unexplored, there are significant uncertainties about the oncogenic potential of IM in the bladder and urethra. This study investigated the presence of potentially oncogenic genetic variants in cases of IM with and without dysplasia. Twenty-three (23) cases of IM (3 urethra, 20 bladder) were sequenced using a solid tumor next-generation sequencing panel. Of these, five contained IM with high-grade dysplasia (including a case with paired IM-adenocarcinoma and another with paired IM-urothelial carcinoma) and 18 lacked dysplasia. Oncogenic genetic variants were found in all cases of IM with high-grade dysplasia and in five non-dysplastic IM cases, including mutations and copy number variants commonly seen in primary adenocarcinoma of the bladder and urothelial carcinoma. This study demonstrates that IM can harbor potentially oncogenic genetic variants, suggesting that it might representa cancer precursor or a marker of increased cancer risk in a subset of cases.

Abstract 2226: Precise stratification of immunotherapy outcomes using response-associated somatic mutations

Abstract Background: Immune checkpoint inhibitors (ICIs) have demonstrated efficacy in a variety of cancer types. However, enhanced predictors of response are needed to identify patients who are likely to respond, while sparing others from unnecessary toxicity. Predictors of response have been identified, including tumor mutation burden (TMB), microsatellite instability (MSI) and PD-L1 expression. Studies looking for somatic mutations associated with ICI response have thus far been limited to single cancer types. Here, we used a clinical-genomic database combining ICI treatment outcomes with next-generation sequencing of tumors to identify somatic mutations associated with ICI response across 12 cancer types. Methods and Results: The cohort consisted 1,525 patients with 12 cancer types, derived from the cohort published by Samstein et al. (Nature Genetics, 2019). All patients had received at least one dose of an ICI (anti-PD-1/PD-L1, anti-CTLA-4 or both) and had a next-generation sequencing panel performed on matched tumor and normal tissue to identify somatic mutations. Using multivariable Cox regression–stratified by cancer type and including age, sex, ICI type, and TMB as covariates–we identified 14 ICI-response associated genes (IRAGs) across 6 cancer types. We verified that these IRAGs were predictive rather than prognostic; they were not associated with overall survival in the non-ICI-treated TCGA cohort. In order to determine whether these IRAGs could be used in conjunction with TMB or MSI to more precisely predict ICI outcomes, we stratified the cohort by MSI and TMB status and repeated the regression analysis. MSI-H was defined as MSIsensor score ≥ 10, while TMB-H was defined as the top 20th percentile within each cancer type. When stratifying by MSI status, we found that RNF43 was independently associated with improved survival among MSI-H COADREAD patients, while 11 IRAGs were associated with worse outcomes in MSI-L tumors. When stratifying by both MSI and TMB status, we found that among TMB-L/MSI-L patients, 3 IRAGs in BLCA and 2 in LUAD were independently associated with worse outcomes. Conclusions: Our results suggest that these IRAGs could be used in combination with MSI and/or TMB status to more precisely predict ICI treatment outcomes. Validation in other ICI-treated cohorts should be pursued. Genes associated with immunotherapy response, stratified by MSI and/or TMB statusCancerGeneSubgroupPatientsMutated (%)HR (95% CI)PCOADREADRNF43MSI-H2919 (66%)0.11 (0.02-0.73)0.023BLCAELF3MSI-L20713 (6%)5.56 (2.03-15.2)<0.001BLCANOTCH1MSI-L20713 (6%)3.72 (1.82-7.61)<0.001BLCAPIK3CAMSI-L20743 (21%)1.99 (1.20-3.32)0.008BRCAKMT2DMSI-L445 (11%)5.33 (1.12-25.4)0.035BRCAPIK3CAMSI-L4414 (32%)3.04 (1.05-8.79)0.04COADREADNCOR1MSI-L815 (6%)8.66 (1.83-41.0)0.007HNSCROS1MSI-L1346 (4%)4.23 (1.35-13.2)0.003HNSCSMARCA4MSI-L1346 (4%)5.13 (1.74-15.1)0.003LUADBRAFMSI-L29322 (8%)2.1 (1.19-3.73)0.011LUADKEAP1MSI-L29364 (22%)1.53 (1.07-2.18)0.021LUADPBRM1MSI-L29316 (5%)2.8 (1.54-.511)<0.001BLCAELF3TMB-L/MSI-L16811 (7%)4.97 (1.63-15.2)0.005BLCANOTCH1TMB-L/MSI-L1686 (4%)5.21 (1.98-13.7)<0.001BLCAPIK3CATMB-L/MSI-L16827 (16%)2.15 (1.17-3.94)0.013LUADBRAFTMB-L/MSI-L23013 (6%)2.65 (1.35-5.18)0.004LUADPBRM1TMB-L/MSI-L2307 (3%)3.2 (1.45-7.08)0.004 Citation Format: Tomi Jun, Tao Qing, Robert Samstein, Carlos Cordon-Cardo, Lajos Pusztai, Kuan-Lin Huang. Precise stratification of immunotherapy outcomes using response-associated somatic mutations [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2226.

Abstract 5456: Clinical validation of NAVIFY® Mutation Profiler for solid tumor NGS variant interpretation

Abstract Introduction: Next-Generation Sequencing (NGS) is a standard of care for identifying actionable mutations in solid tumors. Data analysis and adjudication of variants is a significant barrier to NGS adoption in community settings. NAVIFY® Mutation Profiler (NMP) is a cloud-based software that provides curation, annotation, interpretation and reporting of somatic genomic variants identified by NGS. NMP is agnostic to the sample prep, target enrichment, sequencing, and secondary analysis and will accept Variant Call File (VCF) and BAM (Binary Alignment/Map) files. It provides information on genomic variants based on published literature, public annotations, medical guidelines, drug labels and results of clinical trials. The software reports a tiered classification based on modified AMP guidelines, to aid in interpretation of detected variants, inform available therapies, and create a report. To validate accuracy and reproducibility of the NMP software and curation process when used by the intended users, a usability study was performed. Methods: 15 VCF files were prepared, including 12 semi-contrived VCF files across 4 different solid tumor types (3 lung, 3 colon, 3 melanoma and 3 breast). Each file contained on average 8 variants, 4 AMP Tier I variants (strong clinical significance) and 4 non-Tier I variants including SNVs, Indels, Fusions and CNVs. In addition, each case had filtered out variants (based on a variant allele frequency (VAF) <1% or a read depth < 500) with 1 case containing ~9000 and 2 cases containing ~500 filtered out variants. There were 3 VCF files designed to create error messages (1 > 10,000 variants, 1 VAF filter value missing, 1 incorrect reference genome). The study was performed at 3 external sites with 1, 2, and 3 users (total 6 users). Users downloaded the files onto their computers and processed them according to the instructions provided. Results: Correct variant call lists 100% (486/486), variant call list after filter modification 100% (42/42). Most critical was user agreement of Tier classification (Tier I vs. non-Tier I) for a pre-defined filter set: 99.6% (484/486). One user reclassified a MET amplification (lung case) from Tier I to Tier II and one user reclassified a BRAF V600D mutation (colon case) from Tier II to Tier I. In addition, the three cases designed to produce error messages all failed NMP processing: 100% (18/18). Conclusions: NAVIFY® Mutation Profiler is a robust automated solution for variant reporting. The annotation, curation and interpretation provided is highly concordant with molecular genetics experts. With the increasing number of known clinically relevant variants, software capable of automatically identifying and accurately interpreting variants promises to decrease the manual review time necessary to account for and inform on relevant variants. Citation Format: Sidney Alexander Scudder, Anoop Grewal, Lisha Capucion, Guili Zhang, Leonid Formin, Shuba Krishna, Xi Decker, Joanie Chung. Clinical validation of NAVIFY® Mutation Profiler for solid tumor NGS variant interpretation [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5456.

Abstract 1335: Analytical evaluation of a targeted solid tumor next-generation sequencing assay

Abstract With the growing interest in personalized medicine for cancer patients, next generation sequencing (NGS) variant screening assays are becoming a powerful clinical tool for tumor classification. The AmpliSeqTM for Illumina Focus Panel is a targeted sequencing approach that interrogate 52 of the most common solid tumor genes for single nucleotide variants (SNVs), insertion/deletions (indels), and copy number variants (CNVs) in DNA and gene fusions in RNA concurrently. To evaluate the performance of the Focus assay, an analytical performance study was conducted. Using an automated workflow with high quality DNA reference samples, both intra-assay and inter-assay experiments resulted in high reproducibility (R2 >0.95) for detecting expected variants at various frequencies. Similarly, using high quality RNA reference samples, 100% of all expected fusions were detected in all experiments, demonstrating accuracy and precision of the Focus panel assay with high quality samples. To evaluate assay sensitivity, limit of detection (LOD) studies were carried out by titrating the nucleic acid input into the assay. Using a 5% variant allele frequency threshold (empirically established in the analysis pipeline), the assay detected all expected SNVs and indels across sample across sample input amounts. Using extracted DNA from a human cell line model (HL-60) with a confirmed MYC gene gain, results showed reliable MYC gain detection down to 4 copies in a diploid background. Similarly, extracted RNA from a confirmed EML4-ALK fusion positive cell line (H2228) diluted into background RNA showed 100% positive fusion calling down to 250pg or 133 copies. Finally, to understand the assay performance on clinically-relevant FFPE samples, somatic variant detection in 8 human cell lines and 68 total FFPE samples was evaluated. For FFPE DNA samples, the libraries averaged >90% on-target alignment and coverage uniformity with >3000x mean coverage across all samples. While FFPE RNA showed lower alignment for poor quality samples, the assay showed overall high positive and negative predictive value for variant and fusion calling. In summary, the AmpliSeqTM for Illumina Focus panel is an automation-compatible small panel NGS assay with robust performance to assess somatic variants in solid tumors. Citation Format: Jeff H. Tsai, Valerie Montel, Nathalie Mouttham. Analytical evaluation of a targeted solid tumor next-generation sequencing assay [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1335.

The impact of tumor NGS testing on hereditary cancer risk assessment and population management in an integrated community health care system.

1517 Background: Next-generation sequencing (NGS) for tumor molecular profiling is used in Oncology to identify ‘actionable alterations’ for clinical trials or on/ off-label therapy. Tumor NGS can also reveal potentially heritable germline mutations. The frequency of such incidental germline mutations has been estimated to be 4-15%. The 2015 ASCO Statement supports communication of medically relevant incidental germline findings from somatic mutation profiling to patients (PTS). The impact of tumor NGS testing on hereditary cancer risk assessment programs in the context of a wider population management strategy is unknown. We sought to evaluate this within our Kaiser Permanente Northern California (KPNC) population with ready access to tumor NGS and an ongoing hereditary cancer risk assessment program. Methods: Kaiser Permanente Northern California (KPNC) is part of a large, integrated health care system. NGS at KPNC is performed in collaboration with STRATA Oncology, a precision oncology partnership. All NGS results are reviewed by a multidisciplinary KPNC Genomic Oncology Committee (GOC)which also includes genetic counselors and pathologists. We examined all NGS reports between November 2017 through December 2019 to determine the types of cancers tested, number with a possible germline mutation and number referred for genetic counseling and testing (GCT). Results: 4,825 PTS with advanced cancer underwent STRATA NGS testing. A total of 207 PTS (4.3%) were identified as potential germline mutation carriers, all 207 were recommended for GCT referral. Of these, 92 (45.0%) separately met 2020 NCCN Criteria for Genetic/Familial High-Risk Assessment (2020NG/FA), prior to tumor NGS; 115 (53.6%) did not and 3 (1.4%) had insufficient information. The cancers most frequently meeting NCCN criteria were pancreatic, breast and colon. Of the 92 PTS who met 2020NG/FA, 60 (65%) underwent GCT and 34 (57%) were confirmed to have a germline mutation. Of the 115 PTS that did not meet 2020NG/FA, 47 (41%) underwent GCT and 19 (40%) were confirmed to have a germline mutation. Overall germline mutations were confirmed in 16.5% of patients who did not meet 2020NG/FA and 37% who did. Conclusions: In our community-based integrated healthcare system, systematic review of next-generation sequencing results by an expert GOC led to more robust identification of germline mutation carriers and navigated them to appropriate GCT. Ongoing work will clarify data on cascade testing. We are currently developing automated workflows for GCT.

Advancing precision medicine in clinical oncology: Whole exome paired tumor-normal DNA and RNA sequencing at a single-institution cancer center.

e14006 Background: Next generation sequencing (NGS) allows for reliable, comprehensive and cost-effective identification of clinically actionable genetic and genomic alterations. The increasing adoption of NGS in clinical oncology has increased our ability to identify germline alterations predisposing to cancer development as well as somatic changes enabling prescription of individualized cancer treatment and enhanced clinical trial participation. Here we summarize implementation of an NGS-based precision medicine initiative involving oncology patients from a single institution cancer center. Methods: IRB-approved NGS matched whole exome (WES) germline and solid tumor somatic tumor sequencing together with somatic tumor RNA sequencing (RNA-seq) were performed using germline DNA extracted from peripheral blood lymphocytes and nucleic acids for tumor DNA and RNA sequencing obtained from formalin-fixed, paraffin-embedded tumor specimens. Results of sequencing and analyses were presented to a multi-disciplinary tumor board to establish recommendations for management of germline pathogenic variation, therapeutic drug matching, clinical trials eligibility and molecularly informed patient prognosis. Results: A total of 1,005 patients completed sequencing. Germline and somatic WES exceeded 100X and 250X mean target coverage, respectively; somatic RNA-seq exceeded 200 million mean reads. Patients ranged in age from 17 to 90 years. The study cohort comprised comparable numbers of female (51%) and male (49%) patients. Ethnicities and races were broadly represented with 22% of participants identifying as Hispanic, 14% as Asian, 4% as Black, 55% as Non-Hispanic White and 5% as other. The most common solid tumor histological classification was colorectal (18%), followed by breast (16%), prostate (7%), head and neck (7%), sarcoma (7%), ovarian (5%), melanoma (4%) and lung (3%). Bioinformatic analyses and precision medicine tumor board review established that 12% of patients harbored a germline pathogenic variant and 43% carried clinically actionable genetic/genomic alterations; a majority of patients met molecular requirements for participation in a clinical trial. Conclusions: This study confirms the feasibility and utility of clinical NGS and precision medicine tumor board review in clinical oncology to identify germline genetic pathology, deliver personalized cancer therapeutics, increase clinical trial enrollment and clarify diagnosis and prognosis.

Genetic counseling referrals after next generation sequencing testing.

1515 Background: Next generation sequencing (NGS) testing of tumor tissue or blood is performed to identify ‘actionable’ mutations that might guide patient care. NGS testing might incidentally identify germline mutations associated with cancer syndromes. No distinction is made between germline and somatic alterations on NGS reports, thus confirmatory germline testing is required. In this quality improvement (QI) initiative, we evaluated the frequency of referrals to genetic counseling (GC) for patients with potentially heritable germline mutations identified through NGS testing. Methods: We generated a list of high-risk mutations (HRMs) which merit GC referral based on NCCN guidelines. NGS test results for 3,400 consecutive patients with solid tumor malignancies were reviewed by the molecular tumor board from 1/2014-9/2019 and were screened for pathogenic HRMs. Basic demographic, oncologic, and GC data were retrospectively abstracted for each patient. The outcomes of interest were the frequency of HRMs identified through NGS testing, the proportion of patients subsequently referred to GC, and the proportion of patients ultimately diagnosed with a hereditary cancer syndrome. Results: 472 individual patients (14%) had NGS testing with one or more HRM identified; 465 patients were evaluable which corresponded to 519 HRMs that were included in the analysis (Table). Malignancies included were gastrointestinal 199 (42.8%), lung 83 (17.8%), genitourinary/renal 56 (12.0%), breast 49 (10.5%), gynecologic 35 (7.5%), and other 43 (9.2%). 75 (16.1%) patients had germline testing prior to NGS testing. Of those patients without prior germline genetic testing, 62 (15.9%) were referred to GC, and 19 (4.9%) patients were diagnosed with a hereditary cancer syndrome. Conclusions: Tumor NGS testing identifies HRMs that may represent an undiagnosed heritable germline mutation. Providers ordering NGS tests should review results for HRMs, refer to GC when appropriate, and offer confirmatory germline testing for patients and their families. [Table: see text]

SUN-LB75 The Anti-Tumor Activity of the Selective Ret Inhibitor Selpercatinib (LOXO-292) in Medullary Thyroid Cancer Is Independent of the Specific RET Mutation

The RET receptor tyrosine kinase proto-oncogene is activated by somatic or germline mutations in a majority of medullary thyroid cancers (MTC). However, treatment of MTC has been challenging due to the lack of effective and tolerable RET-specific therapy, thus testing tumors for the presence of somatic RET mutation has not been warranted. In a first-in-human, phase 1/2 clinical trial (LIBRETTO-001, NCT03157128), selpercatinib (LOXO-292), an investigational, highly selective, potent small molecule RET kinase inhibitor, demonstrated significant and durable anti-tumor activity in patients with advanced RET-mutant MTC or with diverse RET fusion-positive cancers (1). Among the primary analysis set of patients with RET-mutant MTC previously treated with cabozantinib and/or vandetanib (N=55), the investigator-assessed objective response rate (ORR) per RECIST 1.1 was 56% (95% CI 42.3-69.7, n=31/55). Duration of response was not reached with a 10.6-months median follow-up (data cutoff date 17-Jun-2019). Here, we evaluated investigator-assessed ORR per RECIST 1.1 and clinical benefit rate (CBR) in this previously treated patient population by RET alteration and by germline or somatic testing used for enrollment. The ORR remained consistent across subgroups with RET M918T (49%, 95% CI 30.8-66.5, n=16/33), V804M/L gatekeeper mutations (60%, 95% CI 14.7-94.7, n=3/5), extracellular cysteine mutations (43%, 95% CI 9.9-81.6, n=3/7), other mutations (90%, 95% CI 55.5-99.7, n=9/10), and germline (50%, 95% CI 6.8-93.2, n=2/4) or somatic (57%, 95% CI 42.2-70.7, n=29/51) testing. The CBR, defined as the proportion of patients with best overall response of confirmed complete response, confirmed or unconfirmed partial response, or stable disease lasting 16 weeks or more, in this patient set was 87% (95% CI 75.5-94.7, n=48/55). The CBR remained consistent across subgroups with RET M918T (88%, 95% CI 71.8-96.6, n=29/33), V804M/L gatekeeper mutations (80%, 95% CI 28.4-99.5, n=4/5), extracellular cysteine mutations (71%, 95% CI 29.0-96.3, n=5/7), other mutations (100%, 95% CI 69.2-100.0, n=10/10), and germline (75%, 95% CI 19.4-99.4, n=3/4) or somatic (88%, 95% CI 76.1-95.6, n=45/51) testing. The primary technologies used to identify RET alterations were tumor next-generation sequencing (n=43) and polymerase chain reaction (n=9). As previously reported, selpercatinib was well tolerated with an acceptable safety profile (1). These results indicate broad anti-tumor activity for selpercatinib in patients with RET-mutant MTC irrespective of the specific RET mutation, and support implementation of RET mutation testing for patients with advanced MTC, including somatic testing, to identify patients who may benefit from selpercatinib. Reference: (1) Wirth et al., Ann Oncol. 2019 Oct; 30(supplement 5): v933.

Comparison of commonly used solid tumor targeted gene sequencing panels for estimating tumor mutation burden shows analytical and prognostic concordance within the cancer genome atlas cohort

BackgroundTumor mutation burden (TMB) is a biomarker frequently reported by clinical laboratories, which is derived by quantifying of the number of single nucleotide or indel variants (mutations) identified by next-generation...

A profile on the FoundationFocus CDxBRCA tests

ABSTRACTIntroduction: Poly(ADP-ribose) polymerase (PARP) inhibitors, including rucaparib, are the only targeted class of therapeutics approved for recurrent epithelial ovarian carcinoma with a predictive biomarker. Currently, three different PARP inhibitors are approved for either the treatment of ovarian cancer or maintenance of remission following chemotherapy. The Foundation Focus CDxBRCA is an FDA-cleared next-generation sequencing tumor tissue assay that detects somatic and sometimes germline mutations in BRCA1 and BRCA2 genes.Areas covered: The authors discuss the evolution of the ovarian cancer genomic testing landscape relative to PARP inhibition, with a focus on Foundation Focus CDxBRCA and CDxBRCA Loss of Heterozygosity (LOH), the complementary diagnostics (CDx) to rucaparib.Expert opinion: Relatively early in PARP inhibitor development, women with somatic and/or germline mutations in the BRCA1 and BRCA2 genes were found to have higher response rates to PARP inhibitors with longer durability than women who were BRCA wildtype. Other measures of homologous recombination deficiency including LOH have proven to be predictive biomarkers also. Because PARP biomarkers are genomic and complex, co-development of high-performance companion diagnostics was a high priority. The Foundation Focus test began as a next-generation sequencing assay capable of detecting germline (gBRCA) and somatic (sBRCA) mutations that predict response to rucaparib treatment. The addition of LOH to the assay was validated by a clinical trial supporting expansion of the Rucaparib FDA label to include maintenance of chemotherapy response.

Molecular profiling of early-stage colorectal cancer (CRC) using targeted next generation sequencing (NGS) to predict signatures of recurrence.

242 Background: While the 5-year recurrence rate in early stage CRC is low (12%) and there is currently limited role of adjuvant chemotherapy in such cases, a unique subset of patients (pts) will have late recurrences. To identify molecular signatures predictive of late recurrence after pts undergo intended curative resection, we employed a 22 targeted gene NGS panel in pts with early CRC. Association between mutation status and recurrence free survival (RFS) was analyzed. Methods: Pts with stage I-II CRC had their tumor prospectively sequenced between 09/2015-12/2018 by an ion torrent targeted 22 gene hotspot NGS panel, including KRAS, EGFR, BRAF, PIK3CA, AKT1, ERBB2/4, PTEN, NRAS, STK11, MAP2K1, ALK, DDR2, CTNNB1, MET, TP53, SMAD4, FBX7, NOTCH1, and FGFR1/2/3. Associations were analyzed with unadjusted p-values (p) and Benjamini & Hochberg adjusted (BHp) shown. Results: Clinical and pathologic data from 180 pts were analyzed: median age 66 (range 24-86), male (47%), stage I (41%), stage II (69%), left (54%) vs right (36%) sided primary tumors, and microsatellite stable (85%). 35 (19%) pts had adjuvant therapy (n = 21 rectal, n = 14 colon). Pathological mutations were found in 160 (89%) of pts, including TP53 (56%), KRAS (44%), PIK3CA (22%), BRAF (12%), SMAD4 (8%), MET (6%) and NRAS (3%). There was only 1 case of ERBB2 mutation. 33 pts (18%) had evidence of recurrence. 36 month RFS was 82%. Common sites of recurrence included liver (13 pts, 39%), lung (10 pts, 30%), and bone (2 pts, 6%). Alterations in MET cDNA and protein were associated with recurrence-free survival (RFS) (HR = 4.1; p =0.0026, BHp= 0.057). Interestingly, while TP53 mutations are typically associated with worse prognosis in metastatic colorectal cancers, it was not associated with RFS (HR = 0.8; p = 0.55, BHp= 0.98). There was also no association between the number of gene alterations and RFS (p = 0.45). Conclusions: These data highlight that targeted NGS tumor profiling of early stage CRC, including sequencing MET among other genes, may be utilized alongside known prognostic pathological factors to predict pts with a higher risk of recurrence and may facilitate tailored adjuvant chemotherapy to mitigate this risk.

Next generation sequencing of cell free circulating tumor DNA in blood samples of recurrent and metastatic head and neck cancer patients.

BackgroundEffective targeted therapy is lacking in head and neck cancer (HNC). The use of next generation sequencing (NGS) has been suggested as a way to potentially expand therapeutic options and improve outcomes. This study was performed in order to further characterize blood sample cell-free circulating tumor DNA (ctDNA) in advanced HNC patients, to determine its ability to identify actionable mutations, and to elucidate its potential role in patient management.MethodsRetrospective analysis of 60 patients with recurrent and metastatic (R/M) HNCs who underwent molecular profiling of blood samples utilizing Guardant360, a 70-gene ctDNA NGS platform. ctDNA sequencing data was compared to tumor NGS data, when available. Best response to therapy was assessed using RECIST measures.ResultsThe most common tumor type was oropharyngeal squamous cell carcinoma (n=21). Other cancer types included salivary gland (n=8) and thyroid (n=4). The most common mutations identified by blood analysis were TP53 (68% of patients), PIK3CA (34% of patients), NOTCH1 (20% of patients), and ARID1A (15% of patients). These findings were consistent with results from tumor sequencing data (n=30) where TP53 (48%) and PIK3CA (24%) were also the most common. Seventy-three percent (n=22) of patients had alterations identified in blood that were not present in tumor specimens. In patients with squamous cell carcinoma, 66% had an off-label option identified and 90% had a trial option identified, while 50% of patients with salivary primaries had off-label option identified and 75% had trial options identified. All patients (n=3, 100%) with thyroid primaries had off-label and clinical trial options identified. Of patients with actionable mutations, 13% (n=8) received matched targeted therapy (MTT). Three patients had stable disease (37.5%), 3 had progressive disease (37.5%), and 2 (25%) were not evaluated at the time of follow up. Of those who did not receive targeted therapy (n=21), 11 patients had stable disease (52.4%), 9 had progressive disease (42.9%), and 1 had a complete response (4.8%).ConclusionsAlterations identified by ctDNA may help inform management decisions in advanced HNC. The majority of patients had unique mutations identified on ctDNA. The role of NGS of ctDNA should be explored in future studies.

Clinical Impact of Next-generation Sequencing in Pediatric Neuro-Oncology Patients: A Single-institutional Experience.

The implementation of next-generation sequencing (NGS) in pediatric neuro-oncology may impact diagnosis, prognosis, therapeutic strategies, clinical trial enrollment, and germline risk. We retrospectively analyzed 58 neuro-oncology patients (31 boys, 27 girls, average age 7.4 years) who underwent NGS tumor profiling using a single commercially available platform on paraffin-embedded tissue obtained at diagnosis (20 low-grade gliomas, 12 high-grade gliomas, 11 embryonal tumors, four ependymal tumors, three meningeal tumors, and eight other CNS tumors) from May 2014 to December 2016. NGS results were analyzed for actionable mutations, variants of unknown significance and clinical impact. Seventy-four percent of patients (43 of 57) had actionable mutations; 26% had only variants of uncertain significance (VUS). NGS findings impacted treatment decisions in 55% of patients; 24% were given a targeted treatment based on NGS findings. Seven of eight patients with low-grade tumors treated with targeted therapy (everolimus, trametinib, or vemurafenib) experienced partial response or stable disease. All high-grade tumors had progressive disease on targeted therapy. Forty percent of patients had a revision or refinement of their diagnosis, and nine percent of patients were diagnosed with a previously unconfirmed cancer predisposition syndrome. Turnaround time between sample shipment and report generation averaged 13.4 ± 6.4 days. One sample failed due to insufficient DNA quantity. Our experience highlights the feasibility and clinical utility of NGS in the management of pediatric neuro-oncology patients. Future prospective clinical trials using NGS are needed to establish efficacy.

Abstract CN03-02: Functional genomics approaches to identify targetable dependencies in cancer cells

Abstract The next-generation sequencing of tumors is providing an increasingly detailed description of the molecular alterations that occur across the diversity of human cancers. An important and challenging next step is to convert this wealth of information into new therapeutic hypotheses to guide patient care. My laboratory integrates cancer genomics, experimental functional genomics and mechanistic studies to elucidate vulnerabilities operative in cancer cells. These efforts are part of our Cancer Dependency Map initiative which aims to assign a dependency to every cancer cell in a patient and which could be exploited to develop new therapies (https://depmap.sanger.ac.uk/). In my presentation, I will discuss recent results from my laboratory and how these are shaping our understanding of the landscape of targetable dependencies in cancer cells. Citation Format: Mathew Garnett. Functional genomics approaches to identify targetable dependencies in cancer cells [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr CN03-02. doi:10.1158/1535-7163.TARG-19-CN03-02