Next-generation Sequencing Cancer Panel Research Articles

Genomic landscape and therapeutic implications in advanced solid cancers: KOSMOS-II (KOrean Precision Medicine Networking Group Study of MOlecular profiling guided therapy based on genomic alterations in advanced Solid tumors, KCSG AL-22-09).

11161 Background: Despite advances in genomic diagnosis and therapeutics, providing precision medicine remains a challenge in the real-world practical application, especially in nations with diverse next-generation sequencing (NGS) cancer panels and a shortage of available targeted agents due to lack of approval or reimbursement. Methods: The KOSMOS-II is an ongoing prospective, nationwide, master observational study with multiple cohorts undergoing molecular-guided treatment (MGT) recommended by a central molecular tumor board based on local NGS cancer panel results for advanced cancer patients (pts) (J Clin Oncol 2023;41:16 suppl TPS 1608). Key genomic features were integrated with clinical data to construct a Clinico-Genomic Database (CGDB). To minimize inter-panel variations, all variant calling format (VCF) data on single nucleotide variants, insertions, and deletions were normalized by lifting over to the same reference genome (hg19). Re-annotation was performed using a consistent pipeline incorporating public databases. This preliminary analysis outlines the genomic landscapes and therapeutic implications in the first half of enrolled patients out of a targeted accrual of 1,000. Results: From September 2022 to December 2023, 489 pts were enrolled with a median age of 60 years (19 - 87), male 56.9%, and a median of 4 lines (1-15) of prior systemic therapy. The most common primary cancer sites were colorectum (n = 96, 19.6%), biliary tract (n = 63, 12.9%), and breast (n=40, 8.2%). CGDB was constructed for 360 pts, employing 20 different panels from 31 participating centers. The most common genomic alterations were TP53 mutation (Mut) (n=183, 50.8%), APC Mut (n=124, 34.4%), and ERBB2 amplification (AMP) (n=90, 25%). MGT was assigned to 59.5% of pts, 45.2% were allocated to investigational drugs targeting genetic alterations approved for other indications (Tier 1), and 12.6 % to clinical trials matching their genetic alterations (Tier 3). The concordance rate between pre-submitted physician choices and the MTB recommendation was 54.6% (Tier 1, 55%; Tier 2 (alternative treatments including palliative care), 46%; Tier 3, 86%). ERBB2 alterations were most frequently led to MGT; trastuzumab emtansine (n = 75, 20.8%; ERBB2 AMP n=36; ERBB2 Mut n=20; ERBB2 AMP & Mut n=17; NA n=2), or trastuzumab plus pertuzumab (n = 17, 7.5%), followed by EGFR gain to bevacizumab plus erlotinib (n = 18, 5%) and high TMB (≥20 mut/Mb) to atezolizumab (n= 46, 12.8%). Response evaluation of Tier 1 treatment was available in 158 patients, with a 16-week clinical benefit rate of 34.8%. Conclusions: The KOSMOS-II study demonstrated the feasibility of a pragmatic, nationwide precision medicine approach using diverse real-world NGS panels and favorable clinical outcomes.

Genomic profiling in advanced NSCLC in Italy: First results of NERoNE (NSCLC Emilia Romagna Next Generation Sequencing Evaluation) project.

e21077 Background: The NERoNE Project evaluates the use of Next Generation Sequencing (NGS) in molecular profiling of advanced NSCLC (aNSCLC) patients (pts) in three Oncology Units (OU) of Emilia Romagna Region aiming to clarify the impact of such extensive tool in characterizing different NSCLC subpopulations. Methods: Since January 2020 to December 2022, 970 sequential aNSCLC pts from three OU (AUSL-IRCCS of Reggio Emilia, Modena University Hospital and IRST Meldola) were profiled by NGS. Samples from Modena and Reggio Emilia were analyzed at Modena University Hospital Molecular Pathology Lab using Myriapod IL- 56G, Cancer Panel DNA, Cancer Panel RNA, Oncomine DX Target Test Assay, Oncomine Focus Assay. Samples from IRST Meldola were processed at its Molecular Diagnostic Unit by Myriapod NGS cancer panel DNA e RNA, Oncomine Focus Assay and Oncomine Comprehensive Assay v3.We considered molecular alterations with available target therapies in Italy: EGFR mutations (mut) (exon 18,19,20,21), KRAS mut, BRAF V600E mut, ALK, ROS1, RET and NTRK rearrangements, MET exon 14 skipping mut, ErbB2 mut, FGFR 1-3 mut. PDL1 expression was assessed by standard immunohistochemistry on 486 pts. Results: Among 970 pts, 501 with at least one mut (52%) were considered for analysis: 266 were female (53%), mean age was 69 ± 11 years (min-max: 33-94) and 473 pts (94%) had adenocarcinoma. Molecular features are listed in table 1. Distribution of mut among ages, categorized as < 60, 60-70 and > 70 y, was similar, EGFR activating mut were most prevalent in females; KRAS mut (G12C and non G12C) were most prevalent in males; 41 pts had co-mutations (8.2%): the most frequent co-mut were EGFR mut (27%). PDL1 expression was < 1% 129 (27%), 1-49% 211 (43,4%) and ≥50% 146 pts (30%). The majority of pts with EGFR activating mut had PDL1 < 50%. Conclusions: NERoNE evaluates data from NGS profiling of three OU mapping key actionable molecular alterations in a large population of aNSCLC also accounting for PDL1 status. This preliminary analysis will be the starting point for further evaluations in order to improve the personalized approach to aNSCLC. [Table: see text]

Microsatellite instability detection using a large next-generation sequencing cancer panel across diverse tumour types

AimMicrosatellite instability (MSI), a hallmark of DNA mismatch repair deficiency, is a key molecular biomarker with multiple clinical implications including the selection of patients for immunotherapy, identifying patients who may...

Cancer Panel Assay for Precision Oncology Clinic: Results from a 1-Year Study

Next-generation sequencing (NGS)-based cancer panel tests are actively being applied in the clinic for precision oncology. Given the importance of NGS panel tests in the palliative clinical setting, it is critical to understand success rates, factors responsible for test failures, and the incidence of clinically meaningful genetic alterations. We performed NGS cancer panel test with tumors from the stomach (n = 234), colorectum (n = 196), and rare tumors (n = 105) from 535 recurrent or metastatic cancer patients for 1 year. Sequencing was successful in 483 (95.3%) archival tumor samples to find single nucleotide variant (SNV), copy number alteration (CNA), and fusion. NGS testing was unsuccessful in 52 (9.7%) specimens due to inadequate tissue (n = 28), low tumor volume (n = 19), and poor quality of nucleic acid (n = 5). According to the Tier system, variants were classified as Tier IA, 0.8%; IIC, 10.3%; IID, 2.0%; III, 66.7% for gastric: Tier IA, 3.6%; IIC, 11.6% for colorectal: Tier IA, 1.6%; IIC, 13.5%; IID, 0.5%; III, 70.8% for melanoma, and Tier IA, 9.1%; IIC, 1.8%; IID, 1.0%; III, 66.4% for GIST. In total, 30.8% of 483 sequenced cases harbored clinically meaningful variants. In Tier IA, KRAS and ERBB2 were the most commonly altered genes. Interestingly, we identified CD274 (PD-L1) amplification, PTPN11 (SHP2) SNV, TPM3-NTRK1 fusion, and FGFR3-TACC3 fusion as a rare (<2%) alteration having therapeutic targets. In conclusion, although small biopsy samples constitute half of cases, informative NGS results were successfully reported in >90% of archival tissue samples, and 30.8% of them harbored clinically meaningful variants.

Targeted next-generation sequencing in the detection of mismatch repair deficiency in endometrial cancers

Mismatch repair deficiency represents a biomarker of immunotherapy response and a phenotypic feature of Lynch syndrome-associated endometrial cancers. Using a targeted next-generation sequencing assay, we identified molecular features of mismatch repair deficiency, specifically insertion and deletion mutations in mononucleotide repeats, and established thresholds for the number of such mutations to classify endometrial cancers as mismatch repair deficient, proficient, or indeterminate. Sequencing classification was compared to the loss of MLH1, MSH2, MSH6, or PMS2 expression by immunohistochemistry. A total of 259 endometrial cancers were classified by sequencing as mismatch repair deficient (n = 48, 19%), proficient (n = 199, 77%), or indeterminate (n = 12, 5%). Sequencing findings were concordant with loss of expression of at least one mismatch repair protein in 47 of 48 (98%) cases classified as deficient and retained expression of all four proteins in 190 of 199 (95%) cases classified as proficient. Of the 12 cases classified as indeterminate, 7 (58%) demonstrated mismatch repair protein loss. Overall, targeted next-generation sequencing exhibited a high rate of concordance with immunohistochemistry for mismatch repair deficiency; however, sequencing was indeterminate in a few cases and demonstrated a false negative rate of 5%. Although we recommend implementation of a mismatch repair deficiency algorithm for laboratories performing next-generation sequencing cancer panels, immunohistochemistry remains a cost-effective screening method for mismatch repair deficiency in endometrial cancer.

Targeted Tissue and Cell-Free Tumor DNA Sequencing of Advanced Lung Squamous-Cell Carcinoma Reveals Clinically Significant Prevalence of Actionable Alterations

BackgroundMajor guidelines do not recommend routine molecular profiling of lung squamous-cell carcinoma (LUSC) because the prevalence of actionable alterations is thought to be low. Increased utilization of next-generation sequencing (NGS), particularly with cell-free circulating tumor DNA, facilitates reevaluation of this premise. Patients and MethodsWe retrospectively evaluated the prevalence of actionable alterations in 2 distinct LUSC cohorts totaling 492 patients. A total of 410 consecutive patients with stage 3B or 4 LUSC were tested with a targeted cell-free circulating DNA NGS assay, and 82 patients with LUSC of any stage were tested with a tissue NGS cancer panel. ResultsIn the overall cohort, 467 patients (94.9%) had a diagnosis of LUSC, and 25 patients (5.1%) had mixed histology with a squamous component. A total of 10.5% of the LUSC subgroup had somatic alterations with therapeutic relevance, including in EGFR (2.8%), ALK/ROS1 (1.3%), BRAF (1.5%), and MET amplification or exon 14 skipping (5.1%). Sixteen percent of patients with mixed histology had an actionable alteration. In the LUSC subgroup, 3 evaluable patients were treated with targeted therapy for an actionable alteration; all of them experienced partial response. ConclusionIn this large, real-world LUSC cohort, we observed a clinically significant prevalence of actionable alterations. Accurate local histopathologic assessment in advanced-stage LUSC can be challenging. Further evaluation of the genomic landscape in this setting is warranted to potentially identify underappreciated treatment options.

Abstract 4651: Development of a targeted NGS panel for solid tumor actionable gene targets using multiplex PCR-based enrichment in an integrated fluidic circuit

Abstract Introduction: Next-generation sequencing (NGS) has been being rapidly adopted in clinical research to align actionable variants in tumors to targeted therapies or clinical trials. Comprehensive NGS-based tumor profiling assays are an efficient and effective method to characterize a variety of clinically relevant somatic mutations. We have developed a targeted NGS cancer gene panel that focuses on actionable gene mutations and employs a NGS library preparation method based on multiplex PCR enrichment in an integrated fluidic circuit (IFC) and the automated JunoTM System. Methods: 53 actionable cancer genes were selected based on both clinical and research knowledge that covers 15 solid tumor types. Assays for the selected regions of the 53 genes were designed by an internal assay design pipeline. Multiplex PCR using target-specific primers is conducted in an LP—48.48 IFC on the Fluidigm Juno™ system, where up to 48 DNA samples can be processed simultaneously. A unique barcode is incorporated in one of the PCR primers to distinguish individual samples. To evaluate the assay performance, three sets of cell line gDNA samples were identified from commercial sources: set 1: 22 commercial reference samples, set 2: 12 samples for single nucleotide variants (SNVs) and indels, and set 3: 12 samples for copy number variations (CNVs). Each sample was tested on the IFC in 4 replicates. PCR products harvested from the IFC were pooled and purified with Agencourt® AMPure® XP magnetic beads. A second PCR step using a universal primer pair was performed to add sequencing adapters. After a second purification, the DNA library was sequenced on a NextSeq™ 500 system. The data analysis was performed by a service provider partner (GenomOncology). Results: A total of 1,508 primer pairs with an average insert size of 155 bp were selected to cover SNVs, indels, and CNVs. The assays were separated into 44 pools to minimize the interaction between assay primers and improve performance. The percentage of reads mapped to the genome was 98.9%, and the percentage of reads that mapped to amplicons was 96.8%. Mutation detection sensitivity was 4% variant allele frequency (VAF.) In 47 selected samples, the total SNVs, CNVs and indels represented are 182, 154 and 28, respectively. For SNVs, the positive percent agreement (PPA) is 1.0 and positive predictive value (PPV) is 0.974. For CNVs, the PPA is 1.0 and PPV is 0.969 and for indels, the PPA and PPV are 1.0 and 0.966, respectively. Overall concordance is 0.99. Conclusions: A targeted NGS cancer panel employing PCR-based enrichment on an IFC has been developed and yielded high quality of libraries generated on the JunoTM system for detecting SNVs, indels and CNV in solid tumor samples. This panel covers actionable targets in 53 cancer genes and utilizes a microfluidic device to provide a simple streamlined workflow for library preparation of up to 48 DNA samples per IFC. Citation Format: Peilin Chen, Jaibiao Gong, David Wang, Devin Do, Lianne McLean, Tom Goralski. Development of a targeted NGS panel for solid tumor actionable gene targets using multiplex PCR-based enrichment in an integrated fluidic circuit [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 4651.

Comparison of the Genetic Alterations between Primary Colorectal Cancers and Their Corresponding Patient-Derived Xenograft Tissues

Patient-derived xenograft (PDX) models are useful tools for tumor biology research and testing the efficacy of candidate anticancer drugs targeting the druggable mutations identified in tumor tissue. However, it is still unknown how much of the genetic alterations identified in primary tumors are consistently detected in tumor tissues in the PDX model. In this study, we analyzed the genetic alterations of three primary colorectal cancers (CRCs) and matched xenograft tissues in PDX models using a next-generation sequencing cancer panel. Of the 17 somatic mutations identified from the three CRCs, 14 (82.4%) were consistently identified in both primary and xenograft tumors. The other three mutations identified in the primary tumor were not detected in the xenograft tumor tissue. There was no newly identified mutation in the xenograft tumor tissues. In addition to the somatic mutations, the copy number alteration profiles were also largely consistent between the primary tumor and xenograft tissue. All of these data suggest that the PDX tumor model preserves the majority of the key mutations detected in the primary tumor site. This study provides evidence that the PDX model is useful for testing targeted therapies in the clinical field and research on precision medicine.

Identification and characterization of a novel adenomatous polyposis coli mutation in adult pancreatoblastoma.

During next generation sequencing (NGS) analysis, many missense mutations were found in a well-known oncogene, many of which were variant of uncertain significance mutations. We recently treated an adult patient with pancreatoblastoma by chemotherapy. Using an NGS cancer panel, we found a previously unreported missense mutation in the 1835 codon of the adenomatous polyposis coli (APC) gene. We also found a heterogeneous mutation in the 1835 codon of the APC gene in the patient's germline by Sanger sequencing. Although this patient did not have a history of familial adenomatous polyposis, functional analysis suggested the R1835G mutant APC showed attenuated repression of Wnt/β-catenin signaling activity. This is the first report showing a novel APC missense mutation involved in the onset of adult pancreatoblastoma.

Abstract 3585: Determination of gene amplifications with a next-generation sequencing cancer panel

Abstract TruSight® Tumor 15* (TST15) uses next-generation sequencing technology to detect somatic point mutations, insertions, and deletions in 15 genes that are commonly mutated in solid tumors. It accurately detects low-frequency mutations with low DNA input and is optimized for formalin-fixed, paraffin-embedded (FFPE) tumor tissue. The test is a multiplexed PCR panel that includes primers to support amplification detection in the EGFR, ERBB2, and MET genes. Here we describe development of a bioinformatics analysis method to enable detection of these gene amplifications. DNA extracted from 31 FFPE samples with amplifications in EGFR, ERBB2, or MET—some with dual amplifications—were diluted with DNA extracted from 18 normal FFPE samples to create a total of 67 unique samples at low-level amplification. Undiluted and diluted samples were assessed for quality based on amplifiability (ΔCq or library yield) and tested with TST15. For comparison, the samples were also tested with droplet digital PCR (ddPCR) to determine the copy ratio of target to wild type. For amplifiable samples, the TST15 algorithm was able to detect amplifications in EGFR and ERBB2 at ddPCR ratios of 1.4 and 1.6, respectively. ddPCR was unable to accurately quantify low MET amplifications; therefore, expected ddPCR ratios were used for comparison. The TST15 algorithm was able to detect amplifications in MET samples at an expected ddPCR ratio of 1.3. Poor sample quality (i.e., high ΔCq or low library yield) impaired amplification detection. In some samples, the presence of multiple amplifications also impaired detection. The addition of this algorithm to the TST15 workflow will allow researchers to more accurately characterize tumor samples by detecting somatic mutations and amplifications simultaneously. *For Research Use Only. Not for use in diagnostic procedures. Citation Format: Ina L. Deras, Aaron Wise, Chen Zhao, Christine Glidewell-Kenney, Phillip Le, Elizabeth Upsall Aderhold, Karen Gutekunst. Determination of gene amplifications with a next-generation sequencing cancer panel [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3585. doi:10.1158/1538-7445.AM2017-3585

Good Laboratory Standards for Clinical Next-Generation Sequencing Cancer Panel Tests.

Next-generation sequencing (NGS) has recently emerged as an essential component of personalized cancer medicine due to its high throughput and low per-base cost. However, no sufficient guidelines for implementing NGS as a clinical molecular pathology test are established in Korea. To ensure clinical grade quality without inhibiting adoption of NGS, a taskforce team assembled by the Korean Society of Pathologists developed laboratory guidelines for NGS cancer panel testing procedures and requirements for clinical implementation of NGS. This consensus standard proposal consists of two parts: laboratory guidelines and requirements for clinical NGS laboratories. The laboratory guidelines part addressed several important issues across multistep NGS cancer panel tests including choice of gene panel and platform, sample handling, nucleic acid management, sample identity tracking, library preparation, sequencing, analysis and reporting. Requirements for clinical NGS tests were summarized in terms of documentation, validation, quality management, and other required written policies. Together with appropriate pathologist training and international laboratory standards, these laboratory standards would help molecular pathology laboratories to successfully implement NGS cancer panel tests in clinic. In this way, the oncology community would be able to help patients to benefit more from personalized cancer medicine.

Comprehensive evaluation and validation of targeted next-generation sequencing performance in two clinical laboratories.

Next-generation sequencing (NGS) has led to breakthroughs for genetic and genomic analyses and personalized medicine approaches for many diseases. More and more clinical laboratories are using NGS as a genetic screening tool for providing mutation information that is used to select the best treatment regimens for cancer patients. However, several obstacles prevent the routine implementation of NGS technology into the clinical molecular diagnosis setting: the sophisticated sample preparation process, high cost, time-consuming data analyses, as well as the reproducibility and accuracy of interpretation. To systematically evaluate the performance and quality of targeted NGS cancer panel analyses in clinical laboratories, we performed three different tests: i) laboratory-to-laboratory accuracy test, ii) intra-laboratory precision validation, and iii) limit of detection test, using formalin-fixed, paraffin-embedded cancer tissue specimens, cell lines and mutation positive DNA. A laboratory-to-laboratory accuracy test performed using 51 samples showed 100% sensitivity and 99.97% specificity. For the intra-laboratory precision test, 100% reproducibility was observed. For the limit of detection test, KRAS mutations from samples diluted from 70 to 2% of mutant allele frequencies were detected correctly. We believe that the present study demonstrated the feasibility of clinical implementation of a targeted NGS cancer panel analysis for personalized medicine.

Next-generation sequencing (NGS) in an advanced hepatocellular carcinoma (HCC) cohort: Analyses of TP53 and CTNNB1.

286 Background: Mutations in TP53 and CTNNB1 are common in early stage HCC resection samples. The frequency and prognostic impact of these mutations in advanced HCC is not known. We conducted this retrospective analysis using a large NGS panel to explore for association between tumor genetics, clinicopathologic features, and prognosis in an advanced HCC cohort. Methods: Eligible cases had diagnosis of unresectable HCC or mixed HCC-cholangiocarcinoma and were enrolled on NCT01008917 or NCT01687673 clinical trials of sorafenib plus temsirolimus with informed consent for specimen banking for future research including genetic testing. Paired tumor and germline (blood) DNA samples were sequenced using a capture-based NGS cancer panel to allow for determination of somatic variants. Analysis was based on the human reference sequence UCSC build hg19. Variants were called using GATK Unified Genotyper software. Somatic, non-synonymous, and exonic calls were curated using COSMIC, cBioPortal, and Pubmed. Results: Cases with HCC (n = 21) and mixed HCC-cholangiocarcinoma (n = 2) comprised the cohort (N = 23). Male/female: 83%/17%. Race: White 56%, Asian 39%. BCLC stage: B 35%, C 65%. Etiology: HBsAg+ 26%, HCV+ 39%. Immune infiltrates ( ≥ 1 on scale 0-3) were present in 7/12 (58%) evaluable tumor samples. TP53 mutations were present in 14/23 (61%, 95% CI: 38.5, 80.0). CTNNB1 mutations were present in 7/23 (30%, 95% CI: 13.2, 52.9). There was no significant difference between HBsAg+ and HCV+. Both TP53 and CTNNB1 mutation were present in 4/23 (17%). CTNNB1 mutation was present in 2/7 (29%) cases with immune infiltrate score ≥ 1, and 1/5 (20%) with score &lt; 1 (not significant). Other mutations and variants will be reported. Conclusions: NGS in this advanced HCC cohort suggests a higher incidence of TP53 and coexisting TP53 plus CTNNB1 mutations than has been reported in early stage HCC which requires confirmation in a larger cohort. There was no clear relationship between these mutations, HCC etiology, or tumor immune infiltrates though interpretation is limited by small sample sizes. Analyses are ongoing to explore for association between TP53 and CTNNB1 mutations and prognosis in this advanced HCC cohort.

Validation of a Next-Generation Sequencing Cancer Panel for Clinical Mutation Profiling in Patients with Diffuse Large B-Cell Lymphoma (DLBCL)

Introduction: A previously developed NGS-based assay (ENGAUGE™-Cancer-DLBCL), which consists of the genes BRAF, BTK, CARD11, CD79A, CD79B, EZH2, KRAS, MYD88, NOTCH1, PIM1, SYK, TNFAIP3 and TP53 was analytically validated for clinical use in patients with Diffuse large B-cell lymphoma. We analyzed the performance of this assay with respect to the reproducibility, accuracy, precision, linearity, as well as limits of detection and quantification in a clinical reference laboratory environment in tumor sections taken from formalin-fixed paraffin-embedded (FFPE) tissue blocks.Methods: Illumina TrueSeq® Custom Amplicon on MiSeq was used for this study. The basic design of the experiments comprised the evaluation of 26 samples in replicate starting from genomic DNA isolated from a pooled sample in which each replicate underwent independent library preparation and was analyzed in a multiplexed run in the same flow cell. We repeated the analysis of the same 26 samples three times on different days (run-to-run reproducibility). All reactions were performed in triplicate with both positive and negative controls, and detection thresholds for each gene were chosen by receiver-operator curve analysis. The sequence data analysis was performed with NextGene, Variant Studio and Diagnovus’s proprietary bioinformatics analysis software. Unsupervised clustering and component analysis were performed to examine the variance and correlation of gene profiles amongst patients and sections within FFPE blocks. Sensitivity and Specificity were also calculated.Results: In all the sequencing runs performed in the validation studies, the average library coverage from clinical FFPE samples was 1847. The allele frequency for 22 out of 23 (96%) previously identified mutations was concordant with those observed by Sanger sequencing. With respect to limits of detection, the NGS assay can detect mutations present at 5-6% allele frequency with at least 95% confidence. Extraction of total DNA yielded high-quality DNA ranging from 250 to 500 ng per sample. Unsupervised clustering and principal component analysis revealed a strong correlation between blocks from the same patient, as well as between tumor samples from the same block. Using only 250ng total dsDNA input, the ENGAUGE™-Cancer-DLBCL NGS assay is capable of detecting all mutations including single base substitutions and small indels, with 5% LOD, ≥95% intra‐run reproducibility (precision), 100% inter‐run reproducibility, and with ≥95% analytical sensitivity and specificity. 17 critical variants with frequencies ranging from 5% to 100% (10 SBS, 4 deletions, and 3 deletions with insertions) were detected in 26 tumor FFPE samples. The most frequently observed mutations were NOTCH1 (27%), CARD11 (19%) and TP53 (19%).Conclusions: Extensive reagent and analytical performance studies were conducted to evaluate the reliability and reproducibility of a gene test panel (ENGAUGE™-Cancer-DLBCL). Our validation study demonstrates that this Illumina TrueSeq® Amplicon panel combined with Diagnovus’s proprietary bioinformatics analysis software provides a highly sensitive and sensitive test with a reportable analytical range of 5% to 100% mutation allele frequency.This assay is suitable for screening diffuse large B-cell patient FFPE tumor specimens for a spectrum of clinically relevant somatic mutations. DisclosuresNo relevant conflicts of interest to declare.

Validation of a next-generation-sequencing cancer panel for use in the clinical laboratory.

Next-generation sequencing allows for high-throughput processing and sensitive variant detection in multiple genes from small samples. For many diseases, including cancer, a comprehensive mutational profile of a targeted list of genes can be used to simultaneously inform patient care, establish eligibility for ongoing clinical trials, and further research. To validate a pan-cancer, next-generation-sequencing assay for use in the clinical laboratory. DNA was extracted from 68 clinical specimens (formalin-fixed, paraffin-embedded; fine-needle aspirates; peripheral blood; or bone marrow) and 5 normal controls. Sixty-four DNA samples (94%; 64 of 68) were successfully processed with the TruSeq Amplicon Cancer Panel (Illumina Inc, San Diego, California) and sequenced in 4 sequencing runs. The data were analyzed at 4 different filter settings for sequencing coverage and variant frequency cutoff. Libraries created from 40 specimens could be successfully sequenced in a single run and still yield sufficient coverage for robust data analysis of individual samples. Sensitivity for mutation detection down to 5% was demonstrated using dilutions of clinical specimens and control samples. The test was highly repeatable and reproducible and showed 100% concordance with clinically validated Sanger sequencing results. Comparison to an alternate next-generation sequencing technology was performed by also processing 9 of the specimens with the AmpliSeq Cancer Hotspot Panel (version 2; Life Technologies, Grand Island, New York). Thirty of the 31 (97%) TruSeq-detected variants covered by the designs of both panels were confirmed. A sensitive, high-throughput, pan-cancer mutation panel for sequencing of cancer hot-spot mutations in 42 genes was validated for routine use in clinical testing.

Validation of next-generation sequencing cancer panels for clinical somatic mutation profilin: Identification of source of variations and artifacts using FFPE tissues.

e22180 Background: Use of a next-generation sequencing (NGS)-based test as a clinical trial assay requires that the test be analytically validated to CLIA/CAP regulations. During the course of anal...

Collaboration of colorado cancer genetic counselors to integrate next generation sequencing panels into clinical practice.

The recent introduction of clinically available next generation sequencing (NGS) cancer panels has presented new challenges for genetic counselors. Determining which patients are appropriate for NGS panel testing is complex. Due to the large number of genes included in the NGS panels, thorough and appropriate pre-test counseling and interpretation of NGS results can be a time-consuming and difficult process. Many of the genes associated with increased cancer risk lack published clinical management guidelines and estimates of cancer risk for individuals with deleterious mutations. In order to efficiently and effectively review the clinical utility of NGS panels, Colorado cancer genetic counselors formed a working group to gain a better understanding of the genes included in NGS cancer panels. This publication reports on the approach of this group, the process used to evaluate a selected NGS panel, future directions for this collaboration, and ideas for other genetic counselors to form similar groups to efficiently evaluate new technologies and improve practice.

Validation of Next Generation Sequencing Cancer Panels for Clinical Somatic Mutation Profiling-- Identification of Source of Variations and Artifacts using FFPE Tissues

Use of a Next Generation Sequencing (NGS)-based test as a clinical trial assay requires that the test be analytically validated to CLIA/CAP regulations. During the course of analytically validating NGS cancer panels for use as patient enrollment assays, we assessed the repeatability, reproducibility and accuracy of commercially available Cancer Panels (Illumina TruSeq Cancer Panel on MiSeq and Life Technologies AmpliSeq v2 Cancer Panel on Ion Torrent PGM). We measured the repeatability and reproducibility by evaluating all variant calls among technical replicates and found in both platforms that variants with higher variant frequency (VF >30%) were called with much higher repeatability and reproducibility than those with lower VF (between 5 and 25%), a level at which many somatic mutations are found. Also, Illumina MiSeq run-to-run reproducibility was significantly higher than that of Ion Torrent PGM. However, Illumina TruSeq library preparation protocol resulted in much lower repeatability than those obtained from Ion Torrent AmpliSeq protocol at the low VF range. To determine the optimal variant call settings, we used different sets of more stringent filters (lower false positive rate, but higher false negative rate), each platform could achieve close to 95% reproducibility and repeatability. Sequenom MassArray was used as a tie-breaker assay for discordant calls between the two NGS platforms to establish the “truth”. Our data provide insight into the steps that contribute most to variability, such as the procedure of library preparation, the sequencingby- synthesis chemistry, the factors that impact mutation calls and sampling variation. We also found very high C to T mutation calls associated with Illumina Cancer Panel using TruSeq library preparation protocol (but not with Ion Torrent using AmpliSeq protocol) when a less stringent filter set was used. The C to T artifact mutation calls from formalin-fixed paraffin-embedded (FFPE) tissue samples observed in this study together with high C to A artifact mutation calls from acoustic shearing of intact DNA observed by others have the potential to negatively impact mutation profiling and mutation signature identification if not carefully addressed. Based on these results we conclude library preparation protocols that start with PCR amplification, such as the AmpliSeq protocol, provide higher repeatability on variant calls with low VF and therefore, are more suitable for mutation profiling and mutation signature studies where somatic mutations (including unknown mutations) are the focus and balanced false positive and false negative rates are critical to success.