Clinical Next-generation Sequencing Assay Research Articles

Abstract 237: Coincidence of InDels with missense mutations: 0, 1 or 2 artifacts

Abstract The purpose of this study was to explain a serendipitously discovered next generation sequencing (NGS) artifact. While reviewing clinical cases, we found a case of a large deletion that also contained multiple missense mutations. This phenotype was unknown to several molecular pathologists in our group. We reviewed 46 clinical cases with short insertions or deletions (InDels) (1-30 bases) or FLT3 internal tandem duplications (ITDs) (6-183 bases) of solid or hematologic malignancy processed with a clinical NGS assay and identified misaligned reads, ranging from 3 to 100% of reads showing mismapped bases. Reads that straddled the InDel with sufficient numbers of bases on both sides were correctly anchored both upstream and downstream of the InDel, and were called correctly. However, reads that ended in the middle of the InDel were incorrectly mapped onto the deletion, thereby producing artifactual missense mutations. The frequency of mismapped bases increased with InDel size, and the VAF of the InDel can be markedly underestimated. The ratio of forward to reverse reads was commonly skewed for the mismapped base compared to the ratio for the wildtype base at that position. ABRA2 was able to correct 41 to 100% of the reads with mismapped bases and led to absolute increases in the VAF from 1 to 61% along with correction of all of the SBSs except for two cases. Knowledge of the various artifacts commonly seen in next generation sequencing is essential to safely sign out molecular pathology cases. Citation Format: Kelly E. Craven, Catherine G. Fischer, LiQun Jiang, Aparna Pallavajjala, Ming-Tseh Lin, James R. Eshleman. Coincidence of InDels with missense mutations: 0, 1 or 2 artifacts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 237.

Optimizing Insertion and Deletion Detection Using Next-Generation Sequencing in the Clinical Laboratory

Detection of insertions and deletions (InDels) by short-read next-generation sequencing (NGS) technology can be challenging because of frequent misaligned reads. A systematic analysis of short InDels (1 to 30 bases) and fms-related receptor tyrosine kinase 3 (FLT3) internal tandem duplications (ITDs; 6 to 183 bases) from 46 clinical cases of solid or hematologic malignancy processed with a clinical NGS assay identified misaligned reads in every case, ranging from 3% to 100% of reads with the InDel showing mismapped bases. Mismaps also increased with InDel size. As a consequence, the clinical NGS bioinformatics pipeline undercalled the variant allele frequency by 1% to 84%, incorrectly called simultaneous single-base substitutions along with InDels, or did not report an FLT3 ITD that had been detected by capillary electrophoresis. To improve the ability of the pipeline to better detect and quantify InDels, we utilized a software program called Assembly-Based ReAligner (ABRA2) to more accurately remap reads. ABRA2 was able to correct 41% to 100% of the reads with mismapped bases and led to absolute increases in the variant allele frequency from 1% to 61% along with correction of all of the single-base substitutions except for two cases. ABRA2 could also detect multiple FLT3 ITD clones except for one 183-base ITD. Our analysis has found that ABRA2 performs well on short InDels as well as FLT3 ITDs that are <100 bases.

FDA-led consortium studies advance quality control of targeted next generation sequencing assays for precision oncology.

Cancer is the second leading cause of mortality worldwide despite tremendous advances in treatment. The promise of precision oncology depends on accurate characterization of tumor mutations and subsequent therapy selection. The lack of tumor reference samples along with the associated next generation sequencing (NGS) technical assessments has hindered the development of NGS assays and the realization of benefits for precision oncology. The summarized results and recommendations of several seminal SEQC2 studies along with a vision of the changing landscape of precision oncology and anticipated next steps by the SEQC2 consortium are reported. Importantly, these studies utilized a new robust reference sample material which was developed and constructed to support multiple DNA and RNA-based NGS assay studies. These studies focused on a wide variety of precision oncology assay scenarios and provided guidelines for standardized analyses and best practice recommendations. The evolving landscape of precision oncology requires insights into critical factors supporting the sensitivity and reproducibility of clinical NGS assays for continued improvement in patient outcomes. Persistent development of robust reference materials, quantitative performance metrics, and actionable data analysis recommendations are needed. This series of SEQC2 studies serve to advance NGS-based assays for precision oncology and support regulatory science endeavors.

TP53 mutations Are Associated with Mutated MYD88 and CXCR4, and Confer an Adverse Outcome in Waldenström Macroglobulinemia

Background: Whole genome sequencing identified highly recurrent somatic mutations in Waldenström macroglobulinemia (WM). Activating somatic mutations in MYD88 and CXCR4 are present in 90-95% and 30-40% of WM patients, respectively, and impact disease presentation, treatment outcome, and overall survival. In contrast, somatic mutations in the tumor suppressor gene TP53 occur less frequently (7%), and the clinical significance remains to be delineated. TP53 aberrations confer an adverse prognosis in related B-cell malignancies, such as chronic lymphocytic leukemia and multiple myeloma, predicting for treatment resistant and inferior survival. As such, we sought to characterize the clinical implications, as well as the clonal architecture, of somatic TP53 mutations in WM. Methods: We searched our database for WM patients with a TP53 mutation identified by a clinical next generation sequencing (NGS) assay using unsorted bone marrow (BM) samples. To validate the findings, CD19+ cells from BM aspirates were isolated, and DNA was extracted for mutational analysis. CD19-depleted peripheral blood (PB) mononuclear cells were used as normal paired samples. All samples were screened for MYD88, CXCR4, and TP53 mutations by Sanger sequencing, and zygosity was determined by establishing the ratio of mutant versus wile-type (WT) allele expression. TP53 copy number was determined using TaqMan Copy Number Assays (Applied Biosystems, Grand Island, NY, USA). Medical files were manually reviewed to gather pertinent clinical data. Results: Fourteen WM patients (14/265; 5.3%) had a TP53 mutation detected by a clinical NGS assay. Sanger sequencing identified somatic TP53 mutations within the WM clone in six patients, including one patient with two somatic mutations. Three patients had a TP53 mutation identified in both CD19+ and CD19- sorted samples, 4 patients were WT, and 1 patient did not have DNA for validation. No recurrent variants were identified. Among the six patients with validated somatic TP53 mutations, four (67%) had biallelic inactivation of TP53 . Three patients had a homozygous TP53 mutation determined by Sanger sequencing and one patient had deletion of TP53 . All six patients harbored both a CXCR4 mutation (4 nonsense, 2 frameshift) and the MYD88 L265P mutation, of which 4 patients (67%) had mutated homozygous MYD88 . At the time the TP53 mutation was detected, the median BM involvement and hemoglobin level was 80% and 9.2 g/dl, respectively. The median serum IgM level was 2,508 mg/dl, and two patients had symptomatic hyperviscosity. Five patients (83%) were high-risk according to the International Prognostic Scoring System for WM. Three patients were untreated, 2 patients were refractory to their most recent therapy (i.e. bortezomib, dexamethasone, rituximab), and 1 patient was relapsing. Four patients went on to receive systemic therapy, to which two patients were refractory. Two patients were treated with ibrutinib and both obtained a major response. After a median follow-up 7.7 months, 2 patients (33%) have died due to progressive disease; both patients had biallelic inactivation of TP53 . Conclusion: Somatic TP53 mutations occur concurrently with both MYD88 and CXCR4 mutations in WM patients and appear to confer an aggressive disease course. Our findings highlight the need to understand the cell-specific origin of mutations detected by clinical NGS assays. DisclosuresCastillo:Pharmacyclics: Consultancy, Research Funding; Janssen: Consultancy, Research Funding; Millennium: Research Funding; Abbvie: Research Funding. Treon:Pharmacyclics: Consultancy, Research Funding.

Assembling and Validating Bioinformatic Pipelines for Next-Generation Sequencing Clinical Assays.

Clinical next-generation sequencing (NGS) is being rapidly adopted, but analysis and interpretation of large data sets prompt new challenges for a clinical laboratory setting. Clinical NGS results rely heavily on the bioinformatics pipeline for identifying genetic variation in complex samples. The choice of bioinformatics algorithms, genome assembly, and genetic annotation databases are important for determining genetic alterations associated with disease. The analysis methods are often tuned to the assay to maximize accuracy. Once a pipeline has been developed, it must be validated to determine accuracy and reproducibility for samples similar to real-world cases. In silico proficiency testing or institutional data exchange will ensure consistency among clinical laboratories. To provide molecular pathologists a step-by-step guide to bioinformatics analysis and validation design in order to navigate the regulatory and validation standards of implementing a bioinformatic pipeline as a part of a new clinical NGS assay. This guide uses published studies on genomic analysis, bioinformatics methods, and methods comparison studies to inform the reader on what resources, including open source software tools and databases, are available for genetic variant detection and interpretation. This review covers 4 key concepts: (1) bioinformatic analysis design for detecting genetic variation, (2) the resources for assessing genetic effects, (3) analysis validation assessment experiments and data sets, including a diverse set of samples to mimic real-world challenges that assess accuracy and reproducibility, and (4) if concordance between clinical laboratories will be improved by proficiency testing designed to test bioinformatic pipelines.

Optimization of Population Frequency Cutoffs for Filtering Common Germline Polymorphisms from Tumor-Only Next-Generation Sequencing Data

Clinical next-generation sequencing assays are often run on tumor specimens without a matched normal specimen, which complicates the differentiation of germline from somatic variants. In tumor-only testing, population data are often used to infer germline status, though no consensus exists on the exact population frequency (PF) cutoff above which a variant should be considered likely germline. In this study, five population databases plus the Catalog of Somatic Mutations in Cancer were used to demonstrate the impact of changing the PF cutoff on assignment of variants as germline versus somatic. The 1% to 2% PF cutoffs widely used in bioinformatic pipelines resulted in high sensitivity for classification of somatic variants, but unnecessarily reduced sensitivity for germline variants. Using optimized PF cutoffs, the source of variants in The Cancer Genome Atlas (TCGA) data could be predicted with >95% accuracy. Further exploration of four TCGA cancer data sets indicated that the optimal cutoff is influenced by both cancer type and the assay region of interest. Comparing TCGA data to data generated from a clinical, hybridization capture test (approximately 615 kb capture space) showed that PF cutoffs may not be transferable between assays, even when the gene set is held constant. Thus, filtering approaches need to be carefully designed and optimized, and should be assay-specific to support tumor-only testing until tumor-normal testing becomes routine in the clinical setting.

Optimization of a microfluidics-based next generation sequencing assay for clinical oncology diagnostics.

Massively parallel, or next-generation, sequencing is a powerful technique for the assessment of somatic genomic alterations in cancer samples. Numerous gene targets can be interrogated simultaneously with a high degree of sensitivity. The clinical standard of care for many advanced solid and hematologic malignancies currently requires mutation analysis of several genes in the front-line setting, making focused next generation sequencing (NGS) assays an effective tool for clinical molecular diagnostic laboratories. We have utilized an integrated microfluidics circuit (IFC) technology for multiplex PCR-based library preparation coupled with a bioinformatic method designed to enhance indel detection. A parallel low input PCR-based library preparation method was developed for challenging specimens with low DNA yield. Computational data filters were written to optimize analytic sensitivity and specificity for clinically relevant variants. Minimum sequencing coverage and precision of variant calls were the two primary criteria used to establish minimum DNA mass input onto the IFC. Wet-bench and bioinformatics protocols were modified based on data from the optimization and familiarization process to improve assay performance. The NGS platform was then clinically validated for single nucleotide and indel (up to 93 base pair) variant detection with overall analytic accuracy of 98% (97% sensitivity; 100% specificity) using as little as 3 ng of formalin-fixed, paraffin-embedded DNA or 0.3 ng of unfixed DNA. We created a targeted clinical NGS assay for common solid and hematologic cancers with high sensitivity, high specificity, and the flexibility to test very limited tissue samples often encountered in routine clinical practice.

Reliable Pan-Cancer Microsatellite Instability Assessment by Using Targeted Next-Generation Sequencing Data.

Microsatellite instability (MSI)/mismatch repair (MMR) status is increasingly important in the management of patients with cancer to predict response to immune checkpoint inhibitors. We determined MSI status from large-panel clinical targeted next-generation sequencing (NGS) data across various solid cancer types. The MSI statuses of 12,288 advanced solid cancers consecutively sequenced with Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets clinical NGS assay were inferred by using MSIsensor, a program that reports the percentage of unstable microsatellites as a score. Cutoff score determination and sensitivity/specificity were based on MSI polymerase chain reaction (PCR) and MMR immunohistochemistry. By using an MSIsensor score ≥ 10 to define MSI high (MSI-H), 83 (8%) of 996 colorectal cancers (CRCs) and 42 (16%) of 260 uterine endometrioid cancers (UECs) were MSI-H. Validation against MSI PCR and/or MMR immunohistochemistry performed for 138 (24 MSI-H, 114 microsatellite stable [MSS]) CRCs, and 40 (15 MSI-H, 25 MSS) UECs showed a concordance of 99.4%. MSIsensor also identified 68 MSI-H/MMR-deficient (MMR-D) non-CRC/UECs. Of 9,591 non-CRC/UEC tumors with MSS MSIsensor status, 456 (4.8%) had slightly elevated scores(≥3 and <10) of which 96.6% with available material were confirmed to be MSS by MSI PCR. MSI-H was also detected and confirmed in three non-CRC/UECs with low exonic mutation burden (< 20). MSIsensor correctly scored all 15 polymerase ε ultra-mutated cancers as negative for MSI. MSI status can be reliably inferred by MSIsensor from large-panel targeted NGS data. Concurrent MSI testing by NGS is resource efficient, is potentially more sensitive for MMR-D than MSI PCR, and allows identification of MSI-H across various cancers not typically screened, as highlighted by the finding that 35% (68 of 193) of all MSI-H tumors were non-CRC/ UEC.

Clonal Evolution Revealed By Exome Sequencing in a Case of Primary Myelofibrosis Associated with Subsequent Development of Aggressive Systemic Mastocytosis/Mast Cell Leukemia

A 77 year-old man initially presented in July 2013 with anemia, splenomegaly, and constitutional symptoms. A bone marrow biopsy revealed a hypercellular marrow with megakaryocytic hyperplasia and atypia and mild reticulin fibrosis, consistent with a diagnosis of primary myelofibrosis (PMF). Cytogenetics revealed a normal karyotype. JAK2 V617F testing was negative. Initiation of treatment with the JAK inhibitor ruxolitinib led to marked symptomatic improvement. The patient was then enrolled in a Phase 2 study with the anti-lysyl oxidase-like-2 (LOXL2) monoclonal antibody simtuzumab, administered via IV infusion every two weeks (while continuing ruxolitinib). He tolerated the simtuzumab infusions well initially, but with the 11th and 12th infusions experienced rigors, hypotension, and hypoxia. This occurred ~8 months after his initial PMF diagnosis. A repeat bone marrow biopsy revealed large aggregates of mast cells comprising 30-40% of the marrow cellularity (with 16% mast cells enumerated on the aspirate). A subset of the mast cells exhibited spindled morphology, and CD25 co-expression was demonstrated by flow cytometry in a subset of CD117-positive cells. Testing for the KITD816V mutation was positive. Tryptase levels were significantly elevated (375 ng/mL). These findings were consistent with a diagnosis of aggressive systemic mastocytosis with an associated hematologic non-mast cell lineage disorder (ASM-AHNMD).Compassionate-use approval for the KIT inhibitor midostaurin was obtained, and treatment with midostaurin (in addition to continuation of ruxolitinib) was initiated. The patient initially reported symptomatic improvement with midostaurin treatment, but after several months his symptoms began to worsen, with a corresponding increase in tryptase (875 ng/mL). A repeat bone marrow biopsy revealed overt evolution to mast cell leukemia (MCL) with > 90% mast cell involvement. Based on the presence of an IDH2mutation identified on a clinical next-generation sequencing assay, the patient was evaluated for a clinical trial with the mutant IDH2 inhibitor AG-221. Unfortunately, the patient decompensated and expired before he could enroll in the study.To identify contributing driver mutations and to delineate clonal hierarchy associated with disease initiation and progression in this unique case of PMF with concomitant ASM, exome sequencing was performed on serial samples obtained at the following disease stages:PMF diagnosis (pre-ruxolitinib) (Day 0)PMF on ruxolitinib (before ASM diagnosis) (Day 181)ASM diagnosis (post-anti-LOXL2 antibody, pre-midostaurin) (Day 394)Progression to MCL (on midostaurin) (Day 519)Matched skin (normal) sampleLikely driver mutations in IDH2 and SRSF2 were identified at ~40-50% variant allele frequency (VAF) in all samples and were therefore likely present in the founding clone. The KIT D816V mutation was found at 23% VAF at Day 0, then ~40% VAF in all other samples, suggesting it was present in a daughter subclone of the IDH2/SRSF2-containing clone that became dominant over time with disease progression. These findings also suggest that targeting KIT with midostaurin would be unlikely to eradicate the founding clone. Rather, selective targeting of IDH2 and/or SRSF2 could potentially ameliorate both diseases (PMF and ASM/MCL).To provide pre-clinical evidence of the potential utility of targeting IDH2, peripheral blood mononuclear cells obtained at the time of ASM diagnosis were plated in liquid culture in the presence or absence of the mutant IDH2 inhibitor AGI-6780. After 14 days in culture, the differentiation status of the cultured cells was examined by mass cytometry (CyTOF). Treatment with AGI-6780 resulted in a marked enhancement of myeloid differentiation (denoted by CD15 and CD66b expression) along with a corresponding decrease in CD34+ progenitor cells. These effects were not seen in cells cultured in the absence of AGI-6780. These results are consistent with prior studies in acute myeloid leukemia indicating that the beneficial effects of mutant IDH2 inhibition are likely related to inducing differentiation of primitive cells. In summary, this study highlights the capacity of serial genomic analysis to define the clonal architecture that drives disease initiation and evolution, and to distinguish founding vs subclonal mutations to identify the most promising targets for therapeutic intervention. DisclosuresOh:Janssen: Research Funding; CTI: Research Funding; Gilead: Membership on an entity's Board of Directors or advisory committees, Research Funding; Incyte Corporation: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Clinical Validation of a Next-Generation Sequencing Genomic Oncology Panel via Cross-Platform Benchmarking against Established Amplicon Sequencing Assays

Next-generation sequencing (NGS) genomic oncology profiling assays have emerged as key drivers of personalized cancer care and translational research. However, validation of these assays to meet strict clinical standards has been historically problematic because of both significant assay complexity and a scarcity of optimal validation samples. Herein, we present the clinical validation of 76 genes from a novel 1212-gene large-scale hybrid capture cancer sequencing assay (University of Chicago Medicine OncoPlus) using full-data comparisons against multiple clinical NGS amplicon-based assays to yield dramatic increases in per-sample data comparison efficiency compared with previously published validations. Using a sample set of 104 normal, solid tumor, and hematopoietic malignancy specimens, head-to-head NGS data analyses allowed for 6.8 million individual clinical base call comparisons, including 2729 previously confirmed variants, with 100% sensitivity and specificity. University of Chicago Medicine OncoPlus showed excellent performance for detection of single-nucleotide variants, insertions/deletions up to 52 bp, and FLT3 internal tandem duplications of up to 102 bp or larger. Highly concordant copy number variant and ALK/RET/ROS1 gene fusion detection were also observed. In addition to underlining the efficiency of NGS validation via full-data benchmarking against existing clinical NGS assays, this study also highlights the degree of performance similarity between hybrid capture and amplicon assays that is attainable with the application of strict quality control parameters and optimized computational analytics.

Next-generation sequencing in patients with advanced cancer: are we ready for widespread clinical use? A single institute's experience.

The next-generation sequencing (NGS) assay targeting cancer-relevant genes has been adopted widely for use in patients with advanced cancer. The primary aim of this study was to assess the clinical utility of commercially available NGS. We retrospectively collected demographic and clinicopathologic data, recommended therapy, and clinical outcomes of 30 patients with a variety of advanced solid tumors referred to Foundation Medicine NGS. The initial pathologic examination was performed at the pathology department of the referring hospital. The comprehensive clinical NSG assay was performed on paraffin-embedded tumor samples using the Clinical Laboratory Improvement Amendments-certified FoundationOne platform. The median number of genomic alterations was 3 (0-19). The median number of therapies with potential benefit was 2 (0-8). In 12 cases, a comprehensive clinical NGS assay did not indicate any therapy with potential benefit according to the genomic profile. Ten of the 30 patients received treatments recommended by genomic profile results. In six of the 10 cases, disease progressed within 2 months and four patients died within 3 months of treatment initiation. Three of the 30 patients benefited from a comprehensive clinical NGS assay and the subsequent recommended therapy. The median PFS was 12 weeks (95% confidence interval 10-57) in patients treated with molecularly targeted agents chosen on the basis of tumor genomic profiling versus 48 weeks (95% confidence interval 8-38) in the control group treated with physician choice therapy (P=0.12). Our study suggests that NGS can detect additional treatment targets in individual patients, but prospective medical research and appropriate clinical guidelines for proper clinical use are vital.

Multiplexed Reference Materials as Controls for Diagnostic Next-Generation Sequencing: A Pilot Investigating Applications for Hypertrophic Cardiomyopathy

Diagnostic next-generation sequencing (NGS)-based gene panels are increasingly used for prevalent disorders with genetic and clinical heterogeneity. Clinical development, validation, and quality management of these panels ideally includes reference samples containing prevalent pathogenic variants; however, clinical domain expertise to select appropriate variants may not be present, samples are often not publicly available, and their inclusion is associated with added cost. Expert-designed, multiplexed controls can remedy some of these challenges. One approach relies on spiking biosynthetic fragments carrying desired variants into human genomic DNA. We piloted the utility of this approach for hypertrophic cardiomyopathy. Data from >3000 previously sequenced probands were used to select 10 common pathogenic and/or technically challenging variants in the top hypertrophic cardiomyopathy genes. Multiplexed controls were constructed across a range of ideal and realistic allelic fractions for heterozygous germline variants. NGS was performed in quadruplicate, and results were compared with diagnostic NGS data for the source patient samples. Overall, results were indistinguishable from patient-derived data with variants being detected at or reasonably close to the targeted allelic fraction ratios. The exception was a common 25-bp deletion in MYBPC3, underscoring the importance of including such variants in test development. These controls may be an attractive addition to the repertoire of materials for development, validation, and quality monitoring of clinical NGS assays.

Abstract 4261: Prospective next generation sequencing (NGS) of rare or poor prognosis cancers

Abstract Background. Genomic screening for somatic alterations in individual tumors is informative for understanding tumor biology and identifying novel treatment approaches and research opportunities. We initiated a research study where patients with rare or poor prognosis cancers were enrolled and underwent targeted sequencing using a CLIA certified assay. Sequencing results were reviewed at a molecular tumor board and therapeutic strategies were suggested if appropriate. Preliminary results of this study are now presented. Experimental procedures. We analyzed the genomic profiles of formalin-fixed paraffin embedded tumor specimens from 72 patients using the FoundationOne™ (Foundation Medicine, Cambridge, MA) platform for targeted sequencing of the entire coding sequence of 236 genes and 47 introns of 19 genes involved in fusions in a CLIA-certified CAP accredited laboratory. A range of evaluated tumor subtypes of epithelial and mesenchymal origin were evaluated (20). The majority of cases were from rare or uncommon histologic classes, though recurrent/refractory cases of colon cancer and triple negative breast cancers were also enrolled. Both clinical history and sequencing data were presented at a multidisciplinary molecular tumor board for development of therapeutic recommendations. Results. At least one genomic alteration was found in 68 cases (95%) and none in four cases (6%). Serial specimens were obtained in six patients. Five had a gain of at least one mutation in serial specimens. The average number of mutations identified was 3.6 (range 0-10). The most common genomic alterations detected were in p53 (40%), KRAS (17%), PI3K (15%) and PTEN (10%). Alterations in the FGF pathway were surprisingly common (16%). A number of mutations occurred at low frequency but included an additional large set of potentially actionable genes: ALK, ERBB2, BRCA2, MET. The majority of cases had mutations for which our molecular tumor board had recommended action: either potential enrollment in clinical trial or off label use of approved therapy. NGS sequencing results led to implemented clinical action in ∼15% of cases. Conclusion. Use of a comprehensive clinical NGS assay in a diverse set of cancers identifies a number of actionable genomic changes associated with targeted therapies. An institutional molecular tumor board is an effective venue to systematically review sequencing data and generate clinical recommendations. Knowledge of genomic alterations has led to referral to clinical trials, off label use of targeted therapy, and planned trials of agents targeting identified pathways. Challenges to effectively implementing clinical recommendations included lack of practical access to clinical trials and limited availability of targeted agents. Expanding clinical trial portfolios, and increasing access to targeted agents is critical for successfully implementing genomic-guided precision cancer therapy. Citation Format: Kim M. Hirshfield, Siraj M. Ali, Vincent A. Miller, Philip J. Stephens, Vassiliki Karantza, Robert S. DiPaola, Lorna Rodriguez-Rodriguez, Shridar Ganesan. Prospective next generation sequencing (NGS) of rare or poor prognosis cancers. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4261. doi:10.1158/1538-7445.AM2014-4261

1024 EGFR single nucleotid polymorphism R521K is a predictor for the occurrence of skin rash

Quality assurance for clinical next-generation sequencing (NGS)–based assays is difficult given the complex methods and the range of sequence variants such assays can detect. As the number and range of mutations detected by clinical NGS assays has increased, it is difficult to apply standard analyte-specific proficiency testing (PT). Most current proficiency testing challenges for NGS are methods-based PT surveys that use DNA from reference samples engineered to harbor specific mutations that test both sequence generation and bioinformatics analysis. These methods-based PTs are limited by the number and types of mutations that can be physically introduced into a single DNA sample. In silico proficiency testing, which evaluates only the bioinformatics component of NGS assays, is a recently introduced PT method that allows for evaluation of numerous mutations spanning a range of variant classes. In silico PT data sets can be generated from simulated or actual sequencing data and are used to test alignment through variant detection and annotation steps. In silico PT has several advantages over the use of physical samples, including greater flexibility in tested variants, the ability to design laboratory-specific challenges, and lower costs. Herein, we review the use of in silico PT as an alternative to traditional methods-based PT as it is evolving in oncology applications and discuss how the approach is applicable more broadly.