Daniel S. Lieber

Orthogonal NGS for High Throughput Clinical Diagnostics.

Next generation sequencing is a transformative technology for discovering and diagnosing genetic disorders. However, high-throughput sequencing remains error-prone, necessitating variant confirmation in order to meet the exacting demands of clinical diagnostic sequencing. To address this, we devised an orthogonal, dual platform approach employing complementary target capture and sequencing chemistries to improve speed and accuracy of variant calls at a genomic scale. We combined DNA selection by bait-based hybridization followed by Illumina NextSeq reversible terminator sequencing with DNA selection by amplification followed by Ion Proton semiconductor sequencing. This approach yields genomic scale orthogonal confirmation of ~95% of exome variants. Overall variant sensitivity improves as each method covers thousands of coding exons missed by the other. We conclude that orthogonal NGS offers improvements in variant calling sensitivity when two platforms are used, better specificity for variants identified on both platforms, and greatly reduces the time and expense of Sanger follow-up, thus enabling physicians to act on genomic results more quickly.

Analytical Validation of a Hybrid Capture-Based Next-Generation Sequencing Clinical Assay for Genomic Profiling of Cell-Free Circulating Tumor DNA.

Genomic profiling of circulating tumor DNA derived from cell-free DNA (cfDNA) in blood can provide a noninvasive method for detecting genomic biomarkers to guide clinical decision making for cancer patients. We developed a hybrid capture–based next-generation sequencing assay for genomic profiling of circulating tumor DNA from blood (FoundationACT). High-sequencing coverage and molecular barcode–based error detection enabled accurate detection of genomic alterations, including short variants (base substitutions, short insertions/deletions) and genomic re-arrangements at low allele frequencies (AFs), and copy number amplifications. Analytical validation was performed on 2666 reference alterations. The assay achieved >99% overall sensitivity (95% CI, 99.1%–99.4%) for short variants at AF >0.5%, >95% sensitivity (95% CI, 94.2%–95.7%) for AF 0.25% to 0.5%, and 70% sensitivity (95% CI, 68.2%–71.5%) for AF 0.125% to 0.25%. No false positives were detected in 62 samples from healthy volunteers. Genomic alterations detected by FoundationACT demonstrated high concordance with orthogonal assays run on the same clinical cfDNA samples. In 860 routine clinical FoundationACT cases, genomic alterations were detected in cfDNA at comparable frequencies to tissue; for the subset of cases with temporally matched tissue and blood samples, 75% of genomic alterations and 83% of short variant mutations detected in tissue were also detected in cfDNA. On the basis of analytical validation results, FoundationACT has been approved for use in our Clinical Laboratory Improvement Amendments–certified/College of American Pathologists–accredited/New York State–approved laboratory.

Blood-based tumor mutational burden as a predictor of clinical benefit in non-small-cell lung cancer patients treated with atezolizumab

Although programmed death-ligand 1–programmed death 1 (PD-L1–PD-1) inhibitors are broadly efficacious, improved outcomes have been observed in patients with high PD-L1 expression or high tumor mutational burden (TMB). PD-L1 testing is required for checkpoint inhibitor monotherapy in front-line non-small-cell lung cancer (NSCLC). However, obtaining adequate tumor tissue for molecular testing in patients with advanced disease can be challenging. Thus, an unmet medical need exists for diagnostic approaches that do not require tissue to identify patients who may benefit from immunotherapy. Here, we describe a novel, technically robust, blood-based assay to measure TMB in plasma (bTMB) that is distinct from tissue-based approaches. Using a retrospective analysis of two large randomized trials as test and validation studies, we show that bTMB reproducibly identifies patients who derive clinically significant improvements in progression-free survival from atezolizumab (an anti-PD-L1) in second-line and higher NSCLC. Collectively, our data show that high bTMB is a clinically actionable biomarker for atezolizumab in NSCLC.A blood-based DNA sequencing assay to infer tumor mutational burden in the absence of tumor biopsy predicts response to PD-L1 blockade in patients with non-small-cell lung cancer.

Abstract B16: Validation and clinical feasibility of a Foundation Medicine assay to identify immunotherapy response potential through tumor mutational burden (TMB)

Background: The ability of tumors to evade immune surveillance by overexpressing immune checkpoint proteins has been exploited for therapeutic intervention through antibodies designed to interrupt their signaling. A number of patients across a range of disease types, including melanoma, lung, renal and bladder cancer, have demonstrated robust and durable responses using checkpoint inhibitor therapies (CPITs). Still, identifying the most likely responders remains an urgent need for proper clinical management. Tumor mutational burden (TMB) measures the overall number of somatic protein coding mutations per area of sequence counted occurring in a tumor specimen. This measure has been associated with both response and survival for multiple CPITs across an array of indications. It is hypothesized that immunotherapies are more effective for tumors with high TMB because these cells are more likely to express immune-reactive neoantigens. In this study we describe Foundation Medicine9s (FMI) work to develop and validate a TMB result as part of the current FoundationOne (F1) and FoundationOne Heme (F1H) comprehensive genomic profiling assays. Methods: We developed an analysis method to determine TMB based on data from both the F1 and F1H comprehensive genomic profiling assays. TMB is calculated by counting all synonymous and non-synonymous somatic variants across 315 or 405 genes. Germline alterations and known and likely driver alterations are excluded to avoid sample bias, as both F1 and F1H specifically target genes with cancer associations. The resulting mutation count is normalized by expressing the number as a mutation density with units of mutations per megabase (mut/Mb) of coding target territory. Analytic validation of TMB focused on accuracy, precision and sensitivity, while initial clinical feasibility was assessed in a cohort of 65 metastatic melanoma patients receiving immunotherapy. To determine accuracy, we compared the TMB values generated from F1 against a CLIA validated whole-exome sequencing (WES) method on 29 patients with TMB values ranging from Results: Foundation Medicine9s TMB measure provides accurate and precise results across a range of tumor mutational burden values on samples with as little as 20% tumor purity. In a cohort of 65 metastatic melanoma patients, the median TMB value was 37.9 mut/Mb in the responder group and 6.6 mut/Mb in the non-responder group (p Conclusions: We have developed and validated a TMB result as part of the FoundationOne and FoundationOne Heme platforms. Initial clinical feasibility results demonstrate that the FoundationOne TMB value can be used to predict the likely response of metastatic melanoma patients to anti-PD1/PD-L1 checkpoint inhibitors, while feasibility in NSCLC and bladder cancer have been presented elsewhere. Citation Format: Daniel S. Lieber, Mark R. Kennedy, Douglas B. Johnson, Joel R. Greenbowe, Garrett M. Frampton, Alexa B. Schrock, Jeffrey S. Ross, Phillip J. Stephens, Siraj M. Ali, Vincent A. Miller, David A. Fabrizio. Validation and clinical feasibility of a Foundation Medicine assay to identify immunotherapy response potential through tumor mutational burden (TMB). [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr B16.

Abstract 2987: Validation and clinical feasibility of a comprehensive genomic profiling assay to identify likely immunotherapy responders through tumor mutational burden (TMB)

Background Patients across a range of disease types have demonstrated robust and durable responses using checkpoint inhibitor therapies (CPITs). Given the limitations of immuno-histochemical based testing, identifying a unified, quantitative metric to determine potential response to CPITs remains an urgent need. Tumor mutational burden (TMB) measures the number of somatic protein coding mutations per target sequence in a tumor specimen. This measure has been associated with response and survival for multiple CPITs across an array of indications. In this study we describe Foundation Medicine’s (FMI) work to develop and validate a TMB result as part of our comprehensive genomic profiling assays and summarize clinical feasibility in NSCLC, melanoma and bladder cancer. Methods We developed an analysis method to determine TMB based on data from our comprehensive genomic profiling assays. TMB is calculated by counting all synonymous and non-synonymous somatic variants across 315 or 405 genes, excluding germline alterations and known or likely driver alterations. The mutation count is normalized by the coding target territory to achieve a mutation density of mutations per megabase (mut/Mb). To determine accuracy, we compared TMB values from our comprehensive genomic profiling assay against a CLIA-validated whole-exome sequencing (WES) method on 29 patients. Precision was assessed over 10 clinical samples replicated 4-6 times. Lower limit of sample tumor purity was determined through dilutions of tumor/normal pairs from 80% to 5% tumor. Clinical feasibility was assessed by analyzing TMB versus immunotherapy-based survival in a cohort of 65 metastatic melanoma patients, 150 urothelial carcinoma patients and 463 NSCLC patients. Additionally, we examined the relationship between TMB and microsatellite instability status (MSI), an independent biomarker associated with response to CPITs. Results Foundation Medicine’s TMB measure provides accurate and precise results across a range of tumor mutational burden values on samples with as little as 20% tumor purity. Using cohort specific thresholds, TMB was significantly associated with improved survival to CPITs in NSCLC, melanoma and bladder cancer. Using data from over 40,000 patient samples, we also show significant overlap between high TMB and high MSI samples and show that MSI-High specimens represent a subset of TMB-High specimens. Conclusions We have developed and validated the tumor mutational burden (TMB) biomarker as part of our comprehensive cancer genomic profiling assays. Initial clinical feasibility results demonstrate that TMB can be used to predict the likely response to anti-PD-1/PD-L1 CPITs across a growing number of indications including NSCLC, melanoma and bladder cancer. Citation Format: Daniel S. Lieber, Mark R. Kennedy, Douglas B. Johnson, Jonathan E. Rosenberg, Marcin Kowanetz, Joel R. Greenbowe, Garrett M. Frampton, Caitlin F. Connelly, Alexa B. Schrock, Jeffrey S. Ross, Philip J. Stephens, Siraj M. Ali, Vincent A. Miller, David A. Fabrizio. Validation and clinical feasibility of a comprehensive genomic profiling assay to identify likely immunotherapy responders through tumor mutational burden (TMB) [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 2987. doi:10.1158/1538-7445.AM2017-2987