Mark Kennedy

Individualized Molecular Analyses Guide Efforts (IMAGE): A Prospective Study of Molecular Profiling of Tissue and Blood in Metastatic Triple-Negative Breast Cancer

Purpose: The clinical utility of next-generation sequencing (NGS) in breast cancer has not been demonstrated. We hypothesized that we could perform NGS of a new biopsy from patients with metastatic triple-negative breast cancer (TNBC) in a clinically actionable timeframe. Experimental Design: We planned to enroll 40 patients onto a prospective study, Individualized Molecular Analyses Guide Efforts (IMAGE), to evaluate the feasibility of obtaining a new biopsy of a metastatic site, perform NGS (FoundationOne), and convene a molecular tumor board to formulate treatment recommendations within 28 days. We collected blood at baseline and at time of restaging to assess cell-free circulating plasma tumor DNA (ptDNA). Results: We enrolled 26 women with metastatic TNBC who had received ≥1 line of prior chemotherapy, and 20 (77%) underwent NGS of a metastatic site biopsy. Twelve (60%) evaluable patients received treatment recommendations within 28 days of consent. The study closed after 20 patients underwent NGS, based on protocol-specified interim futility analysis. Three patients went on to receive genomically directed therapies. Twenty-four of 26 patients had genetic alterations successfully detected in ptDNA. Among 5 patients, 4 mutations found in tumor tissues were not identified in blood, and 4 mutations found in blood were not found in corresponding tumors. In 9 patients, NGS of follow-up blood samples showed 100% concordance with baseline blood samples. Conclusions: This study demonstrates challenges of performing NGS on prospective tissue biopsies in patients with metastatic TNBC within 28 days, while also highlighting the potential use of blood as a more time-efficient and less invasive method of mutational assessment. Clin Cancer Res; 23(2); 379–86. ©2016 AACR.

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.

Beyond microsatellite testing: assessment of tumor mutational burden identifies subsets of colorectal cancer who may respond to immune checkpoint inhibition

Background The clinical application of PD1/PD-L1 targeting checkpoint inhibitors in colorectal cancer (CRC) has largely focused on a subset of microsatellite instable (MSI-high) patients. However, the proposed genotype that sensitizes these patients to immunotherapy is not captured by MSI status alone. Estimation of tumor mutational burden (TMB) from comprehensive genomic profiling is validated against whole exome sequencing and linked to checkpoint response in metastatic melanoma, urothelial bladder cancer and non-small cell lung carcinoma. We sought to explore the subset of microsatellite stable (MSS) CRC patients with high TMB, and identify the specific genomic signatures associated with this phenotype. Furthermore, we explore the ability to quantify TMB as a potential predictive biomarker of PD1/PD-L1 therapy in CRC. Methods Formalin-fixed, paraffin embedded tissue sections from 6,004 cases of CRC were sequenced with a CLIA-approved CGP assay. MSI and TMB statuses were computationally determined using validated methods. The cutoff for TMB-high was defined according to the lower bound value that satisfied the 90% probability interval based on the TMB distribution across all MSI-High patients. Results MSS tumors were observed in 5,702 of 6,004 (95.0%) cases and MSI-H tumors were observed in 302 (5.0%) cases. All but one (99.7%) MSI-H cases were TMB-high (range, 6.3-746.9 mut/Mb) and 5,538 of 5,702 (97.0%) MSS cases were TMB-low (range, 0.0-10.8 mut/Mb). Consequently, 164 of 5,702 (2.9%) MSS cases were confirmed as TMB-high (range, 11.7-707.2 mut/Mb), representing an increase in the target population that may respond to checkpoint inhibitor therapy by 54% (466 vs. 302, respectively). Response to PD-1 inhibitor is demonstrated in MSS/TMB-high cases. Conclusions Concurrent TMB assessment accurately classifies MSI tumors as TMB-high and simultaneously identifies nearly 3% or CRC as MSS/TMB-high. This subgroup may expand the population of CRC who may benefit from immune checkpoint inhibitor based therapeutic approaches.

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 P6-07-08: The complete spectrum of ESR1 mutations from 7590 breast cancer tumor samples

Background: Approximately 70% of newly diagnosed breast cancers express estrogen receptor alpha (ERα), and are treated with agents that block ER signaling. Acquired mutations in ESR1, the gene that encodes ERα, have been associated with resistance to aromatase inhibitor therapy in patients with ER positive metastatic breast cancer (ER+ mBC). The most frequently occurring ESR1 mutations are clustered between amino acids 536 to 538 within the ligand binding domain (LBD), although limited data exists characterizing the full mutation profile in a large number of breast cancer samples. Methods: We surveyed the Foundation Medicine dataset of 7590 primary and metastatic breast cancer tumor samples for ESR1 short variants and copy number alterations. Hormone receptor status was unavailable, therefore two assumptions were made to provide an estimate of prevalence in the ER+ HER2- population: 70% of the tumor samples are from ER+ HER2- patients, and all ESR1 mutations from non-HER2 amplified metastatic sites are from ER+ HER2- patients. In a separate cohort of 48 ER+ mBC patients, circulating tumor DNA (ctDNA) was analyzed for ESR1 mutations using the BEAMing method by Sysmex and with Foundation Medicine9s sequencing assay, FoundationACT (Assay for Circulating Tumor DNA). Results: The prevalence of mutations in ER+ HER2- breast cancer was estimated to be 22% in samples from metastatic sites but less than 3% in samples from primary sites. ESR1 amplification was rare in samples from both primary and metastatic disease sites at 1.3% and 2.0% respectively. A total of 153 unique short variants of known and unknown status were identified. In addition to hotspot mutations at 537 and 538, previously undescribed rare mutations were identified throughout the entire length of the LBD, although 10 alterations at amino acids 380, 463, 536, 537, and 538 account for 86% of all ESR1 mutations in the ER+ HER2- metastatic sites. We also characterized the overlap of ESR1 alterations with commonly altered and clinically relevant genes in breast cancer, including PIK3CA mutations and HER2 amplification, and we report here a landscape of co-occurring alterations. In the cohort of patient samples where ctDNA was analyzed, BEAMing and FoundationAct assays both detected ESR1 mutations in 19 out of 48 samples, and overall concordance of mutation status (wild-type vs mutant) was 100%. A total of 51 individual mutations were detected with the BEAMing assay, 42 of which were detected with the FoundationACT assay. Seven mutations that were undetected by FoundationACT had mutant allele frequencies less than 0.1%. Ten ESR1 mutations were detected only by FoundationACT, 9 of which are not covered with the BEAMing assay. Alterations in PIK3CA, CDH1, TP53, ERBB2, and other breast cancer relevant genes were also detected with FoundationACT. Conclusions: Understanding the mutational landscape of ESR1 and co-occurring alterations is important for diagnostic development in conjunction with the clinical development of novel anti-endocrine therapies. Our data demonstrate a large spectrum of mutations in the LBD in addition to known hotspot mutations. In addition, the FoundationACT assay offers a robust NGS-based method to screen for mutations in ctDNA that is highly concordant with digital PCR methods. Citation Format: Spoerke JM, Schleifman E, Clark TA, Young G, Nahas M, Kennedy M, Young L, Chmielecki J, Otto GA, Lipson D, Wilson TR, Gendreau S, Lackner MR. The complete spectrum of ESR1 mutations from 7590 breast cancer tumor samples [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P6-07-08.

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

Abstract 3965: Rigorous validation of a clinical circulating tumor DNA assay for cancer molecular profiling

Background: Profiling cell-free circulating tumor DNA (ctDNA) for genomic alterations which drive oncogenesis in patients with cancer promises to provide information important for understanding cancer biology, informing therapy selection when conventional FFPE biopsies are unobtainable and potentially monitoring response to therapy. To allow routine use of blood-based ctDNA molecular profiling with clinical samples we developed and performed analytic validation of an accurate, targeted NGS-based assay. The analytic validation included over 400 samples demonstrating ≥99% sensitivity and ≥99% positive predictive value for base substitutions, indels and rearrangements with limit-of-detection below 1%. Methods: To ensure robust performance, the ctDNA assay was developed as part of an integrated workflow including sample collection, storage and transport, and ctDNA purification, followed by optimized construction of adaptor-ligated sequencing libraries and enrichment by solution hybridization and then sequencing to high depth (Illumina HiSeq). Computational methodologies were developed to enable sensitive and specific detection of base substitutions, indels, genomic rearrangements and high-level amplifications from ctDNA. Accuracy and reproducibility were analytically validated in a CLIA-certified laboratory using reference samples with known alterations (117 cell-line mixtures and synthetic constructs) and 268 clinical ctDNA samples. Many alterations found in clinical ctDNA samples were validated with orthogonal reference methods including a CLIA-validated NGS assay, droplet digital PCR and break-point PCR. Results: The ctDNA assay validation demonstrated ≥99% sensitivity and ≥99% positive predictive value for base substitutions, indels and rearrangements with a limit-of-detection below 1% and robust detection of high-level, focal amplifications when present at adequate tumor fraction. In addition, the assay accurately reports the allele frequency of alterations in the sample. In 48 clinical ctDNA samples, 95 alterations of all classes were 100% confirmed by orthogonal testing. As part of our extensive clinical utility study, we report results comparing alterations from patient-matched ctDNA and FFPE biopsies across more than 200 lung, breast and other cancer samples. Conclusions: Accurate clinical profiling of ctDNA enables detection of genomic alterations in patient plasma samples to provide rationale targeted therapeutic options. Our rigorous analytic validation study demonstrates high-sensitivity detection of alterations present in blood at low frequency with a very low rate of false positives, realizing the potential of ctDNA-based molecular profiling for the management of patients with cancer. This validated assay allows us to embark upon a rigorous investigation of clinical best-practices based on tumor-type specific assessment of matched ctDNA and solid biopsy specimens. Citation Format: Travis A. Clark, Mark Kennedy, Jie He, Geneva Young, Mandy Zhao, Mike Coyne, Virginia Breese, Lauren Young, Shan Zhong, Mark Bailey, Bernard Fendler, Erica Schleifman, Eric Peters, Phil J. Stephens, Geoff A. Otto, Doron Lipson. Rigorous validation of a clinical circulating tumor DNA assay for cancer molecular profiling. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3965.

Abstract 2415: Development of a clinical cell-free DNA assay for cancer molecular profiling

Background: Profiling cell-free circulating tumor DNA (ctDNA) for the genomic alterations which drive oncogenesis in patients with cancer promises to provide information important for understanding cancer biology, informing therapy selection when conventional biopsies are unobtainable and monitoring response to therapy. Facile collection of ctDNA samples could enable recurrent molecular profiling of an individual patient9s disease with lower cost, discomfort and risk as compared to conventional biopsies. To assess the clinical validity of profiling cancer genomics in ctDNA, a highly accurate targeted NGS-based assay was developed and ctDNA results were compared to patient-matched FFPE tumor biopsies characterized by comprehensive genomic profiling using the FoundationOne assay from over 150 patients with lung, breast and other cancer types. Methods: To ensure robust performance, the ctDNA assay was developed as part of an integrated workflow including primary sample collection, storage and transport, and ctDNA purification, followed by optimized construction of adaptor-ligated sequencing libraries and enrichment by solution hybridization and then sequencing to high depth (Illumina HiSeq). Computational methodologies were developed to enable sensitive and specific detection of base substitutions, indels, genomic rearrangements and high-level amplificagtions from ctDNA. Accuracy and reproducibility was assessed using cell-line mixtures designed to simulate the limited DNA inputs and low tumor purity expected in routine clinical samples. Results: The ctDNA assay enabled accurate detection of most genomic alterations in cell-line mixtures. Short variants (base substitutions and indels) and genomic rearrangements in ALK and RET were detected with high sensitivity and specificity at Conclusions: Accurate profiling of ctDNA can enable detection of biologically and clinically relevant genomic alterations in clinical plasma samples. These results demonstrate the potential utility of ctDNA molecular profiling for the management of patients with cancer, but prior to integration into routine practice, extensive rigorous tumor-type specific studies of patient-matched ctDNA and solid biopsy specimens are required. Citation Format: Travis Clark, Mark Kennedy, Geneva Young, Lauren Young, Jie He, Roman Yelensky, Siraj Ali, Geoff Otto, Doron Lipson, Vince Miller, Phil Stephens. Development of a clinical cell-free DNA assay for cancer molecular profiling. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2415. doi:10.1158/1538-7445.AM2015-2415