Emily White

Development and validation of a clinical cancer genomic profiling test based on massively parallel DNA sequencing

As more clinically relevant cancer genes are identified, comprehensive diagnostic approaches are needed to match patients to therapies, raising the challenge of optimization and analytical validation of assays that interrogate millions of bases of cancer genomes altered by multiple mechanisms. Here we describe a test based on massively parallel DNA sequencing to characterize base substitutions, short insertions and deletions (indels), copy number alterations and selected fusions across 287 cancer-related genes from routine formalin-fixed and paraffin-embedded (FFPE) clinical specimens. We implemented a practical validation strategy with reference samples of pooled cell lines that model key determinants of accuracy, including mutant allele frequency, indel length and amplitude of copy change. Test sensitivity achieved was 95–99% across alteration types, with high specificity (positive predictive value >99%). We confirmed accuracy using 249 FFPE cancer specimens characterized by established assays. Application of the test to 2,221 clinical cases revealed clinically actionable alterations in 76% of tumors, three times the number of actionable alterations detected by current diagnostic tests.

Identification of new ALK and RET gene fusions from colorectal and lung cancer biopsies

Applying a next-generation sequencing assay targeting 145 cancer-relevant genes in 40 colorectal cancer and 24 non–small cell lung cancer formalin-fixed paraffin-embedded tissue specimens identified at least one clinically relevant genomic alteration in 59% of the samples and revealed two gene fusions, C2orf44-ALK in a colorectal cancer sample and KIF5B-RET in a lung adenocarcinoma. Further screening of 561 lung adenocarcinomas identified 11 additional tumors with KIF5B-RET gene fusions (2.0%; 95% CI 0.8–3.1%). Cells expressing oncogenic KIF5B-RET are sensitive to multi-kinase inhibitors that inhibit RET.

Integrated genomic DNA/RNA profiling of hematologic malignancies in the clinical setting

The spectrum of somatic alterations in hematologic malignancies includes substitutions, insertions/deletions (indels), copy number alterations (CNAs), and a wide range of gene fusions; no current clinically available single assay captures the different types of alterations. We developed a novel next-generation sequencing-based assay to identify all classes of genomic alterations using archived formalin-fixed paraffin-embedded blood and bone marrow samples with high accuracy in a clinically relevant time frame, which is performed in our Clinical Laboratory Improvement Amendments-certified College of American Pathologists-accredited laboratory. Targeted capture of DNA/RNA and next-generation sequencing reliably identifies substitutions, indels, CNAs, and gene fusions, with similar accuracy to lower-throughput assays that focus on specific genes and types of genomic alterations. Profiling of 3696 samples identified recurrent somatic alterations that impact diagnosis, prognosis, and therapy selection. This comprehensive genomic profiling approach has proved effective in detecting all types of genomic alterations, including fusion transcripts, which increases the ability to identify clinically relevant genomic alterations with therapeutic relevance.

Comprehensive Genomic Profiling of Carcinoma of Unknown Primary Site: New Routes to Targeted Therapies

IMPORTANCE For carcinoma of unknown primary site (CUP), determining the primary tumor site may be uninformative and often does not improve outcome. OBJECTIVE To discover opportunities for targeted therapies in patients with CUP not currently searched for in routine practice. DESIGN, SETTING, AND PARTICIPANTS Comprehensive genomic profiling on 200 CUP formalin-fixed paraffin-embedded specimens (mean, 756× coverage) using the hybrid-capture-based FoundationOne assay at academic and community oncology clinics. MAIN OUTCOMES AND MEASURES Presence of targetable genomic alterations (GAs) in CUP and responses to targeted therapies. RESULTS There were 125 adenocarcinomas of unknown primary site (ACUPs) and 75 carcinomas of unknown primary site without features of adenocarcinoma (non-ACUPs). At least 1 GA was found in 192 (96%) of CUP specimens, with a mean (SD) of 4.2 (2.8) GAs per tumor. The most frequent GAs were in TP53 (110 [55%]), KRAS (40 [20%]), CDKN2A (37 [19%]), MYC (23 [12%]), ARID1A (21 [11%]), MCL1 (19 [10%]), PIK3CA (17 [9%]), ERBB2 (16 [8%]), PTEN (14 [7%]), EGFR (12 [6%]), SMAD4 (13 [7%]), STK11 (13 [7%]), SMARCA4 (12 [6%]), RB1 (12 [6%]), RICTOR (12 [6%]), MLL2 (12 [6%]), BRAF (11 [6%]), and BRCA2 (11 [6%]). One or more potentially targetable GAs were identified in 169 of 200 (85%) CUP specimens. Mutations or amplifications of ERBB2 were more frequent in ACUPs (13 [10%]) than in non-ACUPs (3 [4%]). Alterations of EGFR (10 [8%] vs 2 [3%]) and BRAF (8 [6%] vs 3 [4%]) were more common in ACUPs than in non-ACUPs. Strikingly, clinically relevant alterations in the receptor tyrosine kinase (RTK)/Ras signaling pathway including alterations in ALK, ARAF, BRAF, EGFR, FGFR1, FGFR2, KIT, KRAS, MAP2K1, MET, NF1, NF2, NRAS, RAF1, RET, and ROS1 were found in 90 (72%) ACUPs but in only 29 (39%) non-ACUPs (P < .001). CONCLUSIONS AND RELEVANCE Almost all CUP samples harbored at least 1 clinically relevant GA with potential to influence and personalize therapy. The ACUP tumors were more frequently driven by GAs in the highly druggable RTK/Ras/mitogen-activated protein kinase (MAPK) signaling pathway than the non-ACUP tumors. Comprehensive genomic profiling can identify novel treatment paradigms to address the limited options and poor prognoses of patients with CUP.

Abstract A28: Clinical next-generation sequencing (NGS) reveals genomic alterations (GAs) to guide targeted therapy in advanced neuroblastoma patients

Introduction: High-risk neuroblastoma patients have a survival rate below 50% despite dose-intensive chemoradiotherapy. Treatment using molecularly targeted therapy could more effectively manage patients with less toxicity, but would be best deployed through identification of GAs that suggest responsiveness to such therapies. Recent work from the TARGET initiative (Pugh et al., Nature Genetics 2013) demonstrates a near 10% frequency of ALK GAs in high-risk neuroblastoma patients; such patients could benefit from crizotinib or similar agents targeting the ALK kinase. However, the majority of high-risk neuroblastoma patients still lack identifiable options for targeted therapy. We reviewed the GAs in 17 advanced, high-risk neuroblastoma patients who underwent prospective genomic profiling by clinical NGS to identify actionable alterations that might allow successful trials of targeted therapy. Methods: Diagnostic genomic profiling was performed to characterize all classes of GAs (base substitutions, small insertions/deletions, copy number alterations, and rearrangements) on primary tumors or metastatic specimens, either pre- or post-chemotherapy, of 17 advanced neuroblastoma patients. For each specimen, 3,320 exons of 182 cancer-related genes and selected introns of 14 frequently rearranged genes or 3769 exons of 236 cancer-related genes and selected introns of 19 frequently rearranged genes (earlier and current version of assay, respectively) were sequenced to a minimum coverage depth of 250x in a CLIA-certified, CAP-accredited lab (Foundation Medicine, Cambridge, MA). Actionable GAs were defined as those for which there were FDA-approved agents and/or agents being evaluated in clinical trials. Results: The patient population had a median age of 4.6 yrs (range 2 - 18 yrs), 13 were males, and 16 had Stage 4 disease (one Stage 3). Specimens were sequenced to an average depth of 861x, and GAs were present in 100% (17/17) of cases. These 17 cases harbored 27 GAs (1.6 alterations per tumor; range 1 to 5). Ninety-four percent of cases harbored at least one actionable GA, with a mean of 1.6 actionable GAs per tumor (range 1 to 5). Previously described GAs in neuroblastoma were present in this series, such as GAs in ALK (5 cases; 29%), MYCN (5 cases; 29%), and ATRX (2 cases; 12%), which occurred in a largely mutually exclusive fashion. Alterations in ALK were predominantly base substitutions (80%) and included F1174L and R1275Q at frequencies of 40% and 20%, respectively. One ALK alteration was a novel fusion gene predicted to be active in vivo and potentially responsive to crizotinib. Another recurrent actionable alteration is potentially targetable by FGFR inhibitors, as two neuroblastoma cases contained the activating base substitution N546K in FGFR1. Conclusions: Profiling the tumor genomes of 17 high-risk neuroblastoma patients led to the identification of actionable GAs in a high proportion of patients including alterations previously unseen in neuroblastoma. Specifically, novel fusions of ALK and activating alterations in FGFR1 would not be detected by current molecular diagnostic assays employed in neuroblastoma but offer possible immediate benefit from targeted treatment. Such findings suggest the value of rebiopsying progressive or recurrent high-risk neuroblastoma to identify unforeseen avenues for treatment with targeted therapy. Citation Format: Siraj M. Ali, Matthew J. Hawryluk, Kai Wang, Juliann Chmielecki, Gary A. Palmer, Lazaro Garcia, Emily White, Roman Yelensky, Philip J. Stephens, Jeffrey S. Ross, John M. Maris, Vince A. Miller. Clinical next-generation sequencing (NGS) reveals genomic alterations (GAs) to guide targeted therapy in advanced neuroblastoma patients. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr A28.

Abstract A23: Comprehensive next-generation sequencing-based genomic profiling identifies actionable genomic alterations in diverse pediatric tumor types: The Foundation Medicine (FMI) experience

Background: Solid tumor oncology is amidst a paradigm shift with the advent and increasingly successful utilization of targeted therapies that inhibit specific genomic alterations driving an individual patient9s disease. Unfortunately, many pediatric tumors lack approved targeted therapies, and routine genomic profiling of pediatric tumors has yet to be applied in a widespread manner. More comprehensive testing platforms are required to determine the landscape of genomic alterations in pediatric solid tumors and thereby broaden targeted treatment options. We have developed a solid tumor next-generation sequencing (NGS)-based diagnostic test, optimized for routine clinical FFPE specimens including needle biopsies and malignant effusions, and report here on 193 pediatric patients9 tumors analyzed to date in our CLIA-certified and CAP-accredited laboratory. Methods: Hybridization capture of 3,320 exons from 182 cancer-related genes and 37 introns of 14 genes commonly rearranged in cancer (previous version of the test) or 3,769 exons from 236 cancer-related genes and 47 introns of 19 genes commonly rearranged in cancer (current version of the test) was applied to ≥ 50ng of DNA extracted from over 190 pediatric FFPE tumor specimens and sequenced to high, uniform coverage. Genomic alterations (base substitutions, small indels, rearrangements, copy number alterations) were determined and then reported for these patient samples. Results: Successful profiles were generated from 193 pediatric patient specimens from individuals ≤21 years old at the time of biopsy. A total of 361 genomic alterations were identified, with 145/193 (75%, 95% CI 68%-81%) of cases harboring at least one genomic alteration, for an average of 2.5 genomic alterations per case (range 0-14). The number of genomic alterations varied widely depending on tumor type (average alterations per sample): 4 colon cancer samples (9.0), 11 bone sarcoma samples (3.7), 13 leukemia samples (3.1), 23 brain cancer samples (2.4), 27 soft tissue sarcoma samples (2.2), 6 liver samples (2.0), 26 neuroblastoma samples (1.7), 6 lung cancer samples (1.2), 5 kidney cancer samples (1.2), and 24 other tumor types. These alterations included 146 base substitutions, 54 indels, 133 copy number alterations, and 28 rearrangements. This corresponded to alterations within 102 genes with recurrent alterations observed in 61 genes. We identified 249 actionable alterations with 109/193 (56%) of these pediatric patients found to have at least one actionable alteration. Seventy one percent (176/249) of the actionable genomic alterations would not be detected by available tumor type specific tests or hotspot panels. This approach has led to novel insights into pediatric cancers including: novel potentially druggable kinase gene fusions (e.g. ALK, RET) and non-fusion alterations in known drug targets (e.g. EGFR, BRAF, ALK, MET, PTCH1, PIK3CA, KIT, PDGFRA, FGFR1). Conclusions: Comprehensive NGS-based genomic profiling identified alterations in 75% of 193 unselected pediatric cancer clinical cases; and 56% of these patients (N=109) were found to have at least one therapeutically actionable alteration. Widespread deployment of this approach may provide treatment options for pediatric cancer patients. Citation Format: Matthew J. Hawryluk, Kai Wang, Juliann Chmielecki, Siraj M. Ali, Gary Palmer, Lazaro Garcia, Emily White, Roman Yelensky, Philip J. Stephens, Jeffrey S. Ross, Vincent A. Miller. Comprehensive next-generation sequencing-based genomic profiling identifies actionable genomic alterations in diverse pediatric tumor types: The Foundation Medicine (FMI) experience. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr A23.

Abstract 4554: QPCR and sequence analysis of DNA template from a microfluidic CTC isolation platform

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Sequence analysis and quantitative allele specific PCR (QPCR) methods permit genetic profiling of cancer for targeted therapeutic selection; such personalized treatments have been associated with improved outcomes in cancer. Circulating tumor cells (CTC) offer a minimally-invasive opportunity for serial patient sampling, and potentially a means of tracking the molecular evolution that underlies the behavior and response phenotype of the disease including potential therapeutic response markers. Acquiring these types of patient profiles requires a platform and workflow providing reliable detection and recovery of small numbers of mutation-bearing CTC from a blood sample. Availability of such an enabling platform is a necessary prerequisite to the clinical correlation studies needed to demonstrate the utility of mutation-bearing CTC to patient care. We have successfully purified CTCs and converted them into DNA template of sufficient purity and quality to support multiple non-overlapping advanced molecular characterizations. Beginning with whole human blood spiked with defined numbers of cultured cancer cells as surrogates for CTCs, cells were successfully fluid-phase labeled using an anti-EpCAM antibody ferrofluid. Using a proprietary microfluidic sheath flow technology, EpCAM positive, cytokeratin staining cells were selected from 2 to 4 ml of labeled blood. This method produced sufficient DNA template for multiple analyses per patient sample. QPCR analysis of these templates demonstrated reproducible detection of fewer than 1% target cells in a background of non-target cells, allowing detection of the KRAS G12S mutation from as few as 5 recaptured cancer cells. The same DNA templates were then used for hybrid capture and next-generation sequencing of a panel of more than 200 cancer-related genes. This sequencing platform was able to detect multiple somatic mutations in genomic DNA templates produced from samples containing as few as 10 cancer cells per milliliter of blood. Together these data provide initial proof-of-concept for a system capable of detecting and characterizing mutations across any specified set of genes within purified CTC populations. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4554. doi:1538-7445.AM2012-4554