Matthew J. Hawryluk

Emergence of Constitutively Active Estrogen Receptor-α Mutations in Pretreated Advanced Estrogen Receptor–Positive Breast Cancer

Purpose: We undertook this study to determine the prevalence of estrogen receptor (ER) α (ESR1) mutations throughout the natural history of hormone-dependent breast cancer and to delineate the functional roles of the most commonly detected alterations. Experimental Design: We studied a total of 249 tumor specimens from 208 patients. The specimens include 134 ER-positive (ER+/HER2−) and, as controls, 115 ER-negative (ER−) tumors. The ER+ samples consist of 58 primary breast cancers and 76 metastatic samples. All tumors were sequenced to high unique coverage using next-generation sequencing targeting the coding sequence of the estrogen receptor and an additional 182 cancer-related genes. Results: Recurring somatic mutations in codons 537 and 538 within the ligand-binding domain of ER were detected in ER+ metastatic disease. Overall, the frequency of these mutations was 12% [9/76; 95% confidence interval (CI), 6%–21%] in metastatic tumors and in a subgroup of patients who received an average of 7 lines of treatment the frequency was 20% (5/25; 95% CI, 7%–41%). These mutations were not detected in primary or treatment-naïve ER+ cancer or in any stage of ER− disease. Functional studies in cell line models demonstrate that these mutations render estrogen receptor constitutive activity and confer partial resistance to currently available endocrine treatments. Conclusions: In this study, we show evidence for the temporal selection of functional ESR1 mutations as potential drivers of endocrine resistance during the progression of ER+ breast cancer. Clin Cancer Res; 20(7); 1757–67. ©2014 AACR.

New Routes to Targeted Therapy of Intrahepatic Cholangiocarcinomas Revealed by Next-Generation Sequencing

BACKGROUND Intrahepatic cholangiocarcinoma (ICC) is a subtype of primary liver cancer that is rarely curable by surgery and is rapidly increasing in incidence. Relapsed ICC has a poor prognosis, and current systemic nontargeted therapies are commonly extrapolated from those used in other gastrointestinal malignancies. We hypothesized that genomic profiling of clinical ICC samples would identify genomic alterations that are linked to targeted therapies and that could facilitate a personalized approach to therapy. METHODS DNA sequencing of hybridization-captured libraries was performed for 3,320 exons of 182 cancer-related genes and 36 introns of 14 genes frequently rearranged in cancer. Sample DNA was isolated from 40 μm of 28 formalin-fixed paraffin-embedded ICC specimens and sequenced to high coverage. RESULTS The most commonly observed alterations were within ARID1A (36%), IDH1/2 (36%), and TP53 (36%) as well as amplification of MCL1 (21%). Twenty cases (71%) harbored at least one potentially actionable alteration, including FGFR2 (14%), KRAS (11%), PTEN (11%), CDKN2A (7%), CDK6 (7%), ERBB3 (7%), MET (7%), NRAS (7%), BRCA1 (4%), BRCA2 (4%), NF1 (4%), PIK3CA (4%), PTCH1 (4%), and TSC1 (4%). Four (14%) of the ICC cases featured novel gene fusions involving the tyrosine kinases FGFR2 and NTRK1 (FGFR2-KIAA1598, FGFR2-BICC1, FGFR2-TACC3, and RABGAP1L-NTRK1). CONCLUSION Two thirds of patients in this study harbored genomic alterations that are associated with targeted therapies and that have the potential to personalize therapy selection for to individual patients.

Advanced urothelial carcinoma: next-generation sequencing reveals diverse genomic alterations and targets of therapy

Although urothelial carcinoma (UC) of the urinary bladder generally portends a favorable prognosis, metastatic tumors often follow an aggressive clinical course. DNA was extracted from 40 μm of formalin-fixed, paraffin-embedded (FFPE) sections from 35 stage IV UCs that had relapsed and progressed after primary surgery and conventional chemotherapy. Next-generation sequencing (NGS) was performed on hybridization-captured, adaptor ligation-based libraries for 3320 exons of 182 cancer-related genes plus 37 introns from 14 genes frequently rearranged in cancer to at an average sequencing depth of 1164 × and evaluated for all classes of genomic alterations (GAs). Actionable GAs were defined as those impacting the selection of targeted anticancer therapies on the market or in registered clinical trials. A total of 139 GAs were identified, with an average of 4.0 GAs per tumor (range 0–10), of which 78 (56%) were considered actionable, with an average of 2.2 per tumor (range 0–7). Twenty-nine (83%) cases harbored at least one actionable GA including: PIK3CA (9 cases; 26%); CDKN2A/B (8 cases; 23%); CCND1 (5 cases; 14%); FGFR1 (5 cases; 14%); CCND3 (4 cases; 11%); FGFR3 (4 cases; 11%); MCL1 (4 cases; 11%); MDM2 (4 cases; 11%); EGFR (2 cases, 6%); ERBB2 (HER2/neu) (2 cases, 6%); NF1 (2 cases, 6%) and TSC1 (2 cases, 6%). Notable additional alterations included TP53 (19 cases, 54%) and RB1 (6 cases; 17%). Genes involved in chromatin modification were altered by nonsense mutation, splice site mutation or frameshift indel in a mutually exclusive manner in nearly half of all cases including KDM6A (10 cases; 29%) and ARID1A (7 cases; 20%). Comprehensive NGS of 35 UCs of the bladder revealed a diverse spectrum of actionable GAs in 83% of cases, which has the potential to inform treatment decisions for patients with relapsed and metastatic disease.

Clinical next‐generation sequencing successfully applied to fine‐needle aspirations of pulmonary and pancreatic neoplasms

Next‐generation sequencing was performed on pulmonary and pancreatic fine‐needle aspirations (FNAs) and on paired FNAs and resected primary tumors from the same patient.

A High Frequency of Activating Extracellular Domain ERBB2 (HER2) mutation in Micropapillary Urothelial Carcinoma

Purpose: Micropapillary urothelial carcinoma (MPUC) is a rare and aggressive form of bladder cancer. We conducted genomic analyses [next-generation sequencing (NGS)] of MPUC and non-micropapillary urothelial bladder carcinomas (non-MPUC) to characterize the genomic landscape and identify targeted treatment options. Experimental Design: DNA was extracted from 40 μm of formalin-fixed paraffin-embedded sections from 15 MPUC and 64 non-MPUC tumors. Sequencing (NGS) was performed on hybridization-captured, adaptor ligation–based libraries to high coverage for 3,230 exons of 182 cancer-related genes plus 37 introns from 14 genes frequently rearranged in cancer. The results were evaluated for all classes of genomic alteration. Results: Mutations in the extracellular domain of ERBB2 were identified in 6 of 15 (40%) of MPUC: S310F (four cases), S310Y (one case), and R157W (one case). All six cases of MPUC with ERBB2 mutation were negative for ERBB2 amplification and Erbb2 overexpression. In contrast, 6 of 64 (9.4%) non-MPUC harbored an ERBB2 alteration, including base substitution (three cases), amplification (two cases), and gene fusion (one case), which is higher than the 2 of 159 (1.3%) protein-changing ERBB2 mutations reported for urinary tract cancer in COSMIC. The enrichment of ERBB2 alterations in MPUC compared with non-MPUC is significant both between this series (P < 0.0084) and for all types of urinary tract cancer in COSMIC (P < 0.001). Conclusions: NGS of MPUC revealed a high incidence of mutation in the extracellular domain of ERBB2, a gene for which there are five approved targeted therapies. NGS can identify genomic alteration, which inform treatment options for the majority of MPUC patients. Clin Cancer Res; 20(1); 68–75. ©2013 AACR.

RICTOR amplification defines a novel subset of patients with lung cancer who may benefit from treatment with mTORC1/2 inhibitors

UNLABELLED We identified amplification of RICTOR, a key component of the mTOR complex 2 (mTORC2), as the sole actionable genomic alteration in an 18-year-old never-smoker with lung adenocarcinoma. Amplification of RICTOR occurs in 13% of lung cancers (1,016 cases) in The Cancer Genome Atlas and at a similar frequency in an independent cohort of 1,070 patients identified by genomic profiling. In the latter series, 11% of cases harbored RICTOR amplification as the only relevant genomic alteration. Its oncogenic roles were suggested by decreased lung cancer cell growth both in vitro and in vivo with RICTOR ablation, and the transforming capacity of RICTOR in a Ba/F3-cell system. The mTORC1/2 inhibitors were significantly more active against RICTOR-amplified lung cancer cells as compared with other agents targeting the PI3K-AKT-mTOR pathway. Moreover, an association between RICTOR amplification and sensitivities to mTORC1/2 inhibitors was observed. The index patient has been treated with mTORC1/2 inhibitors that led to tumor stabilization for more than 18 months. SIGNIFICANCE RICTOR amplification may define a novel and unique molecular subset of patients with lung cancer who may benefit from treatment with mTORC1/2 inhibitors.

Biological and clinical evidence for somatic mutations in BRCA1 and BRCA2 as predictive markers for olaparib response in high-grade serous ovarian cancers in the maintenance setting

To gain a better understanding of the role of somatic mutations in olaparib response, next-generation sequencing (NGS) of BRCA1 and BRCA2 was performed as part of a planned retrospective analysis of tumors from a randomized, double-blind, Phase II trial (Study 19; D0810C00019; NCT00753545) in 265 patients with platinum-sensitive high-grade serous ovarian cancer. BRCA1/2 loss-of-function mutations were found in 55% (114/209) of tumors, were mutually exclusive, and demonstrated high concordance with Sanger-sequenced germline mutations in matched blood samples, confirming the accuracy (97%) of tumor BRCA1/2 NGS testing. Additionally, NGS identified somatic mutations absent from germline testing in 10% (20/209) of the patients. Somatic mutations had >80% biallelic inactivation frequency and were predominantly clonal, suggesting that BRCA1/2 loss occurs early in the development of these cancers. Clinical outcomes between placebo- and olaparib-treated patients with somatic BRCA1/2 mutations were similar to those with germline BRCA1/2 mutations, indicating that patients with somatic BRCA1/2 mutations benefit from treatment with olaparib.

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 4670: A novel companion diagnostic predicts response to the PARP inhibitor rucaparib in ovarian cancer

Background: Genomic studies suggest that ∼50% of high-grade serous ovarian cancers (OC) have homologous recombination deficiency (HRD). Germline BRCA1/2 mutations (gBRCAmut) are expected to account for 1/3 of HRD in OC, and identification of non-gBRCAmut HRD tumors likely to respond to PARP inhibitors (PARPi) remains a challenge. Using comprehensive next generation sequencing (NGS)-based tumor genomic profiling, we developed a companion diagnostic HRD assay to predict sensitivity to the PARP inhibitor rucaparib by combining tumor BRCA1/2 status (germline and somatic) and genomic loss of heterozygosity (LOH). The HRD assay is being validated in a Phase 2 study (ARIEL2) and will be prospectively applied to the primary analysis of the ongoing Phase 3 study (ARIEL3) of rucaparib. Methods: The HRD assay uses 50-200ng of DNA from tumor FFPE specimens, which undergoes sequencing library construction and hybrid-capture of all coding exons from 100s of cancer-related genes. Libraries are sequenced to high, uniform depth (>500× unique coverage, Illumina® HiSeq) and data are processed by a customized pipeline that accurately detects all classes of genomic alterations, including BRCA1/2 base substitutions, indels, and homozygous deletions. Genomic LOH is assessed by a CGH-like analysis of sequencing coverage and >3,500 genome-wide SNPs and a tumor is classified as HRD with either BRCA1/2 alteration or high genomic LOH (LOH+). Somatic/germline status of discovered BRCA1/2 alterations is assessed by a previously-presented computational approach (“SGZ”, AACR 2014 abstract #1893), and verified against medical records where available. ARIEL2 is an ongoing single-arm (n = 180), open-label study of rucaparib in recurrent, platinum-sensitive OC patients. The primary objective is to evaluate clinical activity of rucaparib among 3 prospectively defined subgroups: tumor BRCAmut, BRCAwt/LOH+ (“BRCAness”) and BRCAwt/LOH-. Response is determined by RECIST and/or GCIG-CA125 criteria. Results: The HRD assay was performed on tumors from 121 patients, of whom 25% were found to be BRCA mutant (17 germline/12 somatic), 42% had the BRCAness signature (BRCAwt/LOH+), and 33% were biomarker negative (BRCAwt/LOH-). Efficacy data available for 61 patients revealed objective response rates (combined RECIST/CA125 criteria) at 70%, 40% and 8%, respectively. Responses were observed for all classes of genomic alterations, and in gBRCAmut and non-gBRCAmut tumors. Conclusions: Preliminary clinical data indicates that the HRD assay identifies OC patients likely to respond to rucaparib and highlights the potential for innovative companion diagnostics enabled by comprehensive genomic profiling based on NGS. Citation Format: James Sun, Iain McNeish, Robert L. Coleman, Amit Oza, Clare Scott, David M. O9Malley, Kevin K. Lin, Christine Burns, Christine Vietz, Philip J. Stephens, Murtaza Mehdi, Matthew Hawryluk, Heidi Giordano, Mitch Raponi, Lindsey Rolfe, Jeff Isaacson, Vincent A. Miller, Andrew Allen, Elizabeth Swisher, Roman Yelensky. A novel companion diagnostic predicts response to the PARP inhibitor rucaparib in ovarian cancer. [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 4670. doi:10.1158/1538-7445.AM2015-4670

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.