Abstract: Rapid advancements in cancer genomics and in the development of targeted therapies provide expanding opportunities to use genomic profiling to improve patient outcomes. However, most patients do not have access to clinical genomic profiling platforms, and currently available assays capture a small set of known mutations or translocations tailored to specific tumor types. The spectrum of somatic alterations in leukemia, lymphoma, and myeloma includes substitutions, insertions/deletions (indels), copy number alterations (CNAs) and gene fusions; no current assay captures the different types of alterations in a single clinical genomic test. We developed a novel, CLIA-certified next-generation sequencing-based assay designed to provide targeted assessment of the genomic landscape of hematologic malignancies, including identification of all classes of genomic alterations using archived FFPE, blood and bone marrow aspirate samples with high accuracy in a clinically relevant timeframe. Methods DNA and RNA were successfully extracted from 350/362 (96%) specimens from 319 patients, including 57 FFPE samples, 150 blood samples and 142 bone marrow aspirates. The initial sample cohort included 20 ALL, 83 AML, 53 CLL, 57 DLBCL, 48 MDS, 32 MPN and 57 multiple myeloma samples. Adaptor ligated sequencing libraries were captured by solution hybridization using a custom bait-set targeting 374 cancer-related genes and 24 frequently rearranged genes by DNA-seq, and 258 frequently-rearranged genes by RNA-seq. All captured libraries were sequenced to high depth (Illumina HiSeq) in a CLIA-certified laboratory (Foundation Medicine), averaging 590x for DNA and 〉 20M total pairs for RNA, to enable the sensitive and specific detection of substitutions, indels, CNAs and gene fusions. Results Sufficient tumor content (≥20%) was present in 317/350 (91%) of the samples (289/319 patients), and a total of 885 alterations were identified (3.1 alterations per sample), including 555 base substitutions, 213 indels, 36 splice mutations, 51 CNAs and 36 fusions/rearrangements. The most frequent alterations across all hematologic malignancies included mutations in TP53 (9%), ASXL1, KRAS, NRAS, IDH2, TET2, SF3B1, JAK2, MLL2, DNMT3A, RUNX1, and SRSF2 (2-5% each); FLT3 ITDs (2%); MLL PTDs (1%); homozygous loss of CDKN2A/B (3%); and focal amplification of REL (1%). Rearrangements in BCL2/6, MYC, MLL, MLL2, NOTCH2, ABL1 and ETV6 were identified using DNA and RNA targeted sequencing, demonstrating the ability of this platform to reliably identify gene fusions with immediate clinical relevance. Overall high accuracy of the assay for substitutions, indels and CNAs was previously demonstrated by extensive validation studies achieving 95-99% across alteration types with high specificity (PPV 〉 99%) [Frampton et al, Nat Biotech, in press]. Comparison of detected alterations to previous molecular testing for JAK2, NPM1, IDH2, FLT3 and CEBPA in MPN/AML samples demonstrated 97% sensitivity (33/34) in our ability to identify known mutations in these clinical samples. We identified additional clinically relevant mutations that were not detected using standard clinical assays, including alterations in JAK2, FLT3 and IDH2, which can inform therapeutic decisions. The use of our content rich sequencing platform allowed us to identify clinically actionable mutations in hematologic malignancies, including IDH1/2 mutations in a spectrum of myeloid/lymphoid malignancies, recurrent BRAF mutations in refractory CLL and myeloma, and mutations in the JAK-STAT signaling pathway in diffuse-large B cell lymphoma. These results demonstrate that a targeted sequencing platform which includes a large set of known disease alleles/therapeutic targets can identify mutations with therapeutic relevance in disease contexts where gene-specific assays are not currently performed in the clinical setting. Conclusions We have developed a sensitive, high throughput assay to detect somatic alterations in hundreds of genes known to be deregulated in hematologic malignancies, which can be used for clinical sequencing of frozen/paraffin samples. We demonstrate that targeted DNA and RNA sequencing can be used to identify all classes of genomic alterations in genes known to be therapeutic targets in a broad spectrum of hematologic malignancies. Disclosures: Lipson: Foundation Medicine, Inc: Employment, Equity Ownership. Nahas:Foundation Medicine, Inc: Employment, Equity Ownership. Otto:Foundation Medicine, Inc: Employment, Equity Ownership. Yelensky:Foundation Medicine, Inc: Employment, Equity Ownership. Wang:Foundation Medicine, Inc: Employment, Equity Ownership. He:Foundation Medicine, Inc: Employment, Equity Ownership. Rampal:Foundation Medicine: Consultancy. Brennan:Foundation Medicine, Inc: Employment, Equity Ownership. Brennan:Foundation Medicine, Inc: Employment, Equity Ownership. Young:Foundation Medicine, Inc: Employment, Equity Ownership. Donahue:Foundation Medicine, Inc: Employment, Equity Ownership. Sanford:Foundation Medicine, Inc: Employment, Equity Ownership. Greenbowe:Foundation Medicine, Inc: Employment, Equity Ownership. Frampton:Foundation Medicine, Inc: Employment, Equity Ownership. Fichtenholtz:Foundation Medicine, Inc: Employment, Equity Ownership. Young:Foundation Medicine, Inc: Employment, Equity Ownership. Erlich:Foundation Medicine, Inc: Employment, Equity Ownership. Parker:Foundation Medicine, Inc: Employment, Equity Ownership. Ross:Foundation Medicine, Inc: Employment, Equity Ownership. Stephens:Foundation Medicine, Inc: Employment, Equity Ownership. Miller:Foundation Medicine, Inc: Employment, Equity Ownership. Levine:Foundation Medicine, Inc: Consultancy.

Abstract: 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 & lt; 0.0084) and for all types of urinary tract cancer in COSMIC (P & lt; 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.

Abstract: Focal amplification and activating point mutation of the MET gene are well-characterized oncogenic drivers that confer susceptibility to targeted MET inhibitors. Recurrent somatic splice site alterations at MET exon 14 (METex14) that result in exon skipping and MET activation have been characterized, but their full diversity and prevalence across tumor types are unknown. Here, we report analysis of tumor genomic profiles from 38,028 patients to identify 221 cases with METex14 mutations (0.6%), including 126 distinct sequence variants. METex14 mutations are detected most frequently in lung adenocarcinoma (3%), but also frequently in other lung neoplasms (2.3%), brain glioma (0.4%), and tumors of unknown primary origin (0.4%). Further in vitro studies demonstrate sensitivity to MET inhibitors in cells harboring METex14 alterations. We also report three new patient cases with METex14 alterations in lung or histiocytic sarcoma tumors that showed durable response to two different MET-targeted therapies. The diversity of METex14 mutations indicates that diagnostic testing via comprehensive genomic profiling is necessary for detection in a clinical setting. Significance: Here we report the identification of diverse exon 14 splice site alterations in MET that result in constitutive activity of this receptor and oncogenic transformation in vitro. Patients whose tumors harbored these alterations derived meaningful clinical benefit from MET inhibitors. Collectively, these data support the role of METex14 alterations as drivers of tumorigenesis, and identify a unique subset of patients likely to derive benefit from MET inhibitors. Cancer Discov; 5(8); 850–9. ©2015 AACR. See related commentary by Ma, p. 802. See related article by Paik et al., p. 842. This article is highlighted in the In This Issue feature, p. 783

Abstract: Gastric cancer (GC) is a major global cancer burden and the second most common cause of global cancer-related deaths. The addition of anti-ERBB2 (HER2) targeted therapy to chemotherapy improves survival for ERBB2-amplified advanced GC patients; however, the majority of GC patients do not harbor this alteration and thus cannot benefit from targeted therapy under current practice paradigms. Materials and Methods. Prospective comprehensive genomic profiling of 116 predominantly locally advanced or metastatic (90.0%) gastric cancer cases was performed to identify genomic alterations (GAs) associated with a potential response to targeted therapies approved by the U.S. Food and Drug Administration or targeted therapy-based clinical trials. Results. Overall, 78% of GC cases harbored one clinically relevant GA or more, with the most frequent alterations being found in TP53 (50%), ARID1A (24%), KRAS (16%), CDH1 (15%), CDKN2A (14%), CCND1 (9.5%), ERBB2 (8.5%), PIK3CA (8.6%), MLL2 (6.9%), FGFR2 (6.0%), and MET (6.0%). Receptor tyrosine kinase genomic alterations were detected in 20.6% of cases, primarily ERBB2, FGFR2, and MET amplification, with ERBB2 alterations evenly split between amplifications and base substitutions. Rare BRAF mutations (2.6%) were also observed. One MET-amplified GC patient responded for 5 months to crizotinib, a multitargeted ALK/ROS1/MET inhibitor. Conclusion. Comprehensive genomic profiling of GC identifies clinically relevant GAs that suggest benefit from targeted therapy including MET-amplified GC and ERBB2 base substitutions.

Abstract: Background: Micropapillary urothelial carcinoma of the urinary bladder (MPUC) encompasses approximately 5% of all bladder cancers and comprises approximately 3,000 to 4,000 new cases diagnosed each year in the US. MPUUPC is a highly aggressive form of bladder cancer associated with distant metastases and shortened patient survival. Once MPUC recurs progresses from loco-regional and progresses to metastatic disease, there is no currently no recognized effective treatment. We conducted a genomic analysis of a series of patients with MPUC to characterize the genomic landscape of MUPUC and identify targeted treatment options for patients with this lethal form of urologic malignancy. Methods: DNA was extracted from 40 microns of formalin-fixed paraffin embedded (FFPE) sections from 15 MPUC and 64 non-MPUC. Sequencing to high, uniform coverage was performed on hybridization-captured, adaptor ligated, hybridization capturedion based libraries for for 3,230 exons of 182 cancer-related genes plus 37 introns offrom 14 genes frequently rearranged in cancer to high, uniform coverage and evaluated for all classes of genomic alteration. Results: Extracellular domain mutations of ERBB2 were identified in 6/15 (40%) MPUC including: S310F (4 cases), S310Y (1 case) and R157W (1 case). All 6 cases of MPUC with ERBB2 mutation were negative for ERBB2 amplification which was confirmed by immunohistochemistry (IHC) in the 3 cases where additional tissue was available. In contrast, 6/64 (9.4%) of non-MPUC harbored an ERBB2 alteration: base substitutions (3 cases), amplifications (2 cases) and gene fusion (1 case), which is higher than the 2/159 (1.3%) of protein changing ERBB2 mutations reported in urinary tract cancer in COSMIC. The enrichment of ERBB2 alterations in MPUC compared to non-MPUC is significant inbetween this series (p & lt; 0.0084) and for all types of urinary tract cancer in COSMIC (p & lt; 0.001). All 9 ERBB2 WT MPUC cases harbored at least 1 actionable alteration, including alterations in AKT1, AKT2, CCND1, EGFR, PIK3CA, PIK3R1 and RAF1. Conclusions: Comprehensive genomic profiling of MPUC revealed actionable genomic alterations in all 15 specimens including a high incidence of ERBB2 extracellular domain mutation. We conclude that genomic profiling of MUPUC samples can reveal actionable alterations that can inform potential targeted treatment decisions for the majority of patients. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B122. Citation Format: Kai Wang, Jeff S. Ross, Laurie M. Gay, Rami N. Al-Rohil, Tipu Nazeer, Christine E. Sheehan, Timothy A. Jennings, Geoff A. Otto, Amy Donahue, Jie He, Gary Palmer, Siraj Ali, Michelle Nahas, Geneva Young, Elaine LaBrecque, Garrett Frampton, Rachel Erlich, John A. Curran, Tina Brennan, Sean R. Downing, Roman Yelensky, Doron Lipson, Matthew J. Hawryluk, Vincent A. Miller, Philip J. Stephens. A high frequency of activating extracellular domain ERBB2 (HER2) mutation in micropapillary urothelial carcinoma. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B122.

Abstract: Xenotransplantation of primary AML samples into immunodeficient mice (PDX models) represents a unique opportunity for pre-clinical testing on a group of primary human samples that possess defined genetic lesions. However, given our recent recognition that multiple genetically distinct subclones can exist in AML, there is a risk that there may be selection for sub-clones from the xenotransplanted sample that might not fully represent the patient’s disease. We sought to establish a collection of genetically defined AML samples capable of engraftment in immunodeficient mice. We transplanted 30 AML patient samples; within 150 days (median 91 days) post transplantation 12 samples produced human CD45+ CD33+ CD19- CD3- engraftment in one or multiple NSG mice. Median patient sample amplification in 25 mice was 21 fold. Genomic DNA and total RNA was isolated from 7 AML patient samples (3 diagnostic samples from patients who remain in remission; 2 diagnostic samples from patients who later relapsed, 2 diagnostic samples from patients with refractory disease) and 14 matched xenotransplanted samples (2 mice per patient sample). Adaptor ligated sequencing libraries were captured by solution hybridization using two custom baitsets targeting 374 cancer-related genes and 24 genes frequently rearranged for DNA-seq, and 272 genes frequently rearranged for RNA-seq. All captured libraries were sequenced to high depth (Illumina HiSeq), averaging 〉 499x for DNA and 〉 20,000,000 total pairs for RNA, to enable the sensitive and specific detection of genomic alterations. The mutations found in the 7 diagnostic samples were consistently identified in the 14 engrafted AML samples, but with some cases showing variation in allele frequency between diagnostic and engrafted samples. This finding shows that the human disease that engrafted in mice mimics the genetic makeup of the disease found in patients. We then assessed for allele frequency (AF) changes from diagnostic to xenografted sample as a measure of clonal progression. Clonal progression was defined as emergence of a clone carrying a novel genetic variant in the xenografted sample as compared to the diagnostic patient sample. Five patient samples (from 10 mice) did not show emergence of novel genetic lesions. In this group 2 patients had refractory disease and 3 patients remain in remission. Two patient samples (from 4 mice) demonstrated apparent emergence of novel genetic lesions not detected in diagnostic patient samples. Both of these patients have relapsed since the diagnostic samples were acquired. In the first case, both xenotransplanted mice engrafted with disease carrying NRAS N12S mutation (AF 0.05 and 0.09), which subsequent evaluation revealed to be present below the limit-of-detection (AF 0.004) in the clinical isolate obtained from patient presentation. We are currently conducting the same analysis on the relapsed sample from this patient. In the second case, both mice engrafted with disease carrying PTPN11 E76V (AF 0.03 and 0.0016) while the patient diagnostic sample did not contain any evidence of the alteration at 718x unique sequence coverage. Of note, one xenografted sample had an IDH1 R132C and another had IDH2 R140Q mutation, both of which have previously been shown to play a role in AML pathogenesis. Available AML cell lines do not carry IDH1/2 mutations, making it challenging to test IDH1/2 inhibitors in pre-clinical settings. These xenografted samples offer an opportunity to test such inhibitors. Overall we conclude that the xenotransplanted samples possess the diversity of genetic abnormalities found in diagnostic AML samples and thus can be used to assess efficacy of novel targeted therapies. We would like to further investigate a model in which the absence of clonal progression in xenografted samples would predict a better patient outcome, while emergence of novel clones might indicate an increased potential for relapse. We are currently expanding the study to include more diagnostic, xenotransplanted and relapsed samples to assess the associations between the ability of a sample to engraft in mice with clinical outcome and genetic/epigenetic lesions. Disclosures: Armstrong: Epizyme Inc.: Has consulted for Epizyme Inc. Other.

Abstract: Patients with multiple myeloma (MM) have realized improved survival with the development of multi-drug combinations using immunomodulatory drugs (IMiDs), proteasome inhibitors, and alkylating agents. Nevertheless, all MM patients eventually become refractory to available therapies, underscoring the importance of identifying additional rational therapeutic targets. Recent genomic studies using exome/copy number analysis have demonstrated that, at presentation, multiple myeloma is characterized by a dominant plasma cell clone and a heterogeneous group of subclones, with resistance emerging due to altered clonal dominance driven by therapeutic selective pressure or clonal evolution through the acquisition of additional mutational events. This suggests oncogenic mutations in dominant plasma cell clones in multiply relapsed disease may not only be involved in resistance, but should also be prioritized for further clinical development. Methods We performed a pilot study by sequencing DNA from cryopreserved whole bone marrow aspirate samples obtained pre-treatment from 28 patients with newly diagnosed myeloma (Cohort A) and 27 heavily pre-treated patients enrolled on a phase II clinical study of infusional carfilzomib (NCT01351623), a selective 2nd generation proteasome inhibitor (Cohort B). Genomic DNA and total RNA was isolated from all patient samples. Adaptor ligated sequencing libraries were captured by solution hybridization using two custom baitsets targeting 374 cancer-related genes and 24 genes frequently rearranged for DNA-seq, and 258 genes frequently rearranged for RNA-seq. All captured libraries were sequenced to high depth (Illumina HiSeq), averaging 712X for DNA and 〉 20,000,000 total pairs for RNA, to enable the sensitive and specific detection of genomic alterations. Results Median follow-up for both cohorts was 21 months (26.3m for A; 15.6m for B). Cohort B patients were treated with a median of 5 prior therapies, with 74% refractory to the non-selective 1st generation proteasome inhibitor bortezomib, 70% refractory to IMiD therapy, and 55% refractory to both therapies. 44% had high-risk cytogenetics. Responses to initial therapy in Cohort A demonstrated that 21%, 7%, and 7%, respectively harbored bortezomib--resistant, IMiD-resistant, or double-resistant myeloma at presentation. 28% of cohort A patients had high risk cytogenetics. We obtained high coverage, high quality sequence data for 54/55 cases and examined alteration prevalence in the 35 samples with sufficient plasma cell content. We observed a high frequency of mutations in the MAPK pathway, including mutually exclusive mutations in NRAS and KRAS in 48% of cases and BRAF V600E mutation in 3%. 14% of cases had TET2 frameshift/nonsense mutations or IDH2 mutations, suggesting the DNA hydroxymethylation pathway is targeted by recurrent somatic mutations in MM. Given that MEK/RAF inhibition has demonstrated efficacy in a spectrum of human tumors and that there are emerging data that epigenetic (decitabine and 5-azacytadine) and targeted (IDH2) therapies offer significant benefit in patients with TET2/IDH mutations, these data demonstrate that mutational profiling can identify patients with actionable mutations that can lead to novel therapies, including mechanism-based clinical trials. Taken together, we identified mutations in epigenetic modifiers in 41% of the patients in our cohort, including mutations in TET2/IDH, in chromatin modifying enzymes/scaffolds (ARID1A, ASXL1), and DNA methyltransferases (DNMT3A). Moreover, we identified novel mutations in DNA repair pathways (ATM, FANCA, FANCD2) and in FAT3, suggesting there are novel disease alleles, which require functional investigation for their role in MM pathogenesis. No differences in mutation frequency were found between bortezomib sensitive vs resistant MM cases present in either cohort. We did not identify mutations, which impacted progression free and overall survival in this small sample set. Conclusions We demonstrate next generation sequencing of unsorted bone marrow samples is feasible in MM and can rapidly identify actionable mutations based on genetic profiling of limited clinical isolates. These include the identification of mutations, which can guide therapeutic trials of clinically targeting specific oncogenic pathways (ex, MAPK or TET2/IDH) on an individual patient level. Disclosures: Lesokhin: Janssen Pharmaceuticals, Inc: Research Funding; Bristol-Myers Squibb: Consultancy, Research Funding; Foundation Medicine, inc: Consultancy. Brennan:Foundation Medicine, Inc: Employment. Wang:Foundation Medicine, Inc: Employment. Sanford:Foundation Medicine, Inc: Employment. Brennan:Foundation Medicine, Inc: Employment. Otto:Foundation Medicine, Inc: Employment. Nahas:Foundation Medicine, Inc: Employment. Lipson:Foundation Medicine, Inc: Employment. Stephens:Foundation Medicine, Inc: Employment. Yelensky:Foundation Medicine, Inc: Employment. Miller:Foundation Medicine, Inc: Employment. Levine:Foundation Medicine, Inc: Consultancy. Dogan:Foundation Medicine, Inc: Consultancy.

Abstract: The frequency with which targeted tumor sequencing results will lead to implemented change in care is unclear. Prospective assessment of the feasibility and limitations of using genomic sequencing is critically important. Methods. A prospective clinical study was conducted on 100 patients with diverse-histology, rare, or poor-prognosis cancers to evaluate the clinical actionability of a Clinical Laboratory Improvement Amendments (CLIA)-certified, comprehensive genomic profiling assay (FoundationOne), using formalin-fixed, paraffin-embedded tumors. The primary objectives were to assess utility, feasibility, and limitations of genomic sequencing for genomically guided therapy or other clinical purpose in the setting of a multidisciplinary molecular tumor board. Results. Of the tumors from the 92 patients with sufficient tissue, 88 (96%) had at least one genomic alteration (average 3.6, range 0–10). Commonly altered pathways included p53 (46%), RAS/RAF/MAPK (rat sarcoma; rapidly accelerated fibrosarcoma; mitogen-activated protein kinase) (45%), receptor tyrosine kinases/ligand (44%), PI3K/AKT/mTOR (phosphatidylinositol-4,5-bisphosphate 3-kinase; protein kinase B; mammalian target of rapamycin) (35%), transcription factors/regulators (31%), and cell cycle regulators (30%). Many low frequency but potentially actionable alterations were identified in diverse histologies. Use of comprehensive profiling led to implementable clinical action in 35% of tumors with genomic alterations, including genomically guided therapy, diagnostic modification, and trigger for germline genetic testing. Conclusion. Use of targeted next-generation sequencing in the setting of an institutional molecular tumor board led to implementable clinical action in more than one third of patients with rare and poor-prognosis cancers. Major barriers to implementation of genomically guided therapy were clinical status of the patient and drug access. Early and serial sequencing in the clinical course and expanded access to genomically guided early-phase clinical trials and targeted agents may increase actionability.