ALBERT

Description: Background: A variety of next-generation sequencing methods have been used to investigate the genomic landscape of primary lymphoma patient samples, including whole-genome, whole-exome, and RNA sequencing. In diffuse large B cell lymphoma (DLBCL), sequencing studies identified numerous genomic alterations (GAs) of potential clinical relevance, but the distribution and frequency of GAs have not been precisely determined. Discrepancies in existing data likely arise from variability in types of specimens examined, sequencing technologies employed, and depth of coverage utilized to identify GAs. In this study, we performed comprehensive DNA/RNA sequencing of genes known to be important across the spectrum of hematologic malignancies in order to determine the nature and prevalence of GAs with potential diagnostic, prognostic, or therapeutic implications in a cohort of 112 well-annotated clinical DLBCL cases. Methods: We performed hybridization capture of 405 cancer-related genes and 31 genes commonly rearranged in cancer (FoundationOne Heme) and 265 frequently rearranged genes for RNA-seq applied to ³50ng of DNA and sequenced to high, uniform coverage. Genomic alterations, including short variants (small indels and base substitutions), rearrangements, and copy number alterations, were determined. All captured libraries were sequenced to high depth (Illumina HiSeq), averaging 〉500x for DNA and 〉20,000,000 total pairs for RNA, to enable the sensitive and specific detection of GAs. Significant non-synonomous variants were identified as mutations from the COSMIC database, amplifications of established oncogenes, or homozygous deletions and/or clear loss-of-function mutations of known tumor suppressors. Results: GAs with diagnostic, prognostic, or therapeutic relevance were identified in 96% of cases, with a median of 5 (range 0 to 12) alterations per sample. Figure 1 shows the frequencies of different GAs. De novo DLBCL tended to exhibit fewer GAs (median 4) than relapsed/refractory (median 6) or transformed disease (median 6) (p=0.179; Wilcoxon rank sum). Compared with reported frequencies ranging from 5 to 44%, we detected alterations in CREBBP/EP300 in 21% of cases, with CREBBP mutations preferentially found in germinal-center B cell-like (GCB) compared to non-GCB DLBCL (26% vs. 8%; p=0.02; Pearson's chi-squared). Although previously described only in Burkitt lymphoma, we identified mutations in ID3 (L70P, P98R, Q100P) and TCF3 (N551K) in GCB-phenotype DLBCL cases with aggressive pathologic features and no MYC expression by IHC. Additional novel findings included alterations of genes involved in cellular metabolism in 18% of cases, including one IDH2 R172M mutation and two cases with SDHA L649fs* truncating mutations. We also identified several mutations more commonly found in solid tumors and leukemias, including BRAF V600E and KRAS G13D. With respect to genomic rearrangements, combined DNA/RNA capture and sequencing detected translocations/fusions in MYC, BCL2, and BCL6, which were concordant with cytogenetics/FISH analysis. We also identified rearrangements involving TBXL1XR1-P63, NOTCH2, SOCS1, and ETV6. Copy number alterations were detected in 26 different genes, including amplifications of CD274/PDCD1LG2 (PD-L1/PD-L2) (n=4) that were restricted to non-GCB cases. Alterations in TP53 were more frequently observed in transformed (42%) and relapsed/refractory (26%) compared to de novo DLBCL (12%; p=0.007; Pearson's chi-squared). TP53 mutations predicted for lack of response to chemotherapy, with chemo-refractory disease occurring in 48% of TP53-mutant patients compared to 10% of patients with intact TP53 (p=0.0004; Fisher's exact). Truncating mutations or deletions of RB1, albeit rare (5% of cases), were the most significant negative prognostic factor and were associated with therapeutic resistance and poor overall survival. Conclusions: Our results demonstrate the utility of comprehensive combined DNA/RNA next-generation sequencing as a promising method to identify clinically relevant GAs in clinical lymphoma specimens. This streamlined approach has the potential to combine multiple molecular and cytogenetic tests into a single platform. Future efforts will be directed at incorporating this approach both retrospectively and prospectively into clinical trials to identify predictive biomarkers to guide therapeutic decisions. Figure 1 Figure 1. Disclosures Intlekofer: Foundation Medicine, Inc: Consultancy. Levine:Foundation Medicine, Inc: Consultancy. Zelenetz:Foundation Medicine, Inc: Consultancy. Dogan:Foundation Medicine, Inc: Consultancy. Palomba:Foundation Medicine, Inc: Consultancy. van den Brink:Foundation Medicine, Inc: Consultancy. Brennan:Foundation Medicine, Inc: Employment. Young:Foundation Medicine, Inc: Employment. He:Foundation Medicine: Employment. Nahas:Foundation Medicine, Inc: Employment. Yelensky:Foundation Medicine, Inc: Employment. Otto:Foundation Medicine, Inc: Employment. Lipson:Foundation Medicine, Inc: Employment. Stephens:Foundation Medicine, Inc: Employment. Miller:Foundation Medicine, Inc: Employment. Younes:Foundation Medicine, Inc: Consultancy.

Description: Background 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.

Description: Background 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.

Description: Whole genome and exome sequencing studies have identified numerous genomic alterations in DLBCL, but these methods have limited applicability for the clinical care of lymphoma patients due to cost, specific tissue requirements, and laborious bioinformatic analysis. FoundationOne-Heme (FOH) is a novel next-generation sequencing platform designed to provide targeted assessment of the genomic landscape of hematologic malignancies, including identification of mutations within specific genes, copy number changes, and translocations. FOH can be performed on small quantities of formalin-fixed paraffin-embedded (FFPE) tissue, detect rare variants due to extensive depth of sequencing coverage, and rapidly provide results via streamlined bioinformatic interpretation. Here we report the first experience using this novel platform to evaluate the genetic landscape of DLBCL. Genomic DNA and total RNA were isolated from FFPE tissue on a cohort of 53 cases of DLBCL, including de novo (n=30), relapsed/refractory (n=12), and large cell transformation from low-grade lymphoma (n=11). The cohort included 25 cases with combined MYC and BCL2 overexpression by IHC (criteria for positivity: 〉40% MYC, 〉70% BCL2), of which only one had a known translocation involving MYC. Adaptor ligated sequencing libraries were captured by solution hybridization using two custom bait sets targeting 374 cancer-related genes and 24 genes frequently rearranged for DNA-seq, and 258 frequently-rearranged genes for RNA-seq. All captured libraries were sequenced to high depth (Illumina HiSeq), averaging 〉658x for DNA and 〉20,000,000 total pairs for RNA, to enable the sensitive and specific detection of genomic alterations. Significant non-synonomous variants were identified as mutations from the COSMIC database, amplifications of established oncogenes, or homozygous deletions and/or clear loss-of-function mutations of known tumor suppressors. The DNA sequencing component of FOH detected translocations in BCL2, BCL6, and MYC, while the RNA sequencing component detected fusion transcripts involving BLC6 and MYC, in agreement with independent cytogenetic analysis via karyotype and FISH where available. The assay detected copy number alterations of 44 different genes, most commonly amplification of REL (15%) or loss of CDKN2A/CDKN2B (17%). The most frequent alterations of known significance are detailed in Figure 1. The most commonly altered gene was CDKN2A, exhibiting either homozygous deletion or loss of function mutation in 28% of cases. Chromatin modifying factors (e.g. MLL2, CREBBP, EZH2) represented the most frequently altered biologic category with alterations occurring in 〉50% of cases. Recurrent alterations in components of the Notch pathway (NOTCH1/2/4, FBXW7, SPEN), each predicted to activate the pathway, were identified in 23% of cases. Cell-of-origin was determined as per the Hans model using IHC for CD10, BCL6, and IRF4/MUM1; CD79B mutations were detected exclusively in non-GCB and EZH2 mutations were found exclusively in GCB-phenotype cases. Furthermore, IHC MYC+/BCL2+ de novo DLBCL cases (n=11) exhibited more frequent hypermutation of PIM1 (46%) compared with the 19 cases of IHC MYC-/BCL2- de novo DLBCL (11%). When comparing the various clinical categories, we found that mutations in tumor suppressors were significantly more common in relapsed/refractory than de novo DLBCL (47% vs 75%, p=0.02). Alterations in TP53 were most frequently observed in transformed lymphoma (55%). Our results demonstrate the feasibility of using a targeted next-generation sequencing platform on FFPE clinical specimens from patients with DLBCL as a means of providing an integrated analysis of gene mutations, copy number alterations, and translocations. This streamlined approach combines multiple molecular and cytogenetic tests into a single platform and uses a small amount of tissue to perform a multifaceted assessment of genomic alterations with potential diagnostic, prognostic, and therapeutic implications. Future efforts will be directed at analyzing additional cases of DLBCL to better establish the biologic and clinical significance of the observed genetic alterations, and to prospectively incorporate this novel platform to select patients for mechanism-based targeted therapy. Disclosures: Intlekofer: Foundation Medicine, Inc: Consultancy. Levine:Foundation Medicine, Inc: Consultancy. Zelenetz:Foundation Medicine, Inc: Consultancy. Palomba:Foundation Medicine, Inc: Consultancy. van den Brink:Foundation Medicine, Inc: Consultancy. Brennan:Foundation Medicine, Inc: Employment. Young:Foundation Medicine, Inc: Employment. He:Foundation Medicine, Inc: Employment. Nahas:Foundation Medicine, Inc: Employment. Yelensky:Foundation Medicine, Inc: Employment. Otto:Foundation Medicine, Inc: Employment. Lipson:Foundation Medicine, Inc: Employment. Stephens:Foundation Medicine, Inc: Employment. Miller:Foundation Medicine, Inc: Employment. Younes:Foundation Medicine, Inc: Consultancy.

Description: Background: The clinical utility of mutation profiling in hematologic malignancies requires robust detection of all classes of genomic alterations in a single analysis across different tumor types. We developed a novel, hybrid capture-based NGS assay (FoundationOne® Heme) designed to comprehensively assess the genomic landscape of hematologic malignancies from archived FFPE, blood and bone marrow aspirate samples, sequencing both DNA and RNA to improve sensitivity for driver fusion events. We have analyzed the genomic alterations including substitutions, indels, copy number alterations and genomic rearrangements of 795 samples with various hematolymphoid malignancies including lymphoma, leukemia and multiple myeloma to demonstrate the diagnostic, therapeutic and prognostic utility of this test in routine clinical practice. Method: Genomic profiles of 746 out of 795 (94%) consecutive samples received in a CLIA-certified laboratory (Foundation Medicine) were successfully characterized by the FoundationOne® Heme test including 527 from bone marrow aspirates, 176 FFPE samples and 92 samples derived from blood. The clinical cohort is composed of 375 multiple myeloma cases, 185 leukemia cases, 150 lymphoma cases, 75 MDS/MPN cases, and 15 unknown hematologic disorder cases (Figure A). Adaptor-ligated sequencing libraries were captured by solution hybridization using a custom bait-set targeting 405 blood cancer-related genes and 31 frequently rearranged genes by DNA-seq, and 265 frequently-rearranged genes by RNA-seq. All captured libraries were sequenced to high depth (Illumina HiSeq), averaging 498x for DNA and ~7M on-target unique pairs for RNA, to enable the sensitive and specific detection of substitutions, indels, copy number alteration and gene rearrangements. Results: 705/746 (95%) patients had at least one alteration reported as a somatic driver mutation, 68% of cases harbored at least one alteration linked to a targeted therapy or would inform enrollment in a clinical trial consistent with therapeutic relevance. These potentially actionable alterations included mutations in KRAS (14% of cases), NRAS (13%), CDKN2A (8%), DNMT3A (6%), IDH1/2 (5%), BRAF (4%) and FLT3 (3%) (Figure B). In addition, 64% of cases harbored at least one alteration that have been shown to predict outcome and have prognostic relevance, including TP53 (14% of cases), TET2 (7%), ASXL1 (7%), CDKN2B (5%), CREBBP (5%), MLL (5%) and NPM1 (3%)(Figure B). We also detected 344 genomic rearrangements in 280/746 (38%) patients. Of the 344 reported rearrangements, 166 were known fusions in various disease types and 178 were novel rearrangements involving known oncogenes and tumor suppressor genes, including a novel RCSD1-ABL2 fusion in a patient with B-cell ALL who will derive benefit from kinase inhibitor therapy as part of their anti-leukemic regimen. Genomic rearrangements (detected in 38% of cases) can lead to various functional consequences: 260 (76%) rearrangements detected in this cohort are predicted to create activating fusions; 19 (6%) rearrangements are predicted to be activating intragenic rearrangements in MLL, FLT3 and CARD11; and 60 (17%) rearrangements are likely truncating events in tumor suppressors. Conclusions: We demonstrated that hybrid capture-based targeted DNA and RNA sequencing can be used to identify all classes of genomic alterations in genes known to be therapeutic targets or prognostic predictors in a broad spectrum of hematologic malignancies. Moreover, our data suggests that the FoundationOne® Heme comprehensive genomic profiling test can alter therapeutic strategy in routine clinical practice. Figure 1 Figure 1. Figure 2 Figure 2. Disclosures He: Foundation Medicine: Employment, Equity Ownership. Nahas:Foundation Medicine, Inc: Employment. Wang:Foundation Medicine, Inc.: Employment, Equity Ownership. Young:Foundation Medicine: Employment, Equity Ownership. Brennan:Foundation Medicine, Inc: Employment. Donahue:Foundation Medicine: Employment. Young:Foundation Medicine, Inc: Employment. Ross:Foundation Medicine, Inc.: Employment, Equity Ownership. Morosini:Foundation Medicine, Inc. : Employment, Equity Ownership. van den Brink:Foundation Medicine, Inc: Consultancy. Intlekofer:Foundation Medicine, Inc: Consultancy. Dogan:Foundation Medicine, Inc: Consultancy. Armstrong:Epizyme, Inc: Consultancy. Yelensky:Foundation Medicine, Inc.: Employment, Equity Ownership. Otto:Foundation Medicine, Inc.: Employment. Lipson:Foundation Medicine, Inc.: Employment, Equity Ownership. Stephens:Foundation Medicine, Inc.: Employment, Equity Ownership. Miller:Foundation Medicine, Inc: Employment. Levine:Foundation Medicine, Inc: Consultancy.

Description: Key Points Novel clinically available comprehensive genomic profiling of both DNA and RNA in hematologic malignancies. Profiling of 3696 clinical hematologic tumors identified somatic alterations that impact diagnosis, prognosis, and therapeutic selection.