Identification Of Actionable Genomic Alterations In Hematologic Malignancies By a Clinical Next Generation Sequencing-Based Assay

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.

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.

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.

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.

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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.