ALBERT

Description: Genomic profiling in AML has led to increased understanding of oncogenic mutations, refined risk stratification, and enhanced identification of alterations that can serve as targets for therapeutic intervention. Comprehensive genomic profiling (CGP) identifies a variety of alterations, including base pair substitutions, insertions, deletions, copy number alterations, and fusions. As distinct age-dependent alterations in AML are being increasingly recognized, we sought to use CGP to gain insight into age-associate variants in pediatric and adult AML. Identification of the significant age-associated biologic differences is critical to improving understanding of the age-dependent drivers of leukemogenesis. This can also advance therapeutic efforts with enhanced risk stratification, disease monitoring, and drug development. Diagnostic and relapse specimens from a total of 934 patients, comprised of 179 pediatric (age 0-18 years) and 755 adult (age 19-87 years) specimens underwent clinical comprehensive sequencing. DNA and RNA integrated next-generation sequencing was performed in a CLIA-certified, CAP-accredited, NYS-approved laboratory for FoundationOne Heme. All captured libraries were sequenced to high depth averaging 569X for DNA (405 genes) and 〉3M unique pairs for RNA 9265 genes). Somatic variants identified included short variants (single nucleotides variants (SNVs) and short insertions, indels), and structural variants (fusions, amplifications, loss of whole genes, or truncations). The total variant prevalence was 571 in 131 genes in the pediatric cohort vs. 3020 variants in 219 genes in the adult cohort. The average number of variants in the pediatric cohort was 3.2 vs. 4.0 in the adults (p

Description: Despite intense efforts, the cure rates of children and adults with AML remain unsatisfactory. Resistance to cytotoxic chemotherapy is the dominant cause of treatment failure. To investigate the molecular basis of primary chemoresistance, we used targeted gene sequencing using the Foundation One Heme platform to profile 405 genes in DNA and 265 genes in RNA known to be somatically mutated in hematologic malignancies in 107 primary specimens obtained at diagnosis from children and adults with cytogenetically normal AML. Comparative analysis revealed mutations of SETBP1 and ASXL1 to be enriched in patients with failure of induction chemotherapy as compared to those who achieved remission (8 out of 50 versus 1 out of 57, p = 0.01). However, the low prevalence of these alterations indicated that other genomic or functional mechanisms must mediate chemoresistance. To identify these mechanisms, we mapped kinase signaling cascades activated in chemoresistant AML cells using functional mass spectrometry proteomics. Phosphopeptides were enriched in primary leukemic blasts isolated from cytogenetically normal AML patients, with relative abundances in each patient determined from iTRAQ 8-plex reporter ions in the associated MS/MS fragment ion spectra. This analysis identified phosphorylation of the leukemogenic transcription factor MEF2C at S222 in the majority of cases. We found MEF2C to be overexpressed in cytogenetically normal AML, as well as MLL-rearranged leukemias, and to be associated with increased risk of relapse (Laszlo et al, ASH submitted). Using reporter assays, we found that phosphorylation of MEF2C S222 was both necessary and sufficient to fully transactivate MEF2C promoter elements (Fig. 1). Expression of mutant S222A MEF2C that cannot be phosphorylated, but not phosphomimetic S222D, induced mitochondrial apoptosis in human AML and genetically-engineered MLL-AF9 mouse leukemia but not in normal mouse hematopoietic progenitor cells. In syngeneic mouse transplant models, we found that MEF2C phosphorylation was required for the maintenance of MLL-AF9 leukemias in vivo. Transcriptome and proteome analysis of gene expression changes and composition of MEF2C transcriptional complexes induced by phosphorylation mutants showed that aberrant leukemia cell survival is caused at least in part by alterations in the expression of apoptotic regulators. Using a recombinant kinase screen, we identified MARK kinases as specific enzymes that phosphorylate MEF2C S222. Treatment of primary patient specimens and human AML cell lines with the MARK kinase inhibitor MRT199665 induced MEF2C downregulation and apoptosis in cells with MEF2C but not those lacking MEF2C expression (Fig. 2). Thus, aberrant MEF2C phosphorylation is associated with primary chemoresistance and induction failure, is required for enhanced leukemia cell survival, and confers susceptibility to MARK inhibition therapy. In addition, genome and proteome profiles should provide additional biomarkers and targeted therapeutic strategies to overcome chemoresistance in AML. Figure 1. MEF2C phosphorylation is required for transactivation of MEF2C promoter elements Figure 1. MEF2C phosphorylation is required for transactivation of MEF2C promoter elements Figure 2. Treatment of AML cells with MRT199665 Figure 2. Treatment of AML cells with MRT199665 Disclosures He: Foundation Medicine, Inc.: Employment, Equity Ownership. Balasubramanian:Foundation Medicine, Inc.: Employment. Zhong:Foundation Medicine, Inc.: Employment, Equity Ownership. Pavlick:Foundation Medicine, Inc.: Employment. Yilmazel:Foundation Medicine, Inc.: Employment. Stone:Merck: Consultancy; AROG: Consultancy; Abbvie: Consultancy; Juno: Consultancy; Amgen: Consultancy; Celator: Consultancy; Agios: Consultancy; Karyopharm: Consultancy; Pfizer: Consultancy; Roche/Genetech: Consultancy; Sunesis: Consultancy, Other: DSMB for clinical trial; Celgene: Consultancy; Novartis: Research Funding. Byrd:Acerta Pharma BV: Research Funding. Levine:CTI BioPharma: Membership on an entity's Board of Directors or advisory committees; Foundation Medicine: Consultancy; Loxo Oncology: Membership on an entity's Board of Directors or advisory committees. Armstrong:Epizyme, Inc: Consultancy.

Description: Background: Lymphoma is a clinically and molecularly heterogenous disease. Next generation sequencing of primary lymphoma samples has identified common recurring genomic alterations (GAs). The distribution and frequency of recurring GAs across lymphoma subtypes remains unknown because prior studies vary in sequencing methods, depth of coverage, and specimen source. In this study, we benchmark the distribution of GAs across different lymphoma subtypes by prospectively analyzing lymphoma cases and performing comprehensive DNA/RNA targeted sequencing of genes commonly found in hematologic malignancies using the Foundation One Heme (F1H) clinical assay. Methods: After obtaining proper consent, archived specimens from 183 samples [formalin fixed paraffin embedded (FFPE) N=141, peripheral blood N=28, BM aspirate N=14] distributed across lymphoma subtypes (including 62 DLBCL, 38 T cell lymphoma, 32 FL, 17 CLL, 13 MCL) were sequenced to high, uniform coverage averaging 〉600x for DNA, 〉20 million pairs for RNA. GAs were determined, including base substitutions, small insertions and deletions, rearrangements, and copy number alterations. Significant non-synonymous 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. Fisher's exact test with Monte Carlo estimation corrected by false discovery rate was used for associations. Results: Samples from prospectively consented patients were banked for a median of 30 days prior to genomic analysis, range 1 day to 6.5 yr. Sequencing data was reported a median of 16 days from sample date receipt. GAs were identified in 95% of samples, with a median of 4 GAs/sample. The most common GAs were TP53 (29%), MLL2 (27%), BCL2 (25%), CDKN2A/B (17%) and CREBBP (14%). Alterations of chromatic modifiers (80%), BCR/NFkB components (51%), and cell cycle pathway (44%) were most common. In our group of unpaired follicular lymphoma samples (N=7 treatment naïve, N=25 treatment exposed), the number of GAs increased with treatment exposure. We found similar gene and biological signatures regardless of prior therapy; however differences emerge in genes of potential clinical relevance. Sequencing profiles augmented or altered the pathologic diagnosis in 11 of 183 (6%) of the cases. Importantly we were able to classify the GAs as actionable, potentially actionable and variants of unclear significance to better define the clinical relevance of targeted genomic sequencing. Conclusions: Integration of comprehensive next generation targeted genomic sequencing and clinical analysis in lymphoma provides an opportunity to describe the spectrum and incidence of GAs across different lymphoma subtypes and provide guidance on application of genomic profiling. This work serves to benchmark GAs across all lymphoma subtypes in a clinically relevant population and enables design of basket trials selecting patients based on shared genomic and biologic similarity instead of lymphoma subtype. To our knowledge, this is the largest repository of clinically annotated genomic sequencing in lymphoma. Table 1. Total Specimens N = 183 Median Age at Diagnosis 57 Range 21 - 84 Median Age at Biopsy 61 Range 21 - 91 Sex • Male • Female 113 70 62% 38% Biospecimen source • Paraffin embedded • Peripheral blood • Marrow aspirate 141 28 14 77% 15% 8% Patient consent • Prospective consent • Retrospective consent 145 38 79% 21% Prospectively consented patients (N=145) Median Days to Result Median Age of Sample 16 30 days 8 - 81 1 day - 6.5 yr Disclosures Palomba: Janssen: Consultancy. Gerecitano:Genentech: Consultancy, Other: Advisory Board; AbbVie: Consultancy, Other: Advisory Board. Matasar:Spectrum: Consultancy; Genentech: Consultancy. Straus:Millenium Pharmaceuticals: Research Funding. He:Foundation Medicine, Inc.: Employment, Equity Ownership. Balasubramanian:Foundation Medicine: Employment, Equity Ownership. Stephens:Foundation Medicine, Inc.: Employment, Equity Ownership. Miller:Foundation Medicine: Employment. Levine:Loxo Oncology: Membership on an entity's Board of Directors or advisory committees; CTI BioPharma: Membership on an entity's Board of Directors or advisory committees; Foundation Medicine: Consultancy. Younes:Celgene: Honoraria; Johnson and Johnson: Research Funding; Novartis: Research Funding; Bayer: Honoraria; Bristol Meyer Squibb: Honoraria; Sanofi-Aventis: Honoraria; Seattle Genetics: Honoraria, Research Funding; Curis: Research Funding; Janssen: Honoraria; Takeda Millenium: Honoraria; Incyte: Honoraria.

Description: Subsets of pediatric acute leukemias are refractory to frontline therapy or relapse with standard treatment. Pediatric leukemias are associated with distinct molecular and genomic alterations. Clinical genomic profiling has been used effectively to improve the diagnosis of adult leukemias. However, its use and utility for pediatric leukemias are not well defined. Here, we used FoundationOne Heme, a clinical grade, high-throughput, hybridization capture-based next-generation sequencing (NGS) assay for targeted sequencing of all exons of 405 genes as well as RNA sequencing of 265 genes. We analyzed the results of samples from 71 patients sequenced on the FoundationOne® Heme assay from children and young adults with acute lymphoblastic leukemia (ALL) (n= 34); early T cell precursor ALL (ETP) (n=3); acute myelogenous leukemia (AML) (n= 19); ambiguous lineage leukemia (n=3); and myelodysplastic syndrome (MDS) (n=12) who were treated at Memorial Sloan Kettering Cancer Center in the Department of Pediatrics over a 26 month period (January 2014-March2016). Age of patients ranged from 1-24 years of age (median =10 years). 42 samples were from patients who had yet to receive cytotoxic therapy (newly diagnosed) and 29 samples were from patients who had relapsed (n=24) or had refractory disease (n=5). The current average turnaround time for FoundationOne® Heme is 12-14 days. Overall 99% (70/71) of specimens were successfully profiled. We identified 230 genomic alterations, including 59.1% (136/230) missense or nonsense mutations of genes, 15.6% (36/230) copy number alterations, and 25.6% (59/230) gene fusions or rearrangements. Molecular aberrations were detected in 81% (9/11) of patients with normal karyotypes; 55% (5/9) of these were cytogenetically cryptic genomic rearrangements including NUP98-NSD1 (n=2) and 1 MLL-PTD (n=1). We identified one novel translocation involving SATB1-PDGFRB in a sample from a patient with pre B ALL. Another patient with pre B ALL was found to have a unique combination of genomic alterations including an MLL-PTD and SSBP2-JAK2. Two patients with MDS were identified as harboring germline GATA2 mutations, indicating the likely benefit of an allogeneic hematopoietic stem cell transplantation. Three patients had therapy modifications based on findings with the addition of dasatinib (ZMIZ1-ABL1), ruxolitinib (PCM1-JAK2) and trametinib (PTPN11mutation), and two patients had risk stratification altered based on MLL rearrangements which were not identifiable by conventional cytogenetics. Overall, the use of comprehensive genomic profiling led to improved accuracy of diagnosis or alteration of therapy in 10% (7/71) cases, including molecular based therapy selection (n=3), escalation of conventional chemotherapy due to high-risk features (n=2), and inclusion of stem cell transplantation (n=2). Thus, clinical genomic profiling of pediatric acute leukemia led to discovery of new pathobiology, improved accuracy of clinical diagnosis, and inclusion of targeted molecular therapies. Figure Figure. Disclosures Kobos: Janssen: Employment. He:Foundation Medicine, Inc: Employment, Equity Ownership. Roshal:BD Biosciences: Consultancy. Zhong:foundation medicine: Employment. Balasubramanian:Foundation Medicine, Inc: Employment, Equity Ownership. Nahas:Foundation medicine: Employment. Vergilio:Foundation Medicine: Employment. Ross:Foundation Medicine, Inc: Employment. Stephens:Foundation Medicine, Inc: Employment, Equity Ownership. Mughal:Foundation Medicine: Employment, Equity Ownership. Miller:Foundation Medicine: Employment, Equity Ownership. Levine:Qiagen: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy.