ALBERT

Description: Key Points FL-associated STAT6 mutations hyperactivate the IL-4/JAK/STAT6 axis.

Description: Introduction Genomic complexity as measured through SNP array-based (SNP-A) genomic profiling is a strong negative predictor of survival outcome in adult acute myelogenous leukemia (AML). The recent discovery of multiple novel recurrently mutated genes in AML has led to the development of several prognostic models based on various combinations of genes along with the well-established risk factors of age and karyotype, but these models do not account for the strong effect of SNP-A-based genomic complexity. In this study, we seek to determine the relative importance of AML genomic complexity and gene mutation status on overall survival (OS) in AML. Methods We employed SNP-A genomic profiling of acquired copy number aberrations (aCNA) and copy neutral LOH (cnLOH) together with sequence analysis of 13 recurrently mutated genes to determine aCNA/cnLOH lesion load and gene mutational status for 156 consecutively collected previously untreated AML patients. AML cell samples were processed with a Ficoll gradient, negatively selected using Miltenyi microbead columns, and then further purified with flow cytometric cell sorting. Processed DNA isolated from highly purified AML blasts and paired buccal DNA was subsequently hybridized to Affymetrix SNP 6.0 arrays. aCNAs were visually identified using the dChip program in paired data displays and corroborated by algorithmic lesion scoring, and cnLOH was detected using internally developed software. Using the same DNA, we resequenced the recurrently mutated exons of NPM1, FLT3, CEBPA, IDH1, IDH2, NRAS, KRAS, and TP53, and all coding exons of RUNX1, ASXL1, TET2, DNMT3A, and BCORL1. Clinical data including age, overall survival, and cytogenetics were ascertained. Cytogenetic risk categories were assigned based on the SWOG criteria. Univariate analyses were performed using Kaplan-Meier estimates of survivor functions, and Cox proportional hazards models were used for multivariate analyses. Results At the time of analysis, 119 (76%) patients had died. aCNA/cnLOH were common with ≥1, ≥2, and ≥3 lesions detected in 62%, 38%, and 26% of cases, respectively. Univariate overall survival analysis of all clinical and molecular variables demonstrated a significantly increased OS for mutations of NPM1 (p=0.01) and decreased OS for mutations of TP53 (p

Description: Introduction Chronic lymphocytic leukemia with elevated genomic complexity (CLL-HGC) is clinically aggressive and is characterized by shortened survival. While it is known that CLL-HGC is enriched for specific aCNAs and that a subset of CLL-HGC carries TP53 mutations, it is currently unclear what other molecular aberrations cause or contribute to genomic instability in CLL with wild type TP53. Methods Within a cohort of 255 CLL cases previously analyzed on SNP 6.0 arrays, we identified 50 CLL-HGC cases with ≥ 3 acquired genomic copy number aberrations (aCNA) and/or acquired uniparental disomy (aUPD). Of these 50 CLL-HGC cases, 26 cases were wild type for TP53, and of these, 23 were subjected to whole exome sequence analysis (WES). Exome capture and WES was performed on DNA isolated from FACS-purified CD19+ and CD3+ cells, and massively parallel sequencing was performed using 96 bp paired-end sequencing on a HiSeq2000 sequencer. Bioinformatics analysis followed validated in-house pipelines. The following genes and exons were re-sequenced in all samples using Sanger sequencing: TP53 (exons 2-10), SF3B1 (exons 13-17), NOTCH1 (exon 34), and POT1 (all coding exons). Genetic data were complemented with assays for p53 protein expression and inducibility (following Nutlin 3 treatment of purified CLL cells), radiation-induced CLL cell apoptosis and ATM Ser-1981 auto-phosphorylation following CLL cell irradiation. Results Sanger sequence validation of all nominated candidate gene mutations in paired samples (T+N) confirmed 192 mutated genes in 23 CLL cases, a mutational load per case that is comparable to published unselected CLL cohorts. A gene mutated at high frequency, akin to TP53, was not identified. Recurrently mutated genes (N=2 or 3 out of 23 cases) in CLL-HGC included MYD88, NCKAP5, EGR2 and NXF1. Mutations in other genes previously suggested to contribute to genomic instability or CLL clinical aggressiveness were largely absent (ATM: N=1; NOTCH1: N=0; SF3B1: N=1 and POT1: N=0). A detailed gene-by-gene review of the mutated genes revealed that some of the genes can be grouped into functional classes that may have relevance to the observed genomic phenotype: nuclear export (XPO, NXF1), apoptosis regulation (BAX, KHDC1, PACS2, FBXW7), RB-E2F-p53 axis (DYRK1A, TRIM16, RB1CC1, E2F7), signaling (MYD88, EGR2), chromosome segregation (BSDC1, DDX46, PDIA4, BOD1L, ZW10) and p53 network (IRF2, SERTAD4, SYVN1, BAX). Notably absent were mutations in other DNA-ds-break response and repair or DNA maintenance pathway genes. Functional data uncovered 3 CLL-HGC cases with impaired p53 protein induction following chemical p53 activation and two cases with impaired ATM-Ser-1981 auto-phosphorylation. Conclusion We describe the results of WES in 23 CLL samples with high genomic complexity and wild type TP53. Overall, the following conclusions can be supported from this work: i) CLL-HGC with wild type TP53 is not associated with a high-frequency mutated gene; ii) the gene mutation load in CLL-HGC with wild type TP53 is similar to unselected CLL cases; iii) most CLL-HGC with wild type TP53 carry mutations in genes that fall into functional classes that may have a role in CLL genome destabilization or a permissive role in the survival of CLL cells with spontaneously occurring DNA lesions. Additional functional studies are in progress testing specific CLL-HGC-associated mutated alleles for effects on chromosomal stability and the p53 network. Overall, the results point to a multi-factorial origin of genomic instability in CLL. Disclosures: No relevant conflicts of interest to declare.

Description: Key Points FL carries mutations in linker histone H1 B, C, D, and E genes in 27% of cases. FL carries recurrent mutations in OCT2 (POU2F2), IRF8, and ARID1A.

Description: Introduction Follicular lymphoma (FL) constitutes the second most common non-Hodgkin’s lymphoma in the Western world. FL carries characteristic recurrent structural genomic aberrations. However despite recent advances, knowledge regarding the coding genome in FL is still evolving and is currently incomplete. Methods To further our understanding of the genetic basis of follicular lymphoma (FL), we used solution exon capture of sheared and processed genomic DNA isolated from FACS-sorted lymphomatous B-cells and paired CD3+ T-cells isolated from twenty three cases of FL and one case of DLBCL (which was transformed from prior FL), followed by paired-end (96-101 base pair read length per side) massively parallel sequencing. The sequence data were characterized by a mean depth of coverage of 41, and 90% of bases in the target region were covered by at least 10 reads. Bioinformatics pipelines developed by our bioinformatics core served as the primary data source to nominate candidate mutated genes in downstream data analysis. Results The bioinformatics pipeline nominated 711 distinct candidate mutations in 24 FL cases. Sanger sequence validation confirmed 39 recurrently (≥ 2 FL cases) mutated genes. Genes with confirmed mutations in ≥ 2 FL cases in the discovery cohort were subsequently selectively expanded into a combined FL validation cohort of 114 cases. In addition to frequent mutations in MLL2, CREBBP, BCL2, TNFRSF14, EZH2, OCT2, ARID1A, IRF8 and MEF2B, we here report novel mutations in STAT6 in FL. STAT6 mutations were identified in 11% (12/114) of FL and predominantly affected the DNA binding domain (DBD; comprising STAT6 amino acids 268-430). Two FL cases each carried two distinct STAT6 mutations, presumably targeting both alleles. Of interest, the majority of FL-associated STAT6 mutations affected a single amino acid codon (codon 419), resulting in the STAT6 mutants p.419D〉D/G or p.419D〉D/H. These FL-associated STAT6 mutations are distinct from mutations previously described in primary mediastinal B-cell lymphoma (PMBCL). Given the involvement of STAT6 in signal transduction pathways activated by multiple cell surface receptors, as well as the recently described involvement of STAT6 in antiviral innate immunity (involving an interaction between the STAT6 DBD and the protein STING), we are currently exploring functional consequences of the novel STAT6 mutations in FL and cell line models. Conclusion We report identification of somatic mutations in STAT6 in 11% of FL. These mutations predominantly affected the STAT6 DNA binding domain. We identify a novel STAT6 mutation hotspot in STAT6 codon 419 (p.419D〉D/G or p.419D〉D/H). Disclosures: Lebovic: Genentech: Speakers Bureau; Allos/Spectrum: Speakers Bureau; Celgene: Speakers Bureau; Onyx: Speakers Bureau.