Key Points• This is the first study to validate the importance of NOTCH1 and SF3B1 gene mutations in the context of a randomized, prospective clinical trial.• Mutations in both genes are independent prognostic biomarkers, and therefore have clinical utility in the accurate risk-adapted stratification of CLL patients. Continuing Medical Education onlineThis activity has been planned and implemented in accordance with the Essential Areas and policies of the Accreditation Council for Continuing Medical Education through the joint sponsorship of Medscape, LLC and the American Society of Hematology. Medscape, LLC is accredited by the ACCME to provide continuing medical education for physicians. Medscape, LLC designates this Journal-based CME activity for a maximum of 1.0 AMA PRA Category 1 Credit(s) ™ . Physicians should claim only the credit commensurate with the extent of their participationin the activity. All other clinicians completing this activity will be issued a certificate of participation. To participate in this journal CME activity: (1) review the learning objectives and author disclosures; (2) study the education content; (3) take the post-test with a 70% minimum passing score and complete the evaluation at http://www.medscape.org/journal/blood; and (4) view/print certificate. For CME questions, see page 566. Disclosures Associate Editor John G. Gribben served as an advisor or consultant for Celgene and Roche and as a speaker or a member of a speakers bureau for Roche, Jensen, and Celgene. The authors and CME questions author Laurie Barclay, freelance writer and reviewer, Medscape, LLC, declare no competing financial interests. For personal use only. on May 9, 2018. by guest www.bloodjournal.org From Continuing Medical Education online Learning objectivesUpon completion of this activity, participants will be able to:1. Describe the frequency of NOTCH1 and SF3B1 mutations in patients with chronic lymphocytic leukemia (CLL), and their correlations with other genetic markers.2. Describe survival in CLL patients with NOTCH1 mutations, and the prognostic value of this mutation. 3. Describe survival in CLL patients with SF3B1 mutations, and the prognostic value of this mutation.

© F e r r a t a S t o r t i F o u n d a t i o ntreatment. However, the relative frequency and clinical significance of ATM and BIRC3 abnormalities is unclear. This study addresses this issue in a large cohort of 11q-deleted patients detected by SNP6 profiling and screened for ATM and BIRC3 mutations. As a consequence, in the context of a phase III clinical trial of chemotherapy, we show that ATM mutational status remains the most clinically informative genomic lesion in 11q-deleted CLL, identifying cases with outcome comparable to TP53 deletion and/or mutation patients, and that the presence of BIRC3 deletion and/or mutation is associated with outcome comparable to other 11q-deleted CLL cases. Methods Patients and molecular diagnostic assaysA total of 166 untreated CLL patients diagnosed according to standard morphological and immunophenotypic criteria were included in this study (Table 1 and Online Supplementary Table S1). This cohort principally included patients from the UK LRF CLL4 trial 9 (n=133) which allowed accurate clinical correlations to bemade. An additional 33 11q-deleted patients were also included to allow more significant associations between the 11q deletion and other genomic variables to be made, such as with mutational status of ATM and BIRC3. The additional del11q patients were sampled subsequent to the development of progressive disease, with a median time from diagnosis of 3.2±5.2 years (±1 standard deviation (SD); range 1 month-27 years). For the entire cohort of 166 patients, mutational data were available for TP53 (n=125), SF3B1 (n=140) and NOTCH1 (n=146). 28,29 Details on the molecular diagnostic assays 30,31 are available in the Online Supplementary Methods. Informed consent was obtained from all patients in accordance with the Declaration of Helsinki and our local ethics committee gave their approval for the study. DNA extraction, SNP6 array hybridization, data extraction and analysisGenomic DNA was extracted from CLL B cells (n=166) and buccal swabs (n=32), prior to being purified, amplified, labeled and hybridized to the Affymetrix SNP6.0 platform (Affymetrix, Santa Clara, CA, USA) as previously described 32 (Online Supplementary Methods).11q23 deletions, ATM and BIRC3 mutations in CLL haematologica | 2014; 99(4) 737 © F e r r a t a S t o r t i F o u n d a t i o n Mutational analysis of ATM and BIRC3 genesThe presence or absence of somatically-acquired single nucleotide variants (SNVs or mutations) in ATM and BIRC3 were successfully ascertained in 105 (CLL4 cases: n=79 of 133) and 162 (CLL4 cases: n=131 of 133) patients, respectively. Denaturing high-performance liquid chromatography (DHPLC), high-resolution melt (HRM) polymerase chain reaction (PCR) analysis and Sanger sequencing were utilized. 20,26,27,29 More details of methods and the strategy adopted for assigning the somatic nature of each SNV are provided in the Online Supplementary Methods and Online Supplementary Table S2. Statistical analysisStatistical analysis was performed using SPSS (v.20 Results Incidence ...

The pathogenesis of splenic marginal zone lymphoma (SMZL) remains largely unknown. Recent high-throughput sequencing studies have identified recurrent mutations in key pathways, most notably NOTCH2 mutations in >25% of patients. These studies are based on small, heterogeneous discovery cohorts, and therefore only captured a fraction of the lesions present in the SMZL genome. To identify further novel pathogenic mutations within related biochemical pathways, we applied whole exome sequencing (WES) and copy number (CN) analysis to a biologically and clinically homogeneous cohort of seven SMZL patients with 7q abnormalities and IGHV1-2*04 gene usage. We identified 173 somatic non-silent variants, affecting 160 distinct genes. In additional to providing independent validation of the presence of mutation in several previously reported genes (NOTCH2, TNFAIP3, MAP3K14, MLL2 and SPEN), our study defined eight additional recurrently mutated genes in SMZL; these genes are CREBBP, CBFA2T3, AMOTL1, FAT4, FBXO11, PLA2G4D, TRRAP and USH2A. By integrating our WES and CN data we identified three mutated putative candidate genes targeted by 7q deletions (CUL1, EZH2 and FLNC), with FLNC positioned within the well-characterized 7q minimally deleted region. Taken together, this work expands the reported directory of recurrently mutated cancer genes in this disease, thereby expanding our understanding of SMZL pathogenesis. Ultimately, this work will help to establish a stratified approach to care including the possibility of targeted therapy.

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Despite advances in chronic lymphocytic leukaemia (CLL) treatment, globally chemotherapy remains a central treatment modality, with chemotherapy trials representing an invaluable resource to explore disease-related/genetic features contributing to long-term outcomes. In 499 LRF CLL4 cases, a trial with >12 years follow-up, we employed targeted resequencing of 22 genes, identifying 623 mutations. After background mutation rate correction, 11/22 genes were recurrently mutated at frequencies between 3.6% (NFKBIE) and 24% (SF3B1). Mutations beyond Sanger resolution (<12% VAF) were observed in all genes, with KRAS mutations principally composed of these low VAF variants. Firstly, employing orthogonal approaches to confirm <12% VAF TP53 mutations, we assessed the clinical impact of TP53 clonal architecture. Whilst ≥ 12% VAF TP53mut cases were associated with reduced PFS and OS, we could not demonstrate a difference between <12% VAF TP53 mutations and either wild type or ≥12% VAF TP53mut cases. Secondly, we identified biallelic BIRC3 lesions (mutation and deletion) as an independent marker of inferior PFS and OS. Finally, we observed that mutated MAPK-ERK genes were independent markers of poor OS in multivariate survival analysis. In conclusion, our study supports using targeted resequencing of expanded gene panels to elucidate the prognostic impact of gene mutations.