SUMMARY Which genetic alterations drive tumorigenesis and how they evolve over the course of disease and therapy are central questions in cancer biology. We identify 44 recurrently mutated genes and 11 recurrent somatic copy number variations through whole-exome sequencing of 538 chronic lymphocytic leukemia (CLL) and matched germline DNA samples, 278 of which were collected in a prospective clinical trial. These include previously unrecognized cancer drivers (RPS15, IKZF3) and collectively identify RNA processing and export, MYC activity and MAPK signaling as central pathways involved in CLL. Clonality analysis of this large dataset further enabled reconstruction of temporal relationships between driver events. Direct comparison between matched pre-treatment and relapse samples from 59 patients demonstrated highly frequent clonal evolution. Thus, large sequencing datasets of clinically informative samples enable the discovery of novel cancer genes and the network of relationships between the driver events and their impact on disease relapse and clinical outcome.

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Key Points• Long-term remissions after FCR chemoimmunotherapy in previously untreated patients with CLL.• Updated results on safety and efficacy of the CLL8 trial.Despite promising results with targeted drugs, chemoimmunotherapy with fludarabine, cyclophosphamide (FC), and rituximab (R) remains the standard therapy for fit patients with untreated chronic lymphocytic leukemia (CLL). Herein, we present the long-term follow-up of the randomized CLL8 trial reporting safety and efficacy of FC and FCR treatment of 817 treatment-naïve patients with CLL. The primary end point was progressionfree survival (PFS). With a median follow-up of 5.9 years, median PFS were 56.8 and 32.9 months for the FCR and FC group (hazard ratio [HR], 0.59; 95% confidence interval [CI], 0.50-0.69, P < .001). Median overall survival (OS) was not reached for the FCR group and was 86.0 months for the FC group (HR, 0.68; 95% CI, 0.54-0.89, P 5 .001). In patients with mutated IGHV (IGHV MUT), FCR improved PFS and OS compared with FC (PFS: HR, 0.47; 95% CI, 0.33-0.68, P < .001; OS: HR, 0.62; 95% CI, 0.34-1.11, P 5 .1). This improvement remained applicable for all cytogenetic subgroups other than del(17p). Long-term safety analyses showed that FCR had a higher rate of prolonged neutropenia during the first year after treatment (16.6% vs 8.8%; P 5 .007). Secondary malignancies including Richter's transformation occurred in 13.1% in the FCR group and in 17.4% in the FC group (P 5 .1). First-line chemoimmunotherapy with FCR induces long-term remissions and highly relevant improvement in OS in specific genetic subgroups of fit patients with CLL, in particular those with IGHV MUT. This trial was registered at www.clinicaltrials.gov as #NCT00281918. (Blood. 2016;127(2):208-215)

Key Points• Independent prognostic impact of biological markers, notably TP53 and SF3B1 mutations, in CLL patients requiring therapy.• NOTCH1 mutation as a predictive factor for reduced benefit from the addition of rituximab to FC chemotherapy.Mutations in TP53, NOTCH1, and SF3B1 were analyzed in the CLL8 study evaluating firstline therapy with fludarabine and cyclophosphamide (FC) or FC with rituximab (FCR) among patients with untreated chronic lymphocytic leukemia (CLL). TP53, NOTCH1, and SF3B1 were mutated in 11.5%, 10.0%, and 18.4% of patients, respectively. NOTCH1 mut and SF3B1 mut virtually showed mutual exclusivity (0.6% concurrence), but TP53 mut was frequently found in NOTCH1 mut (16.1%) and in SF3B1 mut (14.0%) patients. There were few significant associations with clinical and laboratory characteristics, but genetic markers had a strong influence on response and survival. In multivariable analyses, an independent prognostic impact was found for FCR, thymidine kinase (TK) ‡10 U/L, unmutated IGHV, 11q deletion, 17p deletion, TP53 mut , and SF3B1 mut on progression-free survival; and for FCR, age ‡65 years, Eastern Cooperative Oncology Group performance status ‡1, b2-microglobulin ‡3.5 mg/L, TK ‡10 U/L, unmutated IGHV, 17p deletion, and TP53 mut on overall survival. Notably, predictive marker analysis identified an interaction of NOTCH1 mutational status and treatment in that rituximab failed to improve response and survival in patients with NOTCH1 mut . In conclusion, TP53 and SF3B1 mutations appear among the strongest prognostic markers in CLL patients receiving current-standard first-line therapy. NOTCH1 mut was identified as a predictive marker for decreased benefit from the addition of rituximab to FC. This study is registered at www.clinicaltrials.gov as #NCT00281918. (Blood. 2014;123(21):3247-3254)

Through the European Research Initiative on chronic lymphocytic leukemia (CLL) (ERIC), we screened 3490 patients with CLL for mutations within the NOTCH1 (n=3334), SF3B1 (n=2322), TP53 (n=2309), MYD88 (n=1080) and BIRC3 (n=919) genes, mainly at diagnosis (75%) and before treatment (>90%). BIRC3 mutations (2.5%) were associated with unmutated IGHV genes (U-CLL), del(11q) and trisomy 12, whereas MYD88 mutations (2.2%) were exclusively found among M-CLL. NOTCH1, SF3B1 and TP53 exhibited variable frequencies and were mostly enriched within clinically aggressive cases. Interestingly, as the timespan between diagnosis and mutational screening increased, so too did the incidence of SF3B1 mutations; no such increase was observed for NOTCH1 mutations. Regarding the clinical impact, NOTCH1 mutations, SF3B1 mutations and TP53 aberrations (deletion/mutation, TP53ab) correlated with shorter time-to-first-treatment (P<0.0001) in 889 treatment-naive Binet stage A cases. In multivariate analysis (n=774), SF3B1 mutations and TP53ab along with del(11q) and U-CLL, but not NOTCH1 mutations, retained independent significance. Importantly, TP53ab and SF3B1 mutations had an adverse impact even in U-CLL. In conclusion, we support the clinical relevance of novel recurrent mutations in CLL, highlighting the adverse impact of SF3B1 and TP53 mutations, even independent of IGHV mutational status, thus underscoring the need for urgent standardization/harmonization of the detection methods.

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In chronic lymphocytic leukemia (CLL), TP53 gene defects, due to deletion of the 17p13 locus and/or mutation(s) within the TP53 gene, are associated with resistance to chemoimmunotherapy and a particularly dismal clinical outcome. On these grounds, analysis of TP53 aberrations has been incorporated into routine clinical diagnostics to improve patient stratification and optimize therapeutic decisions. The predictive implications of TP53 aberrations have increasing significance in the era of novel targeted therapies, i.e., inhibitors of B-cell receptor (BcR) signaling and anti-apoptotic BCL2 family members, owing to their efficacy in patients with TP53 defects. In this report, the TP53 Network of the European Research Initiative on Chronic Lymphocytic Leukemia (ERIC) presents updated recommendations on the methodological approaches for TP53 mutation analysis. Moreover, it provides guidance to ensure that the analysis is performed in a timely manner for all patients requiring treatment and that the data is interpreted and reported in a consistent, standardized, and accurate way. Since next-generation sequencing technologies are gaining prominence within diagnostic laboratories, this report also offers advice and recommendations for the interpretation of TP53 mutation data generated by this methodology.

Summary Background Bendamustine and rituximab (BR) has been a standard of care for the management of patients with relapsed/refractory (R/R) chronic lymphocytic leukemia (CLL). We evaluated the efficacy and safety of adding idelalisib, a first-in-class targeted PI3Kδ inhibitor, to BR in patients with R/R CLL Methods This trial was a global, multicenter, double-blind, placebo -controlled trial in adult patients (≥18 years) with R/R CLL requiring treatment for their disease. Patients had to have measurable lymphadenopathy (≥1 nodal lesion ≥2.0 cm in the longest diameter and ≥1.0 cm in the longest perpendicular diameter) by computer tomography or magnetic resonance imaging, disease progression within <36 months since last prior therapy, a Karnofsky Performance Status score ≥60 and adequate bone marrow, liver and kidney function. Key exclusion criteria included histological transformation to an aggressive lymphoma (eg, Richter transformation) or disease refractory to bendamustine. Patients were randomised 1:1 using a central interactive web response system that assigned a unique treatment code for each patient, to receive intravenous BR infusions for a maximum of 6 cycles in addition to blinded study drug matching the assigned treatment of either twice-daily oral idelalisib 150 mg or placebo administered continuously until disease progression or intolerable study drug-related toxicity. Randomisation was stratified based on high-risk features (IGHV, del(17p)/TP53 mutation) and refractory vs relapsed disease. The primary endpoint was progression-free survival (PFS) assessed by an independent review committee in the intent-to-treat population. Overall survival was a key secondary endpoint. Crossover was not permitted to the idelalisib arm at progression. The trial is ongoing (ClinicalTrials.gov # NCT01569295). Findings Between 26 June 2012 and 21 August 2014, 416 patients with R/R CLL were enrolled; 207 patients were randomised to the idelalisib and 209 to the placebo arm. After the prespecified interim analysis, the Independent Data Monitoring Committee (IDMC) recommended discontinuation and unblinding of the trial due to efficacy. Updated data are presented in this manuscript with a cutoff date of 07 October 2015. Median (95% CI) PFS was 20·8 (16·6, 26·4) and 11·1 (8·9, 11·1) months in the idelalisib and placebo arms, respectively (hazard ratio [HR], 0·33; 95% CI, 0·25, 0·44; P<0·0001) at a median (Q1, Q3) follow-up of 14 (7, 18) months. The most frequent grade 3 or greater AEs were neutropenia (124/207 [60%]) and febrile neutropenia (48/207 [23%]) in the idelalisib arm and neutropenia (99/209 [47%]) and thrombocytopenia (27/209 [13%]) in the placebo arm. Serious AEs included febrile neutropenia, pneumonia and pyrexia and were common in both treatment arms. An increased risk of infection was observed in the idelalisib vs placebo arm. Interpretation Idelalisib plus BR is superior to BR alone, improving PFS and OS. This regimen represents an important new treatment option for patients with R/R CLL.