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.

Chronic lymphocytic leukaemia (CLL) has several unique features that distinguish it from other cancers. Most CLL tumour cells are inert and arrested in G0/G1 of the cell cycle and there is only a small proliferative compartment; however, the progressive accumulation of malignant cells will ultimately lead to symptomatic disease. Pathogenic mechanisms have been elucidated that involve multiple external (for example, microenvironmental stimuli and antigenic drive) and internal (genetic and epigenetic) events that are crucial in the transformation, progression and evolution of CLL. Our growing understanding of CLL biology is allowing the translation of targets and biological classifiers into clinical practice.

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)

Charting differences between tumors and normal tissue is a mainstay of cancer research. However, clonal tumor expansion from complex normal tissue architectures potentially obscures cancer-specific events, including divergent epigenetic patterns. Using whole-genome bisulfite sequencing of normal B cell subsets, we observed broad epigenetic programming of selective transcription factor binding sites coincident with the degree of B cell maturation. By comparing normal B cells to malignant B cells from 268 patients with chronic lymphocytic leukemia (CLL), we showed that tumors derive largely from a continuum of maturation states reflected in normal developmental stages. Epigenetic maturation in CLL was associated with an indolent gene expression pattern and increasingly favorable clinical outcomes. We further uncovered that most previously reported tumor-specific methylation events are normally present in non-malignant B cells. Instead, we identified a potential pathogenic role for transcription factor dysregulation in CLL, where excess programming by EGR and NFAT with reduced EBF and AP-1 programming imbalances the normal B cell epigenetic program.

To identify genomic alterations in chronic lymphocytic leukemia (CLL), we performed single-nucleotide polymorphismarray analysis using Affymetrix Version 6.0 on 353 samples from untreated patients entered in the CLL8 treatment trial. Based on paired-sample analysis (n ‫؍‬ 144), a mean of 1.8 copy number alterations per patient were identified; approximately 60% of patients carried no copy number alterations other than those detected by fluorescence in situ hybridization analysis. Copy-neutral loss-ofheterozygosity was detected in 6% of CLL patients and was found most frequently on 13q, 17p, and 11q. Minimally deleted regions were refined on 13q14 (deleted in 61% of patients) to the DLEU1 and DLEU2 genes, on 11q22.3 (27% of patients) to ATM, on 2p16.1-2p15 (gained in 7% of patients) to a 1.9-Mb fragment containing 9 genes, and on 8q24.

The prognosis of fludarabine (F)-refractory chronic lymphocytic leukemia (CLL) is very poor, and underlying mechanisms are only partly understood. To assess the contribution of p53 abnormalities to F-refractory CLL, we studied TP53 mutations in the CLL2H trial (subcutaneous alemtuzumab; n ‫؍‬ 99). We found TP53 mutations in 37% of patients. Twelve of 67 (18%) patients without the 17p deletion showed a TP53 mutation and 50% showed evidence of uniparental disomy. A total of 75% of cases with TP53 mutation (without 17p؊) showed clonal evolution/expansion. TP53 mutations had no impact on overall survival (P ‫؍‬ .48). CLL with the 17p deletion or TP53 mutation showed very low miR-34a expression. To investigate the mechanisms underlying refractory CLL beyond p53, we studied cases without 17p؊/TP53 mutation in detail. In several paired samples before and after F-refractory disease, no change in p21/ p53 induction was observed after DNA damage. Although TP53 mutations and 17p deletions are found in a high proportion of F-refractory CLL, more than half of the cases cannot be explained by p53 defects (deletion or mutation), and alternative mechanisms need to be investigated. Alemtuzumab is effective irrespective of genetic high-risk subgroups with TP53 mutations. These clinical trials are registered at www.clinicaltrials. gov as #NCT00274976. (Blood. 2009;114: 2589-2597)

17p (TP53) deletion identifies patients with chronic lymphocytic leukemia (CLL) who are resistant to chemotherapy. The members of the miR-34 family have been discovered to be direct p53 targets and mediate some of the p53-dependent effects. We studied miR-34a and miR-34b/c expression in a large cohort to define their potential role in refractory CLL. While no expression of miR-34b/c could be detected, we found variable expression levels of miR-34a. miR-34a levels were upregulated after DNA damage in the presence of functional p53, but not in cases with 17p deletion (P < .001). We found a strong correlation of low miR-34a levels with impaired DNA damage response, TP53 mutations (without 17p deletion), and fludarabine-refractory disease (also in the absence of 17p deletion). Up-regulation of miR-34a after irradiation was associated with induction of Bax and p21, but not Puma. CLL cells with reduced miR-34a expression showed increased viability after DNA damage independently of 17p status. Therefore, low expression of miR-34a in CLL is associated with p53 inactivation but also chemotherapy-refractory disease, impaired DNA damage response, and apoptosis resistance irrespective of 17p deletion/TP53 mutation. The elucidation of mechanisms underlying miR-34a regulation and overcoming its role in chemotherapy resistance warrant further study. IntroductionChronic lymphocytic leukemia (CLL) is the most frequent type of leukemia in the Western world and is characterized by a highly variable clinical course. 1-3 Traditionally, therapy has been used for advanced stage or symptomatic disease and consists of chemotherapy with alkylating agents, purine analogs, and more recently antibody-chemotherapy combinations. Different molecular prognostic factors have been used to predict time to treatment, likelihood of responding to chemotherapy and survival time. [4][5][6] A central role of the DNA damage-response pathway and particularly p53 has been suggested by the prognostic role of 17p and 11q deletions in CLL. In the critical regions, 2 prominent genes are located that are involved in the cells' response to DNA damage (eg, induced by chemotherapy). ATM and TP53 have been shown to be deleted in virtually all cases with deletion 11q and 17p, respectively. In contrast, TP53 and ATM are mutated in different proportions. [7][8][9][10][11] There is growing evidence that mutations of TP53 or ATM also in the absence of deletion of 17p or 11q are associated with poor prognosis as a result of impaired response to chemotherapy. 12 Particularly loss of 17p has been associated with failure to respond to chemotherapy and short event-free and overall survival. 8,13,14 However, in chemotherapy-refractory CLL, only 30% to 40% percent of cases will have a deletion or mutation of TP53, whereas approximately one-third of the remaining cases have a deletion of 11q. 15 This suggests that almost half of the refractory cases cannot be explained by a direct defect of p53 or loss of 11q. A hypothesis explaining resistance in these cases might be defects ...

433 Novel gene mutations have been found in CLL by next generation sequencing including mutations of NOTCH1 and SF3B1 in 5–20% of cases. In initial studies, both have been associated with advanced disease and poor outcome. We assessed the incidence and impact of gene mutations in the CLL8 trial (1st line FC vs. FCR, n=817). TP53 (exons 2–11) was analyzed by a re-sequencing chip (Amplichip, Roche Molecular Systems) with confirmatory Sanger sequencing. NOTCH1 was analyzed by Sanger sequencing exon 34, chr9:139,390,619–139,391,290 (PEST domain). SF3B1 (exons 13–16) was analyzed by DHPLC (WAVE® 3500HT, Transgenomic Inc.) with subsequent Sanger sequencing. Baseline samples were available for analysis of genetic markers in 619 (75.8%) to 645 (78.9%) patients. All markers were available for 573 (70.1%) patients and this cohort was representative of the full trial population. Mutations (mut) were found in TP53, NOTCH1, and SF3B1 in 11.5%, 10.0%, and 18.4%, respectively. At least one mutation was identified in 35.2% patients, while 30.6% had one, 4.4% had two and 0.2% had three mutations. Concurrent NOTCH1mut and SF3B1mut were found in only 0.5% patients. TP53mut was observed in 16.7% of NOTCH1mut cases (p=.528) and in 14.5% of SF3B1mut patients (p=.472). Regarding baseline characteristics, there were significant associations of TP53mut with CIRS>1, unmutated IGHV and 17p-; of NOTCH1mut with Binet A/B, no B-symptoms, unmutated IGHV, and 17p-; and of SF3B1mut with TK>10, and no +12. Regarding response to therapy, TP53mut was significantly associated with refractory disease in both arms (FCR: 25.0% vs. 1.8%, p<.001, FC: 48.4% vs. 7.8%, p<.001,); while NOTCH1mut showed only a trend in the FCR arm (FCR: 10.9% vs. 3.4%, p=.109, FC: 11.9% vs. 12.9%, p=.775); and SF3B1mut did not impact response to therapy (FCR: 3.6% vs. 3.7%, p=1.00, FC: 12.3% vs. 10.9%, p=1.00). At extended follow-up (median 69.97 months), FCR resulted into significantly improved PFS (HR 0.586, p<.001) and OS (HR 0.678, p=.001). TP53mut was associated in both treatment arms with significantly decreased PFS (FC: HR 4.295, p<.001; FCR: HR 3.173 p<.001) and OS (FC: HR 4.642 p<.001; FCR: HR 4.447, p<.001). In contrast, NOTCH1mut was only in the FCR arm associated with significantly decreased PFS (FC: HR 0.931, p=.741; FCR: HR 1.718, p=.013) and a trend to inferior OS (FC: HR 0.854, p=.605; FCR: HR 1.610, p=.112). SF3B1mut was associated in both treatment arms with significantly decreased PFS (FC: HR 1.520, p=.009; FCR: HR 1.463, p=.033) and a trend to inferior OS (FC: HR 1.338, p=.178; FCR: HR 1.305, p=.301). To evaluate the independent prognostic impact, we performed multivariable analyses by Cox regression for PFS and OS including the following variables: treatment, age, sex, stage, ECOG status, B-symptoms, WBC, TK, β2-MG, 11q-, +12, 13q-, 17p-, IGHV, TP53, NOTCH1 and SF3B1. Regarding PFS, the following independent prognostic factors were identified: FCR (HR 0.510, p<.001), TK>10 (HR 1.367, p=.019), IGHV<98% (HR 1.727, p<.001), 11q- (HR 1.536, p<.001), 17p- (HR 2.949 p<.001), TP53mut (HR 2.113 p<.001), and SF3B1mut (HR 1.348, p=.024). Regarding OS, the following independent prognostic factors were identified: FCR (HR 0.701, p=.049), ECOG>0 (HR 2.202, p<.001), TK>10 (HR 2.707, p<.001), IGHV<98% (HR 1.547, p=.055), 17p- (HR 3.546 p<.001) and TP53mut (HR 3.032 p<.001). To identify a predictive impact of gene mutations for a specific treatment effect by the addition of rituximab, we performed multivariable analyses including the treatment arms, the gene mutations and the interaction of both. Regarding PFS, FCR (HR 0.544, p<.001), TP53mut (HR 3.607, p<.001), SF3B1mut (HR 1.355, p=.012) and NOTCH1mut interaction with FCR (HR 1.652, p=.022) were identified as independent factors. Regarding OS, FCR (HR 0.654, p=.002) and TP53mut (HR 4.470, p<.001) were identified as independent factors while NOTCH1mut interaction with FCR (HR 1.331, p=.344) showed a trend. The interaction between NOTCH1mut and FCR treatment is illustrated in univariate PFS analysis, in which the addition of rituximab led to a benefit only among patients without NOTCH1mut (Figure). In conclusion, gene mutations show independent prognostic value for PFS (TP53, SF3B1) and OS (TP53) in patients receiving 1st line FC and FCR treatment. Of note, NOTCH1mut appears to identify a subset of CLL patients that does not benefit from the addition of rituximab to FC. Disclosures: Stilgenbauer: Roche: Consultancy, Honoraria, Research Funding. Patten:Roche: Employment. Wenger:Roche: Employment. Mendila:Roche: Employment. Hallek:Roche: Consultancy, Honoraria, Research Funding.