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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 KRASmutations principally composed of these low VAF variants. Firstly, employing orthogonal approaches to confirm <12% VAF TP53mutations, we assessed the clinical impact of TP53clonal architecture. Whilst ≥ 12% VAF TP53mut cases were associated with reduced PFS and OS, we could not demonstrate a difference between <12% VAF TP53mutations and either wild type or ≥12% VAF TP53mut cases. Secondly, we identified biallelic BIRC3lesions (mutation and deletion) as an independent marker of inferior PFS and OS. Finally, we observed that mutated MAPK-ERKgenes 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.

NOTCH1and SF3B1mutations have been previously reported to have prognostic significance in chronic lymphocytic leukemia but to date they have not been validated in a prospective, controlled clinical trial. We have assessed the impact of these mutations in a cohort of 494 patients treated within the randomized phase 3 United Kingdom Leukaemia Research Fund Chronic Lymphocytic Leukemia 4 (UK LRF CCL4) trial that compared chlorambucil and fludarabine with and without cyclophosphamide in previously untreated patients. We investigated the relationship of mutations in NOTCH1(exon 34) and SF3B1(exon 14-16) to treatment response, survival and a panel of established biologic variables. NOTCH1and SF3B1mutations were found in 10% and17% of patients, respectively. NOTCH1mutations correlated with unmutated IGHVgenes, trisomy 12, high CD38/ ZAP-70 expression and were associated with reduced overall (median 54.8 vs 74.6 months, P= .02) and progression-free (median 22.0 vs 26.4 months, P= .02) survival. SF3B1mutations were significantly associated with high CD38 expression and with shorter overall survival (median 54.3 vs 79.0 months, P< .001). Furthermore, multivariate analysis, including baseline clinical variables, treatment, and adverse prognostic factors demonstrated that although TP53alterations remained the most informative marker of dismal survival in this cohort, NOTCH1(HR 1.58, P= .03) and SF3B1(HR 1.52, P= .01) mutations have added independent prognostic value.

Chronic lymphocytic leukemia (CLL) cells have variably low surface IgM (sIgM) levels/signaling capacity, influenced by chronic antigen engagement at tissue sites. Within these low levels, CLL with relatively high sIgM (CLLhigh) progress more rapidly than CLL with low sIgM (CLLlow). During ibrutinib therapy, surviving CLL cells redistribute into the peripheral blood and can recover sIgM expression. Return of CLL cells to tissue may eventually recur, where cells with high sIgM could promote tumor growth. We analyzed time to new treatment (TTNT) following ibrutinib in 70 CLL patients (median follow-up of 66 months) and correlated it with pre-treatment sIgM levels and signaling characteristics. Pre-treatment sIgM levels correlated with signaling capacity, as measured by intracellular Ca2+mobilization (iCa2+), in vitro(r=0.70; p<0.0001). High sIgM levels/signaling strongly correlated with short TTNT (p<0.05), and 36% CLLhighversus 8% CLLlowprogressed to require a new treatment. In vitro,capacity of ibrutinib to inhibit sIgM-mediated signaling inversely correlated with pre-therapy sIgM levels (r=-0.68, p=0.01) or iCa2+(r=-0.71, p=0.009). In patients, sIgM-mediated iCa2+and ERK phosphorylation levels were reduced by ibrutinib therapy, but not abolished. The residual signaling capacity downstream of BTK was associated with high expression of sIgM, while it was minimal when sIgM expression was low (p<0.05). These results suggested that high sIgM levels facilitated CLL cell resistance to ibrutinib in patients. The CLL cells, surviving in the periphery with high sIgM expression, include a dangerous fraction, able to migrate to tissue and receive proliferative stimuli, which may require targeting by combined approaches.

With the application of high-throughput sequencing, our understanding of the somatic mutations that contribute to the pathogenesis of CLL has expanded significantly. Amongst these mutations, NOTCH1and SF3B1are the most prevalent and at diagnosis are associated with reduced survival independent of clinical and biological variables. However, we have limited understanding of the prognostic impact of other, less prevalent mutations. To address this question, we investigated the distribution, clinical significance and clonal acquisition of SF3B1, NOTCH1, XPO1, POT1, BIRC3, MYD88 and FBXW7mutations in a single-centre diagnostic cohort of 140 patients with CLL, of whom 115 had Binet stage A disease and 91 patients with clinical MBL based on the 2008 IWCLL/NCI diagnostic criteria. Ninety patients (35 MBL and 55 CLL) also had DNA samples analyzed at a second time point (median 72 months, 24-145 months from diagnosis). We defined progressive disease as either presentation with stage B/C disease, evolution of MBL or stage A to stage B/C and/or the need for treatment. With high-resolution melt (HRM) analysis and subsequent Sanger sequencing, we identified mutations of SF3B1in 9% (21/225), NOTCH1in 5% (12/228), POT1in 6 %(7/123), XPO1in 2 %(4/196), MYD88in 1% (3/223) and BIRC3mutations in 0.5 % (1/222) of patients. FBXW7mutations were identified in 8% (3/36) of the trisomy 12 patients from our cohort. In the patients studied sequentially we confirmed acquired mutations of SF3B1(n=5) and XPO1(n=1) in 3 patients with MBL and in 3 with CLL. Whilst we confirmed the impact of NOTCH1mutations on treatment free survival (76 vs. 107 months, p=0.002) and SF3B1mutations on overall survival (96 vs. 117 months, p=0.004), the prevalence of mutations in the other genes was too low to assess individually. We therefore assessed the collective burden of mutations in our cohort (excluding the MYD88 cases which had long term stable disease ) and demonstrated the following: 1) In both the MBL and CLL cohorts, the frequency of a gene mutation was higher in patients with progressive disease than in those with long-term, clinically stable disease (MBL: 36 vs. 12% p=0.02, CLL: 41 vs. 10%, p<0.001),and also independent of SF3B1 and NOTCH1 mutations and deletions of 17p (MBL and CLL: 11 vs. 3%; p=0.031). 2) All patients and those in the MBL cohort, with mutations targeting genes other than SF3B1 and NOTCH1were more likely to require treatment (Entire cohort: OR 3.1, p=0.008, MBL cohort: OR 20, p= 0.006). This resulted in a reduced treatment-free survival (Entire cohort: 40 vs. 102 months, HR 10, 95%CI 3-30, p<0.001) and overall survival. (Entire cohort: 97 vs. 120 months HR 2.7, 95% CI 1.2-5.9, p=0.013) which was independent of the presence of a 17p deletion (p=0.008) and of IGHVstatus in bivariate analysis (p=0.02). 4/6 patients who acquired a mutation at 38-208 months (median 127 months) after initial analysis required treatment. Taken together, our study suggests that identifying mutations in genes other than NOTCH1and SF3B1may have utility in the clinical management of MBL and CLL as they predict for progressive disease, treatment requirement and reduced survival. Much larger studies with sequential data post therapy will be required to elucidate whether specific rare gene mutations predict a poor outcome or whether they either result in, or are a marker of genomic instability and subsequent genomic complexity.Frequencies of mutations and cytogenetic aberrationsMBL stable (n=34)MBL evolving to stage A CLL (n=29)MBL progressive (n=22)Binet A CLL stable (n=70)Binet A CLL progressive (n=45)Binet B CLL (n=20)NOTCH1 (%)0/340/293/22 (14)1/69 (1.4)4/43 (9.3)1/19 (5)SF3B1 (%)1/34 (2.9)4/29 (14)2/22 (9)3/69 (4.3)11/45 (24)5/20 (25)total including all other screened genes (%)1/34 (2.9)4/29 (14)8/22 (36)7/70 (10)18/45 (40)7/20 (35)11q23 (%)1/34 (2.9)1/27 (3.7)2/20 (10)1/66 (1.5)9/40 (23)3/16 (19)17p (%)1/32 (3.1)1/27 (3.7)1/19 (5)2/66 (3)0/400/16IGHV unmutated/mutated (% / %)5/29 (15/85)5/24 (17/83)15/7 (68/32)10/60 (14/86)25/20 (66/44)18/2 (90/10)tri12 (%)7/24 (29)5/22 (23)7/18 (32)9/57 (16)10/39 (26)8/16 (50)biallelic del13q14 (%)9/21 (43)4/14 (29)2/11 (9)20/40 (50)6/19 (32)2/12 (17)