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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.

NOTCH1 and SF3B1 mutations 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. NOTCH1 and SF3B1 mutations were found in 10% and17% of patients, respectively. NOTCH1 mutations correlated with unmutated IGHV genes, 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. SF3B1 mutations 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 TP53 alterations 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.

A population of metachromatic cells with mast cell (MC) and basophil features was identified recently in the peripheral blood of patients with several allergic disorders. This study now shows that these metachromatic cells express on their surface the high-affinity IgE receptor (FcεRI), CD4, and the chemokine receptors CCR3, CCR5, and CXCR4, but not the T-cell surface protein CD3 and the monocyte/macrophage surface protein CD68. This population of MCs/basophils can be maintained ex vivo for at least 2 weeks, and a comparable population of cells can be generated in vitro from nongranulated hematopoietic CD3−/CD4+/CD117− progenitors. Both populations of MCs/basophils are susceptible to an M-tropic strain of human immunodeficiency virus 1 (HIV-1). Finally, many patients with acquired immunodeficiency syndrome have HIV-1–infected MCs/basophils in their peripheral blood. Although it is well known that HIV-1 can infect CD4+ T cells and monocytes, this finding is the first example of a human MC or basophil shown to be susceptible to the retrovirus.

A population of metachromatic cells with mast cell (MC) and basophil features was identified recently in the peripheral blood of patients with several allergic disorders. This study now shows that these metachromatic cells express on their surface the high-affinity IgE receptor (FcεRI), CD4, and the chemokine receptors CCR3, CCR5, and CXCR4, but not the T-cell surface protein CD3 and the monocyte/macrophage surface protein CD68. This population of MCs/basophils can be maintained ex vivo for at least 2 weeks, and a comparable population of cells can be generated in vitro from nongranulated hematopoietic CD3−/CD4+/CD117−progenitors. Both populations of MCs/basophils are susceptible to an M-tropic strain of human immunodeficiency virus 1 (HIV-1). Finally, many patients with acquired immunodeficiency syndrome have HIV-1–infected MCs/basophils in their peripheral blood. Although it is well known that HIV-1 can infect CD4+T cells and monocytes, this finding is the first example of a human MC or basophil shown to be susceptible to the retrovirus.

Even in the era of the targeted therapies, there remains clinical value in exploring the impact of disease characteristics in chemo-immunotherapy (CIT) trials, namely to identify features that contribute most to long-term outcomes, thereby pinpointing patients destined to benefit from these therapies. One such feature is the CLL DNA methylome, that recapitulates normal B cell maturation, with IGHV mutated (M-CLL) and unmutated CLL (U-CLL) retaining an imprint of the DNA methylation signature of memory (m-CLL) and naive B cells (n-CLL), respectively, with a third intermediate epigenetic subgroup (i-CLL) with borderline mutation status. The pyrosequencing analysis of 5 CpG sites can divide CLL into these three subgroups (Queiros 2015, Leukemia 29:598-605), each with different clinico-biological features.

Despite the excitement of targeted therapies such asibrutinib and idelalisib, the longer-term follow-up offered by early immuno/-chemotherapy trials presents opportunities to explore the disease features that contribute most to long-term outcomes.

No relevant conflicts of interest to declare.

Introduction

Anagnostopoulos: Gilead Sciences: Research Funding. Fazi:Rhizen Pharmaceuticals SA: Research Funding. Ghia:Merck: Consultancy; GSK, Roche Italia: Consultancy; Gilead, Pharmacyclics, Boehringer Ingelheim, Celgene, Roche Italia: Membership on an entity's Board of Directors or advisory committees.

No relevant conflicts of interest to declare.

Most CLL is diagnosed with a low tumor burden with no indication for therapy. Biomarkers, such as unmutated IGHVgenes, TP53 loss/mutation and raised β2M predict short time to first treatment and overall survival; however there remain patients with good risk biomarkers who nevertheless develop progressive disease. Advances in genomics and immunogenetics have lead to the discovery of new biomarkers and their integration with cytogenetic data refines outcome prediction. However these novel markers are predominantly found in IGHV unmutated cases (U-CLL).

No relevant conflicts of interest to declare.

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)

No relevant conflicts of interest to declare.

Abstract 658

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Abstract 757

Epidemiological data and family studies have indicated that inherited factors may predispose to the development of myeloproliferative neoplasms (MPN). It has also been suggested that single nucleotide polymorphisms (SNPs) within JAK2 are associated with specific MPN subtypes. To explore the role of inherited factors in more detail, we initially performed quantitative analysis of a series of JAK2 SNPs in homozygous PV cases (%V617F >50%; n=73). Most mutant haplotypes could be read directly from the distorted allele ratios brought about by expansion of the homozygous clone. In many cases with 50–90% V617F, the residual wild type haplotype could also be read. Strikingly, of the 144 V617F alleles that could be determined, 111 (77%) had an identical core haplotype (subsequently designated 46/1) whereas only 9/76 (12%) residual wild type alleles were 46/1 (P = 1.9e-21, Fisher’s exact test). To explore this observation in more detail we first determined the haplotype structure of JAK2 using 14 SNPs genotyped by the Wellcome Trust Case Control Consortium (WTCCC) in 1500 UK blood donors. Nine haplotypes were inferred using the program PHASE that accounted for 94% of alleles, with a frequency of 0.24 for haplotype 46/1. Haplotype inference and tag SNP analysis revealed that 46/1 was also more frequent in heterozygous V617F positive MPD cases (135/354 alleles) compared to 188 locally sourced healthy controls (92/376 alleles; P = 0.0001) as well as the WTCCC cohort (P = 3.3e-8). Haplotype 46/1 was more frequent in all V617F positive disease entities compared to controls: PV (n=203; P=1.2e-16), ET (n=81; P=1.2e-9) and MF (n=41; P=8.0e-5) however there was no difference in the frequency of 46/1 between controls and V617F negative MPD / idiopathic erythrocytosis (n=123). To determine if heterozygous V617F also preferentially arose on a 46/1 allele as seen for homozygous cases, we developed an allele specific PCR between V617F and a SNP that tags this haplotype. In an analysis of 67 informative heterozygous V617F cases, 50 V617F alleles were 46/1 compared to only 17 residual wild type alleles (P=9.4e-9). We conclude that the 46/1 JAK2 haplotype is a strong predisposition factor for development of V617F associated MPDs (RR=2.6; 95% CI 2.3–2.9). The reason for this predisposition is currently unknown but it is likely that 46/1 is in linkage disequilibrium with an unknown constitutional functional variant that interacts with V617F JAK2.

Epidemiological data and family studies have indicated that inherited factors may predispose to the development of myeloproliferative neoplasms (MPN). It has also been suggested that single nucleotide polymorphisms (SNPs) within JAK2 are associated with specific MPN subtypes. To explore the role of inherited factors in more detail, we initially performed quantitative analysis of a series of JAK2 SNPs in homozygous PV cases (%V617F >50%; n=73). Most mutant haplotypes could be read directly from the distorted allele ratios brought about by expansion of the homozygous clone. In many cases with 50–90% V617F, the residual wild type haplotype could also be read. Strikingly, of the 144 V617F alleles that could be determined, 111 (77%) had an identical core haplotype (subsequently designated 46/1) whereas only 9/76 (12%) residual wild type alleles were 46/1 (P = 1.9e-21, Fisher's exact test). To explore this observation in more detail we first determined the haplotype structure of JAK2 using 14 SNPs genotyped by the Wellcome Trust Case Control Consortium (WTCCC) in 1500 UK blood donors. Nine haplotypes were inferred using the program PHASE that accounted for 94% of alleles, with a frequency of 0.24 for haplotype 46/1. Haplotype inference and tag SNP analysis revealed that 46/1 was also more frequent in heterozygous V617F positive MPD cases (135/354 alleles) compared to 188 locally sourced healthy controls (92/376 alleles; P = 0.0001) as well as the WTCCC cohort (P = 3.3e-8). Haplotype 46/1 was more frequent in all V617F positive disease entities compared to controls: PV (n=203; P=1.2e-16), ET (n=81; P=1.2e-9) and MF (n=41; P=8.0e-5) however there was no difference in the frequency of 46/1 between controls and V617F negative MPD / idiopathic erythrocytosis (n=123). To determine if heterozygous V617F also preferentially arose on a 46/1 allele as seen for homozygous cases, we developed an allele specific PCR between V617F and a SNP that tags this haplotype. In an analysis of 67 informative heterozygous V617F cases, 50 V617F alleles were 46/1 compared to only 17 residual wild type alleles (P=9.4e-9). We conclude that the 46/1 JAK2 haplotype is a strong predisposition factor for development of V617F associated MPDs (RR=2.6; 95% CI 2.3–2.9). The reason for this predisposition is currently unknown but it is likely that 46/1 is in linkage disequilibrium with an unknown constitutional functional variant that interacts with V617F JAK2.