ALBERT

Description: Analysis of the chronic lymphocytic leukemia (CLL) coding genome has recently disclosed that the NOTCH1 proto-oncogene is recurrently mutated at CLL presentation. Here, we assessed the prognostic role of NOTCH1 mutations in CLL. Two series of newly diagnosed CLL were used as training (n = 309) and validation (n = 230) cohorts. NOTCH1 mutations occurred in 11.0% and 11.3% CLL of the training and validation series, respectively. In the training series, NOTCH1 mutations led to a 3.77-fold increase in the hazard of death and to shorter overall survival (OS; P 〈 .001). Multivariate analysis selected NOTCH1 mutations as an independent predictor of OS after controlling for confounding clinical and biologic variables. The independent prognostic value of NOTCH1 mutations was externally confirmed in the validation series. The poor prognosis conferred by NOTCH1 mutations was attributable, at least in part, to shorter treatment-free survival and higher risk of Richter transformation. Although NOTCH1 mutated patients were devoid of TP53 disruption in more than 90% cases in both training and validation series, the OS predicted by NOTCH1 mutations was similar to that of TP53 mutated/deleted CLL. NOTCH1 mutations are an independent predictor of CLL OS, tend to be mutually exclusive with TP53 abnormalities, and identify cases with a dismal prognosis.

Description: Abstract 283 The clinical course of chronic lymphocytic leukemia (CLL) ranges from very indolent, with a nearly normal life expectancy, to rapidly progressive leading to death and occasionally undergoing transformation to Richter syndrome (RS). TP53 disruption identifies a fraction of high risk CLL destined to experience a very short survival. High risk CLL, however, cannot be fully recapitulated by TP53 disruption and other lesions of cancer genes may be implicated in this aggressive phenotype. Analysis of the CLL coding genome has recently disclosed that the NOTCH1 proto-oncogene is recurrently mutated at CLL presentation. Here we assessed the prognostic role of NOTCH1 mutations in CLL. Two series of previously untreated CLL were utilized as training (n=309, median follow-up 6 years) and validation (n=230, median follow-up 7 years) cohorts. NOTCH1 mutations were analyzed by DNA Sanger sequencing in blind with respect to clinical data. In the training series, NOTCH1 mutations occurred in 34/309 (11.0%) patients, being mostly represented (26/34, 76.5%) by a recurrent two bp frameshift deletion (c.7544_7545delCT). The remaining NOTCH1 mutations (8/34, 23.5%) were frameshift deletions other than c.7544_7545delCT (n=7) and frameshift insertions (n=1). All mutations were predicted to disrupt the NOTCH1 PEST domain. CLL with NOTCH1 mutations preferentially carried unmutated IGHV genes (76.5%, p

Description: RS occurs in 5–15% CLL, depending on follow-up length and re-biopsy policy. The value of biological and clinical risk factors in predicting RS is unknown and represents the aim of this study. The analysis was based on a consecutive series of 185 CLL. Diagnosis of RS was based on lymph node or extranodal tissue biopsy. RS was diagnosed in 17 cases all represented by diffuse large B-cell lymphoma (DLBCL). Cumulative incidence of RS at 5 and 10 years was 13.6% (95%CI: 7.0–20.1%) and 16.2% (95%CI: 8.0–24.4%), respectively. Transformation plateaued at 82 months of follow-up. Median time to transformation was 23.0 months (95%CI: 15.9–30.1 months) from CLL diagnosis. IGHV studies documented clonal relationship between CLL and DLBCL in 15/17 (88.2%) cases. At the time of CLL diagnosis, molecular variables predicting RS were: IGHV4–39 usage (5-year risk IGHV4-39: 56.2% vs IGHVnon-VH4-39: 11.2%; p

Description: BCL2 expression is increased in CLL cells and associates with decreased survival of CLL patients. BCL2 C938A polymorphism is localized in the inhibitory P2 promoter of BCL2 and modulates BCL2 expression. Accordingly, BCL2 expression is significantly increased in CLL carrying BCL2 938 AA genotype, compared to CLL carrying BCL2 938 CC genotype. We aimed at testing the prognostic impact of BCL2 C938A in a series of 182 consecutive CLL. Genotyping of BCL2 C938A was performed by SNP minisequencing. Biological covariates were CD38 expression, IGHV mutation, FISH karyotype, and p53 mutations. Clinical covariates were age, sex, Rai and Binet stages, number of nodal areas, largest lymph node size, splenomegaly, lymphocyte count, Hb, platelet count, bone marrow lymphocytes, pattern of bone marrow involvement, lymphocyte doubling time (LDT), beta-2-microglobulin (B2M), LDH, alkaline phosphatase, albumin. Genotype distribution of BCL2 C938A in CLL was 47/182 (25.8%) AA, 96/182 (52.7%) AC, and 39/182 (21.5%) CC. Allele frequencies were in accordance with Hardy-Weinberg equilibrium (A: 0.522; C: 0.478; p=0.749, χ2). Biological and clinical variables at diagnosis distributed without significant differences among AA, AC and CC genotypes. Treatment free survival (TFS) did not differ in AA, AC and CC genotypes when analyzed separately (5-year TFS AA=58.1%; AC=46.4%; CC=50.0%; p=.228), after pooling C-allele carriers (5-year TFS AA=58.1%; AC/CC=47.6%; p=.414), or after pooling A-allele carriers (5-year TFS AA/AC= 50.1%; CC=50.0%; p=.247). Overall survival (OS) did not differ in AA, AC and CC genotypes when analyzed separately (5-year OS AA=80.1%; AC=76.8%; CC=76.1%; p=.900), after pooling C-allele carriers (5-year OS AA=80.1%; AC/CC=76.5%; p=.837), or after pooling A-allele carriers (5-year OS AA/AC= 77.9%; CC=76.1%; p=.649). The independent prognostic value of BCL2 C938A was assessed separately for biological and clinical variables by Cox multivariate analysis. When tested along with biological covariates, BCL2 C938A was not an independent predictor of TFS or OS. Biological covariates independently predicting TFS were del11q22–23 (HR 5.52; 95%CI 2.26–11.87; p

Description: Abstract 466 The genetic lesions identified to date do not fully recapitulate the molecular pathogenesis of chronic lymphocytic leukemia (CLL) and do not entirely explain the development of severe complications, such as chemorefractoriness. BIRC3, along with TRAF2 and TRAF3, cooperate in the same protein complex that negatively regulates MAP3K14, the central activator of non-canonical NF-kB signaling. Following the initial observation of recurrent BIRC3 mutations in splenic marginal zone lymphomas, we performed targeted re-sequencing of the BIRC3 coding sequence and splicing sites across the spectrum of B-cell neoplasia. By this screening, BIRC3 mutations were identified only in CLL (2/20), while were absent in diffuse large B-cell lymphoma (0/30), Burkitt lymphoma (0/38), follicular lymphoma (0/20), extranodal marginal zone lymphoma (0/65), hairy cell leukemia (0/19), and multiple myeloma (0/22). This observation prompted the investigation of prevalence and clinical impact of BIRC3 genetic lesion in CLL. The study population included 4 clinical CLL panels representative of different disease phases: i) a cohort of fludarabine-refractory CLL (n=49); ii) a cohort of fludarabine-sensitive CLL (n=62); iii) clonally related Richter syndrome (RS) (n=33; all diffuse large B cell lymphomas); and iv) a consecutive series of newly diagnosed and previously untreated CLL (n=308). BIRC3 was analyzed for mutations by Sanger sequencing (exons 2–9) and for copy number abnormalities by FISH (probe RP11-177O8). BIRC3 was affected in 12/49 (24%) fludarabine-refractory CLL by inactivating mutations (7 frameshift and 1 non-sense) and gene deletions (n=7) that distributed in a mutually exclusive fashion with TP53 disruption (p=.004) (Fig 1A and 1B). Two fludarabine-refractory CLL carried a biallelic inactivation of the BIRC3 gene by mutation of one allele and deletion of the second allele. All inactivating mutations were somatically acquired, were predicted to generate aberrant transcripts carrying premature stop codons, and caused elimination or truncation of the C-terminal RING domain, whose E3 ubiquitin ligase activity is essential for MAP3K14 proteosomal degradation by BIRC3. Western blot analysis confirmed the expression of aberrant bands corresponding in size to the predicted truncated BIRC3, and revealed constitutive non-canonical NF-kB activation in fludarabine-refractory CLL harboring BIRC3 disruption by mutations or deletion, as documented by NFkB2 processing from p100 to p52 (Fig. 1). BIRC3 was the sole gene of the TRAF3/MAP3K14-TRAF2/BIRC3 complex targeted by molecular lesions after extensive mutation and FISH analysis of TRAF2, TRAF3 and MAP3K14 in the fludarabine-refractory CLL cohort (n=49). To investigate whether BIRC3 genetic lesions were restricted to chemorefractory cases, we analyzed BIRC3 for mutations and deletions in other CLL subsets. Fludarabine-sensitive CLL were consistently devoid of BIRC3 disruption in all cases (n=62), suggesting that BIRC3 genetic lesions specifically associate with a chemorefractory phenotype among CLL requiring treatment (Fig. 1A). BIRC3 disruption was restricted to 1/33 (3.0%) clonally-related RS, corroborating the notion that RS is molecularly distinct from chemorefractory progression without transformation (Fig. 1A). In a consecutive series evaluated at CLL diagnosis, BIRC3 disruption was rare (13/308, 4%, all TP53 wild type), associated with primary chemorefractoriness among cases requiring treatment, and predicted poor outcome (Fig. 1A). By univariate analysis, the crude impact of BIRC3 disruption on survival was a ∼5.3 fold increase in the hazard of death (HR: 5.31; 95% CI: 2.62–10.73) and a significant overall survival (OS) shortening (median OS in BIRC3 disrupted cases: 3.1 years vs not reached in BIRC3 wild type cases; p

Description: Background Almost 60% cases of splenic marginal zone lymphoma (SMZL) cases harbor molecular lesions affecting signalling pathways involved in normal marginal zone (MZ) differentiation, including the NOTCH pathway, the NF-kB pathway, the toll-like receptor (TLR) pathway and the B-cell receptor (BCR) pathway. However, little is known with regard to these lesions in other indolent B-cell lymphoproliferative disorders mimicking SMZL. Methods Candidate gene mutations (NOTCH2, NOTCH1, BIRC3, TNFAIP3, TRAF3, IKBKB, MYD88, CD79A, CD79B, CARD11) were investigated in 60 indolent B-cell lymphoproliferative disorders, including nodal marginal zone lymphoma (NMZL=33), monoclonal B-cell lymphocytosis showing a MZL-like phenotype (MZL-like MBL=16), and variant hairy cell leukemia (vHCL=11). In all cases, tumor representation was 〉50% in order to allow the detection of clonal lesions. All cases lacked the BRAF V600E mutation as assessed by AS-PCR. Results Overall, the genetics of NMZL and MZL-like MBL was consistent with that of SMZL, suggesting the involvement of a common oncogenic pathway in these disorders. Among NMZL, 51% (17/33) of cases were characterized by mutually exclusive genetic lesions affecting MZ differentiation genes, including NOTCH2 stabilizing mutations in 24% (8/33) of cases, TNFAIP3 disrupting mutations in 12% (4/33), MYD88 activating mutations in 12% (4/33), and NOTCH1, TRAF3 and BIRC3 mutations in 3% (1/33) of cases each. Among MZL-like MBL, 37% (6/16) of cases harbored mutually exclusive lesions of MZ genes, including MYD88 mutations in 25% (4/16) of cases, NOTCH2 mutations in 12% (2/16), and TNFAIP3 and CD79B mutations in 6% (1/16) of cases each. On the contrary, all cases (n=11) of vHCL lacked mutations of NOTCH, NF-kB, TLR or BCR genes, suggesting that none of these signaling pathways plays a relevant role in this disease. Among NMZL, MYD88 mutations were enriched among cases harboring an IgM type monoclonal component (3/4, 75% vs 1/18, 5%, p=.010) and showing cytological clues of plasma cell differentiation of lymph node tumor cells (3/6, 50% vs 0/9; p=.040). NOTCH2 mutations did not correlate with any pathologic feature (i.e. lymph node pattern of infiltration, presence of monocytoid B cells, immunoglobulin gene usage or mutation status). Among MZL-like MBL, neither MYD88 mutations nor NOTCH2 mutations correlated with specific clinico-pathologic features (i.e. presence of an IgM type monoclonal component, intrasinusoidal bone marrow infiltration, plasma cell differentiation). Conclusions These data suggest that: i) SMZL, NMZL and MZL-like MBL share a similar genotype and are all promoted by the same molecular deregulation of MZ differentiation genes; and ii) vHCL stands as a genetically different entity among indolent B-cell lymphoproliferations. These data might help to refine the differential diagnosis of indolent B-cell lymphoproliferations mimicking SMZL. Disclosures: No relevant conflicts of interest to declare.

Description: Splenic marginal zone lymphoma (SMZL) is one of the few B-cell lymphoma types that remain orphan of molecular lesions in cancer-related genes. Detection of active NF-κB signaling in 14 (58%) of 24 SMZLs prompted the investigation of NF-κB molecular alterations in 101 SMZLs. Mutations and copy number abnormalities of NF-κB genes occurred in 36 (36%) of 101 SMZLs and targeted both canonical (TNFAIP3 and IKBKB) and noncanonical (BIRC3, TRAF3, MAP3K14) NF-κB pathways. Most alterations were mutually exclusive, documenting the existence of multiple independent mechanisms affecting NF-κB in SMZL. BIRC3 inactivation in SMZL recurred because of somatic mutations that disrupted the same RING domain that in extranodal marginal zone lymphoma is removed by the t(11;18) translocation, which points to BIRC3 disruption as a common mechanism across marginal zone B-cell lymphomagenesis. Genetic lesions of NF-κB provide a molecular basis for the pathogenesis of more than 30% of SMZLs and offer a suitable target for NF-κB therapeutic approaches in this lymphoma.

Description: Background. Nodal marginal zone lymphoma (NMZL) is one of the few B-cell tumors still remaining orphan of cancer gene lesions. By combining whole exome sequencing (WES), deep sequencing of tumor-related genes, high resolution SNP array and RNAseq, here we aim at characterizing the coding genome of NMZL and at disclosing the pathways that are molecularly deregulated in this lymphoma. Methods. The study was based on 35 NMZL (tumor representation 〉70%) with a diagnosis confirmed by: i) pathological revision of lymph node histology; and ii) lack of clinico-radiological evidence of extranodal or splenic disease either at diagnosis or during follow-up. Consistent with NMZL, the cases investigated: i) lacked CD5, CD10 and cyclin D1 expression, 7q deletion, t(11;14), t(14;18), t(11;18) and t(1;14) translocations; and ii) recurrently harbored +3 (14%), +12 (14%) and preferential usage of the IGHV4-34 gene (17%). WES (HiSeq 2500, Illumina; mean coverage per sample: 38x-114x) and high density SNP array (Cytoscan HD, Affymetrix) of tumor/normal DNA pairs from 18 discovery NMZL identified 557 non-synonymous somatic mutations (average: 30.9/case) affecting 504 genes and 61 copy number abnormalities (CNA) (average 3.4/case). To further characterize mutation recurrence, the 504 discovered genes were investigated in an independent validation panel of 17 NMZL by targeted sequencing of tumor/normal DNA pairs (MiSeq; target region: 1.6 Mb; mean coverage per sample: 171x-386x). The 17 validation NMZL were also assessed for CNA by high density SNP arrays. RNAseq of 11 discovery NMZL did not identify any recurrent gene fusion. Results. By compiling the results of WES and high resolution SNP array, 39 genes were recurrently affected in 〉3/35 (9%) NMZL by mutations (n=30 genes) or focal CNA (n=9 genes). Among these, MLL2 (34%), PTPRD (20%) and NOTCH2 (20%) were most frequently mutated. Overall, recurrently mutated genes pointed to the molecular deregulation of specific programs in NMZL, namely JAK/STAT, NOTCH, NF-κB and toll-like receptor (TLR) signaling, cell cycle, chromatin remodeling/transcriptional regulation and immune escape (Fig. 1A). JAK/STAT signaling was targeted by mutually exclusive lesions in 43% of NMZL, and the protein tyrosine phosphatase receptor delta (PTPRD) tumor suppressor was the most frequently affected gene of this system in 20% of NMZL (Fig. 1B-E). PTPRD inhibits JAK/STAT signaling through the dephosphorylation of active p-STAT3. PTPRD lesions in NMZL were represented by somatic mutations that truncated or modified the tyrosine phosphatase domain, as well as deletions of the entire gene locus, including focal and biallelic losses (Fig. 1B-C). Interrogation of institutional and public genomic datasets revealed that PTPRD mutations are specific for NMZL, being rare or absent in other mature B-cell tumors, including splenic marginal zone lymphoma (Fig. 1D). Other JAK/STAT signaling genes affected in NMZL were JAK2, CXCR4 (6%), PTPN2, JAK3, STAT2, SH2B3 and CUL3 (3%) (Fig. 1E). NF-kB signaling was altered in 54% of NMZL by lesions of TNFAIP3 (14%), BCL10, REL (11%), CARD11 (9%), TRAF3 and BIRC3 (6%). NOTCH signaling was targeted in 40% of NMZL by mutations that alternatively involved NOTCH2 (20%), SPEN (11%), RBPJL (6%), FBXW7, DTX1, ITCH and MAML2 (3%). TLR signaling was targeted in 17% of NMZL, including mutations of MYD88 (9%), IRAK1BP1, PELI2 and SEMP6 (3%). Several cell cycle genes were molecularly deregulated in 43% of NMZL, including CDKN2A, PARK2, PARKG (9%), CDC16, CDCA2 (6%), CCNA1, CCNT2, CDK5, CDK13, CDK20, BTG2, HECA and PLK2 (3%). Most (71%) NMZL harbored genetic lesions affecting epigenetic modifiers (MLL2: 34%; CREBBP: 9%; EP300: 6%; TRRAP: 6%), histones (20%) or transcriptional co-repressors (TBL1XR1: 14%; ARID1A: 14%; RCOR1: 11%; NCOR2: 9%, ARID1B: 9%). Finally, the TNFRSF14 and FAS genes, involved in T cell-mediated tumor surveillance, were disrupted by mutations and/or deletions in 17% and 14% NMZL, respectively. Conclusions. A number of actionable cellular programs are molecularly deregulated in NMZL, including JAK/STAT, NOTCH, NF-κB and TLR signaling, cell cycle and chromatin remodeling. PTPRD lesions are among the most recurrent alterations in NMZL and appear to be specific for this lymphoma type across mature B-cell tumors. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Description: The genetic lesions identified to date do not fully recapitulate the molecular pathogenesis of chronic lymphocytic leukemia (CLL) and do not entirely explain the development of severe complications such as chemorefractoriness. In the present study, BIRC3, a negative regulator of noncanonical NF-κB signaling, was investigated in different CLL clinical phases. BIRC3 lesions were absent in monoclonal B-cell lymphocytosis (0 of 63) and were rare in CLL at diagnosis (13 of 306, 4%). Conversely, BIRC3 disruption selectively affected 12 of 49 (24%) fludarabine-refractory CLL cases by inactivating mutations and/or gene deletions that distributed in a mutually exclusive fashion with TP53 abnormalities. In contrast to fludarabine-refractory CLL, progressive but fludarabine-sensitive patients were consistently devoid of BIRC3 abnormalities, suggesting that BIRC3 genetic lesions associate specifically with a chemorefractory phenotype. By actuarial analysis in newly diagnosed CLL (n = 306), BIRC3 disruption identified patients with a poor outcome similar to that associated with TP53 abnormalities and exerted a prognostic role that was independent of widely accepted clinical and genetic risk factors. Consistent with the role of BIRC3 as a negative regulator of NF-κB, biochemical studies revealed the presence of constitutive noncanonical NF-κB activation in fludarabine-refractory CLL patients harboring molecular lesions of BIRC3. These data identify BIRC3 disruption as a recurrent genetic lesion of high-risk CLL devoid of TP53 abnormalities.

Description: The genetic lesions identified in chronic lymphocytic leukemia (CLL) do not entirely recapitulate the disease pathogenesis and the development of serious complications, such as chemorefractoriness. While investigating the coding genome of fludarabine-refractory CLL, we observed that mutations of SF3B1, encoding a splicing factor and representing a critical component of the cell spliceosome, were recurrent in 10 of 59 (17%) fludarabine-refractory cases, with a frequency significantly greater than that observed in a consecutive CLL cohort sampled at diagnosis (17/301, 5%; P = .002). Mutations were somatically acquired, were generally represented by missense nucleotide changes, clustered in selected HEAT repeats of the SF3B1 protein, recurrently targeted 3 hotspots (codons 662, 666, and 700), and were predictive of a poor prognosis. In fludarabine-refractory CLL, SF3B1 mutations and TP53 disruption distributed in a mutually exclusive fashion (P = .046). The identification of SF3B1 mutations points to splicing regulation as a novel pathogenetic mechanism of potential clinical relevance in CLL.