Splenic Marginal Zone Lymphoma Cases Research Articles

The Proliferative History Index, Epicmit, Derived from Genome-Wide Epigenomic Profiling, Is a Key Driver of Clinical Survival in Splenic Marginal Zone Lymphoma

The epiCMIT (epigenetically-determined Cumulative MIToses) mitotic clock (Durran-Ferrer, 2020) tracks DNA hypo- and hyper-methylation changes in heterochromatin and H3K27me3-containing chromatin, respectively, providing a comprehensive record of B cell mitotic history (pre- and post-malignant transformation). Higher epiCMIT levels correlate with poor survival in CLL and MCL, suggesting that a greater mitotic history predicts future proliferative capacity. However, no studies have investigated epiCMIT in splenic marginal zone lymphoma (SMZL). We studied 142 SMZL cases, a rare lymphoid malignancy with specific genetic characteristics [del(7q), KLF2and NOTCH2 mutations, biased use of IGHV1-2*04]. After calculating epiCMIT scores from 450/850K Illumina arrays we compared these data with gene mutations from targeted resequencing and WGS, copy number variations, telomere length, RNA-Seq, miRNA-Seq data, and clinical outcome data. By defining epiCMIT-hyper [mean: 0.6, range: 0.29-0.91] and -hypo [mean: 0.71, range: 0.39-0.90] values in our cohort, we demonstrate a strong positive correlation between hyper- and hypo- scores, as previously observed in MCL and CLL (Durran-Ferrer, 2020). The highest score obtained for epiCMIT-hyper or -hypo per sample was then selected as the epiCMIT score per patient [mean: 0.72, range: 0.39-0.91]. We identified significant correlations between epiCMIT and key clinico-biological characteristics, including a significant negative correlation between epiCMIT and telomere length (R 2=0.67 p<0.001) supporting cell proliferation and concomitant telomere attrition. High epiCMIT scores were associated with female gender (p=0.02), IGHV1-2*04 alleles (p<0.001) with intermediate levels of IGHV somatic hypermutation (SHM) load (97-99.9%, p=0.04), driver somatic gene mutations, including those in KLF2 (p<0.001), NOTCH2 (p<0.01), TP53 (p=0.01) and KMT2D (p<0.001) and deletion of 7q (p=0.01) ( Fig1). Using previously defined classification systems (Arribas, 2015 and Bonfiglio, 2022), we show enrichment of high-risk subgroups [Arribas- high global methylation subgroup (High-M, p<0.001) and Bonfiglio- NFKB, NOTCH and KLF2 genetic subgroup (NNK, p=0.01)] in cases with high epiCMIT. In a sub-cohort of 42 SMZL cases, with a spectrum of epiCMIT values and WGS/ (mi)RNA-Seq data available, we demonstrated 1) a significantly higher genome-wide somatic mutational burden in patients with high epiCMIT-total scores (25 v 17mut/Mb, p=0.001), and 2), correlations between epiCMIT and specific gene/miRNA expression associated with cellular survival and proliferation ( CARD11 [R 2=0.6, p<0.01], MAP2K1 [R 2=0.61, p<0.01], miRNA-155 [R 2=0.58, p=0.018]), apoptosis ( BCOR [R 2=0.61, p<0.01]), and epigenetic regulation ( EZH2 [R 2=0.58, p<0.01]). We then conducted univariate Cox Proportional Hazards analysis to investigate the prognostic significance of clinical and molecular features on time to first treatment (TTFT; most prevalent treatments: splenectomy, n=51; rituximab, n=20) and overall survival (OS). The epiCMIT score and epiCMIT-hyper values showed the highest hazard ratios (HR) for TTFT (HR=26.6, p=0.001) and OS (HR=9.45, p=0.043) respectively ( Fig2). Other factors associated with shorter TTFT included TP53 mutation, 3q gain, IGHV1-2*04 usage, High-M, short telomeres and female gender. Shorter OS was significantly (p<0.05) linked to mutations in key genes (including TP53 and NOTCH2), genomic complexity and del(7q). To avoid redundancy and collinearity, only the most significant epiCMIT metric was included in a multivariate Cox Proportional Hazards model (backwards selection, 94 patients/63 events), and epiCMIT-total emerged as an independent marker for shorter TTFT (HR=44.4, p=0.004), together with gain of chromosome 3q (HR=2.6, p=0.002). Kaplan-Meier analysis further confirmed the importance of epiCMIT-total, with higher scores (>median: 0.73) associated with significantly shorter TTFT (median 23 vs 3.5 months) and higher mortality (p=0.004). To conclude, we demonstrate the potential clinical utility of epiCMIT score in SMZL patients, identifying that SMZL patients with high epiCMIT harbour specific genetic characteristics, and exhibit reduced treatment-free survival. EpiCMIT could be valuable in clinical practice, helping to identify those patients destined to progress and require closer monitoring.

Favorable Outcomes of Splenic Marginal Zone Lymphoma in an International Study of 934 Patients with Long Follow-up

Introduction: Splenic marginal zone lymphoma (SMZL) is a rare, indolent disease. There is no universally accepted standard therapy and long-term outcome data are limited. Rituximab monotherapy is increasingly used over splenectomy in the first-line setting despite a paucity of comparative studies. In the absence of randomized trials, large-scale international real-world data studies are critical to evaluate outcomes of different treatment interventions and to understand the natural history of SMZL in the rituximab-era. Methods: Adult patients with SMZL diagnosed between Jan 1 st 2000 and Dec 31 st 2018 were identified using regional/nationwide population-based registries or hospital registries in Europe, the UK, North America, and Australia. Medical records and pathology reports from eligible patients were reviewed locally by clinicians with specialty experience in hemato-oncology. SMZL cases were required to meet the diagnostic criteria by Matutes et al. Detailed data on clinico-pathological features, comorbidities, treatments (including treatment intention), treatment responses and survival outcomes were entered in a central study database. Overall survival (OS) was defined as time from diagnosis until death from any cause or censoring at last follow-up unless stated otherwise. Event-free survival (EFS) was defined as the time from diagnosis until histological transformation, initiation of new chemotherapy or death from any cause. Survival probabilities were estimated using the Kaplan-Meier estimator. Cumulative risk of transformation was estimated with death as a competing risk. Primary endpoints were 10-year OS and 5-year EFS respectivly. Results: A total of 934 patients from 25 sites were included. Median age at diagnosis was 68 years (IQR 60-76) and 54% were female. Bone marrow involvement was present in 96% (564/590) of those assessed, anemia (Hb <12g/dL for women or <13g/dL for men) in 58%, LDH >UNL in 46%, lymphocytosis (>5 x10 9/L) in 34%, and B-symptoms in 34%. Treatment data were available for 97% of patients. Median follow-up was 9.1 years (range 0-23). Watch and wait was the initial strategy in 28% and 88% of patients received at least one line of treatment during follow-up with the most common being splenectomy (45%), immuno-chemotherapy (19%) or rituximab monotherapy (8%). Fifteen percent received “other” treatments (i.e., combinations of listed therapies and/or radiation). The overall response rate to first-line treatment was 88% (95%CI: 85-90%) with 61% (95%CI: 57-64%) achieving CR. Second line (2L) therapy was needed in 34% during follow-up, 3L in 20% and 4L+ in 9%. The 5-year EFS and OS for all patients were 61% (95%CI: 57-65%) and 77% (95%CI: 74-80%) respectively. The 10-year OS and lymphoma-specific mortality (with non-SMZL deaths as competing risk) estimates were 60% (95%CI: 57-64%) and 11% (95%CI: 9-14%) respectively. The 5-year OS after 2L and 3L were 61% (95%CI: 55-68%) and 51% (95%CI: 42-61%) respectively (figure 1). The 10-year cumulative risk of histological transformation was 17% (95%CI: 13-21%), with 5-year post-transformation OS of 42% (32-54%). At the time of transformation, 60% were chemo-naive and 52% had never been treated for SMZL. The 5-year OS for chemo-naive patients with transformation was 54% (95%CI: 41-71%) versus 24% (95% CI: 13-44%) for transformations after (immuno)chemotherapy (p = 0.01). When comparing patients intended for treatment in first line with splenectomy (n=312) versus rituximab (n=70), patients treated with splenectomy more frequently presented with extra-hilar lymphadenopathy (35% vs 18%), splenomegaly (92% vs 87%) and anemia (64% vs 48%). OS was similar for the two groups (Figure 2, p = 0.53). The 5-year EFS were 63% (95%CI: 57-68%) and 64% (95%CI: 54-77%), respectively (p = 0.31). Conclusions: To date, this is the largest real-world study of SMZL with detailed treatment and outcome data. The study shows that histological transformation was not uncommon with 10-year risk of 17%. However, outcomes for the entire cohort were excellent irrespective of initial treatment and both OS and EFS were similar after first line rituximab versus splenectomy. Long-term survival remained relatively high even following multiple lines of therapy.

Clinicopathological features of CD5-positive splenic marginal zone lymphoma

AimsTo investigate the clinicopathological features, immunophenotypes and differential diagnosis of CD5-positive splenic marginal zone lymphoma (SMZL).MethodsWe retrospectively analysed 16 CD5-positive cases of SMZL. Assess their clinicopathological features and survival outcomes...

Distinct Modes of Ongoing Antigen Interactions Shape Intraclonal Dynamics in Splenic Marginal Zone Lymphoma

Almost one-third of all splenic marginal zone lymphoma (SMZL) cases express B cell receptor immunoglobulin (BcR IG) encoded by the IGHV1-2*04 gene. Such cases display a distinctive profile of genomic aberrations (e.g. higher incidence of NOTCH2 and KLF2 mutations) and a more aggressive clinical course compared to SMZL cases utilizing other IGHV genes. Such skewing of the BcR IG gene repertoire implicates antigen selection in SMZL ontogeny. Although the supportive evidence is compelling, it mostly derives from low-throughput approaches, which are inherently limited in their capacity to capture the complexity of the BcR IG gene repertoire. This hinders the comprehensive assessment of the subclonal architecture of SMZL that could offer insight into the dynamics of antigen-IG interactions. Here, we sought to overcome this limitation through a high-throughput immunogenetic investigation of SMZL, focusing on the detailed characterization of somatic hypermutation (SHM) and intraclonal diversification (ID) profiles. Our study included 22 cases utilizing the IGHV1-2*04 gene and 36 cases utilizing other IGHV genes. IGHV-IGHD-IGHJ (IGH) gene rearrangements were PCR-amplified and libraries were sequenced on the Illumina MiSeq platform. Data was analyzed with the IMGT/HighV-QUEST and TRIP software as well as a novel bioinformatics/biostatistics pipeline. Clonotypes were defined as unique combinations of a given IGHV gene+VH CDR3 amino acid (aa) sequence. Only IGH gene rearrangement sequences assigned to the dominant clonotypes of each case were assessed. In detail, all nucleotide variants (nt vars, i.e. all sequences clustered in the same dominant clonotype yet displaying distinct SHM profiles) were identified and further analyzed. Starting from the most abundant nt var, a network was built representing its connections with all other nt vars. For this analysis, we introduce the terms ‘most relevant pathway’ (MRP) corresponding to the pathway including connected nt vars with the highest total number of IGH sequences; and ‘longest mutational pathways’ (LMP) corresponding to the pathways with the highest number of nt vars (Fig. 1). Different graph metrics assessed the impact of ID in different SMZL subgroups: the first one focuses on the ‘most relevant pathway’ and quantifies SHM convergence [ratio of the total number of IGH sequences corresponding to the nt vars of this pathway to the number of IGH sequences in the most abundant nt var]; while the second refers to the length of the ‘longest mutational pathways’.Cases lacking additional connected nt vars [length of the LMP=1; 3 IGHV1-2*04 cases and 4 non-IGHV1-2*04 cases] were excluded. Consequently, the analysis included 19 IGHV1-2*04 cases and 32 non-IGHV1-2*04 cases. Significant differences were noted in the SHM and ID profiles between groups; the IGHV1-2*04 group had significantly (p<0.01) higher convergence values ranging from 0.009 to 1.243 (median: 0.102), as opposed to the non-IGHV1-2*04 group (range: 0.002-1.13, median: 0.014), overall suggesting that stronger selective pressures act in SMZL cases expressing the IGHV1-2*04 versus others. Moreover, IGHV1-2*04 cases displayed significantly (p<0.01) longer mutational pathways (length range: 2-6, median: 3) compared to the other group (range: 2-5, median: 2), alluding to more pronounced ID arising due to ongoing SHM.Finally, all mutations leading to aa changes were analyzed in the context of ID networks. More recurrent aa mutations were identified amongst cases with higher levels of convergence. For instance, the VH FR2 M39I change, one of the most prominent recurrent SHMs in the IGHV1-2*04 group, was found in the most abundant nt var in 13/19 IGHV1-2*04 cases, while it was identified in nt vars with extra mutations in another 3 cases. Of interest, it was present at the end of the mutational pathways in these 16 cases, whilst in the other group it was present only in one case using the IGHV1-2*02 gene, and absent in the rest (p<0.01).In conclusion, in the first large-scale high-throughput immunogenetic analysis of SMZL we provide strong evidence for more pronounced antigenic pressure in cases utilizing IGHV1-2*04 versus other IGHV genes. Our findings highlight a unique subclonal architecture for IGHV1-2*04 SMZL and corroborate the hypothesis that this group may represent a distinct molecular variant of SMZL. [Display omitted] DisclosuresRossi: Abbvie: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; AstraZeneca: Honoraria, Research Funding; Gilead: Honoraria, Research Funding; Verastem: Honoraria, Research Funding; Roche: Honoraria, Research Funding; Cellestia: Honoraria, Research Funding. Chatzidimitriou: Janssen: Honoraria, Research Funding; Abbvie: Honoraria, Research Funding. Stamatopoulos: Janssen: Honoraria, Research Funding; Gilead: Honoraria, Research Funding; Abbvie: Honoraria, Research Funding; AstraZeneca: Honoraria, Research Funding.

Microenvironment-Specific Activation of Notch2 in Splenic Marginal Zone Lymphoma

Notch receptors are transmembrane proteins that participate in a signaling pathway in which ligand binding induces cleavage and nuclear translocation of the Notch intracellular domain (NICD). This leads to formation of a transcriptional activation complex that turns on Notch target genes and mediates downstream effects. Gain-of-function mutations leading to Notch hyperactivation are common in a subset of hematologic malignancies. However, the distribution of mutations within Notch receptors varies. In T-ALL, mutations involving the extracellular domain of NOTCH1 that lead to ligand independent receptor cleavage and activation are frequently observed. By contrast, mutations in NOTCH1/2 in B-cell lymphomas are largely restricted to the C-terminal PEST domain, which normally ensures rapid turnover of ICD. This implies that oncogenic Notch signaling in B-cell lymphomas is ligand- dependent, with PEST deletions serving to turn up signaling within particular ligand-rich microenvironments.We previously observed that NOTCH1 activation in CLL was sharply increased in the lymph node microenvironment and was not confined to NOTCH1 mutated tumors, though NOTCH1 mutated CLLs did have higher levels of NICD1. By analogy, we hypothesized that NOTCH2 activation in splenic marginal zone lymphoma (SMZL) would be increased in splenic marginal zones, a microenvironment rich in the Notch ligand DLL1, and that NOTCH2 activation would be stronger in, but not restricted to, NOTCH2 mutated tumors. We tested these predictions by doing immunohistochemistry (IHC) using an antibody directed against NICD2 in archival formalin-fixed paraffin embedded cases of SMZL (39), chronic lymphocytic leukemia (CLL, 43), non-splenic marginal zone lymphoma (MZL, 57), and mantle cell lymphoma (MCL, 47). Two experienced pathologists scored the stained slides for intensity (0-3) and the percentage of positive cells. Positive staining was defined as at least focal nuclear staining in ³5% of cells. Positive and negative controls were included in all staining runs, which were performed on an automated Leica Bond immunostainer. Image analysis with a customized IHC nuclear V9 algorithm (Aperio technologies, Inc.) was used to quantify nuclear staining in different histologic compartments. Targeted next-generation sequencing (BWH OncoPanel 447-gene panel) was performed on a subset of SMZL cases (n=20).The vast majority of SMZL cases (36/39; 92%) were positive for NICD2 staining. By contrast, only weak NICD2 staining was seen in small subsets of other B cell tumors - 2% of CLL (1/42), 19% of MCL (9/38) and 16% of non-splenic MZL (9/48) cases. Most SMZL cases showed nuclear staining of variable intensity (weak to strong) in a variable number of neoplastic cells (5-80%). The majority of NICD2-positive cases (26/36, 72%) showed weak NICD2 staining in a subset of neoplastic cells (< 40% of cells), while a minority (10/36, 28%) showed more diffuse NICD2 staining (³ 40% of cells). In addition, NICD2 staining was accentuated in marginal zones relative to the inner half of white pulp nodules and the red pulp (1.6x, p<0.05). Targeted next-generation sequencing performed on subsets of NICD2-high and NICD2-low cases (n=10 of each from n=19 patients) revealed gain-of-function NOTCH2 PEST domain mutations in 9 cases. All but one of the cases (8/9) with high NICD2 staining had a gain-of-function NOTCH2 PEST domain mutations. Overall, the pattern of NICD2 staining correlated with NOTCH2 mutation status in 85% (17/20) cases. There was no significant difference (p>0.05) in overall survival between NOTCH2-wild type and -mutated or NICD2-high and -low groups.In conclusion, among low-grade B cell lymphomas, NOTCH2 activation is much more prevalent in SMZL than in CLL, MCL, and nonsplenic MZL and is accentuated in (but not confined to) tumors with NOTCH2 PEST domain mutations. These findings suggest that NOTCH2 signaling is broadly important in the pathogenesis of SMZL, where it likely acts following ligand-mediated activation in the microenvironment of splenic marginal zones to drive expression of currently unknown target genes that support SMZL growth and survival. DisclosuresBenhadji:Eli Lilly and Company: Employment. Oakley:Eli Lilly and Company: Employment. Aster:Eli Lilly and Company: Membership on an entity's Board of Directors or advisory committees.

Whole Genome Sequencing in Hairy Cell Leukemia-Variant (HCL-v) and Splenic Marginal Zone Lymphoma (SMZL)

Background: The WHO 2016 classification defines a variety of lymphomas. Among the mature B-cell neoplasms different types of splenic lymphomas are separated from each other. In particular, hairy cell leukemia-variant (HCL-v) and splenic marginal zone lymphoma (SMZL) are defined as distinct entities, however, while they are readily separated from hairy cell leukemia based on cytomorphology, immunophenotype and lack of BRAF V600E mutation, HCL-v and SMZL feature partial overlaps of findings in cytomorphology and immunophenotype. Cytogenetic changes are rare and nonspecific. Thus, a comprehensive molecular work up may provide insight into shared and differentiating mutation.Aims: Molecular characterization of HCL-v and SMZL through whole genome sequencing (WGS) to provide a more distinct disease category.Patient cohort and methods: Diagnostic peripheral blood or bone marrow samples of 14 patients (7 HCL-v and 7 SMZL, median age 82, range 59-94; 8 female, 6 males) were selected for WGS with 90x coverage. Infiltration by mature B-lymphatic population ranged from 6%-72% (median 52%) by immunophenotyping. HCL-v cases were negative for BRAF . Library preparation was performed using Truseq DNA PCR-Free HT sample preparation kit (Illumina, San Diego, CA) according to manufacturer's protocol using fresh/frozen samples and sequenced on NovaSeq sequencing instruments. Tumor-only data was aligned by BaseSpace (BS) WGS app with default parameters and (structural) variant calling was performed with Tumor/Normal app using an unmatched reference DNA (Promega, Fitchburg, WI). Data was subsequently loaded into BS Variant Interpreter to filter and prioritize variants of interest. For the structural variants we looked at all high confidence variant calls passing filters with at least 5 paired reads and 3 split reads with a somatic QScore of >30. In addition, all calls were eliminated with a population frequency of >1% according to the DGV (Database of Genomic variants). For mutation analysis we filtered down the passed variant list to the coding regions of the CancerGeneCensus genes, with a variant read frequency of >0.2, ExAC population frequency of <1% and a damaging/deleterious mutation prediction score with PolyPhen-2 and SIFT. We performed mutation signature analysis by looking at the distribution of base substitutions across the sample with a 3mer context to decompose somatic mutation patterns and compared the results to the COSMIC signature database.Results: We observed a heterogeneous structural variation pattern with no obvious recurrent aberrations across both cohorts. The high diversity in molecular pattern was further confirmed on variant level. Mutation analysis yielded 164 mutations with a median of 12.5 (range 3-18) mutations per sample involving 122 of the 600 investigated genes. 19 genes were mutated in both entities, 44 were exclusive to HCL-v and 59 exclusive to SMZL. Recurrent mutations (n=3/14) were observed in the following genes: ASXL1, ATM, KMT2C, PCSK7, SMO, SPEN, TP53, TRIP11, WRN . PCSK7 is a transcriptional regulator and has been described to be associated with B-cell lymphoma. WRN is a helicase enzyme involved in DNA repair and associated with AML. Strikingly 3/7 (42%) of SMZL cases were mutated in these two genes. SPEN is known to block the differentiation of precursor B-cells into marginal zone B-cells. Additional exclusive recurrent mutations (n=2) were observed in ABL2, ARID1B, CREBBP for HCL-v, and BCL11A, NACA, NSD1, SF3B1 for SMZL. ABL2 is a tyrosine kinase and thus may provide a treatable target. Pathway analysis revealed that 30% of genes mutated (n=36) are involved in the MAPK/ERK pathway. We subsequently conducted a context-sensitive mutation signature analysis with the SNV data, using 96 substitution patterns rather than 6 to increase the complexity. In all instances we found a highly similar pattern across all 14 samples with a strong bias towards C>T substitutions.Conclusion: HCL-v and SMZL are highly diverse on the molecular level. Mutation signature analysis creates patterns associated with cancer and age. More data and further refinement of mutational entity specific signatures are needed to increase the granularity of these signatures. We identified several putative genes to be involved in HCL-v and SMZL, which were not associated with the two entities before. In two instances tyrosine kinase proteins were involved revealing a target for drugs. DisclosuresHaferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Nadarajah:MLL Munich Leukemia Laboratory: Employment. Meggendorfer:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Frequency of MYD88 L256P mutation and its correlation with clinico-hematological profile in mature B-cell neoplasm

Objective/backgroundB-cell neoplasms are clonal tumors of B cells at various stages of maturation, including diffuse large B-cell lymphoma (DLBCL), chronic lymphocytic lymphoma (CLL), Burkitt lymphoma (BL), lymphoplasmacytic lymphoma (LPL)/Waldenström’s macroglobulinemia (WM), splenic marginal zone lymphoma (SMZL), nodal marginal zone lymphoma (NMZL), mantle cell lymphoma (MCL), follicular lymphoma (FL), and hairy cell leukemia (HCL). In this study, we analyzed the frequency of MYD88 L265P mutation and its correlation with clinico-hematological profile in mature B-cell neoplasms. MethodsA total of 110 consecutive cases of B-cell neoplasms showing peripheral blood and/or bone marrow infiltration were included. MYD88 L265P mutation was detected by polymerase chain reaction amplification of exon 5 of MYD88 gene, followed by restriction fragment length polymorphism analysis. ResultsAmong the 110 cases, the major group was of CLL (54.5%, n = 60), followed by HCL. Other cases included MCL, LPL, DLBCL, SMZL, NMZL, FL, and BL. MYD88 L265P mutation was seen in 21 (19.1%) cases of B-cell neoplasm, whereas 89 (80.9%) cases were negative for MYD88 L265P mutation. It was most commonly seen in LPL/WM cases followed by HCL, SMZL, CLL, and MCL cases. No case of DLBCL, FL, and BL showed MYD88 L265P mutation. Statistically significant difference was seen for hemoglobin level in CLL cases, with MYD88 L265P mutated cases showing higher mean hemoglobin levels than MYD88 wild-type cases (p = .001). For other parameters, no statistically significant difference was noted between mutated and unmutated cases. ConclusionMYD88 L265P mutation is seen in various B-cell neoplasms; it is most commonly seen in LPL/WM cases but not specific for it.

Splenic marginal zone lymphoma: A case report and review of the literature

Splenic marginal zone lymphoma (SMZL) is a low-grade B-cell non-Hodgkin’s lymphoma characterized by massive splenomegaly, moderate lymphocytosis with or without villous lymphocytes, rare involvement of peripheral lymph nodes, and indolent clinical course. SMZL has a distinct immunologic profile which when correlated with the morphologic findings of peripheral blood and bone marrow aspirates as well as clinical and hematologic features enables accurate diagnosis and obviates the need for further invasive diagnostic procedures. Here, we present a case of SMZL who presented with massive splenomegaly and was diagnosed on the basis of clinical, hematological, and characteristic immunophenotypic findings on bone marrow aspirate and biopsy.

Clonal lineage analysis of CLL research samples by IGH chain sequencing to reveal novel CLL subgroups defined by ongoing class-switch recombination and somatic hypermutation.

e20001 Background: B cell somatic hypermutation (SHM) and class switch recombination (CSR) are mechanistically related but distinct processes requiring precisely targeted generation and repair of single and double strand DNA breaks. Within the context of chronic lymphocytic leukemia (CLL), the presence of ongoing SHM or CSR may reveal the functionality of DNA damage repair pathways, with potential relevance to therapeutic strategies involving the generation of DNA breaks or inhibition of DNA repair machinery. Here we apply clonal lineage analysis of IGH chain sequencing data to evaluate CSR and SHM in a cohort of CLL and splenic marginal zone lymphoma (SMZL) research samples. We present evidence of ongoing CSR and SHM in a significant subset of samples. Methods: Multiplex primers targeting the IGH framework 1 region and isotype region (Oncomine IGH-LR assay; detection of all nine isotypes) were used for IGH repertoire sequencing via the Ion Gene Studio S5 from 25ng peripheral blood total RNA derived from 63 individuals with CLL and 4 individuals with SMZL. Clonotyping and clonal lineage analysis was performed by Ion Reporter, whereby clonal lineages are defined as sets of unique rearrangements having a shared variable and joining gene, the same CDR3 length, and a minimum CDR3 nucleotide similarity of 85%. Ongoing CSR was defined as the presence of IgM/IgD and at least one switched isotype (IgG, IgA, or IgE), or a combination of switched isotypes, within the same lineage. Ongoing SHM was defined as the presence of subclones that differ within the VDJ region sequence compared to other clonal lineage members. Results: 11/68 cases showed evidence of ongoing CSR or SHM. Of the 57 cases showing no evidence of ongoing CSR or SHM, variable gene mutation analysis revealed the presence of three distinct subgroups having either no SHM, intermediate SHM (average 98% sequence identity) or high SHM &lt; 94% identity). 3 of 4 SMZL cases showed evidence of ongoing CSR or SHM. Conclusions: These results reveal previously underappreciated heterogeneity within CLL and suggest the subdivision of CLL based on a combination of IGHV mutation level and presence of ongoing SHM or CSR. The described heterogeneity may serve as a valuable criterion for stratifying CLL patients in the future.

Identification of a Splenic Marginal Zone Lymphoma Signature: Preliminary Findings With Diagnostic Potential.

Splenic marginal zone lymphoma (SMZL) is a rare, indolent non-Hodgkin's lymphoma that affects 0. 13 per 100,000 persons annually. Overall survival of SMZL is estimated to reach 8–11 years in most cases, but up to 30% of SMZL cases develop aggressive presentations resulting in greatly diminished time of survival. SMZL presents with a very heterogeneous molecular profile, making diagnosis problematic, and accurate prognosis even less likely. The study herein has identified a potential diagnostic gene expression signature with highly specific predictive utility, coined the SMZL-specific Gene Expression Signature (SSGES). Additionally, five of the most impactful markers identified within the SSGES were selected for a five-protein panel, for further evaluation among control and SMZL patient samples. These markers included EME2, ERCC5, SETBP1, USP24, and ZBTB32. When compared with control spleen and other B-cell lymphoma subtypes, significantly higher expression was noticed in SMZL samples when stained for EME2 and USP24. Additionally, ERCC5, SETBP1, USP24, and ZBTB32 staining displayed indications of prognostic value for SMZL patients. Delineation of the SSGES offers a unique SMZL signature that could provide diagnostic utility for a malignancy that has historically been difficult to identify, and the five-marker protein panel provides additional support for such findings. These results should be further investigated and validated in subsequent molecular investigations of SMZL so it may be potentially incorporated into standard oncology practice for improving the understanding and outlook for SMZL patients.

Splenic Marginal Zone Lymphoma: A U.S Population-Based Survival Analysis (1997-2016)

INTRODUCTION: Splenic marginal zone lymphoma (SMZL) is a rare and indolent form of NHL with no standard treatment due to scarcity of clinical trials. Watch and wait approach, single agent rituximab or with chemotherapy and splenectomy are valid treatment approaches. No population-based studies examining the impact of chemotherapy and splenectomy on survival of SMZL patients are available. Thus, we decided to compare survival outcomes by clinical factors in SMZL patients deposited in the SEER database, emphasizing in the role of splenectomy on survival outcomes. METHODS: Retrospective analysis of SMZL cases in the SEER database from 1997 to 2016.SEER collects cancer incidence and survival data from population-based cancer registries covering 34% of the U.S. population. PATIENTS: SEER database was queried for SMZL patients at all ages from 1997 to 2016. Cases without histologic confirmation or no survival data were excluded. Demographic and clinical variables (age, sex, race, ethnicity, B-symptoms, Ann-Arbor stage, DLBCL transformation, first malignancy, chemotherapy with or without rituximab, splenectomy and radiotherapy) were summarized by descriptive statistics. Logistic regression used to determine variables associated with splenectomy. Overall survival (OS) and Cancer-specific survival (CSS) were the major end-points, estimated by Kaplan-Meier method. Cox regression used to calculate Hazard Ratios. RESULTS: 2212 patients met inclusion criteria, with a median age of 68yo (25-96). Patient characteristics were a follows: males (n=1152, 52.1%); whites (n=1991, 90.0%), blacks (112, 5.1%); Non-Hispanics (n=2049, 92.6%); B-symptoms (n=461, 20.8%); first malignancy (n=1754, 79.3%); Ann-Arbor Stage I (429, 19.4%), Stage IV (n=1351, 61.1%); DLBCL transformation (n=84, 3.8%). Adjusted Odds ratio (aOR) were used to compare splenectomy versus non-splenectomy treated patients: ≥60yo (aOR 0.49;0.40-0.62), males (aOR 0.77;0.64-0.93). Median survival of all the patients was 120 months (95%CI:111-132); On univariate analysis: shorter OS was significantly associated with age ≥60(HR 3.55;2.87-4.38) and non-first malignancy(HR 1.6;1.36-1.88), while shorter CSS was significantly associated with age ≥60(HR 1.76;1.33-2.34), B-symptoms (HR 1.93;1.44-2.60), Stage IV (HR 1.62;1.15-2.28), DLBCL transformation (HR 2.27;1.55-3.33) and treatment with chemotherapy (HR 1.54;1.22-1.94). On multivariate analysis, shorter OS was significantly associated with age ≥60(HR 3.52;2.83-4.37), male sex (HR 1.15;1.00-1.33), B-symptoms (HR 1.23;1.01-1.51) and non-first malignancy (HR 1.42;1.20-1.67). Shorter CSS was significantly associated with age ≥60(HR 2.15;1.61-2.87), Hispanic ethnicity (HR 1.44;1.00-2.07), B-symptoms (HR 1.77;1.30-2.39), DLBCL transformation (HR 2.03;1.38-2.99) and treatment with chemotherapy (HR 1.38;1.07-1.77). Ann Arbor staging and splenectomy were not associated with OS or CSS. CONCLUSIONS: Older age, Hispanic ethnicity, B-symptoms, DLBCL transformation, and treatment with chemotherapy were associated with poor lymphoma-specific survival. Although, age&lt;60 and female gender were associated higher odds to undergo splenectomy, splenectomy had no impact on the risk of lymphoma-related death or OS. This retrospective study demonstrates that chemotherapy treatment (excluding rituximab) was associated with inferior outcomes in SMZL, while advanced Ann-Arbor stage was not associated with shorter survival as is observed in other indolent lymphomas that commonly present at advanced stage. Table Disclosures Alderuccio: Targeted Oncology: Honoraria; Foundation Medicine: Other: Immediate family member; Agios: Other: Immediate family member; Puma Biotechnology: Other: Immediate family member; OncLive: Consultancy; Inovio Pharmaceuticals: Other: Immediate family member. Lossos:NIH: Research Funding; Seattle Genetics: Membership on an entity's Board of Directors or advisory committees; Janssen Scientific: Membership on an entity's Board of Directors or advisory committees.

Clonal B-Cell Lymphocytosis with Mariginal-Zone Features: Comparison with Overt Splenic Marginal-Zone Lymphomas in 77 Patients from a Monocentric Series

Diagnosis of non-CLL B Cell Chronic Lymphoproliferative Disorders (BCLPD), defined by the detection of peripheral blood (PB) B-Cell clone with flow cytometry (FC) Matutesscore≤3, is not an infrequent event in clinical daily practice. Only a portion of these cases may be actually classified according to the 2016 revision of WHO classification of lymphoid neoplasms, as in these cases a non-bone marrow (BM) tissue biopsy is rarely available due to "liquid-only disease" (BM, PB, splenomegaly) or difficult accessible sites. The two most likely diagnosis of BCLPD according to WHO 2016 are splenic marginal-zone lymphoma (SMZL) and monoclonal B Cell lymphocytosis (MBL) with non-CLL phenotype (CD19+, CD20+, SIg+, CD5-), sometimes reported as MZL-like, defined by monotypic clonal B lymphocytes (CBL) &lt;5 x 109/l. A recent study pointed out a similar outcome regardless CBL value exceeding or not this arbitrary cut-off in strictly defined cases (absence of cytopenias, splenomegaly and adenopathy) of clonal B cell lymphocytosis with marginal-zone features (CBL-MZ) and confirmed the really indolent behaviour of this entity (Xochelli, Blood 2014). On these bases we decided to retrospectively collect clinical, immunophenotypic, molecular features and outcome of all consecutive patients (pts) with CD5- CBL-MZ owing to the Department of Hematology of University of Insubria (Varese, Italy) from 2011 to 2019 and to compare them with overt SMZL cases. Excluding 5 SMZL pts who were splenectomized, all cases were diagnosed by means of non-BM tissue biopsy, namely BM histology and PB/BM FC, FISH and molecular studies, when appropriate, and complete laboratory and imaging staging procedures (CT scan or US). The primary objective of the study was time to progression (TTP) for CBL-MZ group. Secondary objectives were overall survival (OS), progression-free survival (PFS) for treated pts and evaluation of early progression &lt;24 months (Early POD) (Luminari, Blood 2019). Overall we collected complete data of 33 pts with CD5- CBL-MZ, and 44 with SMZL (Table 1). Focusing on CBL-MZ, FC immunophenotypic findings showed a B-Cell population with Matutes score 0 to 2 in all cases, uniform expression of B Cell antigens, namely CD19, CD20 strong, FMC7 (93%), SIg (intermediate/bright in 71%), while CD10 and CD5 were consistently negative. CD23 was expressed in 18%, CD38 in 9% and CD49b in 95%. BM sinusoidal infiltration pattern was detected in 50% of cases. TP53 mutation/deletion was detected in 4/16 pts (25%) while trisomy 12 and del13q in 1 pt each. Twenty pts presented with &lt;5000/mmc CBL (MBL-MZ) and 13 with ≥5000/mmc CBL. With a median follow-up of 1.8 years, 6 pts (18%) progressed (4 in SMZL, 1 NMZL and 1 SLL; Fig 2) with a median TTP of 1.7 yrs (range 0.3- 12.3), while the remaining 27 remained completely stable. Among 4 pts treated, 2 received Rituximab-based regimen, both achieving CR and 2 with Chlorambucil both experiencing progression. Overall, 5 pts died (2 for progressive lymphoma), with an estimated 5-yrs of 80%. Concerning prognostic factors, after adjusting by competing risk method, at univariate analysis age &gt;60 yrs, CBL &gt;5000/mmc and albumin &lt;3.5 g/dl, were associated to shorter TTP, while age &gt;60 yrs, Hb &lt;11 g/dl, ECOG PS ≥2 and albumin &lt;3.5 g/dl resulted predictive of worse OS (Table 2). By applying multivariate Cox regression model, only age &gt;60 yrs and CBL&gt;5000/mmc retained prognostic significance for TTP (p&lt;0.0001), while ECOG PS ≥2 and albumin &lt;3.5 g/dl remained independently associated with inferior OS (p&lt;0.0001). Considering the SMZL group, 34 pts underwent systemic treatment (rituximab-based in 32, including R-Bendamustine in 17). With a median follow-up time of 2.8 yrs, 9 pts progressed (4 of whom within 24 months from treatment initiation), with a median PFS 2.9 yrs, and 8 died (7 due to progression, including 3 DLBCL transformations) with an estimated 5-yrs OS of 79%. Notably, Early POD SMZL pts exhibited a significant increased risk of death (p&lt;0.0001, fig.2), while in CBL-MZ did not (p=0.4). In conclusion, CBL-MZ confirmed to be associated with indolent course and preferential evolution toward SMZL, of which it can be viewed as an initial phase or a precursor entity. In contrast with previous findings, the WHO 16 CBL cut-off defining MBL (5 x 109/l), together with advanced age, retained prognostic relevance for TTP. Finally, we confirmed for the first time in an independent series of SMZL the highly prognostic impact of Early POD24. Disclosures Passamonti: Roche: Consultancy; Janssen: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau; Novartis: Consultancy, Honoraria, Speakers Bureau.

Detailed Functional Characterization of Splenic Marginal Zone Lymphoma: Uncovering Links between the Epigenetic and the Signaling Machinery

Splenic marginal-zone lymphoma (SMZL) ontogeny and evolution is a complex process, involving, amongst others, (super)antigenic stimulation, molecular deregulation of genes involved in the physiological differentiation of splenic marginal zone B cells (e.g. NOTCH2 and KLF2), and epigenetic alterations leading to silencing of different tumor suppressor genes and overexpression of oncogenes, including the PRC2-complex (EZH2, EED, and SUZ12). However, despite important recent progress, advances in the fundamental understanding of SMZL have not yet been translated in improved patient management, highlighting the need for further molecular investigations. In this context, here we aimed at detailed characterization of: (i) epigenetic modifiers, particularly the histone methyltransferase EZH2, a marker of clinical aggressiveness and druggable target in lymphoma; (ii) immune signaling capacity of SMZL B cells; and, (iii) antigen reactivity profile of the SMZL clonotypic B Cell Receptor immunoglobulin (BcR IG). Quantification of EZH2 mRNA levels using quantitative Real-time PCR (n=26 SMZL patients) and protein levels using western blotting (WB) (n=14) revealed that EZH2 was expressed in all cases yet in a heterogeneous manner (2^-ΔCt range: 0.079-1.38; ΕΖΗ2+ cells: 3.11%-44.8%). Interestingly, higher proliferation rates (Ki67+ cells) were observed in cases with high EZH2 protein levels (n=4) compared to cases with low EZH2 (n=6) (FD=2.6; p&amp;lt;0.05). Stimulation of SMZL CD19+ B cells (n=8) through i) the BcR with anti-IgM ii) Toll-Like receptor 9 (TLR9) with CpG and iii) BcR/TLR for 60 minutes affected pBTK, pERK and pNF-kB levels as revealed by WB and flow cytometry (FCM). Although, great heterogeneity was observed in the responses between different cases, categorization of SMZL cases based on EZH2 levels showed that, at basal level, EZH2high cases expressed higher pBTK, pERK and pNF-κB compared to EZH2low cases (FC=2.4, FC=6,1, FC=12.7, respectively; p&amp;lt;0.05 for all comparisons). BcR and TLR9 activation and, especially, co-stimulation with anti-IgM/CpG induced the expression of pBTK, pERK and pNF-κB in EZH2low cases, while the opposite was observed in EZH2high cases. Moreover, stimulation through both the BcR and BcR/TLR9 for 24 hours could also induce EZH2 expression compared to the unstimulated control cells (FC=1.6; p&amp;lt;0.05 for both comparisons). To gain insight into the type of antigenic stimuli that could activate BcR signaling through the BcR we investigated the antigen binding profile of 10 SMZL clonotypic BcR IGs expressed as recombinant human IgM monoclonal antibodies (mAbs). Cases were representative of the most common IGHV genes in SMZL (IGHV1-2*04, IGHV4-34 and IGHV3-23). Reactivity (at 15 μg/ml) against various autoantigens was evaluated by ELISA. Five of 9 (56%), 4/7 (57%), 5/10 (50%), 2/9(22%), 2/9 (22%), 3/9 (33%), 3/8 (38%), 4/8 (50%) and 4 of 9 (44%) SMZL IgM mAbs displayed reactivity against native DNA, nuclear Hep-2 cell extract, actin, myosin, thyroglobulin, β-amyloid, carbonic anhydrase, F(ab')2 and the hapten trinitrophenyl respectively, with optical density (OD) values greater than the cut-off point. Moreover, 5/10 (50%) SMZL IgM mAbs recognized more than 2 antigens and were characterized as polyreactive. Each of these SMZL mAbs exhibited a distinct antigen polyreactivity profile, while heterogeneity was observed even amongst cases expressing the same gene or cases with similar IGHV gene mutational load. Recognition of epitopes on the surface of viable MEC1 B and HEK293 cells was assessed by FCΜ. Two of 3 and 5/11 mAbs bound to viable MEC1 B cells and HEK293 cells, respectively: binding to HEK293 cells was more pronounced for minimally mutated/unmutated versus mutated mAbs (p&amp;lt;0.05). Importantly, stimulation of SMZL CD19+ B cells with the SMZL BcR cognate antigens actin and myosin resulted in regulation of pERK/pPLCγ2 in 3/6 and 1/2 cases, respectively, as revealed by WB. Overall, SMZL cells can be activated by microenvironmental signals albeit in a different manner depending on EZH2 levels, highlighting links between the epigenetic and the signaling machinery with potential therapeutic implications. Moreover, the recognition of a wide range of autoantigens by the SMZL mAbs indicates that SMZL B cell clones may arise from polyreactive B cells, likely resident in the normal splenic marginal zone. Disclosures Stamatopoulos: Janssen: Honoraria, Research Funding; Abbvie: Honoraria, Research Funding.

Systematic Review of Somatic Mutations in Splenic Marginal Zone Lymphoma

The aims of this systematic review are to refine the catalogue of somatic variants in splenic marginal zone lymphoma (SMZL) and to provide a well-annotated, manually curated database of high-confidence somatic mutations to facilitate variant interpretation for further biological studies and future clinical implementation. Two independent reviewers systematically searched PubMed and Ovid in January 2019 and included studies that sequenced SMZL cases with confirmed diagnosis. The database included fourteen studies, comprising 2817 variants in over 1000 genes from 475 cases. We confirmed the high prevalence of NOTCH2, KLF2 and TP53 mutations and analysis of targeted genes further implicated TNFAIP3, KMT2D, and TRAF3 as recurrent targets of somatic mutation based on their high incidence across studies. The major limitations we encountered were the low number of patients with whole-genome, unbiased analysis and the relative sensitivities of differing sequencing approaches. Overall, we showed that there is little concordance between whole exome sequencing studies of SMZL. We strongly support the continuing unbiased analysis of the SMZL genome for mutations in all protein-coding genes and provide a valuable database resource to facilitate this endeavour that will ultimately improve our understanding of SMZL pathobiology.

Surgical management of splenic marginal zone lymphoma.

Splenic marginal zone lymphoma (SMZL) is a rare indolent B-cell lymphoma with variable prognosis. As a result, there is sparse knowledge on the role of splenectomy and best management approaches. We aim to explore management strategies and outcomes amongst the cohort of SMZL patients at our centre. A retrospective review of all splenectomies performed at a tertiary referral unit over a 23-year period was assessed. Immunohistochemical and pathological results of splenic samples, bone marrow biopsies, and peripheral blood were compiled. Operative management, surgical, and survival outcomes were assessed. Prognostic stratifications were applied and survival rates were calculated. Eight cases of SMZL from a database of 693 splenectomies were identified. All patients had intermediate/high-risk disease. All patients underwent splenectomy with one patient receiving preoperative rituximab. All patients had progression-free survival and resolution of disease. Based on the data obtained, current practice requires defined guidelines and centralised care.

Splenic Marginal Zone Lymphoma with Acquired von Willebrand Syndrome Diagnosed via Splenic Bleeding

An 85-year-old woman underwent emergent splenectomy due to left abdominal pain and active bleeding in a massively enlarged spleen. The histological diagnosis was splenic marginal zone lymphoma (SMZL). A prolonged activated partial thromboplastin time (APTT) was noted, and additional tests led to the diagnosis of type 2A-like acquired von Willebrand syndrome (AVWS). An APTT cross mixing test ruled out the presence of inhibitors. She received eight courses of rituximab monotherapy. The coagulation data showed no improvement, possibly because the lymphoma showed a poor response to the treatment. AVWS rarely causes bleeding in solid organs. This is the first case of SMZL with AVWS diagnosed via splenic bleeding.

Characteristics of splenic marginal zone lymphoma: Clinical, hematologic, and flow cytometry findings of 34 cases

Background: Splenic marginal zone lymphoma (SMZL) is a low-grade disorder that regularly presents with peripheral blood (PB) involvement. A precise description of clinical, laboratory features and immunophenotypic characterization of SMZL are still lacking. Here, we reviewed 34 patients presenting with SMZL to describe the clinical, hematologic features, and flow cytometry immunophenotypic findings of this type of lymphoma at diagnosis. Objectives: The aim of this study is to confirm that SMZL has a specific immunologic profile which enables the hematopathologist and clinician to differentiate this low-grade B-cell lymphoma from other B-cell lymphoproliferative disorder, especially chronic lymphocytic leukemia and hairy cell leukemia, which could sometimes stimulate SMZL morphologically and to emphasize that a correlation of immunophenotypic findings, clinical, and hematologic features of patients plus careful morphological examination of PB and/or bone marrow (BM) aspirate can lead confidently to the correct diagnosis. Materials And Methods: Flow cytometry immunophenotypic findings of 34 cases of SMZL were reviewed. The analysis was performed by BD FACS Calibur™ and FACSCanto II flow cytometers. B lymphocytes were identified according to their Side-Scattered (SSC)/CD19 distribution. A marker was considered positive when expressed in more than 20% of cells above the control. Results: Median age was 60 years, range (35–84 years), both sexes were affected equally. All patients presented with splenomegaly, 71% of patients had absolute lymphocytosis and 88% of patients showed PB involvement. Seventy-four percent of patients had anemia and (53%) of them had thrombocytopenia. Cells from all cases expressed pan B-cell antigens (CD19, CD20), 74% of cases expressed CD79b and Human Leukocyte Antigen – antigen D Related (HLA-DR) expressed in nearly almost all cases (97%). Half of the patients expressed CD11c and SIgD, 41% expressed CD5 and FMC7 while CD25 and CD103 showed positivity in less than 5% of cases. Preferential expression of Kappa light chain was demonstrated, CD10 and CD38, SIgG were negative. Conclusion: SMZL has a distinct immunologic profile which if correlated with morphologic findings of PB or BM aspirates, clinical and hematologic features can help to make the accurate diagnosis and lessens the need of further invasive diagnostic procedure.

Targeting the Epigenome and the Cell Signaling As Novel Therapeutic Approaches for Splenic Marginal Zone Lymphoma

Background: DNA promoter methylation is a well-studied major regulatory machinery of gene expression. Aberrant DNA methylation is not only reported in many cancers but may have utility as a prognostic and/or diagnostic implications in lymphoma. Since pharmacological intervention may revert the aberrant methylation program, new therapeutic opportunities may be explored. We have previously published data that point to epigenetic aberrant reprogramming in the pathogenesis of splenic marginal zone lymphoma (SMZL) (Arribas, Rinaldi et al, Blood 2015). One quarter of SMZL cases are characterized by a high degree of DNA promoter methylation and this associates with inferior survival, a higher risk of histological transformation, IGHV1-02*04 allele usage, mutations of NOTCH2 gene, and 7q31-32 loss. Exposure of SMZL cell lines and primary cells to the demethylating agent decitabine is able to partially revert the aberrant methylation program. Here, we further explored the therapeutic implications of these early observations.Materials and methods: SMZL cell lines (VL51, Karpas-1718, SSK41) were treated with decitabine with a short (48 h, 2µM) or long exposure (5 days, 100nM). For in vivo experiments, NOD-Scid (NOD.CB17-Prkdcscid/NCrHsd) mice were subcutaneously inoculated with 15x106 SSK41 cells and divided in two groups (n= 8 each). Intraperitoneal treatment with decitabine started with tumors of 100-110 mm3 volume. For in vitro combinations, cell lines were exposed to increasing doses of agents alone or in combination for 72h and synergy was assessed with the Chou-Talalay combination index (CI): <0.3, very strong synergy 0.3-0.9, synergy; 0.9-1.1 additive effect; >1.1, antagonism/no benefit.Results: SMZL cell lines, bearing the aberrant methylation program observed in clinical SMZL samples, showed an important reduction in cell growth upon exposure to decitabine (48 h, 2µM or 5 days, 100nM). In particular, the long exposure to low concentrations of decitabine induced G0/G1 cell cycle arrest and apoptosis in all the SMZL cell lines. We the validated the anti-tumor effect of decitabine in a SMZL xenograft model with the SSK41 cell line. Decitabine was given at the dose of 2 mg/kg on day 1 and day 3. The drug led to a complete eradication of the tumors in all the treated mice, that lasted for 15 days, while the mice of control developed tumors with an average volume of 1500 mm3.Since the aberrant methylation program is associated with activation of NOTCH, EZH2, NF-κB and PI3K/AKT signaling pathway (Arribas, Rinaldi et al, Blood 2015), we have been assessing possible combinations schemes with additional targeted agents. The sequential combination of demethylating agent (decitabine 50 nM, 5 days) followed by a gamma-secretase inhibitor (PF-03084014, 200 nM for 6 days) resulted in much higher rate of cell death than the single agents. Also, combinations of gamma-secretase inhibitor given concomitantly to the BTK inhibitor ibrutinib, the PI3Kδ inhibitor idelalisib and the EZH2 inhibitor DZNep were tested. All the combinations were active in the three cell lines, achieving strong synergy in two cell lines and synergy in the remaining one.Conclusions: The demethylating agent decitabine showed in vitro and in vivo anti-tumor activity in SMZL, providing the rationale for possible clinical studies. Moreover, combinations of agents targeting pathways deregulated in SMZL showed a very promising in vitro synergism and are worth of further investigations. DisclosuresArcaini:Gilead: Research Funding; Roche: Membership on an entity's Board of Directors or advisory committees; Bayer: Membership on an entity's Board of Directors or advisory committees; Sandoz: Membership on an entity's Board of Directors or advisory committees. Rossi:Gilead: Honoraria, Research Funding; Abbvie: Honoraria; Janseen: Honoraria.

Splenic Marginal Zone Lymphoma: A Case Report with Review of Literature Running Title: A Rare Case Report On Splenic Marginal Zone Lymphoma

Splenic marginal zone lymphoma(SMZL) is a rare B-cell neoplasm comprising less than 2% of lymphoid neoplasms ,composed of small lymphocytes which surround and replace the splenic white pulp ,germinal centres,follicle mantle and merge with marginal zone.We report a case of SMZL in a 40 year old male patient presenting with complaints of low grade fever and left sided abdominal discomfort for 6 months and was noted to have massive splenomegaly on physical examination. Laboratory evaluation revealed leucocytosis and presence of atypical lymphocytes which were larger than mature lymphocytes having moderately basophilic cytoplasm with cytoplasmic processes at the poles of the cell with round to oval nucleus with clumped chromatin and indistinct nucleoli.Immunophenotypic analysis revealed a B-cell population with CD -20 positivity.

IGHV mutational statue in patients with splenic marginal zone lymphoma

目的研究脾边缘区淋巴瘤（SMZL）患者免疫球蛋白重链可变区（IGHV）基因突变状态及典型模式的分布情况，并与国外报道的数据进行对比，以探讨其差异。方法对40例SMZL患者资料进行回顾性分析。采用克隆测序法检测IGHV的VDJ序列并进行比对及聚类分析，明确是否存在B细胞受体的典型模式，分析IGHV突变患者与未突变患者的临床特征。结果40例患者中，IGHV突变者30例（75.0%），未突变者10例（25.0%），两者比例与国外报道相当。在V区基因，V2-70的使用频率高于国外报道（10.3％对0.8%，P=0.002），而V3-23明显减低（2.6％对18.0%，P=0.006）。在D区基因中，D2-21和D6-13均高于国外报道（17.9％对2.3%，12.8%对3.8%，P值分别为0.000、0.046）。40例患者中发现1对新的典型模式，同时SMZL特异性的V1-2基因使用频率也最高（25.6%）。与IGHV突变组相比，未突变组患者的IgG、IgA表达水平显著增高[10.70（5.28~15.50）g/L对12.90 (7.71~23.50) g/L，1.06（0.21~3.13) g/L对1.66 (0.81~2.93) g/L，P值分别为0.038、0.040）]，2例17p缺失患者的IGHV均呈未突变状态。与IGHV未突变组相比，突变组患者的无进展生存期显著延长（P=0.009），但总生存期差异无统计学意义（P=0.430）。结论在SMZL患者中，IGHV突变与未突变患者比例与国外报道相当，但V区和D区基因的使用频率仍存在差异，而且V1-2基因的使用呈现疾病特异性，同时发现1例新的典型模式。IGHV突变可降低患者的IgG、IgA表达水平。