ALBERT

Description: Background: The salivary gland is one of the most common sites of involvement by non-gastric extranodal marginal zone lymphomas of mucosa-associated lymphoid tissue (MALT). This multicenter, international trial sought to characterize the clinical course, treatment and outcome of patients with salivary gland MALT lymphoma. Methods: Patients were identified from multiple international sites including the Mayo Clinic Lymphoma Database (n=89), three International Extranodal Lymphoma Study Group (IELSG) cohorts (n=138), and the University of Athens (n=21). All patients had biopsy-confirmed MALT lymphoma of the salivary gland by WHO criteria. Clinical characteristics, treatment, and outcomes were obtained from the respective patient databases. Survival probability was estimated using the Kaplan-Meier method and compared between groups with the Wilcoxon or log-rank. The impact of variables on survival was assessed by the Cox proportional hazards model. Results: Patients and clinical features: From 1983 to 2012, 247 patients with MALT lymphoma were included. Clinical characteristics are shown in Table 1. The median age at diagnosis was 59, and the male-to-female ratio was 1:3. The majority of patients (76%) presented with limited stage disease, and the parotid gland was the most common site of involvement (78%). There was a history of antecedent autoimmune disease in 41% of patients, and Sjögren’s disease was the most common (83%). Treatment: Information on initial treatment was available on 242 patients. 137/242 patients (57%) initially received local therapy with either surgery (n=81), radiation (n=26), or both (n=30). Of these, 25 received adjuvant systemic therapy. 90/242 patients (37%) were treated initially with systemic therapy, of whom 54% had localized and 46% had stage IV disease. Rituximab was used alone (n=16) or in combination with chemotherapy (n=26) in 42 of the 90. 15/242 patients (6%) were initially observed. Survival: The median OS for all patients was 18.3 years. PFS following primary therapy was 9.3 years. There was no difference in the outcomes between patients receiving local or systemic therapy in first line for all patients, or for those with stage I or II disease. On univariate analysis age

Description: We have recently shown that serum free light chain ratio (sFLCR) provides independent prognostic information in patients with newly diagnosed MM (Kyrtsonis et al, Br J Haematol, 137: 240–243, 2007). The aim of the present study was to extend our previous observations in a multicenter setting and to investigate the potential additive effect of sFLCR to the ISS system, in determining the prognosis of patients with MM. We analyzed 214 newly diagnosed MM patients (125 kappa-, 89 lambda-). Serum free light chain levels were measured in sera drawn at diagnosis, using a latex-enhanced immunoassay (The Binding Site, Birmingham, UK). Then, the sFLCR was calculated, accordingly as kappa/lambda or lambda/kappa, depending on the monoclonal light chain type of the patient. Based on our previous study “high” sFLCR was defined as ratios ≥3.57 and ≥45.09 for kappa- and lambda- MM respectively. The median age of the patients was 68 years (33–92), 51% were males, 28%, 30%, and 42% had Durie-Salmon stages I, II, and III, 14% creatinine 〉2 mg/dl, and 13% had Bence-Jones MM. ISS stage was 1, 2, or 3 in 33%, 33%, and 34% of the patients, 48% had CRP ≥4 mg/l, 18% elevated LDH, 31% hemoglobin

Description: Background: MACOP-B or even chemotherapy (CT) with consolidation high dose therapy with autologous stem cell support (HDT-ASCT) have been considered superior to CHOP in PMLBCL. However, in the absence of randomized trials, there is no established optimal treatment for these patients. The role of R-CHOP in PMLBCL, which usually affects young patients, has not been established. Aims: To evaluate the efficacy of R-CHOP±RT in PMLBCL and to compare this approach with CHOP±RT administered to historical controls. Patients and Methods: Between 1994 and 2006, 74 patients with PMLBCL were treated in 6 participating centers. R-CHOP displaced CHOP in the treatment of PMLBCL at a given timepoint in each center. Thus 31 consecutive patients who received R-CHOP, were compared with 43 consecutive historical controls, who had been treated with CHOP prior to that point. Results: The median age of the patients was 30 years (17–82), only 2 patients (3%) were older than 60 years, and 47/74 (64%) were females. All individual IPI parameters as well as B-symptoms were also balanced between the two groups, with the exception of performance status. The median follow-up of currently alive patients was 28 and 73 months for patients treated with R-CHOP±RT and CHOP±RT respectively, the complete response (CR/CRu) rate was 97% vs 67% (p=0.002), and the overall response rate was 100% vs 79%, respectively (p=0.007). All relapses after CHOP occurred within 22 months from diagnosis. The 3-year failure free survival (FFS) was 93±5% vs 53±8% for patients who received R-CHOP±RT vs CHOP±RT (p=0.0006). Within the subgroup of patients with L/LI risk IPI, the corresponding 3-year FFS rates were 95±5% vs 58±10% (p=0.007), while they were 90±9% vs 45±12% (p=0.03) among patients with HI/H risk IPI. The 3-year event free survival (EFS) for all patients was 90±5% vs 51±8% (p=0.001). The 3-year overall survival (OS) was 97±3% vs 67±7% (p=0.008), while the 3-year lymphoma specific survival (LSS) was 100% vs 67±7% (p=0.002). Conclusions: R-CHOP and RT provided impressive results with no cases of primary refractory disease, no lymphoma-related deaths and only 2 failures recorded so far after a median follow-up of 28 months among 31 patients. Patients treated with R-CHOP had significantly higher CR, FFS, EFS, OS, and LSS rates, when compared with CHOP-treated historical controls. Based on these results we continue to treat PMLBCL patients with R-CHOP and RT, avoiding more intensive strategies. Further studies are warranted to investigate whether RT is needed after R-CHOP, especially in the case of a negative post-chemotherapy PET-scan.

Description: Introduction: An ISSWM was recently proposed (Morel et al, ASH 2006), which was based on a large number of patients treated primarily with alkylating agents and /or nucleoside analogues. The ISSWM based on 5 adverse covariates wich defined 3 risk groups: low, intermediate and high risk with 5-years survival rates of 87%, 68% and 36% respectively. In our current analysis, we assessed the impact of this system in patients with WM who received primary treatment with rituximab-based regimens. Patients and methods: Ninety-three previously untreated, symptomatic patients who received treatment either with single agent rituximab (21 patients) or with the combination of dexamethasone, rituximab, and cyclophosphamide (72 patients) were classified according to the ISSWM, which is based on 5 adverse covariates: age〉 65 years, hemoglobin ≤11.5 g/dl, platelet count ≤ 100 x 109/L, β2- microglobulin 70g/L. Low risk is defined by the presence of ≤ 1 adverse characteristics except age, high risk by the presence of 〉2 adverse characteristics and intermediate risk by the presence of 2 adverse characteristics or age 〉65 years. Results: The disease features of the 93 patients were typical of symptomatic WM: age 〉 65 years in 63%, males 65%, B-symptoms in 22%, splenomegaly in 29%, lymphadenopathy in 34%. 15% of patients were rated as low risk, 65% as intermediate risk and 20% as high risk. Criteria for initiation of therapy included cytopenia, hyperviscosity, constitutional symptoms, organomegaly or IgM-related disorders. Overall, 62% of patients were alive at 6 years. Median survival was not reached for low and intermediate risk and was 38 months for high risk patients (p=0.006). There was a clear separation of the survival curves in the three groups. At the time of last follow-up the percentage of patients alive was 100%, 82% and 58% for patients classified as low, intermediate and high-risk group respectively. Conclusions: The recently proposed ISSWM is applicable in patients with WM who receive primary treatment with rituximab-based regimens and may serve as a basis to compare outcomes in different studies.

Description: Introduction: Combination chemoptherapy regimens such as CHOP and MACOP-B with or without RT are considered the standard first-line treatment for PMLBCL patients. Consolidation with high dose therapy and autologous stem cell support (HDT-ASCT) at first remission is an alternative approach for this young population. However results are not optimal. Given the superiority of R-CHOP over CHOP in elderly patients with diffuse large B-cell lymphoma, the role of Rituximab added to first-line chemotherapy in younger patients is not clear. Aims: In this study we evaluate the effectiveness of R-CHOP±RT in PMLBCL and we compare the results of this strategy to CHOP±RT in historical controls. Patients and Methods: A total of 46 patients with PMLBCL were treated in two participating centers between 1994 and 2004. At a given timepoint R-CHOP replaced CHOP in both centers. Thus, 31 consecutive historical controls were treated with CHOP prior to that point and were compared to 15 patients who received R-CHOP thereafter. Results: The median age of the patients was 31 years (17–58) and 32/46 (70%) were females. Baseline characteristics between the R-CHOP and CHOP groups were well balanced, including age-adjusted IPI [ ≥2 in 40% of R-CHOP and 42% of CHOP-treated patients (p=0.90)]. Complete response (CR) was achieved in 100% in the R-CHOP±RT vs 61% in the CHOP±RT group (p=0.009). No patient has relapsed after R-CHOP, while all relapses after CHOP occurred within 22 months from diagnosis. The 3-year failure free survival (FFS) was 100% and 47±9% for patients treated with R-CHOP±RT and CHOP±RT respectively(p=0.005). Within the subgroup of patients with L/LI risk IPI the corresponding 3-year FFS rates were 100% vs 61±11% (p=0.059), while they were 100% vs 26±13% (p=0.02) among patients with HI/H risk IPI for R-CHOP±RT and CHOP±RT respectively. The 3-year event free survival (EFS) was 93±7% vs 47±9% (p=0.02). The 3-year overall survival was 93±7% vs 47±9% (p=0.27), while the 3-year lymphoma specific survival was 100% vs 67±9% (p=0.049) for the R-CHOP and CHOP groups respectively. Conclusions: R-CHOP±RT exhibited impressive efficacy with no failures among 15 patients. CR and FFS rates were significantly better in favor of R-CHOP compared to CHOP-treated historical controls. EFS and lymphoma specific survival were also improved. Based on these data, our standard approach for PMLBCL patients is the application of R-CHOP±RT. Furthermore the addition of Rituximab to front-line treatment might overcome the need for more aggressive strategies such as consolidation with HDT-ASCT in this patient population.

Description: Abstract 1560 Background Epstein-Barr virus (EBV) is a ubiquitous pathogen that chronically infects B lymphocytes and is implicated in the pathogenesis of lymphoproliferative diseases. Latent membrane protein 1 (LMP1), the major oncoprotein of the virus, has been shown to inhibit apoptosis and trigger survivin expression in malignant cell lines. Although EBV has not been implicated in the pathogenesis of low grade B-cell lymphomas, LMP1-mRNA has been detected in a significant proportion of patients with chronic lymphocytic leukemia (CLL). LMP1 is known for its antiapoptotic properties, but recent data show that LMP1 can simultaneously induce and inhibit apoptosis in B-cells. These opposite functions of LMP1 have not been studied in patients with low grade B-cell lymphomas. Objectives Our objectives were to detect LMP1-mRNA in patients with leukemic low grade B-cell lymphomas ant to investigate the postulated apoptotic properties of the protein, by correlating its expression to survivin levels. Patients and Methods Peripheral whole blood from 64 patients with leukemic low grade B-cell lymphomas was tested by qRT-PCR for the presence of BXLF-1 gene of EBV. The patients' characteristics are shown in table 1. All positive samples were tested by conventional PCR for LMP1-mRNA. Subsequently, survivin m-RNA levels were measured by qRT-PCR in all samples and compared between LMP1 positive and negative patients (Mann-Whitney U Test). Results The BXLF-1 gene was detected in 27/64 (42.1%) patients. LMP1-mRNA was detected in 23/64 (35.9%) patients and in 23/27 (85.2%) EBV-positive patients. Among CLL patients, LMP1-mRNA was detected in 19/44 (43.2%). Finally, surviving-mRNA levels were found to be 8.37 times higher in EBV-negative vs EBV-positive patients, (p=0.002) and 7.19 times higher in LMP1-negative vs LMP1-positive patients (p=0.009). The results are reported in detail in Table 1. Discussion Data from this year's studies suggest that LMP1 may exert both antiapoptotic and apoptotic functions. While the carboxy-terminal domain of LMP1 drives the proliferation and survival of EBV-infected B cells in vitro and in vivo, LMP1 may activate, through its amino-terminal six-transmembrane domains (6TM), the transmembrane receptor proteins PERK, ATF6 and IRE-1, leading to unfolded protein response (UPR) induction. UPR is a cellular stress response that promotes apoptosis. In different environments, LMP1 signaling may show differences regarding its apoptotic effects on B lymphocytes. In our study, we detected LMP1-mRNA in 43.2% of CLL patients, a proportion significantly higher than previously reported (14%). Moreover, for the first time, LMP1-mRNA was detected in patients with other than CLL low grade B-cell lymphomas (Table 1). In patients with leukemic low grade B-cell lymphomas, in the pathogenesis of which EBV is not causally implicated, LMP1 may have apoptotic instead of anti-apoptotic properties, as evidenced by the lower survivin m-RNA levels in LMP1-positive patients. This finding deserves further investigation, in order to reveal the clinical significance of the different functions of LMP1 in non-EBV related lymphomas. Disclosures: No relevant conflicts of interest to declare.

Description: Whilst the spectrum and clinical significance of gene mutations and immunogenetic features in common mature B-cell malignancies is well established, their incidence, biological and clinical importance in splenic marginal zone lymphoma (SMZL) remains uncertain. Accordingly we screened 175 well-characterised SMZL cases for mutations in 768 genes (Haloplex Target Enrichment System) with a known or postulated role in B-cell physiology, B-cell malignancies in general and SMZL pathophysiology in particular. After sequencing (HiSeq 2000) we achieved a mean depth across our gene panel of 297-fold (range 128-702x), with more than 85% of all bases covered at 〉50-fold. After comprehensive filtering, 1,374 single nucleotide variants and insertions/deletions were identified. 221 genes were recurrently mutated at a gene frequency of 2-16% [n=2-28]. Sanger sequencing confirmed 86/86 selected variants in our recurrent genes, and showed 99% concordance between our Haloplex and Sanger sequencing of NOTCH2 exon 34, which was performed in all patients. Comprehensive validation of both germ-line Haloplex [n=18 patients] and Sanger sequencing established the sensitivity and specificity of our approach, and confirmed the biological importance and somatic origin of the genes described herein. To identify biologically relevant genes, we employed MutSigCV analysis, an algorithm that identifies significantly mutated genes by accounting for background mutation rate, DNA replication time and the gene size. 18 mutated genes were identified with TP53 [n=28], KLF2 [n=21], MYD88 [n=12], NOTCH2 [n=17], TNFAIP3 [n=13] and CCND3 [n=15] being the most significant; genes that encode components of pathways important in the regulation and differentiation of mature B-cells were also identified, including CREBBP [n=9], MAP3K6 [n=5], KDM2B [n=7], SETD1B [n=6], TRAF3 [n=9], ARID1A [n=10], BIRC3 [n=3], BCL10 [n=5], BTG1 [n=3], ATM [n=10], NFKBIE [n=4] and DDI1 [n=4]. Then, we searched for significant pairwise gene correlations and mutually exclusive relationships between our mutated genes demonstrating the following: (1) independent events, such as MYD88, where a mutation is invariably observed as an isolated event; (2) cancer drivers that have a similar proportion of co-occurring and mutually-exclusive relationships, such as NOTCH2, TP53, TNFAIP3 and CREBBP, and (3) genes such as KLF2, CCND3 and ARID1A that have proportionally more co-occurring relationships, thus suggesting a synergistic function to promote tumorigenesis. Finally, we studied clonal evolution, by differentiating between early, clonal events, and later, subclonal mutations (ABSOLUTE algorithm), and we were able to classify clonal or subclonal mutation in 6/18 of our MutSigCV genes. Paradigmatically, we observed that all the CREBBPmutations were fully clonal. Amongst our most novel findings was KLF2, or Krüppel-like factor 2, mutations that were distributed across the entire protein, with a cluster in the C2H2 domain and were all somatically acquired. All mutations tested were clonal, significantly associated with del(7q) (P=0.001), IGHV1-2*04 gene usage (P

Description: Introduction Poly [ADP-ribose] polymerase 1 (PARP-1) has a central role in the repair of single-stranded DNA (ssDNA) breaks, thus protecting the cell from genomic instability. This enzyme is a promising target in anticancer treatment. The aim of the present study is to investigate the role of PARP1 in B chronic lymphocytic leukemia (B-CLL) patients pre- and post- treatment. Patients and Methods Blood samples were collected from B-CLL patients before treatment and following 3 cycles of immunochemotherapy (chosen by the treating physician according to common clinical practice). Quantification of PARP1-mRNA levels was performed by a SYBR-green based PCR performed on BIORADCFX96 (BIORAD). The results were expressed in correlation to the mRNA levels of the endogenous housekeeping gene of beta actin (ACTB). The detection of the gene product was performed by Western Blot using anti-PARP1 antibody (clone 47-258; Calbiochem). We detected the 70 KD protein segment that results from the in vivo fractionation of PARP1 by caspase-3. The protein bands were visualized using an enhanced chemiluminescence detection system (Amersham Biosciences) and quantitated by densitometry using the gel analysis software ImageJ. We correlated the PARP1 protein levels to those of a group of healthy volunteers. We also studied the differences between PARP1-mRNA and protein levels before and after treatment and correlated them to clinical response. The related samples Wilcoxon Signed Rank test (IBM SPSS statistics, version 19.0) was used for the statistical analysis of the results. Results Samples were obtained from 24 B-CLL patients before treatment and from 15/24 following 3 cycles of immunochemotherapy. The patients' characteristics are shown in Table 1. Pre-treatment PARP1 protein levels were similar to those of 15 healthy volunteers (p=0.426). No statistically significant difference was noted between the PARP1-mRNA and protein levels following treatment (p=0.507 and 0.650 respectively) (Table 1). Multivariate analysis did not reveal statistically significant differences in the mRNA and protein levels in correlation to the stage of disease, the peripheral blood lymphocyte count, the LDH levels, the response to treatment and the overall prognosis of the patients. Conclusions The expression of PARP1 in patients with B-CLL seems to be comparable to that of healthy persons. We could not distinguish patients displaying reduced levels of PARP1, as previously reported by others. Moreover, PARP1 expression is not affected by immunochemotherapy, irrespective of stage and response to treatment. Thus, this molecule seems not to participate in the mechanisms involved in DNA repair and apoptosis that are impaired in B-CLL. The study is still ongoing with the addition of more subjects. Disclosures: No relevant conflicts of interest to declare.

Description: Key Points Clonal B-cell lymphocytosis of potential marginal-zone origin (CBL-MZ) rarely progresses to a well-recognized lymphoma. CBL-MZ does not require treatment in the absence of progressive disease.