ALBERT

Description: Abstract 2793 Background: DLBCL is the most frequent aggressive non Hodgkin's lymphoma in adults. The International Prognostic Index (IPI) is currently the most used tool to identify different risk patients. The role of an interim PET to early identify patients with bad response to chemotherapy is still controversial. Aims: The aim of this analysis is to evaluate the predictive value for event-free survival of an interim PET (after 2 chemotherapy cycles) and at the end of therapy (after 6 chemotherapy cycles) of dose-dense R-CHOP in patients with DLBCL. Methods: This is a prospective clinical trial for patients with DLBCL older than 65 with IPI 0–5 or younger than 65 with IPI 0–2. Treatment consists on 6 cycles of R-CHOP administered every 14 days followed by pegfilgrastim (6 mg per cycle) on day 2. In this sub-analysis we have included 69 patients who completed the 6 cycles of chemotherapy and who have PET evaluation at diagnosis, after 2 cycles of R-CHOP and at the end of treatment. All evaluations were made by combined PET/CT, except for 14 patients who were evaluated by PET alone. PET was determined to be positive or negative based on the local radiology reports. Interim PET results did not change the planned treatment. Results: 124 patients were included in the trial, 19 (15.3%) did not finish 6 cycles of treatment: 2 due to serious adverse events, 2 due to progression disease/stabilization, 7 due to investigators decision and 8 due to death. Over 105 patients who completed 6 cycles of treatment, 69 patients with complete PET evaluation (at diagnosis, after 2 cycles and at the end of treatment) were included in the analysis. Median age was 61.6 years old (range 18.2–82.8), 34 (49.3%) were older than 65 yo, 37 (53.6%) were male, 59 (85.5%) had ECOG 0–1. Characteristics of the disease at diagnosis were as follows: stage III-IV: 45 (65.2%), bulky disease: 17 (24.64%), 〉2 extra-nodal sites involvement: 4 (5.8%), B symptoms: 18 (26.1%), elevated LDH: 36/67 (53.7%), elevated beta-2-microglobulin: 23/62 (37.1%), IPI 3–5: 23 (33.3%). The median relative dose intensity (RDI) for cyclophosphamide and adriamycin calculated according to 3-week interval as a reference was 139.8% (≥130% in 75.2% of patients): 141.6% in younger patients and 138.6% in the elderly. Thirty-five (50.7%) patients achieved a negative PET evaluation after 2 R-CHOP cycles and 57 (82.6%) at the end of therapy. Patients with bulky disease had a positive interim PET more frequently (64.7% vs 44.2%), but most of them turned negative at the end of treatment (positive end-of-treatment PET for bulky disease: 11.7%). With a median follow-up of 28 months (limits 2.3–49), event-free survival (EFS) was 70.6% for patients with positive interim PET and 92.9% for patients with negative interim PET (p=0.14). The negative predictive value (NPV) of a negative interim PET was 94.3% and the positive predictive value (PPV) of a positive interim PET was 14.7%. EFS was 47.6% for patients with positive end-of-treatment PET and 95.6% for patients with negative end-of-treatment PET (p

Description: Abstract 1561 In recent years, the microRNA (miRNA) pathway has emerged as a crucial regulation system in tumorogenesis. miRNA expression is deregulated in the tumor, and miRNAs can function both as oncogenes and tumor suppressor genes. miR-SNPs are a novel class of single nucleotide polymorphisms that can affect miRNA biogenesis and target sites and can alter the expression and functions of miRNAs. We have evaluated 9 miR-SNPs and investigated whether a distinct haplotype of miR-SNPs predicts clinical outcome in HL. One hundred and forty-one adult patients (median age, 32 yrs; range, 13–89; males 51.1%) diagnosed with HL in our institution between September 1995 and June 2005 were included in the study. Distribution according to histology: nodular sclerosis (58.9%), mixed cellularity (17.7%), lymphocyte rich (6.4%), lymphoid depletion (4.3%), and nodular lymphocyte predominant (7.1%). Epstein-Barr Virus was present in 38.1% of the samples. SNP analysis was performed by allelic discrimination on ABI Prism 7500 (TaqMan assays) in DNA obtained from formalin-fixed, paraffin-embedded lymph nodes. We examined 9 miR-SNPs: 4 in miRNA genes (MIR196A2 rs11614913; MIR149 rs2292832; MIR423 rs6505162; MIR146 rs2910164); 2 in miRNA binding sites (KRT81 rs3660; FAM179B rs1053667); and 3 in miRNA-processing machinery (XPO5 rs11077; RAN rs14035; TRBPrs784567). miR-SNP genotypes were correlated with probability of treatment failure, treatment-related toxicity, disease-free survival (DFS) and overall survival (OS). The median follow-up was 50 months (range, 1–143). Of 141 patients, 119 (84.4%) achieved complete response, 7 (5%) showed a partial response, and 14 (9.9%) were chemoresistant. We observed an increased probability of treatment failure in patients carrying the XPO5 AA or CC genotype (P=0.036). In 14 patients with neurological toxicity, an association was observed with the KRT81 genotype (P=0.047). In 7 patients with bleomicine-associated pulmonary toxicity, we observed an association with the XPO5 genotype (P=0.048). XPO5 and TRBP genotypes emerged as significant markers for DFS. Mean DFS for 57 patients (56%) with the XPO5 AC genotype was 111 months vs 82 months for patients with the AA or CC genotype (P=0.044). Mean DFS for 37 patients (31.6%) with the TRBP CC genotype was 124 months vs 90 months for patients with the TT or TC genotype (P=0.022). A trend towards an association between the MIR196A2 genotype and DFS was also observed (P=0.07). Only the XPO5 genotype was associated with OS. Mean OS for 66 patients (54%) with the XPO5 AC genotype was 134 months vs 111 months for patients with the AA or CC genotype (P=0.038). Given the evidence for the influence of TRBP and XPO5 as individual markers, we then investigated the combined effect of these miR-SNPs on DFS and OS. We found a significant correlation between the TRBP/XPO5 haplotype and DFS (P=0.005) and OS (P=0.005). Patients with the XPO5 AA/CC and TRBP TT/TC genotypes had the worst prognosis (DFS: 71 vs 114 months; OS: 95 vs 135 months). In the multivariate analyses, the TRBP/XPO5 haplotype (OR, 2.977; 95%CI, 1.1–7.4; P=0.01) emerged as an independent variable for DFS. Only the Hasenclever prognostic index (OR, 5.7; 95%CI, 2–18; P=0.004) emerged as an independent variable for OS, but we observed a trend towards significance for the TRBP/XPO5 haplotype as well (P=0.06). In conclusion, miR-SNPs are a novel class of SNPs that can add useful prognostic information on the clinical outcome of HL, specifically in the detection of chemoresistant patients and patients likely to relapse. The TRBP/XPO5 haplotype has surfaced as a promising prognostic factor that warrants further investigation to confirm its role as a biomarker in HL. Figure. Kaplan-Meier curves for DFS (A) and OS (B) according to the TRBP/XPO5 haplotype. Figure. Kaplan-Meier curves for DFS (A) and OS (B) according to the TRBP/XPO5 haplotype. Disclosures: No relevant conflicts of interest to declare.

Description: Human bone marrow contains a rare population of mesenchymal stroma stem cells (BM-MSC) that are capable of generating all skeletal lineages such as osteoblasts, adipocytes, chondrocytes and fibroblastic reticular cells. In vivo, BM-MSC are essential constituents of the hematopoietic stem cell niche, thus playing an important role in supporting, maintaining and controlling hematopoiesis. Despite their central role in bone marrow physiology, little is known about the primary MSC. This is mainly due to the fact that a precise phenotypical definition of BM-MSC has been lacking so far. We were first to report that low/negative expression of PDGFRα on linneg/CD45neg/CD271pos cells allowed identify the candidate primary stromal stem cell population in adult human bone marrow, i.e linneg/CD45neg/CD271pos/PDGFRαlow/neg cells were highly enriched in CFU-F (1 in 4) and showed all typical BM-MSC properties in-vitro and in-vivo (Li et al. Blood, 2012, 120:3460). This key finding now facilitated to characterize the molecular signature and hematopoiesis-supporting function of primary BM-MSC. Illumina Human HT-12 expression v4 BeadChips containing 48,107 probes were utilized to perform gene expression profiling analysis comparing linneg/CD45neg/CD271pos/PDGFRαlow/neg and linneg/CD45neg/CD271pos/PDGFRαpos cells (five independent samples in each group). The expression of 365 genes, including 22 novel surface antigens, such as ITGB5, ANTXR2, CD63, CD230, APLNR and CD74 was significantly higher in the PDGFRαlow/neg subset, whereas 65 genes showed reduced expression. The expression of cell cycle inhibitor genes, such as cyclin-dependent kinase inhibitor 1 (CDKN1A), B cell translocation gene family, member 3 (BTG3) and dual specificity protein phosphatase 3 (DUSP3), was increased in the PDGFRαlow/neg population, indicating a quiescent status of the majority of the cells, which was confirmed by cell cycle analysis (Ki67/DAPI staining; 96.8 ± 2.9% in G0 phase, n=3). In accordance with the hematopoiesis maintenance function, expression of genes encoding ECM proteins, such as laminin subunit alpha-4 (LAMA4), adrenomedullin (ADM) and collagen type I alpha 1 (COL1A1) was also significantly higher in PDGFRαlow/neg cells. Additionally, quantitative RT-PCR analysis revealed that PDGFRαlow/neg cells expressed high levels of CXCL12, VCAM1 and osteopontin, i.e. genes known to be related to hematopoietic stem cell (HSC) supportive function. In order to test the hematopoiesis-supporting capacity of primary BM-MSC, 7-day co-culture experiments with human cord blood CD34+ cells were performed in SCF, TPO, and FL-supplemented serum-free medium. CD34+ cells were evenly distributed and mainly found in close contact with stromal cells when cocultured with linneg/CD45neg/CD271pos/PDGFRαlow/neg cells, compared to cultures with linneg/CD45neg/CD271neg/PDGFRαneg cells and linneg/CD45neg/CD271pos/PDGFRαpos cells. Co-culture with PDGFRαlow/neg cells did not only amplify total hematopoietic cells (38.8 ± 14.4 fold) but, importantly, also expanded CD34+ cells very effectively (10.2 ± 3.8 fold, compared to 2.4 ± 0.2, 3.6 ± 0.7, and 3.2 ± 0.8 fold for no stroma, CD271neg/PDGFRαneg, and PDGFRαpos cells, respectively; p

Description: Key Points Mx1 + stromal cells and/or their descendants provide functional niches for HSPCs and regulate their localization. Targeting Ext1 or HSPG can mobilize more potent reconstituting cells and enable engraftment without cytotoxic conditioning.

Description: Abstract 1382 The effectiveness of rituximab maintenance in the treatment of CLL has been investigated in a phase II clinical trial that includes two treatment parts. First, patients were given R-FCM up to 6 cycles as induction therapy, achieving an overall response rate of 93% and 46% of CR with negative minimal residual disease (MRD) (Bosch et al. JCO, etc). Second, three months after concluding R-FCM, patients having achieved CR or PR receive rituximab maintenance (375 mg/m2) every three months for two years (up to 8 cycles). We present here the preliminary results of the second part of the study, namely the efficacy of rituximab maintenance. Evaluation of response was performed three months after the last cycle of maintenance and included bone marrow (BM) examination, MRD assessment in peripheral blood and BM by four-color flow cytometry. Patients in whom rituximab maintenance was prematurely interrupted due to toxicity were considered as failures. Fifty-six patients (median age 60 years, 70% female) responding to R-FCM were evaluable for response to rituximab maintenance. Median number of cycles of maintenance given was 8 (range, 3 to 8), 77% of patients completed the entire planned treatment, whereas 91% received 6 or more cycles. Treatment was delayed due to insufficient hematological recovery in 12 cycles (2.7%). Toxicity was mainly hematological, with neutropenia being observed in 31.8% of cycles (Grade 3&4 in 8.9%), thrombocytopenia in 3.4% and anemia in 3.9%. Hypogammaglobulinemia occurred in 38% of patients (low levels of IgA in 50%, IgG in 34%, and IgM in 60%). Eight patients, three of them with hypogammaglobulinemia, experienced grade 3&4 infectious episodes (4 pneumonia, 2 gastrointestinal, 1 myositis, and 1 cerebral abscess). Herpes virus (I/VZ) reactivation was observed in 8 patients. Two patients died due to multifocal leukoencephalopathy and hemophagocytic syndrome, respectively. After rituximab maintenance, 44.6% of patients were in CR MRD negative, 41% in CR, 3.6% in PR, and 10.7% failed to treatment. Failures were due to disease progression (two patients), development of severe neutropenia (two patients), and death (two patients). Among 28 patients that were in CR MRD (-) at the onset of the maintenance part, 19 held the MRD negative status at the end of maintenance, 5 (18%) turned negative into positive MRD (probability of conversion, 40% at 30 months), whereas 4 failed to treatment (2 neutropenia, 1 progression, 1 death). Moreover, 5 of 24 patients (22%) in CR MRD(+) after R-FCM became MRD negative after rituximab maintenance, 17 maintained the CR, one patient achieved a PR, and one patient progressed under maintenance (Table 1). In conclusion, rituximab maintenance after chemoimmunotherapy seems to prolong duration of response and, in some cases, improves the quality of response towards a CR with negative MRD. Maintenance with rituximab had the major benefit in patients in CR with positive MRD. The exact role and the best dosage and treatment schedule of rituximab as maintenance therapy in CLL should be now investigated in randomized clinical trials. Disclosures: Bosch: Hoffman La Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Off Label Use: Rituximab is currently not approved as maintenance therapy for patients with chronic lymphocytic leukemia. Garcia-Marco:ROCHE: Consultancy, Honoraria, Research Funding.

Description: Abstract 706 Thrombocytopenia is a common clinical problem among neonates, affecting 30% of all infants admitted to the neonatal intensive care unit, and up to 70% of those born prematurely. The neonatal predisposition to develop thrombocytopenia has been largely attributed to the increasingly recognized substantial differences between fetal/neonatal and adult megakaryocytes (MKs). However, little is known about overall platelet production rates and platelet survival during neonatal life. To address this question, we evaluated the sites of thrombopoiesis, platelet counts, reticulated platelet percentages (RP%), blood volume, and platelet survival in C57/B6 mice during the first 2 wks of life. During that period, the MK concentration decreased sharply in the liver (the main site of platelet production during fetal life), while it increased steadily in the BM, where it reached nearly adult levels by day 14. During this transition, the spleen helped support thrombopoiesis through a 6-fold increase in MK concentration (which peaked at day 10), prior to returning to baseline levels. Consistent with our recent findings in human MKs (Liu et al., Blood), murine neonatal MKs were small but cytoplasmically mature by immunofluorescence and electron microscopy. In regard to platelet production, platelet counts in one day old pups were approximately 50% of adults, but then increased and reached nearly adult values by day 14. During the same time period, the body weight and total blood volume increased ∼5-fold. Combined, these changes led to a 10-fold increase in the total platelet number over the first 2 wks of life. The RP%, a measure of new platelet release, was ∼2-fold higher in 3- and 10-day old pups compared to adults, suggesting that increased platelet production contributes to the rapid increase in total platelet number during this period of development. To evaluate for potential developmental differences in platelet survival, we conducted in vivo biotinylation studies in newborn and adult mice. These showed that neonatal platelets have a significantly longer half life than adult platelets (90 vs. 50 hrs, respectively; p

Description: Myeloproliferative neoplasms (MPNs) are originated by mutations in a hematopoietic stem cell (HSC), frequently in the Janus kinase 2 (JAK2) gene. Different outcomes of this common event and limited efficacy of JAK2 inhibitors suggest the contribution of other factors. Additional HSC mutations and HSC-niche interaction might influence MPN progression, characterized by sequential expansion of HSCs, blood cells and megakaryocytes. Ensuing bone marrow (BM) fibrosis and osteosclerosis, which are contributed by osteoblastic lineage cells in a BCR/ABL CML model (Schepers et al Cell Stem Cell 2013), impede normal hematopoiesis. We have previously shown that BM nestin+ mesenchymal stem cells (MSCs) innervated by sympathetic nerve fibers regulate HSCs (Méndez-Ferrer et al Nature 2008 & 2010). Here we demonstrate that damage to this regulatory network is required for MPN manifestation. Nestin+ MSCs and NESTIN mRNA expression were rapidly reduced in the BM of MPN patients and mice expressing the human JAK2-V617F mutation. This reduction was not due to nestin+ MSC differentiation into fibroblasts or osteoblasts, as shown by 25-week lineage-tracing studies using Nes-CreERT2;RCE-loxP mice, but instead caused by early MSC apoptosis. In turn, nestin+ cell reduction stimulated MPN progression; selective nestin+ cell depletion using Nes-CreERT2;iDTA mice increased peripheral white and red blood cells, megakaryocytic invasion of spleen germinal centers and BM osteosclerosis. Our recent results indicate that the neural crest contributes during development to BM MSCs with specialized HSC niche function and that postnatal murine BM Nestin-GFP+ cells do not only contain MSCs but also Schwann cell precursor-like cells (Isern et al, ISSCR Annual Meeting 2013). BM Nestin-GFP+ cells from MPN mice showed reduced expression of HSC maintenance and mesenchymal genes, and increased expression of genes related to axon guidance and Schwann cell differentiation. Principal component analyses of independent biological samples further showed that control BM nestin+ cells clustered together with MSCs, whereas MPN BM nestin+ cells resembled Schwann cell precursors. These data suggested alterations to the neural component of the BM HSC niche in MPN. Indeed, BM sympathetic nerve fibers and Schwann cells, closely associated but different from Nestin-GFP+ cells, were rapidly reduced in the BM of diseased animals. Symptomatic MPN mice were treated with selective β3-adrenergic agonists to compensate for the loss of sympathetic stimulation of nestin+ MSCs. Treatment with BRL37344 or the recently FDA approved drug Mirabegron prevented MPN-associated neutrophilia and thrombocytosis, while it did not affect peripheral blood counts of wild-type mice. While vehicle-injected animals showed severe BM fibrosis, long-term BRL37344 treatment led to virtual absence of focal reticulin deposits or excessive fibroblasts. To further confirm the contribution of BM neural damage to MPN pathogenesis, diseased mice were treated with a neuroprotective agent. Sympathetic nerve-ensheathing Schwann cells were strongly reduced in the BM of vehicle-injected animals but preserved in 4-methylcatechol-treated mice. Like in BRL37344-treated animals, this was associated with prevention of very early MPN events, including neutrophilia and BM overproduction of the pro-inflammatory cytokine interleukin-1β. Since MPN is originated by a mutant HSC, we reasoned that sympathetic neuropathy might contribute to MPN pathogenesis through early disruption of the HSC niche. The chemokine Cxcl12 regulates HSC migration and proliferation. At early MPN stage, HSC expansion and mobilization correlated with decreased BM Cxcl12 expression and protein levels. Concomitantly, BM nestin+ MSC number and their Cxcl12 expression were significantly reduced. BRL37344 treatment completely restored the number of BM nestin+ cells and improved Cxcl12 BM levels. Treatment with 4-methylcatechol or BRL37344 prevented the early expansion of mutant hematopoietic progenitors, whereas long-term BRL37344 treatment efficiently reduced mutant hematopoietic progenitor numbers in BM and peripheral blood. These results demonstrate that damage of the niche, induced by the mutated HSCs, critically contributes to JAK2-V617F+ MPN pathogenesis. They also unravel HSC niche-forming MSCs and their neural regulation as promising novel therapeutic targets in MPN. Disclosures: Arranz: Centro Nacional de Investigaciones Cardiovasculares (CNIC): National patent application number 201330677 entitled “Neuroprotective/neurocompensatory therapy for the treatment of myeloproliferative diseases”, with priority date May 10, 2013, National patent application number 201330677 entitled “Neuroprotective/neurocompensatory therapy for the treatment of myeloproliferative diseases”, with priority date May 10, 2013 Patents & Royalties. Off Label Use: Beta-3-adrenergic agonists (e.g. FDA-approved Mirabegron) and neuroprotective drugs for the treatment of myeloprolifeative diseases. Isern:Centro Nacional de Investigaciones Cardiovasculares (CNIC): National patent application number 201330677 entitled “Neuroprotective/neurocompensatory therapy for the treatment of myeloproliferative diseases”, with priority date May 10, 2013, National patent application number 201330677 entitled “Neuroprotective/neurocompensatory therapy for the treatment of myeloproliferative diseases”, with priority date May 10, 2013 Patents & Royalties. Méndez-Ferrer:Centro Nacional de Investigaciones Cardiovasculares (CNIC): National patent application number 201330677 entitled “Neuroprotective/neurocompensatory therapy for the treatment of myeloproliferative diseases”, with priority date May 10, 2013, National patent application number 201330677 entitled “Neuroprotective/neurocompensatory therapy for the treatment of myeloproliferative diseases”, with priority date May 10, 2013 Patents & Royalties.

Description: Key Points Besides maintaining short telomeres, telomerase is required for cell proliferation and tumor growth in CTCL.

Description: Abstract 4631 BACKGROUND The precursor of nucleotide biosynthesis acadesine or 5-aminoimidazole-4-carboxamide (AICA) riboside induces apoptosis in CLL cells, and other lymphoproliferative diseases such as splenic marginal zone lymphoma and mantle cell lymphoma. This effect is selective for B-cells, at least ex vivo, however there is no evidence of whether this cytotoxic effect depends on well known prognostic variables such as ZAP-70 expression or IgVH mutational status. AIM To analyse ex vivo the cytotoxic effect of acadesine on peripheral CLL cells and correlate it with prognostic variables. METHODS Cryopreserved cells from 62 CLL patients were incubated ex vivo with acadesine at 0.2, 0.5, and 1 mM for 24 hours. Viability was determined by flow cytometry using Annexin V-FITC and DAPI staining combined with CD19-PE and CD3-PerCP to differentiate cell viability of B- and T-cells. Cells were considered sensitive to the drug when the percentage of acadesine-induced apoptosis was equal to or higher than 15% with respect to the viability of control cells. The mutational status of IgVH genes was determined by RT-PCR amplification using a set of six VH family-specific primers (VH1 through VH6) along with primers complementary to the constant region (IgM and IgG). Products were directly sequenced from both strands using the Big Dye Terminator Cycle Sequencing Ready Reaction (version 3.1, Applied Biosystems). Sequencing analysis and alignments were performed with use of V-QUEST software and the online international immunogenetics information (IMGT) data library. Samples in which fewer than 2 percent of base pairs differed from those of the consensus sequence have been considered unmutated. ZAP-70 expression was quantified by flow cytometry (cut-off:20%) and cytogenetic alterations associated with CLL (trisomy 12, del13q, del 17p and del11q) were determined by FISH. RESULTS After 24h of ex vivo incubation, 0.2 mM acadesine induced a significant cytotoxic effect (〉 15%) in 31 out of 62 patients (50%). Higher concentrations, 0.5 mM and 1 mM, induced a significant effect in 91.4% (57 of 62 patients) and 98% (61 of 62 patients), respectively. The viabilities (mean ±SD) of the different culture conditions are shown in the table. The cytotoxic effect induced by acadesine was analyzed with respect to the IgVH mutational status and ZAP-70 expression. No significant differences were observed between cases with unmutated IgVH (n=20) and mutated IgVH(n=37), or between ZAP-70 positive (n=24) and ZAP-70 negative cases (n=34). Interestingly, 5 cases showing deletion of 17p were sensitive to treatment with acadesine, in agreement with previously published studies showing that acadesine-induced apoptosis is independent of p53. Only one case showing deletion 17p and VH3-21 usage was not sensitive to acadesine. CONCLUSIONS Acadesine induces apoptosis in B-cells from CLL regardless of ZAP-70 expression, IgVH mutational status and 17p status. Disclosures: Campàs: Advancell: Employment. de Frías:Advancell: Employment.

Description: Abstract 2003 Background: Tumor microenvironment has been shown to play a critical role in the biology and treatment response of some types of non Hodgkin's lymphomas. The significance of the presence of Treg cells (FOXP3+) in MALT lymphomas of the stomach remains unknown. The main aim of this study was to analyze by immunohistochemistry the presence of Treg cells in the microenviroment of gastric MALT (gMALT) lymphomas at the time of diagnosis and during follow-up after treatment, and to evaluate their possible impact in outcome. Methods: Patients were included in the study if they had gMALT lymphoma diagnosed according to the 2008 WHO criteria and diagnostic paraffin-embedded blocks were available for review. Thirty-three patients met the criteria for inclusion in the study. Sections were immunostained for CD20, CD3, CD4, CD8, CD68, FOXP3, PD-1, bcl-10 and Ki67. t(11;18) was studied by FISH and/or PCR and t(1;14) by FISH. Clonality study of the B cell receptor was done according to the BIOMED-2 protocol. The number of CD20+ tumor cells and FOXP3+ infiltrating cells was quantified using a micrometric ocular (WPK 10 × mn) that has a 10 mm linear scale divided to 100 parts. Results: The median age was 63 y (range 32–83) with 52% being male. Stage: I in 66%, II in 25% and IV in 9%; B-symptoms in 6%. Translocation t(11;18) was present in 9 (27%) pts and 2 additional cases had strong nuclear expression of BCL-10 without t(11;18) or t(1;14). At diagnosis, the mean (± standard deviation) number of CD20+ tumor cells and FOXP3+ infiltrating cells was 680±232 cells/cm2 and 30±29 cells/cm2, respectively. The mean number of CD20+ tumor cells and FOXP3+ infiltrating cells was similar between gMALT with or without t(11;18). Number of treatments analyzed was 37 (treatment not available in 2 pts and 5 pts received more than 1 treatment). Treatment regimens included: eradication therapy (n=11); interferon+ribavirin (n=1); single or combined agent chemotherapy without rituximab (n=5); rituximab alone or CHOP-like with rituximab (n=4); fludarabine (n=8); fludarabine or bendamustine with rituximab (n=8). The first response evaluation showed an overall clinical response rate of 84% (CR 76%) with persistence of residual disease by histology or molecular (IGHV) studies in 31% and 56%, respectively. Overall, the mean (±SD) CD20+ tumor cells and FOXP3+ infiltrating cells was significantly reduced after treatment (730±208 vs 285±343, p