ALBERT

Description: INTRODUCTION. Hairy cell leukemia (HCL) is a rare indolent B-cell malignancy, mainly characterized by peripheral cytopenias and splenomegaly. The current standard of treatment for HCL are Nucleoside Analogs (NA) cladribine and pentostatin, which produce remarkably high remission rates and durable responses. Aim of this study was to evaluate efficacy, short- and long-term toxicity of NA in HCL pts treated outside clinical trials. PATIENTS AND METHODS. We retrospectively analyzed 86 HCL patients (pts) treated with NA between 1996 and 2015 in two Hematologic Centers in Italy. The study was conducted in accordance to the Helsinki Declaration of 1964, as revised in 2000. Cladribine and pentostatin were administered according to standard schedules. Response criteria published by Jones et al. (Br J Haematol 2012) were retrospectively applied. Molecular assessment of BRAF-V600E mutation before and after NA therapy using quantitative real-time polymerase chain reaction (qRT-PCR)-based allelic discrimination assay (sensitivity 0.1%) was performed in 10 pts. RESULTS. The median follow-up of pts (71 males and 15 females, median age 53 years) was 5.8 years (range 0.5-28). During the disease course, 86 pts were treated with NA (cladribine n=76, 88%; pentostatin n=10, 12%); 59 pts received NA front-line (cladribine in 56/59 pts, 95%). Among the other 27 pts, receiving NA as second or subsequent line of therapy, 25 had been previously treated with interferon. Median time from diagnosis to the first NA was 3.3 months (range 0-315). Hematological toxicity was observed in 53 of 77 evaluable pts (69%), and was not significantly different with cladribine (72%) or with pentostatin (37%) (p=0.1). Grade 3-4 neutropenia, anemia and thrombocytopenia were observed in 50 (65%), 7 (9%) and 7 (9%) pts respectively. Extra-hematological toxicity was reported in 48 of 79 evaluable pts (61%). The incidence of extra-hematological toxicity with cladribine (63%) and pentostatin (37%) was not statistically different (p=0.2). Grade 3-4 febrile neutropenia was observed in 24 pts (30%); 9 pts (11%) had grade 3-4 infections; 4 pts (5%) had grade 3-4 skin toxicity, 1 pt (1%) had grade 3 hepatic toxicity. Four of 86 pts (5%) developed a second malignancy (prostatic adenocarcinoma n=2, colon adenocarcinoma n=1, diffuse large B cell lymphoma n=1). The median time from NA to second malignancy was 58 months (range 49-111). Four of 86 pts (5%) developed a skin cancer (basal-cell carcinoma n=3, squamous cell carcinoma n=1), after a median time of 59 months (range 16-126). Response was assessed at a median time of 3 months after the end of therapy. Overall Response Rate (ORR) and Complete Remission (CR) Rate were respectively 93% and 63% in the entire cohort, and 98% and 72% in pts treated with NA front-line. The allelic burden of BRAF before and after therapy with NA was available in 10 cases; none of the pts in clinical CR after NA achieved a complete molecular response (Table 1). The median Progression-Free Survival (PFS) for pts treated with NA frontline was 6.5 years. There was a trend toward a longer PFS in pts receiving NA as first-line therapy as compared to those treated in second or subsequent lines (p=0.05). PFS curves according to type of NA and line of therapy are shown in Figure 1. The 5- and 10-year Overall Survival (OS) rates were 96% (95% CI: 85% - 99%) and 90% (95% CI 76%-96%) respectively. OS was similar in pts treated with cladribine orpentostatin (p=0.49). CONCLUSIONS. This study shows that: i) NA are associated with an acceptable toxicity, neutropenia and febrile neutropenia being the main complications of therapy; ii) timing of response assessment may account for a CR rate that is slightly lower than reported in the literature, confirming the importance of waiting at least 4 months for response evaluation as recommended by current guidelines; iii) the persistence of molecular disease also in pts achieving a clinical CR supports the implementation of consolidation strategies aimed at eradicating minimal residual disease to prolong disease-free survival. Table 1. BRAF allelic burden before and after therapy Patient Age (years) Sex Line of Therapy Allelic Burden (%) Clinical Response Pre- Post- 1 33 F 1 21,8 0,1 CR 2 36 M 2 5,4 0,4 CR 3 39 M 1 24,2 0,7 CR 4 54 M 1 10,8 0,3 PR 5 42 F 1 44 1,2 CR 6 53 F 1 7,9 0,2 NV 7 56 M 1 6,5 0,3 CR 8 54 M 1 12,2 0,2 CR 9 32 M 1 35,8 9,5 PR 10 38 M 2 54,4 12,2 PR Figure 1. Progression-free survival according to type of NA and line of therapy Figure 1. Progression-free survival according to type of NA and line of therapy Disclosures Arcaini: Gilead: Consultancy, Other: Advisory Board; Celgene: Consultancy, Other: Advisory Board; Roche: Consultancy, Other: Advisory Board.

Description: Abstract 2638 Although patients with early-stage Hodgkin's lymphoma (HL) overall have a high rate of cure, they cannot be considered as a homogeneous group. In fact, a portion of these patients are resistant to or may relapse after standard treatment. Current prognostic criteria based on clinical and laboratory parameters at diagnosis do not allow to accurately identify the subset of patients with less favorable clinical outcome. In a study aimed at defining new biomarkers for risk stratification, an increased number of tumor-associated macrophages was found to be strongly associated with shortened survival in patients with classic Hodgkin's lymphoma [N Engl J Med. 2010 Mar 11;362(10):875-85]. The aim of this study was to evaluate the clinical significance of the proportion of CD68-positive infiltrating macrophages in patients with early-stage Hodgkin's lymphoma. By using an immunohistochemistry method, we analyzed diagnostic biopsies of 63 patients followed at our institution between 2006 and 2010, and uniformly treated with ABVD (adriamycin, bleomycin, vinblastine, dacarbazine) chemotherapy. Thirty-nine (62%) patients were males and 24 (38%) were females; median age at diagnosis was 30 years (range 17–85). Histological subtype was nodular sclerosis HL in 55 cases, mixed cellularity HL in 3 cases, lymphocyte-rich HL in 3 cases, and not classified in 2 cases. Five patients had subdiaphragmatic disease while 58 had supradiaphragmatic localizations. Forty-four patients with supradiaphragmatic disease were classified in the EORTC unfavorable subset: in detail, 25 patients had B symptoms and ESR ≥30, or ESR ≥50 despite the absence of B symptoms, 29 patients had bulky disease, 7 patients were older than 50 years, and 6 patients had more than 3 nodal areas involved. Thirty-six out of 63 (59%) patients received radiotherapy as a consolidation treatment after chemotherapy. After completion of the therapeutic program, 54 out of 63 (86%) patients obtained complete remission, while 9 (14%) had refractory disease; 15 out of 54 (28%) patients in complete remission relapsed during follow up. Diagnostic biopsies were reviewed by expert hematopathologists and classified into 3 groups according to the intensity of CD68 expression [N Engl J Med. 2010 Mar 11;362(10):875-85]. CD68-positive infiltrate was lower than 5% in 14 patients (group A), between 5 and 25% in 43 patients (group B), and greater than 25% in 6 patients (group C). Overall survival (OS) and progression-free survival (PFS) were estimated using the Kaplan-Meier method with observation time calculated from diagnosis. Comparison between survival curves was performed by means of the Gehan-Wilcoxon test. The 2-year OS and PFS in the entire cohort were 98% and 79%, respectively. There was a difference in the 2-year PFS between patients with favorable and those with unfavorable prognosis according to the EORTC risk criteria (100% vs 72%, P

Description: Background Mutant calreticulins carrying the sequence translated after a +1 frameshift at the C-terminus are major drivers of myeloproliferative neoplasms (MPNs). These mutant CALRs bind and activate TpoR/MPL in cells co-expressing TpoR and mutant CALRs, resulting in persistent JAK2-STAT5 signaling. Whether mutant CALR proteins are secreted, thus acting in trans on other cells, is not known. Aims Our objectives were to: 1) assess the direct TpoR-mutant CALR interaction both when expressed in the same or in different cells; 2) determine whether mutant CALRs are secreted; and 3) determine whether mutant CALR can act as extracellular cytokines. Methods Engineered CALR and TpoR mutants were analyzed by a combination of biochemical approaches (bioluminescence resonance energy transfer, recombinant protein production), functional assays (cell growth and transcriptional assays, flow cytometry, primary megakaryocytic clonogenic assay, analysis of CALR del52 knock-in mice) and cell imaging (confocal microscopy, flow cytometry and immuno-gold electron microscopy). Secreted CALRs were determined by ELISA using mutant specific antibodies. Results 1) Two systems provided evidence that mutant CALRs and TpoR directly interact. First, using Nano-BRET in cells co-expressing N-terminally fused TpoR or EpoR with Nano-luciferase and mutant or WT CALR C-terminally tagged with HaloTag that is bound to the 618-ligand fluorophore, we show that TpoR and mutant CALRs interact in a complex whether the two proteins are within 10 nm. The interaction does not occur between TpoR and WT CALR, or between EpoR and mutant or WT CALRs. Second, expressing mutant CALR and TpoR extracellular domain in S2 Drosophila Schneider cells showed that stable complex formation requires immature high mannose structure on TpoR. Lastly, we could detect surface expression of the TpoR/CALRdel52 complex using proximity ligation assay with anti-TpoR and anti-mutant CALR antibodies in CRISPR/Cas9 engineered UT7/Tpo cells that express endogenous mutant CALR and TpoR levels. 2) We used flow cytometry, confocal immunofluorescence and immunogold electron microscopy and could show that mutant CALRs are trafficking via cis-, medial- and trans-Golgi to the cell-surface and are secreted, independently from TpoR expression. Importantly, mutant CALRs are also secreted in CALR mutated MPN patients as determined by mutant CALR-specific ELISA assay in patient plasma (mean plasma level 24.6 ng/ml, range 0-156.5 ng/ml). In the 113 evaluated CALR mutated patients from different centers the plasma mutant CALR levels correlated with CALR mutant allele burden (P

Description: Abstract 2720 Introduction: Bendamustine is a promising agent that has been showed high efficacy in combination with Rituximab in indolent lymphomas; however experiences on its use in combination with other cytotoxic agents are scant, particularly in FL. A previous FIL trial showed that a brief R-FND induction chemoimmunotherapy with only 4 courses of R-chemotherapy followed by 4 rituximab doses as consolidation can achieve high CR and PFS rates in FL patients, supporting the feasibility of this regimen in the elderly (Vitolo et al, ASH 2011). These promising results prompted FIL to investigate safety and efficacy of a similar combined brief regimen substituting Bendamustine for Fludarabine aimed at reducing toxicity and maintaining efficacy. Patients and methods: From September 2009 to November 2011, 76 elderly patients (age 65–80) with advanced stage FL at diagnosis were enrolled. Inclusion criteria were: untreated grade I, II and IIIa FL; advanced disease requiring treatment (stage III/IV) or stage II including at least one of the following conditions: bulky disease (〉7 cm), active disease with rapid lymph node progression, lactate dehydrogenase or β2 microglobulin above normal, systemic symptoms and extranodal involvement. A comprehensive geriatric assessment (ADL, I-ADL, CIRS) was also performed at diagnosis and only “FIT” patients were enrolled into the study. Treatment plan was: 4 monthly courses of R-BM (375 mg/m2 Rituximab day 1, 90 mg/m2 Bendamustine days 1–2, 8 mg/m2 Mitoxantrone day 1), followed by consolidation with 4 weekly Rituximab infusions. Polymerase chain reaction (PCR) for BCL2/IgH rearrangement was performed on bone marrow samples at diagnosis, after R-BM and after consolidation. Results: At the time of analysis, 69/76 patients are evaluable for clinical characteristics, response to treatment and toxicity. Median age was 71 years (range 65–80); 26 males, 43 females; WHO grading was as follow: I 16%, II 55% and IIIa 29%; 19% had advanced stage II disease, 27% stage III and 54% stage IV; 49% had BM involvement, 19% B symptoms and 7% leukemic dissemination; 58% patients had no comorbidity, 20% one and 22% two or more comorbidities. According to FLIPI patients were: 12% at low, 30% at intermediate and 58% at high risk. PCR analysis was carried out in 64 patients at diagnosis: 58% were Bcl-2 positive. Sixty three patients completed the planned treatment and the whole program was completed according to the planned time in 60/63 patients. Six patients did not complete the treatment: 1 for progressive disease, 4 for adverse events (2 haematological toxicity with prolonged neutropenia; 1 CMV colitis and 1 for infection and concomitant worsening of pre-existing oral pemfigo) and 1 patient for worsening of performance status. Overall response after R-BM + Rituximab was observed in 64 (96%) patients with 52 (75%) patients in complete remission and 12 (18%) in partial remission (PR). Response to 4 cycles of R-BM was as follow: 27 (39%) in CR, 37 (54%) in PR and 5 (7%) patients in stable (SD) or progressive disease. Of the 40 patients in PR or SD after 4 R-BM, 26 (65%) converted to CR following further Rituximab consolidation. At the time of this analysis, of the 37 patients Bcl-2 rearranged at diagnosis, 19 (51%) were evaluable for molecular response during and after treatment. PCR negativity was achieved in 16/19 (84%) patients after R-BM and in 18/19 (95%) patients at the end of treatment. A total of 521 courses of R-BM and Rituximab were given. The most frequent severe toxicity (CTC grade 3–4) was neutropenia reported in 18% of the courses; nevertheless only four serious infections were recorded (all due to bacterial agents). Pulmonary and cardiac toxicities were 〈 1%. Two deaths were recorded: one due to lymphoma after second line treatment and one due to hepatic carcinoma occurred 3 months after completion of therapy. Conclusions: A brief course of chemo-immunotherapy R-BM followed by Rituximab consolidation is safe and effective with a high CR rate in elderly patients with untreated advanced stage FL. Planned future analysis of the entire study will give further information on late toxicity and outcome. Disclosures: Off Label Use: In Europe use of Bendamustine in first-line treatment of follicular lymphoma is off-label. Drug was provided free by Mundipharma. Vitolo:Roche: Membership on an entity's Board of Directors or advisory committees.

Description: Abstract 3951 Poster Board III-887 Background An increased incidence of second cancers has been reported in some lymphoproliferative disorders. Whether the predisposition to other malignancies is due to disease-related immune-suppression rather than to the carcinogenic role of therapy given to treat the hematologic disease is still poorly understood. Purpose The aims of this study were to assess the frequency, characteristics and factors predicting second cancers in patients with WM and to evaluate whether WM patients are at increased risk of developing other malignancies as compared to an age- and sex-matched control population. Patients and methods The clinical records of 220 consecutive WM patients seen in two Hematologic centres of Northern Italy from 1980 to 2008 were reviewed. All cancers, with exception of non-melanoma skin cancers, were considered for this analysis. Standardized incidence ratios (SIRs), defined as the ratio between observed and expected cases, were used to compare the incidence of second cancers in WM with that of the general population. The number of expected cancers was obtained from age, sex and calendar-specific incidence rates reported by AIRTUM (Associazione Italiana Registri Tumori) for Northern Italy. A multivariate Cox proportional hazards regression model was used to examine risk factors for the development of second cancers. Therapy was analyzed as a time-dependent covariate. Results The median age of patients was 61 years (range: 26-86), 131 (60%) were males and 89 females (40%). The median follow-up was 4.8 years (range: 0.5-25). Overall, 136 patients (62%) with symptomatic WM received therapy, whereas a watch-and-wait policy was adopted in 84 patients (38%) with asymptomatic WM. Among treated patients, first-line therapy consisted of chlorambucil in 93 cases (68%), cyclophosphamide-based regimens in 15 (11%) and nucleoside analogs-containing regimens in 17 (13%). Rituximab was associated to chemotherapy in 19 patients (14%). Details on therapy were not available in 11 cases (8%). Overall, 52 second cancers were observed in 49 patients (22%). Forty-six patients (94%) had one malignancy and 3 (6%) developed two cancers. The types of cancer were: gastrointestinal (n=9, 17%), lung (n=8, 15%), breast (n=7, 13%), urinary tract (n=6, 11%), prostate (n=5, 10%), diffuse large B cell lymphoma (n=5, 10%), myelodysplastic syndrome/acute leukemia (n=3, 6%), brain (n=3, 6%), thyroid (n=2, 4%), mouth (n=2, 4%), other cancers (n=2, 4%). The diagnosis of cancer preceded that of WM in 13 cases (27%), was concomitant (within 3 months) in 6 (12%) and subsequent in 30 (61%). The median time from diagnosis of WM to the occurrence of a subsequent cancer was 4.3 years (range: 0.2-12.9). The cumulative probability of developing a second cancer after WM was respectively 11% at 5 years, 21% at 10 years and 33% at 15 years. As compared to the control population, the risk of second cancer in WM was 1.66 times higher than expected (95% CI: 1.16-2.37, p=0.005), without significant difference between males and females (p=0.7). In multivariate analysis, the risk of second cancer was not influenced by age (p=0.91), sex (p=0.45), reduction of IgG (p=0.91) or IgA (p=0.58) levels. In a time-dependent analysis, therapy given for WM did not increase the risk of developing a second malignancy (hazard ratio: 1.12, p=0.76). Conclusions This study shows that WM are at higher risk of developing second cancers as compared to the general population. We did not find an association between the occurrence of second cancers and treatment of WM. Disease-related immune-suppression may predispose WM patients to develop other malignancies. Disclosures: No relevant conflicts of interest to declare.

Description: Polycythemia vera (PV) is a chronic myeloproliferative disorder with a propensity to develop myelofibrosis, a condition named post polycythemia vera myelofibrosis (post-PV MF). Survival and prognostic factors after transition to MF remain to be defined. We studied 68 patients with post-PV MF to define survival and prognostic factors for survival at diagnosis of post-PV MF. We also developed a dynamic prognostic model to predict survival at any time from diagnosis of post-PV MF. The median interval between the diagnosis of PV and that of post-PV MF was 13 years (range, 4–29.6 years). Patients with post-PV MF were observed for 181 person-years of follow-up. At diagnosis of post-PV MF, 43 (63%) of 68 patients had less than 65 years. During the follow-up, the incidence of thrombosis was 42 × 1000 person-years (95% CI: 19–93.5) and the incidence of leukemia was 50.3 × 1000 person-years (95% CI: 26–115). The median survival was 5.7 years. Multivariable Cox proportional hazard regression including age, hemoglobin value, platelet count, leukocyte count, and spleen size, showed that hemoglobin 〈 10 g/dL (P 〈 .001) and platelet count 〈 100 × 109/L (P= .026) were independent risk factors for survival. We stratified patients at diagnosis of post-PV MF, according to these factors, obtaining two risk groups with significantly different survival (P = .003): low risk (Hb 〉 10 g/dL and platelet count 〉 100 × 109/L) with a median survival of 7 years, and high risk (Hb 〈 10 g/dL or platelet count 〈 100 × 109/L) with a median survival of 2 years. The prognostic model retained significance after adjustment for age in a multivariable Cox proportional hazard regression (HR: 4.3, 95% CI: 1.6–11.4; P= .003). To assess whether this prognostic model may predict survival at any time from diagnosis of post-PV MF, we evaluated in a time-dependent analysis 64 patients who had longitudinal blood cell counts during follow-up. As first step, we evaluated univariate survival analysis with hemoglobin value 〈 10 g/dL and platelet count 〈 100 ×109/L as time-dependent covariates. Both time-dependent parameters affected survival (HR for hemoglobin 5.8, 95% CI: 2.2–15.2, P 〈 0.001; HR for platelets 4.5, 95% CI: 1.67-12, P=.002). As second step, we evaluated the prognostic model assessed at diagnosis as time-dependent covariate, to define whether the acquisition of one risk factor during follow-up may affect survival. The HR was 7.5 (95% CI: 2.4-23.4; P 〈 .001). The time-dependent prognostic model retained statistical significance after adjustment for age (P 〈 .001). In conclusion, in patients developing post-PV myelofibrosis, a prognostic model based on hemoglobin level 〈 10 g/dL and platelet count 〈 100 × 109/L may predict survival at diagnosis of post-PV MF and at any time thereafter.

Description: In splenic marginal zone B-cell lymphoma (SMZL) no specific genetic alterations are known. Abnormalities of chromosome 7p and of p53 are reported as adverse prognostic factors. In a recent multicentre study (Arcaini et al Blood 2006), a prognostic model based on hemoglobin, albumin and LDH identified 3 risk categories. HCV infection was present in nearly 20% of patients (pts). At now, no data are available on genetic alterations in the HCV-positive subset of SMZL and in the different prognostic categories. The aims of the study were: a) to analyze copy number alterations (CNAs) by means of array comparative genomic hybridization (array-CGH) with a resolution of ∼100 kb; b) to compare CNAs in HCV-positive and HCV-negative pts; c) to identify potential genetic alterations related to the clinical features and to the prognostic categories. We analyzed marrow and blood samples from 34 pts with SMZL: 22 were HCV-negative (serology and HCV-RNA) and 12 were HCV-positive (genotype 2a/2c in 10 pts, genotype 1 in 2). DNA was extracted from bone marrow (16) and peripheral blood lymphocytes (18) and was hybridized with pooled blood lymphocyte reference DNA on Agilent’s 44K oligonucleotide microarray (kit 44B). Images and data were analyzed using Agilent’s Feature Extraction (v9.1) and CGH analytics (v3.4.27) softwares. Ten cases (4 HCV+ and 6 HCV-) did not show CNAs. A single alteration was present in 7 pts, 2 to 5 alterations in 11 and 〉5 in 6. All CNAs were detected in mosaicism (from 20% to 90%). A median of 5.6 (range 1 to 20) and 3.8 (range 1 to 13) CNAs were detected in HCV+ and in HCV- cases, respectively. The most frequent CNAs were hetereogeneous in size with the following common regions: losses of 1p36.21-p35.3 (3 pts), 7q31.1-q32.3 (7 pts), 8p21.3-p12 (6 pts), 13q14.2-q14.3 (6 pts), 14q32.12-q32.13 (4 pts) and 17pter-p12 (8 pts); gains of 3q21.1-q29 (5 pts), 12q13.1-q21.31 (5 pts), 17q24.1-qter (4 pts), Xpter-p11.23 (4pts). A homozygous 13q14.2 deletion, overlapping that found in CLL and including Rb1 gene, was found in one HCV- pt. The del(7)(q31.1-q32.3) was the more frequent and it ranges from 14,1Mb to 34Mb. No difference in number of CNAs and in specific common regions alterations was found between HCV+ and HCV- cases except for dup(X)(pter-p11.23) (p=0.01, 4 HCV+ pts and none HCV- pt). High-risk prognostic category was significantly associated with del(7)(q31.1-q32.3) (p=0.01) and del(17)(pter-p12) (p=0.02). Mutational status of immunoglobulin variable heavy-chain gene was related to del(7)(q31.1-q32.3) (p=0.04) and dup(12)(q13.1-q21.31) (p=0.03). The presence of villous lymphocytes was associated with del(1)(p36.21-p35.3) (p=0.02); del(8)(p21.3–p12) was related to an autoimmune background in the HCV+ subset (p=0.04). The number of CNAs was associated to leukemic disease (p=0.02) and to the presence of villous lymphocytes (p=0.04). In conclusion, array-CGH in SMZL does not show specific genetic abnormalities for pts with HCV-positive or HCV-negative SMZL. 7q and 17p deletions are significantly associated with the high-risk prognostic category, clinically and biologically identifying a group of pts with aggressive disease.

Description: Splenic marginal zone B-cell lymphoma (SMZL) is a rare clinical and pathological entity recognized by the WHO classification. SMZL usually presents with isolated splenomegaly, bone marrow involvement, and leukemic picture. Nodal disease is generally rare at diagnosis. When lymphocytes with villous projections are found in peripheral blood, the disease is termed splenic lymphoma with villous lymphocytes (SLVL). High HCV-seroprevalence is frequently reported in SMZL. The disease commonly pursues an indolent course; however, about a third of pts follow a more aggressive course. SMZL is also heterogeneous with respect to the preferential usage of IgVH genes, as well as the percentage of mutation load. No previous studies correlated IgVH rearrangement features with the clinical characteristics of SMZL. The aim of the present study was to determine the tumor-related IgVH gene rearrangement and compare the gene usage and the mutation status with clinical features of 59 pts. Diagnosis was made according to the WHO classification and to the diagnostic criteria for SMZL (Matutes et al., Leukemia 2008). Paraffin sections from lesional tissues (14 spleens, and 59 bone marrow trephines) were available for all pts. Histology and blood smears were reviewed. Total RNA was extracted from PB (n=13) or BM (n=46) mononuclear cells. IgVH gene rearrangements were amplified using 6 family-specific VH leader primers coupled with a 3′ heavy chain joining (JH) primer or with a constant region of cμ chain primer. The sequence obtained from direct sequencing of the clonal PCR product was compared with germline in the IMGT database. Sixty VDJ rearrangements were amplified and 54 were functional. VH1 family was used in 19 rearrangements (32%), VH3 family in 33 (55%) and VH4 family in 8 (13%). The most frequent VH genes were VH1-02 (n=13), VH3-23 (n=15), VH3-30 (n=7) and VH4-34 (n=5) (67% of all sequences). VH was unmutated in 25%. DH segments were assignable in 56/60 rearrangements (all of the VH unmutated and 41/45 of VH mutated). The most frequent DH families were DH2 (n=9) and DH3 (n=21); the most frequent DH genes were DH3-03 (n=8), DH3-22 (n=6) and DH2-02 (n=6). Most rearrangements used JH4 family (n=21), JH5 (n=8), and JH6 (n=19), accounting for 80% of all rearrangements. JH4b (n=14), JH6b (n=10), and JH6c (n=8) were the most common JH genes. Median length of HCDR3 region was 17 aa (range 11–32). For antigen selection analysis P value was 〈 0.05 in 41% for CDRs and in 55% for FRs. No correlation was found among VH and DH families (p=0.1), among VH and JH families (p=0.09) and among DH and JH families (p=0.4). Forty-two % of pts were unmutated in VH1 family, 15% in VH3 family and 25% in VH4 family (p=0.09). Unmutated cases were 7% in VH3-23 group, 46% in VH1-02 group and 14% in VH3-30 group (p=0.03). For clinical correlations we considered only the 54 functional rearrangements. Villous lymphocytes 〉10% were detected in 53% of VH1 pts, in 57% of VH4 pts, and in 17% of VH3 pts (p=0.01). Villous lymphocytes 〉10% were detected in 21% of VH3-23 pts, in 50% of VH1-02 pts and in no VH3-30 pt (p=0.05). Liver involvement was present in 9% of VH3-23 pts, in no VH1-02 pt and in 50% of VH3-30 pts (p=0.009). HCV serology was positive in 7% of VH3-23 pts, 17% of VH1-02 pts and 50% of VH3-30 pts (p=0.04). Serum MC was detected in 50% of VH3-23 pts, in 9% of VH1-02 pts and in 17% of VH3-30 pts (p=0.06). BM was involved in 86% of VH3-23 pts, in 100% of VH1-02 pts and in 50% of VH3-30 pts (p=0.02). A sinusal localization was detected in 67% of VH3-23 pts, in 20% of VH1-02 and in 100% of VH3-30 (p=0.03). Unmutated status was significantly related to a higher percentage of BM infiltration (p=0.003). The proportion of intermediate and high risk patients according to the SMZL score (Arcaini et al. Blood 2006) was higher in the unmutated respect to the mutated group (69% vs 32%, p=0.05). After a median F-UP of 3 yrs, 10 pts died (7 of lymphoma, 3 of causes non related to lymphoma). The median OS was 12 yrs and the median PFS was 2 yrs. OS did not differ among VH families (p=0.9). Median PFS was 2 yrs for mutated pts and 1 yr for unmutated pts. We performed clustering of pts by applying a 2-means clustering algorithm, using as parameters nodal disease, villous lymphocytes 〉10%, HCV serology positive, BM involvement: in cluster 1 VH1 family pts accounted for 20%, VH3 family for 71%, VH4 family for 9%; in cluster 2 VH1 family pts accounted for 47%, VH3 family for 29% and VH4 family for 24% (p=0.01). In conclusion, VH rearrangement analysis in SMZL reveals a non-random preference for VH1-02, VH3-23 and VH3-30 genes, whose use differs according to distinctive clinical features at presentation.

Description: Post–polycythemia vera myelofibrosis (post-PV MF) is a late evolution of PV. In 647 patients with PV, we found that leukocytosis leukocyte count 〉 (15 × 109/L) at diagnosis is a risk factor for the evolution of post-PV MF. In a series of 68 patients who developed post-PV MF, median survival was 5.7 years. Hemoglobin level less than 100 g/L (10 g/dL) at diagnosis of post-PV MF was an independent risk factor for survival. The course of post-PV MF, however, is a dynamic process that implies a progressive worsening of clinical parameters. Using a multivariate Cox proportional hazard regression with time-dependent covariates, we found that a dynamic score based on hemoglobin level less than 100 g/L (10 g/dL), platelet count less than 100 × 109/L, and leukocyte count more than 30 × 109/L is useful to predict survival at any time from diagnosis of post-PV MF. The resulting hazard ratio of the score was 4.2 (95% CI: 2.4-7.7; P 〈 .001), meaning a 4.2-fold worsening of survival for each risk factor acquired during follow up. In conclusion, leukocytosis at diagnosis of PV is a risk factor for evolution in post-PV MF. A dynamic score based on hemoglobin level, and platelet and leukocyte count predicts survival at any time from diagnosis of post-PV MF.

Description: According to evidence- and consensus-based practice guidelines (Haematologica2002;87:1286–306), red cell transfusion is the therapy of choice for the majority of patients with myelodysplastic syndrome (MDS) and symptomatic anemia. Previous studies have shown that widespread organ dysfunction can result from transfusion iron overload developing in non-thalassemic adults (Schafer et al, N Engl J Med1981;304:319–24; Cazzola et al, Blood1988;71:305–12). In this study, we evaluated the effect of transfusion dependency and secondary iron overload on survival of MDS patients classified according to WHO criteria. Four hundred and sixty-seven consecutive patients with a diagnosis of de novo MDS made at the IRCCS Policlinico San Matteo, University of Pavia Medical School, Pavia, Italy, between 1992 and 2002 were retrospectively evaluated and reclassified according to the WHO criteria. The effects that developing transfusion dependency or secondary iron overload had on survival were evaluated by applying Cox proportional hazards regression with time-dependent covariates. Transfusion-dependent patients had a significantly shorter overall survival (OS) and leukemia-free survival (LFS) than did patients who did not become transfusion-dependent (HR=2.16, P