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Genome studies of diffuse large B-cell lymphoma (DLBCL) have revealed a large number of somatic mutations and structural alterations. However, the clinical significance of these alterations is still not well defined. In this study, we have integrated the analysis of targeted next-generation sequencing of 106 genes and genomic copy number alterations (CNA) in 150 DLBCL. The clinically significant findings were validated in an independent cohort of 111 patients. Germinal center B-cell and activated B-cell DLBCL had a differential profile of mutations, altered pathogenic pathways and CNA. Mutations in genes of the NOTCH pathway and tumor suppressor genes (TP53/CDKN2A), but not individual genes, conferred an unfavorable prognosis, confirmed in the independent validation cohort. A gene expression profiling analysis showed that tumors with NOTCH pathway mutations had a significant modulation of downstream target genes, emphasizing the relevance of this pathway in DLBCL. An in silico drug discovery analysis recognized 69 (46%) cases carrying at least one genomic alteration considered a potential target of drug response according to early clinical trials or preclinical assays in DLBCL or other lymphomas. In conclusion, this study identifies relevant pathways and mutated genes in DLBCL and recognizes potential targets for new intervention strategies.

Patients with diffuse large B-cell lymphoma (DLBCL) who develop lymphomatous meningitis (LM) during or after first-line treatment have a poor prognosis, with CNS relapse occurring within 1 year in approximately 80%. Risk factors have been defined and prophylaxis is recommended in patients with high-risk DLBCL. Liposomal cytarabine (DepoCyte®), a sustained-release preparation of cytarabine for intrathecal (IT) injection, has been shown to be well tolerated and effective in the treatment of LM. Its long CSF half-life allows liposomal cytarabine to be given less frequently than conventional therapy, reducing discomfort for patients and the risks associated with repeated lumbar punctures. The potential of liposomal cytarabine to improve the outcome of prophylaxis against LM is being investigated into a multicenter and prospective trial in patients with high-risk DLBCL in Spain. Preliminary safety results are reported in 22 patients (median age 67 years; range 18–79; 14 male) with newly diagnosed DLBCL at high risk of developing LM (defined as the presence of at least one of the following criteria: retroperitoneal mass >10 cm, Waldeyer’s ring or paranasal involvement, involvement of >30% bone marrow, testicular involvement) who received prophylactic IT liposomal cytarabine during treatment with R-CHOP14 regimen between June 2006 and July 2007 at 10 centers in Spain. Liposomal cytarabine 50 mg was administered during the first day of treatment at first, second and sixth cycles of R-CHOP14 scheme (study days 1, 15 and 71). The median number of doses administered was 3 (range 1–3). Seventeen patients received corticosteroid as prophylaxis for chemical arachnoiditis: 16 dexamethasone (4 mg IT [n = 9] or PO at varying dosages [n = 7]); and 1 patient received IT hydrocortisone (20 mg). The remaining patients did not receive specific corticosteroid prophylaxis for chemical arachnoiditis. Overall, liposomal cytarabine was well tolerated. Six patients experienced minor side effects including headache (Grade 1/2, n = 4; grade 3/4, n = 2) and nausea/vomiting (Grade 3/4, n = 1). No signs of neurological progression or relapsed were observed. These preliminary observations indicate that IT injection of liposomal cytarabine (DepoCyte®) is well tolerated and can be administered safely in combination with dose-dense regimens. Longer-term follow-up will be needed to confirm these encouraging observations.

Patients with diffuse large B-cell lymphoma (DLBCL) who develop lymphomatous meningitis (LM) during or after first-line treatment have a poor prognosis, with CNS relapse occurring within 1 year in approximately 80%. Risk factors have been defined and prophylaxis is recommended in patients with high-risk DLBCL. Liposomal cytarabine (DepoCyte®), a sustained-release preparation of cytarabine for intrathecal (IT) injection, has been shown to be well tolerated and effective in the treatment of LM. Its long CSF half-life allows liposomal cytarabine to be given less frequently than conventional therapy, reducing discomfort for patients and the risks associated with repeated lumbar punctures. The potential of liposomal cytarabine to improve the outcome of prophylaxis against LM is being investigated into a multicenter and prospective trial in patients with high-risk DLBCL in Spain. Preliminary safety results are reported in 22 patients (median age 67 years; range 18–79; 14 male) with newly diagnosed DLBCL at high risk of developing LM (defined as the presence of at least one of the following criteria: retroperitoneal mass >10 cm, Waldeyer's ring or paranasal involvement, involvement of >30% bone marrow, testicular involvement) who received prophylactic IT liposomal cytarabine during treatment with R-CHOP14 regimen between June 2006 and July 2007 at 10 centers in Spain. Liposomal cytarabine 50 mg was administered during the first day of treatment at first, second and sixth cycles of R-CHOP14 scheme (study days 1, 15 and 71). The median number of doses administered was 3 (range 1–3). Seventeen patients received corticosteroid as prophylaxis for chemical arachnoiditis: 16 dexamethasone (4 mg IT [n = 9] or PO at varying dosages [n = 7]); and 1 patient received IT hydrocortisone (20 mg). The remaining patients did not receive specific corticosteroid prophylaxis for chemical arachnoiditis. Overall, liposomal cytarabine was well tolerated. Six patients experienced minor side effects including headache (Grade 1/2, n = 4; grade 3/4, n = 2) and nausea/vomiting (Grade 3/4, n = 1). No signs of neurological progression or relapsed were observed. These preliminary observations indicate that IT injection of liposomal cytarabine (DepoCyte®) is well tolerated and can be administered safely in combination with dose-dense regimens. Longer-term follow-up will be needed to confirm these encouraging observations.

Treatment with FCM results in responses rates of 60% in relapsed or refractory CLL patients. Against this background, we started a trial of FCM in untreated patients diagnosed with CLL younger than 65 yrs. FCM consisted of fludarabine 25 mg/m2 i.v. on days 1 to 3, cyclophosphamide 200 mg/m2 on days 1 to 3, and mitoxantrone 6 mg/m2 i.v. on day 1, given at a 4-week intervals up to six courses. Patients received support with G-CSF and prophylaxis with cotrimoxazole. Response was assessed two months after treatment and included bone marrow and minimal residual disease (MRD) analysis by four-color flow cytometry and PCR. Out the 64 evaluable patients (74% male, median age 58 years), 83% were in advanced (B and C) Binet’s clinical stage and 62% had increased (>20%) ZAP-70 expression. FISH analysis disclosed del(13q) in 25%, +12 in 22%, del(17p) in 10% of cases and del(11q) in 23%. Eighty-three per cent of the patients received the entire planed treatment. Overall response rate was of 88%. MRD-negative CR was obtained in 24%, MRD-positive CR in 23%, nPR 22% and PR 11%. Two out of 14 nPR cases were MRD-negative. Duration of response was 55% at 36 months. Initial parameters associated with CR achievement were the presence of del(17p) (p=0.003), increased serum LDH (P=0.014), and splenomegaly (p=0.04). Hematological toxicity was mild, with grade III-IV neutropenia in 8% of the cases, and moderate infections in 12%. Two patients developed fulminant B hepatitis, one of them dying as a direct consequence of it. In conclusion, in untreated CLL patients, FCM induces a high CR rate, including an important number of MRD negative CR. This places FCM among the most effective regimens for CLL and serves to build up a new immunochemotherapy regimen for CLL (R-FCM) currently under investigation.

Introduction: Epstein-Barr virus (EBV) infection is a common life event that has been consistently associated to several lymphoproliferative malignancies. It is unclear to what extent EBV can be associated to clinical presentation and outcome of lymphomas. Objectives: To evaluate the association between the seroresponse to EBV with the clinical presentation and survival in the different lymphoid malignancies. Methods: As part of the EPILYMPH_Spain case-control study, 414 newly diagnosed lymphoma cases and 488 controls, age and sex matched to the cases, were systematically recruited in a single center during 20 months (starting in 1998). From all subjects, a blood sample was drawn at diagnosis and EBV serostatus was evaluated through ELISA assays to measure IgG reactivity against immunodominant epitopes of EBNA1(BKRF1) and VCA-p18 (BFRF3). Further, immunoblot analysis was performed to evaluate distinct antibody patterns to EBNA1, VCA18, VCA-p40 (BdRF1) and Zebra (BZLF1). These patterns were classified into two groups: reactive (with wide responsiveness to the different EBV proteins) and non-reactive (with an IgG reactivity restricted to a limited number of EBV proteins). Statistical differences between each group were analyzed using Chi-square test for categorical data and T-Student test for continuous data. Survival analysis was performed by Kaplan-Meier techniques and compared with the log rang test. Values of p<0.05 were considered to be statistically significant. All data were adjusted for age and sex. Results: Twenty-four percent (101/414) of the cases and 17% (86/488) of the controls had an EBV reactive response pattern (p= 0.012). Among cases, 29% females versus 20% of males presented an EBV reactive pattern (p= 0.031), with no differences regarding median age. When we agrupated the different lymphoid malignancies into the 3 most important entities, differences in the prevalence of EBV seropositivity were found: B-cell neoplasms (25%), Hodgkin lymphomas (12%) and T-cell neoplasms (34%), p=0.05. When analyzing the different WHO entities, in myeloma patients, EBV reactive pattern was found to be associated to an early stage disease (29% Stage 1 vs 5% Stage 2–3; p= 0.038) and a longer median survival (not reached vs 38 months; p= 0.02). Conclusions: EBV seroprevalence is higher in lymphoma patients, with a slight predominance in females. EBV reactive pattern seems to be associated to early stage myeloma, which implicates a better survival.

Treatment with FCM results in responses rates of 60% in relapsed or refractory CLL patients. Against this background, we started a trial of FCM in untreated patients diagnosed with CLL younger than 65 yrs. FCM consisted of fludarabine 25 mg/m2i.v. on days 1 to 3, cyclophosphamide 200 mg/m2on days 1 to 3, and mitoxantrone 6 mg/m2i.v. on day 1, given at a 4-week intervals up to six courses. Patients received support with G-CSF and prophylaxis with cotrimoxazole. Response was assessed two months after treatment and included bone marrow and minimal residual disease (MRD) analysis by four-color flow cytometry and PCR. Out the 64 evaluable patients (74% male, median age 58 years), 83% were in advanced (B and C) Binet's clinical stage and 62% had increased (>20%) ZAP-70 expression. FISH analysis disclosed del(13q) in 25%, +12 in 22%, del(17p) in 10% of cases and del(11q) in 23%. Eighty-three per cent of the patients received the entire planed treatment. Overall response rate was of 88%. MRD-negative CR was obtained in 24%, MRD-positive CR in 23%, nPR 22% and PR 11%. Two out of 14 nPR cases were MRD-negative. Duration of response was 55% at 36 months. Initial parameters associated with CR achievement were the presence of del(17p) (p=0.003), increased serum LDH (P=0.014), and splenomegaly (p=0.04). Hematological toxicity was mild, with grade III-IV neutropenia in 8% of the cases, and moderate infections in 12%. Two patients developed fulminant B hepatitis, one of them dying as a direct consequence of it. In conclusion, in untreated CLL patients, FCM induces a high CR rate, including an important number of MRD negative CR. This places FCM among the most effective regimens for CLL and serves to build up a new immunochemotherapy regimen for CLL (R-FCM) currently under investigation.

Introduction: Family clustering of hematological tumours has largely been reported in chronic lymphocytic leukemia (CLL) patients. Objectives: To identify clinical and biological parameters at diagnosis that could be associated to family history of haematological neoplasms in CLL patients. Methods: Eighty-five unselected consecutive CLL patients were included in the case-control multicenter study Epilymph_Spain. Immunophenotype data, including CD38 expression, genetics and clinical presentation were performed at diagnosis. Personnel interviews were conducted to collect data on family history of cancer, including site, age at diagnosis, and status of the affected relative. Statistical differences between each group were analysed using Chi-Square tests. Results: Out of the 85 CLL patients included in the study, 50 (59%) reported a first-degree relative with cancer, from which 7 (8%) were haematological tumours. In the following table, the most relevant variables related with the family history of hematological neoplasms in CLL patients are presented. Conclusions: Family history of haematological neoplasms, CD38 expression and younger age at presentation of the disease seems to identify a subgroup of CLL patients that could have a genetic origin. Family history of Haematological neoplasm p NO (n=78) YES (n=7) Median age (range) 71 (45–87) 62 (53–74) 0,061 Gender (M/F) 51 /27 4 /3 0,476 CD38 + 21 (27%) 5 (71%) 0,026 Rai 0–1/2–4 67 /11 7 /0 0,369

Introduction:Family clustering of hematological tumours has largely been reported in chronic lymphocytic leukemia (CLL) patients.

Introduction:Epstein-Barr virus (EBV) infection is a common life event that has been consistently associated to several lymphoproliferative malignancies. It is unclear to what extent EBV can be associated to clinical presentation and outcome of lymphomas.

Purpose: to analize the response rate and survival of a series of patients with disseminated large B-cell lymphoma treated with R-CHOP/21 x 8 cycles and to evaluate the prognostic factors and the prognostic value of gallium-67 imaging. Patients and Methods: 128 patients with DLCL have been prospectively treated with R-CHOP/21 from March 2002 to March 2004, ages between 18 and 85 years. Survival curves were expressed as Kaplan-Meier plots. Prognostic factors were evaluated by the Chi-square test. Survival curves were compared by the Log-rank test. Results: the median age was 65 years (limits: 27–83), 31% older than 70 years. 59 (54%) patients were males. Gallium imaging was positive at diagnosis in 91%. Clinical presentation of DLCL was: bulky disease: 40%, extra-nodal disease: 66% (³ 2 sites: 33%), B symptoms: 41%, ECOG ³ 2: 53%, LDH>N: 67%, IPI ³ 3: 60%. Chemotherapy cycles received were: <6: 20%; 6: 34%; 8: 46%. The intention-to-treat response was: CR 72%, PR 11%, failures 11%, not evaluable: 6%. Survival, with a median follow-up of 13 months, was: OS: 74%, and EFS: 70%. There are no differences between patients who have been treated with 6 or 8 cycles. EFS of patients with normal gallium imaging at the end of treatment was 90% (p=0.03). EFS was not different between patients older or younger than 70 years. EFS of patients with age adjusted IPI 0 was 100%, IPI 1:70%; IPI 3: 82% and IPI 4: 48%. Three (5%) patients relapsed. Prognostic factors for OS and EFS were: LDH > Normal (p=0.01 y p=0.005 respectively) and ECOG ³ 2 (p=0.0008 y p=0.02). Conclusion: Rituximab associated with CHOP improves the results of historical series treated with CHOP for patients of all ages, but in patients with age-adjusted IPI=3 results have to be improved with other therapeutic strategies. Normalization of gallium-67 imaging at the end of treatment is predictive of a significantly better EFS. Age is not a prognostic factor in patients who receive the same treatment. For patients with disseminated disease, LDH and ECOG are the only significant prognostic factors.

Introduction: Mantle cell lymphoma (MCL) is a CD20+ malignancy comprising up 5% of non-Hodgkin’s lymphomas, and has a poor prognosis under standard chemotherapy. The HyperCVAD-M/A regimen (fractionated high-dose cyclophosphamide, vincristine, doxorubicin and prednisolone alternated with methotraxate and cytarabine) has yielded encouraging results when combined with autologous stem cell transplantation (ASCT) in MCL, with 5-year failure-free survival of 54% and overall survival 72%. In an effort to improve these results further, we have combined rituximab in vivo purging and post-transplant consolidation with HyperCVAD-M/A plus ASCT. Methods: Patients aged <65 years with previously untreated or relapsed MCL were treated with four courses of HyperCVAD-M/A followed by four once-weekly doses of rituximab 375mg/m2 as purging prior to stem cell mobilization and harvesting, high-dose chemotherapy (ICT-CY or BEAM), stem cell reinfusion and four further doses of rituximab immunotherapy post-transplant. Results: Of the 34 patients enrolled so far, 15 (12 male, 3 female; 12 previously untreated) have been transplanted. The median age was 52 years (range 47–63 years). After the final post-ASCT immunotherapy all 15 patients were in clinical complete remission. With a median follow-up of 30 months from diagnosis (range 7–52 months), 14 patients remain alive with 13 in first complete remission. One patient died 15 months post-ASCT without evidence of disease recurrence. Kaplan-Meier estimates of 4-year overall and event-free survival are 93.3% and 86.6% respectively. Conclusions: This approach seems safe and feasible and produces durable remissions; longer follow-up of a more patients will be required to assess the effect of the procedure on survival.

Purpose: to compare the response rate and survival between patients diagnosed of PTLD and treated with front-line rituximab and those not treated with rituximab. Patients and Methods: 108 patients with PTLD have been studied from January 1996 to January 2004. Survival curves were expressed as Kaplan-Meier plots and were compared by the Log-rank test. A multivariate Cox regression analysis was performed to asses the effect of prognostic factors on survival. Results: median age was 55 years (limits: 18–73). 70% were males. The transplanted organ was: kidney 46%, liver 28%, heart 16%. Median time between transplant and PTLD was 59 months, 25% were diagnosed during the first year after the transplant. The most frequent histological subtypes were: large B-cell lymphoma 53% and polymorphic SLPT 13%. 70% were EBV +. Clinical characteristics at diagnosis were: disseminated disease: 52%, extra-nodal disease: 81%, ECOG 3 2: 43%, LDH >N: 60%, IPI 3 3: 40%. Treatments used were: reduction of immunosuppression 91%, chemotherapy 59%, rituximab 33%, antiviral 13%. Response to treatment was: CR 46%, PR 13% failure 11%, not evaluable (early deceased): 29%. With a median follow-up of 15,2 months, survival was: OS 21% and EFS 15%. Forty-six (43%) patients died. The causes of death were: lymphoma progression 15 (33%), infection 15 (33%), toxicity 16 (34%). Survival of patients treated with rituximab was significantly better than the general group: OS 76% (p=0.007) and EFS 70% (p=0.02). Among patients treated with rituximab, 8 (23%) patients died. The significant bad prognostic factors for EFS in the multivariate analysis were: disseminated disease (RR: 2, 95% IC:1,02–3,8; p=0,04), ECOG 3 2 (RR: 5, 95% IC:2,6–9,8; p=0,0001), not been treated with rituximab (RR: 3,8, 95% IC: 1,7–10; p=0,001). IPI did not have prognostic impact. Conclusions: survival of patients with PTLD is low with conventional therapy, and the main causes of death are toxicity and infections. Treatment with Rituximab significantly improves their survival. Patients with disseminated disease and bad performance status have worse prognosis. IPI is not a useful index of prognosis in patients with PTLD.

Purpose:to analize the response rate and survival of a series of patients with disseminated large B-cell lymphoma treated with R-CHOP/21 x 8 cycles and to evaluate the prognostic factors and the prognostic value of gallium-67 imaging.

Purpose:to compare the response rate and survival between patients diagnosed of PTLD and treated with front-line rituximab and those not treated with rituximab.

Introduction: Mantle cell lymphoma (MCL) is a CD20+ malignancy comprising up 5% of non-Hodgkin's lymphomas, and has a poor prognosis under standard chemotherapy. The HyperCVAD-M/A regimen (fractionated high-dose cyclophosphamide, vincristine, doxorubicin and prednisolone alternated with methotraxate and cytarabine) has yielded encouraging results when combined with autologous stem cell transplantation (ASCT) in MCL, with 5-year failure-free survival of 54% and overall survival 72%. In an effort to improve these results further, we have combined rituximab in vivo purging and post-transplant consolidation with HyperCVAD-M/A plus ASCT.