ALBERT

Description: Abstract 2647 New data concerning the important role of microenvironment on lymphoma growth are emerging, and in recent years surrogate biomarkers have been identified as prognostic factors for survival in non-Hodgkin lymphoma (NHL). Unlike follicular (FL), diffuse B-cell lymphoma (DLBCL) and Hodgkin lymphoma (HL), there is still no specific internationally accepted risk stratification scoring system for peripheral T-cell lymphomas (PTCL), and the International Prognostic Index (IPI) or the Prognostic Index for T-cell lymphomas (PIT) model are been used to identify higher risk cases of PTCL. Here we retrospectively analyzed the relevance of the well recognized prognostic parameters for T-NHL in 172 patients with different types of PTCL. In 94 cases in whom peripheral blood monocyte count (PBMC) at diagnosis was available, we evaluated whether monocytosis (PBMC 〉800/mm3) could be used as a simple prognostic factor for overall survival (OS) and outcome in PTCL. For the entire group with a median follow-up of 19 months (range 1–168 months), the 5-years OS was 42%, and the median OS 48 months. Monocytosis was present in 23% of the evaluable cases and patients with high PBMC (〉800/mm3) at diagnosis had a worse OS (median 12 months) compared to those with PBMC 〈 800/mm3.This difference showed strong statistical significance (p=0.003) (Fig 1) and the Hazard ratio (HR) for PBMC 〉800/mm3, stratified by histopathological subtype, was 2.81. In particular 3-years OS of patients with PBMC 〉800/mm3 with anaplastic large cell lymphoma, angioimmunoblastic T-cell lymphoma, and peripheral T-cell lymphoma not otherwise specified was 50%, 25%, and 12%, respectively compared to 86%, 65%, and 50% for those patients with PBMC 60y, advanced stage, bone marrow involvement, ECOG PS 〉1, LDH〉UNL, PBMC 〉800/mm3, hemoglobin 800/mm3) has a negative impact on OS (p=0.003) Disclosures: No relevant conflicts of interest to declare.

Description: Background and purpose Successful treatment of T cell lymphoma (TCL) is still problematic and identification of new pharmacological targets in this malignancy is urgently needed. Histone deacetylase (HDAC) inhibitors are emerging as an exciting new therapeutic option for lymphoid malignancies. These drugs increase the acetylation status and modulate the activity of a wide range of non-histone proteins, and effects on both histone and non-histone proteins may contribute to their anti-cancer activity. Romidepsin (depsipeptide) is a potent and specific inhibitor of class 1 HDACs that has shown remarkable activity in the treatment of TCL in preclinical studies and early-phase clinical trials. Lenalidomide  belongs to the immunomodulatory agents (IMID®) and it is has been demonstrated to be very active for the treatment of several types of hematological neoplasia. Purpose of the present study was to determine whether lenalidomide potentiates romidepsin activity in TCL cell lines and if so, by which mechanisms. Methods TCL cell lines (Hut-78: cutaneous TCL cells and Karpas-299 anaplastic lymphoma cells) were treated with increasing concentrations of either romidepsin (0.5 - 25 nM) or lenalidomide (1 - 100 µM), and IC50 at 24-48 and 72 hours was calculated. The interaction between romidepsin (0.5, 1, 2.5 nM) and lenalidomide (2, 4, 10 µM) was evaluated using the Chou-Talalay method to determine if the combination had additive or synergistic effects. The cell cytotoxicity was assessed by MTT assay and apoptosis was measured with annexin-V/propidium iodide (PI) by flow cytometry. Caspase activation was confirmed by Western blot analysis. The effect of the combination on AKT/PI3K and MAPK/ERK signaling pathways and cyclin D1 expression was evaluated by Western blot. Results Treatment with romidepsin alone resulted in time- and dose-dependent cytotoxicity in both cell lines. The IC50 of romidepsin at 24-hour was 5.87 nM and 6.36 nM in Hut-78 and Karpas-299 respectively. Lenalidomide alone did not induce a cytotoxic effect, and we were unable to reach IC50 even after 72 h of treatment. However, after 24 hours, the combination of romidepsin (2.5 nM) and lenalidomide (10µM) (ratio 1:4) showed a strong synergistic interaction with a CI (combination index) of 0.14 in Hut-78 cells, and an additive effect with a CI of 1.08 in Karpas-299 cells. In HUT-78 cells, the combination of romidepsin and lenalidomide enhanced apoptosis compared with each drug alone by the activation of caspases-3, -9 and -8. No change in the expression of Bcl-2 was observed with either treatment alone, or in combination. These events were associated with dephosphorylation of PI3K/Akt and MAPK/ERK pathways, decreased expression of cyclin D1 and Bcl-xL, and an accumulation of acetylated alpha-tubulin. The combination had relatively modest effect on cell cycle parameters. The analysis of the cell cycle showed an increase percentage of cells in sub G0/G1 and G2/M phase, and decrease of S phase. Conclusion These preliminary results indicate that the combination of romidepsin with lenalidomide has a synergistic effect in TCL cell lines and induces apoptosis through signaling events involving pro-survival pathways PI3K/AKT and MAPK/ERK, down-regulation of cyclin D1 and accumulation of acetylated alpha-tubulin. Data look promising and further investigations are required to better define the molecular mechanisms of cell death induced by the combination of romidepsin and lenalidomide in T cell lymphoma. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 2754 Background. Therapy for patients with non-Hodgkin's Lymphomas (NHL) have significantly improved over the last decade, especially since the discovery of monoclonal antibodies and other biologic therapies. Although patients with B-cell NHL usually respond to conventional chemotherapy, they often relapse in spite of salvage therapy and stem cell transplantation. Early clinical studies of Bortezomib-based combinations, showed encouraging results both in Follicular Lymphoma (FL) as well as in Mantle Cell Lymphomas (MCL). In this study we hypothesize that combining Bortezomib with Enzastaurin or Lenalidomide would target separate signaling pathways increasing tumor-cell death. Methods. Bortezomib, Lenalidomide and Enzastaurin alone and their combinations were tested in WSU-NHL, RL (FL cell lines) and Granta-519 and Jeko-1 (MCL cell lines) and primary cells from lymphoma patients. B-NHL cell lines were treated for 24–48 hours. The cell proliferation was determined by using the CellTiter 96® Aqueous One Solution Cell Proliferation Assay kit and cell cytotoxicity with MTT-assay. The interaction between drugs was evaluated by isobologram analysis using the STACorp 8.2 software program based upon the Chou-Talalay method to determine if the combination were additive or synergistic. Apoptosis was evaluated by flow cytometry using Annexin V/Propidium Iodide (PI) staining. The effect on cell cycle was analyzed using PI by flow cytometry. Western blotting experiments were performed to determine whether the drugs combinations affected PI3K/Akt, PKC and MAPK/ERK pathways. Results. In the present study we have shown that Enzastaurin and Lenalidomide enhanced the cytotoxicity of Bortezomib in all B-NHL cell lines and primary cells from lymphoma patients. A clear synergistic interaction, confirmed by the Chou-Talalay method (combination index

Description: Abstract 1645 Background: Indolent non follicular B-Cell Lymphomas (INFL) are an eterogenous group of lymphomas and include small lymphocityic lymphoma (SLL), lymphoplasmacytic lymphoma (LPL), and marginal zone lymphoma (MZL). Relapsed INFLs in advanced stage have a relatively poor prognosis, with low complete response to conventional chemotherapy and short survival. Lenalidomide (R®) is an immunomodulatory drug with effects on the innate immune system that may enhance antibody-dependent cell mediated cytotoxicity as well as the development of specific anti-tumor immune responses. These immunologic effects may synergize with the action of rituximab (R). To test the efficacy of R® combined with R (R2), we have conducted a multi center, open label phase II clinical trial in patients (pts) with relapsed INFL. Methods: Eligible pts must have indolent non follicular B-cell lymphoma relapsed after at least 2 but less than 4 prior lines of R-containing immuno-chemotherapy with measurable disease. Patients received oral R® 20 mg once daily on days 1–21. R is administered at a dose of 375 mg/m2 at day 14 of every course. Treatment is repeated every 28 days for up to 6 courses. The primary objectives of the study were to evaluate the antitumor activity of oral R® when given in combination with R and to assess the safety of R2 evaluated by standard criteria (CTC-NCI 3.0). The secondary objectives were progression free survival (PFS), event free survival (EFS) and duration of remission (DR). Results: From July 2010 and June 2012, 39 patients were enrolled: 19 had SLL, 11 had LPL, 4 splenic MZL, 3 extranodal MZL and a 2 nodal MZL. Median age was 68 years (51–76) and 58% were male. LDH value was increased in 17% of pts and β-2-microglobulin in 87%; 41% of pts had Hb

Description: Background Advanced-stage, relapsed indolent non follicular lymphomas (INFLs) have a relatively poor prognosis, with low complete response to conventional chemotherapy and short survival. Thus, there is a need for innovative treatment with high efficacy and a good safety profile. Lenalidomide (R®) is an immunomodulatory drug with a direct tumoricidal effect and action on T, NK and stromal cells that may enhance antibody-dependent cell mediated cytotoxicity as well as the development of specific anti-tumor immune responses. These immunologic effects may synergize with the action of rituximab (R). To test the efficacy of R® combined with R (R2), we have conducted a multi center, open label phase II clinical trial in patients (pts) with relapsed INFL. Methods Eligible pts must have indolent non follicular B-cell lymphoma relapsed after at least 2 but less than 4 prior lines of R-containing immuno-chemotherapy with measurable disease. Patients received oral R® 20 mg once daily on days 1-21. R is administered at a dose of 375 mg/m2 at day 14 of every course. Treatment is repeated every 28 days for up to 6 courses. The primary objectives of the study were to evaluate the antitumor activity of oral R® when given in combination with R and to assess the safety of R2 regimen evaluated by standard criteria (CTC-NCI 3.0). The secondary objectives were the evaluation of progression free survival (PFS) and overall survival (OS). Results From July 2010 and March 2013, 44 pts entered the protocol. Six out of 44 pts were excluded from this analysis as 2 withdrew informed consent and 4 refused to start treatment immediately after signing the informed consent. Enrolled pts (38 cases) had: 18 small lymphocytic lymphoma (SLL), 12 lymphoplasmacytic lymphoma (LPL) and 8 marginal zone lymphoma (MZL). Median age was 68 years (51-75) and 58% were male. LDH value was increased in 21% of pts and β-2-microglobulin in 75%; 51% of pts had Hb

Description: Abstract 5185 BACKGROUND. In the last 10 years retrospective studies and randomized clinical trial showed an improvement of survival in patients with Diffuse Large B Cell Lymphoma (DLBCL) and Follicular Lymphoma (FL) treated with chemotherapy regimens containing rituximab (R). However, clinical trials based data refer to a very selective subgroup of patients that does not necessarily correspond to the general population. Therefore we analysed data available from the cancer registry of the province of Modena (MCR), Italy, where R was introduced as standard treatment of DLBCL starting from 2003. Aim of this study was to evaluate if the survival benefit of R treatment observed in clinical trials in patients with DLBCL was confirmed in a study population METHODS. From 1996 to 2008 we identified 662 metachronous DLBCL (ICDO3 histology codes 9678–9680 6984/3) that were divided in two groups: 345 patients diagnosed between 1996 and 2002 (before the use of R), and 317 from 2003 to 2008 (when R was routinely used in the province of Modena). Relative survival (RS) was estimated using the Hakulinen method, with the complete cohort approach and age-adjusted by Corazziari method. The relative risks (RR) by gender and year of diagnosis (1996–2002 vs 2003–2008) was estimated assuming Poisson distribution for excess deaths. Moreover, by means of incidence based mortality approach (IBM) we estimated the overall effect of introduction of the R with the rate mortality ratio (RMR) between the cohort 2004–2007 (last follow-up 2008, N=198) and 1998–2001 (last follow-up 2002, N=200), and the effect by gender and age at diagnosis (

Description: Abstract 2651 In non-Hodgkin lymphoma (NHL), individual characteristics of the tumor cells alone are not the only parameters which determine prognosis and overall survival. Non-malignant immune cells and the tumor microenvironment also play an important contributory role in determining the eventual clinical outcome. There is an increasingly growing interest in the role of monocytes and their precursors in the pathogenesis of lymphoproliferative disorders. Recently, elevated peripheral blood monocyte counts have been shown to be an independent prognostic parameter associated with poor prognosis and decreased overall survival in patients with both non- Hodgkin lymphoma and Hodgkin. The main aim of this collaborative multicenter study was to re-evaluate and attempt to verify the prognostic significance of monocytosis (〉 800 cells/mm3) at diagnosis, in a large cohort of 1026 newly diagnosed patients with diffuse large B-cell lymphoma (DLBCL) followed at different medical centers both in Israel and Italy. Additional aims of this study were to define possible correlations with a number of other well recognized prognostic factors in DLBCL. We reviewed clinical and laboratory data of consecutive untreated patients with DLBCL, followed and eventually treated in different centers in Haifa, Israel and in Italy between 1993–2010. Median age at diagnosis was 59 years (range 18–95 yrs) and the 5-years OS after a median follow up of 51 months (range 0.2–180 months) was 69% for the entire cohort. Monocyte counts were available for 852 patients. Those with peripheral blood monocyte count (PBMC) 〈 800 mm3 had a 5 years OS of 71%, compared to 59% for those with PBMC 〉 800 mm3 (p=0.0002). In univariate analysis age, IPI score and monocyte counts were associated with a worse OS. In multivariate analysis, monocytosis retained a negative prognostic value even when adjusted for IPI (HR 1.53, CI95% 1.16–2.02, p=0.003). Of the 852 patients, 496 (58%) were treated with chemotherapy alone, while 356 patients (42%) received chemo-immunotherapy with similar regimens including rituximab. The addition of rituximab to the combination chemotherapy did not abrogate the negative adverse effect of monocytosis (p=0.0015) observed in patients receiving chemotherapy alone. This large validation study confirms earlier very recent results and shows that the monocyte level (monocytosis), a simple prognostic parameter, can easily be used routinely to evaluate newly diagnosed patients with DLBCL and identify those with a higher risk of poor survival. The addition of monocytosis as an additional parameter to the basic IPI, may possibly generate a more comprehensive score incorporating both patient, tumor and immune parameters. Disclosures: No relevant conflicts of interest to declare.

Description: Cytomegalovirus (CMV) is an important cause of morbidity and mortality in patients who have undergone severe immunosuppressive therapy. Ganciclovir continues to be the first choice for pre-emptive therapy, but it needs multiple intravenous daily administration for three weeks and may cause myelosuppression. Cidofovir is a non myelotoxic nucleotide analogue effective against CMV; its favourable pharmacokinetic profile allows a once-a-week dosing. We reviewed a database on 110 consecutive Autologous Stem Cell Transplant (ASCT) and that of 15 Chronic Lymphocytic Leukemia (CLL) patients treated with alemtuzumab. All patients were virologically monitored by quantification of pp65 antigenemia in peripheral blood. Cytomegalovirus infections were identified respectively, in 13 of 110 (12%) ASCT group and in 10 of 15 (66%) CLL group. Nine out 23 CMV reactivation showed manifestation of the infection. All patients were treated on outpatient basis. Patients with a positive pp65 assay were treated with cidofovir 5 mg/kg once-a-week for two weeks followed by one or two doses every two weeks. Twenty-three patients (13 autologus, 10 alemtuzumab) had 23 episodes of CMV-pp65 detection treated with cidofovir. The first positive antigenemia occurred after a median of 36 days from starting treatment (range 5–20) and the median antigenemia level at first appearance was 2 (range 1– 89). The treatment produced regression of symptoms in all cases and clearance of the virus in 21 (11 post-transplant 84%; 10 post alemtuzumab 100%), stained by CMV antigenemia. Median duration of therapy was 21 days (range 14–30 days) and the time to the first undetectable antigenemia was seven days (range7–28). We did not observe any further CMV reactivations, also in six of the ten patients who restarted treatment with alemtuzumab after the end of pre-emptive therapy. We did not observe any of the side effects potentially related to cidofovir administration: notably, none of the patients experienced renal toxicity, proteinuria, nausea or vomiting, ophthalmological or neurological toxicity. In our experience, pre-emptive therapy of CMV infection with cidofovir is safe and effective. In our opinion it could be considered an interesting alternative to ganciclovir for pre emptive therapy, particularly advantageous for treatment of CLL and ASCT ambulatory patients at low risk of developing CMV disease.

Description: Abstract 1801 Background: Velcade ® (V) and Mabthera ® (M) have demonstrated an individual considerable efficacy in the treatment of non Hodgkin's lymphoma. The aim of this study was to evaluate the efficacy and safety of the combination of V and M in patients with relapsed Follicular lymphoma (FL). Methods: Patients (pts) with histologic documentation of CD20+ FL, measurable and active disease, received : 1.3 mg/m2 of V on days 1–4-8-11 every 21 days for 6 cycle and M 375 mg/m2 on day 1 of each cycle from cycle III to VI. Two additional doses of M were administered alone after cycle VI, every 21 days (cycle VII and VIII). Response was assessed after 2 and 8 cycles using the NCI recommendations for Non-Hodgkin's Lymphomas. Results: At the time of current analysis, initial planned accrual of 41 evaluable pts was not completed. From 2007 to now 37 pts entered into the trial. The pts characteristics at baseline were: median age 66 years (range: 46–84), male 51%, stage IV 43 % elevated values of LDH, 27%, and of Beta2microglobulin 41%. The FLIPI score was calculated in 34 pts (92%) and 11 pts (31%) had a poor prognostic score (〉 3). The median number of previous immuno/chemotherapy regimens was 2 (range:1-3), and the median duration of last remission before registration into trial was 23 months (range: 3–67). In four out of the 37 pts who entered into the trial, the treatment is ongoing and thirty-three pts were evaluable for response. The overall response rate in the intent to treat analysis was 58% (19 pts), of which 16 pts (49%) obtained complete response (CR) and 3 (9%) partial response (PR). Stable disease was seen in 1 pt (3%). Eight pts (24%) had progressive disease and 2 (6%) pts were lost at follow-up. Three pts (9%) had to stop the treatment: one pt (3%) for grade IV peripheral neuropathy, one pt (3%) refused to continue the treatment after 2 cycle and one pt (3%) died during the treatment for toxicity . After a median follow up of 14 month (0-44), the median overall survival and the event free survival were not reached. Overall, 2 pts relapsed (10 %) and 1 pt (5 %) showed a progression of disease. A total of five pts died, four because of lymphoma progression, and one for toxicity during treatment. Complete response are ongoing in 14 pts . Toxicity was evaluable in 33 patients. We observed the following grade 1/2 adverse events: neuropathy (10 pts), neutropenia (2 pts), infection (5 pts), constipation (4 pts), rash (2 pts), fatigue (1 pt). Further we saw the following grade 3/4 adverse events: thrombocytopenia (5 pts), neuropathy (5 pts), neutropenia (1 pt) and infection with fever(1 pt). Three patient interrupted the treatment due to severe neuropathy. Conclusions: The combination of V+M is associated with acceptable toxicity and a promising percentage of response. Further follow-up is required to evaluate the response duration and survival in the whole group of patients Disclosures: Sacchi: Janssen-Cilag: Research Funding. Off Label Use: Velcade is not approved in Italy for the treatment of Follicular lymphomas. However,we have perfomed with study has I have thought that the association with Mabthera could have efficacy and low toxicity in the treatment of NHL.

Description: Abstract 5134 BACKGROUND: Bisphosphonates (BP) are standard supportive care in patients affected by symptomatic multiple myeloma (MM) with skeletal lesions. Despite the long term use of BP in the clinic, many of the effects of this category of drugs and their optimal schedule of administration are still matter of debate. In the recent past the identification of osteonecrosis of the jaw induced clinicians and researchers to reevaluate the schedule of administration of BP in myeloma, questioning about their effects on other cells than osteoclasts. Therefore a better definition of risks and benefits of anti-catabolic agents may help addressing future studies in this field. Recently a growing number of publications alerted orthopedics and endocrinologists about a rare but serious event called “atypical low energy fractures” (LEF) in patients affected by osteoporosis treated with long term BP (1–3). LEF refer to stress fractures, mainly localized in the subtrochanteric region, spontaneous or secondary to minor trauma, often preceded by local pain, with specific radiologic patterns and sometimes delayed healing. Localizations in bones different than femur have been reported. Still debated is the association with BP and the pathophysiology of this condition. So far three cases of fractures with the characteristics of LEF have been described in MM patients. METHODS: in order to evaluate the possible existence of other cases of LEF in patients with MM we started a retrospective survey in hematological centers, collecting the cases of MM followed by each center between January 2005 and December 2010, and any case of atypical fracture not related to MM or major trauma. Inclusion criteria for LEF were so defined: diagnosis of MM; treatment with BP; fractures induced by minor trauma or spontaneous, not associated with MM localization; radiological aspect of stress fracture; +/− prodromic pain. Central revision of patients history and radiology will be conducted with the support of an orthopedic in the patients with atypical fractures, followed by bone histomorphometry on bone marrow biopsy. RESULTS: The study is ongoing. At present seven centers reported a total of 1065 patients affected by MM followed between 2005 and 2010 and five cases of suspect LEF. The first patient is a woman diagnosed with anaplastic myeloma in 2002, apparently in complete remission after 4 lines of treatment. In June 2008 the patient was diagnosed with an atraumatic left fracture of the fifth metatarsal bone, followed by a right metatarsal fracture one month later and second right metatarsal stress fracture in 2011. The fractures were not related to myeloma localization. Other four cases of fractures are under investigation in other two center. CONCLUSIONS: MM patients are exposed to high rate of bone fractures related to the hematological disease and at the best of our knowledge the frequency of stress fractures in this population is unknown. With the present study, over a population of more than one thousand patients, we observed five cases of possible LEF that will undergo detailed analysis through central revision of patients history, radiology and bone histomorphometry with the aim to identify individual risk factors. Despite LEF is a rare and still controversial condition, the identification of individual risks to develop fractures not secondary to MM, may help clinicians tailoring the treatment for bone disease, much needed in an era of new drug discoveries for bone treatment. Disclosures: Palumbo: Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Merck: Honoraria; Amgen: Honoraria.