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The clinical significance of concurrent expression of MYC and BCL2 protein, known as "double-expressor lymphoma" (DEL), among patients with relapsed or refractory aggressive B-cell lymphomas, remains unclear. A retrospective analysis was performed of 38 patients treated with a salvage treatment consisting of rituximab, ifosfamide, etoposide, cytarabine and dexamethasone followed by consolidative high-dose chemotherapies. A total of 17 cases (45%) were categorized as DEL using immunohistochemical assay with a cut-off value of positivity of 40% for MYC and 50% for BCL2, respectively. DEL was associated with a lower overall response rate (35% vs 71%, p = 0.0481), worse 2-year progression-free survival (9% vs 67%, p = 0.001) and overall survival (35% vs 71%, p = 0.037). This analysis suggests that DEL is common among patients with relapsed/refractory aggressive B-cell lymphomas and that such patients require novel treatment strategies.

We retrospectively evaluated the safety and efficacy of high-dose chemotherapy consisting of cyclophosphamide, etoposide and ranimustine (CEM) with autologous peripheral blood stem cell transplant (PBSCT) in 55 adult patients with relapsed or high-risk de novo diffuse large B-cell lymphoma (DLBCL) or DLBCL associated with follicular lymphoma. This included 36 patients in the upfront setting in their first complete remission. The median follow-up of 42 patients surviving at the time of the analysis was 52 months (range 1-159). Relapse or disease progression after PBSCT was a frequent cause of death, but no therapy-related mortality associated with PBSCT was observed. The 5-year overall survival and progression-free survival were 70.6% (95% confidence interval [CI], 54.0-82.1) and 57.0% (95% CI, 39.5-71.2), respectively. Chronic renal impairment, therapy-related myelodysplastic syndrome and prostate cancer were the major late complications. The CEM regimen is a tolerable, effective conditioning regimen for autologous PBSCT for DLBCL, with no therapy-related mortality observed.

Even after the advent of rituximab, clinical outcomes of conventional immuno-chemotherapy for high-risk diffuse large B-cell lymphoma (DLBCL) remain unsatisfactory. We retrospectively evaluated the efficacy and safety of R-Double-CHOP (R-D-CHOP), consisting of rituximab (375 mg/m(2), day -2), cyclophosphamide (750 mg/m(2), day 1, 2), doxorubicin (50 mg/m(2), day 1, 2), vincristine [1.4 mg/m(2) (maximum 2.0 mg/body), day 1], and prednisolone (50 mg/m(2), day 1-5), followed by consolidation high-dose chemotherapy. This treatment was given to 51 de novo DLBCL patients with a median age of 54 (range 19-65), who were categorized as high/high-intermediate risk by the age-adjusted International Prognostic Index. Treatment was given every 3 weeks up to three courses. The overall response and the complete response rate for R-D-CHOP were 94 and 78 %, respectively. A total of 30 responders proceeded to high-dose chemotherapy followed by autologous stem cell transplantation (HDC/ASCT), whereas 16 received high-dose methotrexate (HD-MTX) alternatively. The 3-year overall survival and the event-free survival for all patients were 78 and 61 %, respectively. Major adverse events included hematological toxicities, but there were no treatment-related deaths during the observation period. We conclude that the R-D-CHOP regimen followed by HDC/ASCT or HD-MTX is a promising treatment option for younger patients with highly advanced DLBCL.

Calpain-7 (CAPN7) is a unique intracellular cysteine protease that has a tandem repeat of microtubule interacting and trafficking (MIT) domains and lacks a penta-EF-hand domain. Although the MIT domains of CAPN7 were previously shown to interact with a subset of endosomal sorting complex required for transport (ESCRT)-III and ESCRT-III-related proteins, including charged multivesicular body protein 1 and increased sodium tolerance (IST)1, knowledge of the involvement of the protease in membrane trafficking has been limited. In the present study, compared with control cells, we found that epidermal growth factor receptor (EGFR) degradation was mildly delayed in CAPN7-knockdown HeLa cells and mouse embryonic fibroblast cells established from CAPN7 knockout (Capn7−/−) mice. Re-expression of wild-type CAPN7 but not a protease-inactive mutant of CAPN7 (CAPN7C290S) resulted in a recovery of the rate of EGFR degradation. We found, by immunofluorescence microscopic analysis, that monomeric GFP fused with the protease-inactive mutant of CAPN7 [monomeric green fluorescent protein (mGFP)-CAPN7C290S] was mobilized to EGFR-positive endosomes upon epidermal growth factor stimulation in HeLa cells. Although mGFP-CAPN7C290S exhibited dominant-negative effects on EGFR degradation, a deletion mutant of MIT domains in mGFP-CAPN7C290S did not have such properties, suggesting that the interaction between the MIT domains and ESCRT proteins is important for the function of CAPN7. Moreover, we found that epidermal growth factor stimulation induces translocation of IST1 from the cytosol to endosomes positive in both EGFR and mGFP-CAPN7C290S. When IST1 was knocked down, mGFP-CAPN7C290S lost its co-localization with EGFR. These results demonstrate for the first time that the proteolytic activity of CAPN7 is important for the acceleration of EGFR degradation via the endosomal sorting pathway utilizing a part of the ESCRT system. Structured digital abstract EGFR and CAPN7 colocalize by fluorescence microscopy (View interaction) EGFR, CAPN7 and IST1 colocalize by fluorescence microscopy (View interaction) EEA1 and CAPN7 colocalize by fluorescence microscopy (View interaction) CAPN7 and LAMP1 colocalize by fluorescence microscopy (View interaction)

Calpain-7 is a mammalian ortholog of a fungal non-classical calpain named PalB, which is an intracellular cysteine protease and functions in fungal alkaline adaptation in association with the endosomal sorting complex required for transport (ESCRT) system. Despite our previous finding [Osako Y et al. (2010) FEBS J 277, 4412–4426] of autolytic activity, neither physiological nor non-physiological substrates of calpain-7 have yet been identified, and experimentally useful substrates that show robust evidence of intermolecular proteolytic activity of calpain-7 are required. In this study, we found limited proteolysis of C-terminally truncated ALG-2-interacting protein X (ALIX; (ALIXΔC), but not full-length ALIX, when the mutant was co-over-expressed with calpain-7 in HEK293T cells and analyzed by western blotting. The extent of ALIXΔC cleavage by calpain-7 was enhanced by co-expression with several ESCRT proteins. We investigated whether fusion of casein, a commonly used substrate for a variety of proteases including calpains, to the Bro1 domain confers the ability to serve as a substrate of calpain-7, but no specific cleavage was observed. However, when domain 1 of calpastatin, an endogenous inhibitory protein of ubiquitous classical calpains, was fused with the Bro1 domain, the fusion protein was cleaved at the C-terminal border of subdomain B (an inhibitory center for calpains) of calpastatin. These results demonstrate for the first time that calpain-7 has limited proteolytic activity and substrate preference. Moreover, the proteolytic assay system developed enabled us to perform mutational analysis of calpain-7, which revealed the importance of not only the N-terminal microtubule-interacting and trafficking (MIT) domains but also the C-terminal C2 domain-like domains for proteolytic activity.

Abstract 4855 Background: Peripheral T-cell lymphomas (PTCLs) are a rare and heterogeneous group of non-Hodgkin lymphomas, often resulting in poor prognoses. The CHOP chemotherapy regimen, which includes cyclophosphamide, doxorubicin, vincristine, and prednisone, had been used previously to treat other types of lymphomas. Here, we examined the efficacy and safety of a dose-intensified CHOP regimen (Double-CHOP), which was followed by autologous stem-cell transplantation (ASCT) or high-dose methotrexate (HDMTX), in PTCL patients. Patients and Methods: Twenty-eight PTCL patients, who received 3 courses of Double-CHOP at our institution, were retrospectively studied from 1996 to 2012. Patients with anaplastic lymphoma-kinase-positive anaplastic large-cell lymphoma (ALK+–ALCL) were excluded from this study. The Double-CHOP regimen consisted of 3 courses of intravenous (iv) administration of cyclophosphamide (750 mg/m2, days 1–2, over 2 h), doxorubicin (50 mg/m2, days 1–2, over 30 min), vincristine (1.4 mg/m2, day 1, max 2 mg/Kg body weight), and per os (p.o.) prednisone (50 mg/m2, days 1–5). For patients aged >60, cyclophosphamide and doxorubicin doses were modified. The third cycle of Double-CHOP regimen was used for stem cell mobilization. Consolidating high-dose therapy (HDT) regimen consisted of cyclophosphamide (60 mg/kg, day 6 and 7, iv, over 3 h), etoposide (500 mg/m2; day 4, 5, and 6; iv; over 6–8 h), and ranimustine (250 mg/m2, day 2 and 3, iv, over 1 h). ASCT was performed on day 0 and G-CSF administered from day 1 until neutrophil engraftment. As an alternative to HDT/ASCT, HDMTX (8 g/m2, day 1, iv, over 4 h) was indicated for patients who could not yield a sufficient number of stem cells or were ineligible for HDT/ASCT. Results: Patients' median age was 58 years (range: 17–69). They had low-intermediate (n = 11), high-intermediate (n = 10), or high (n = 7) risk according to the International Prognostic Index (IPI). The overall complete remission (CR) rate following Double-CHOP treatment was 68%. Of all the CR patients, 10 could successfully tolerate a consolidated HDT followed by ASCT, and 7 received HDMTX. Only a single case of treatment-related mortality was recorded during the study. On a median 31-month follow-up, the estimated 3-year overall survival (OS) rate and 3-year relapse-free survival (RFS) rate after CR were 68% and 60%, respectively. Conclusion: Although this study included elderly and excluded low-risk IPI and ALK+–ALCL patients, both RFS and OS results were superiorly favorable, indicating the efficacy of this Double-CHOP regimen and Double-CHOP followed by ASCT/HDMTX consolidations is safe and may achieve prolonged EFS, especially in patients with poor prognostic factors. However, an effective treatment strategy for refractory or relapsing patients needs to be validated and established. Disclosures: No relevant conflicts of interest to declare.

The addition of rituximab to cyclophosphamide, doxorubicin, vincristine and prednisone [CHOP (i.e., R-CHOP)] is considered to be the standard regimen for treating localized, primary gastric diffuse large B-cell lymphoma (PG-DLBCL). However, few studies have reported the long-term efficacy of R-CHOP therapy in the management of localized PG-DLBCL. In the present study, we performed a retrospective analysis of 11 patients with localized PG-DLBCL, who were treated with R-CHOP at Nihon University Itabashi Hospital and Kasukabe Municipal Hospital (Japan) from 2001 to 2008. Limited stage cancer was defined as stage I/II according to the Lugano staging system for gastrointestinal (GI) lymphomas. The relative dose intensity (RDI) of CHOP therapy was calculated for each patient. The median age of the patients was 68 years (range, 48–82). Gastralgia and anemia were common symptoms at initial presentation. All patients except 1 received 6 cycles of R-CHOP treatment without consolidative radiation therapy or prior surgery. RDI was maintained at over 80% in 9 out of 11 patients. All patients achieved complete remission and the estimated overall survival with a median follow-up of 54 months (range, 39–103) was 100%, without relapse or significant GI adverse effects, such as perforation or bleeding during R-CHOP treatment. No long-term adverse effects of rituximab were recorded during the observation period. Helicobacter pylori infection was diagnosed in 72.7% (8 cases) of the patients, but was eradicated in a limited number of patients. Our data suggest the feasibility and effectiveness of the addition of rituximab to conventional CHOP therapy in the management of localized PG-DLBCL.

We evaluated the efficacy and toxicity of a new salvage regimen, consisting of rituximab (375 mg/m(2), day 1), ifosfamide (1500 mg/m(2) on days 3-7), etoposide (150 mg/m(2), days 3-5), cytarabine (100 mg/m(2), days 3-5) and dexamethasone (40 mg/body, days 3-5) (R-IVAD) for relapsed or refractory aggressive B-cell lymphoma. In this study, a total of 32 patients with a median age of 64 years (range 38-79) who received an average of 2.6 cycles of R-IVAD from 2001 to 2009 in our institution were retrospectively analyzed. R-IVAD was given every 3 weeks up to a total of three courses with support by granulocyte colony stimulating factor. The overall response rate was 72%, with 56% complete response. On a median follow-up of 16 months (range 2-99), estimated 2-year overall survival (OS) and event-free survival were 55% and 36%, respectively. Of these patients, 10 successfully proceeded to consolidating high-dose chemotherapy followed by autologous stem cell transplantation, accounting for 90% of the 2-year OS. No treatment-related mortality was observed during the investigation. We, therefore, conclude that R-IVAD regimen is a safe and efficacious alternative for patients with relapsed or refractory aggressive B-cell lymphoma.

Introduction Patients with hematological malignancies are at high-risk for severe infections with drug-resistant gram-positive bacteria, as typified by methicillin-resistant staphylococcus aureus (MRSA). Vancomycin (VCM) is widely used as empirical therapy for these high-risk infections, but the global decline in their susceptibilities to VCM is at issue. Arbekacin sulfate (ABK), a unique aminoglycoside with anti-MRSA activity, has not yet been evaluated in this setting. Patients and methods This is a phase 4 study to evaluate the efficacy and safety of ABK for adult patients with hematological malignancies complicated with infections including febrile neutropenia, who are at high-risk for MRSA. All participant provided written informed consent and this study was approved by the institutional review board of the hospital. ABK was administered intravenously at a dose of 100–400mg every 24–48 hours based on the patients’ body weight and the renal function. Therapeutic drug monitoring (TDM) using Habekacin TDM software (Meiji Seika Pharma, Tokyo, Japan) was performed 48–96 hours after the initiation of ABK. The clinical response was rated as “effective” if the symptoms of infection disappeared or significantly improved, taking into account the vital signs, the serum C-reactive protein (CRP) concentration, and other clinical parameters at baseline and after completion/discontinuation of the treatment. Results From December 2010 to June 2012, 35 eligible patients were treated with ABK up to 4 times with intervals of at least 2 weeks. Consequently, a total of 54 febrile or infectious episodes were registered. The median age, serum creatinine concentration, absolute neutrophil count, axillary temperature, and serum CRP concentration for all cases at baseline were 59 (range 34–77) years, 0.64 (range 0.34–1.31) mg/dl, 0.2 X 109/L (range 0–15.1), 38.4°C (range 37.2–40.5), and 8.2 mg/dl (range 0.7–40.6), respectively. Primary diseases consisted of 34 cases of acute myeloid leukemia, 12 cases of relapsed/advanced non-Hodgkin lymphoma, 3 cases of acute lymphoblastic lymphoma/leukemia, 3 cases of myelodysplastic syndrome/aplastic anemia, and 2 cases of multiple myeloma. Diagnosis of infections consisted of 26 cases of febrile neutropenia, 18 cases of septicemia (methicillin-resistant coagulase negative staphylococci in most cases), 7 cases of pneumonia (MRSA pneumonia in one case), 2 cases of cellulitis, and 1 case of neutropenic colitis. Fifty-two cases (96%) had already received oral prophylaxis using fluoroquinolones and/or intravenous therapy with broad-spectrum antibacterials, and 47 (87%) had received cytotoxic chemotherapies. ABK was initiated with a median of 2 days (range 0–14) after the onset of fever/infection, concomitantly with a variety of broad-spectrum beta-lactams. Overall, 43 cases (80%) attained afebrile states with the absence of symptoms and rated as “effective” at the end of treatment with a median of 10 days (range 4–35). All grade renal toxicity was observed in 6 cases (11%), but they were generally mild and reversible. There were 3 deaths within 30 days of the treatment, but all of them were associated with the progression of the primary disease. Conclusion This study demonstrated for the first time the high efficacy of ABK for persistent or high-risk infections in patients with hematological malignancies. Further evaluations­—e.g. randomized controlled trials comparing ABK with VCM for these populations—are therefore warranted. Disclosures Miura: Meiji Seika Pharma: Consultancy, Research Funding. Iriyama:Meiji Seika Pharma: Research Funding. Kobayashi:Meiji Seika Pharma: Research Funding. Hatta:Meiji Seika Pharma: Research Funding. Takei:Meiji Seika Pharma: Research Funding.

Abstract 3121 Background We evaluated the safety and efficacy of the CEM regimen containing ranimustine (MCNU) with autologous peripheral blood stem cell transplantation (PBSCT) in adult patients with relapsed or high-risk de novo DLBCL or DLBCL associated with follicular lymphoma (FL/DLBCL) in our institution. Patients and methods We retrospectively analyzed 55 consecutive patients who underwent autologous PBSCT following the CEM regimen between March 1999 and June 2011 for the treatment of relapsed or high-risk DLBCL and FL/DLBCL. High-risk DLBCL was defined as partial or no response to initial treatment or high-intermediate/high risk disease according to international prognostic index (IPI) at initial diagnosis. Age-adjusted IPI was used for patients under 60. The CEM regimen consisted of CY (60 mg/kg on days -7 and -6), etoposide (500 mg/m2 on days -6 to -4) and MCNU (250 mg/m2on day -3 and -2), followed by PBSCT. Results Median age at transplant was 51 years (range, 23–66), and median time from diagnosis to transplant was 5 months (range, 3–241). Of them, 7 patients received radiation therapy before transplant, and 11 had no history of rituximab use before transplant. The 36 patients in CR1 at transplant were with high or high-intermediate risk according to international prognostic index at diagnosis. Other 15 patients in CR2, and 4 patients who were not in CR1/2 at transplant were included. CY dose was reduced in five patients by physicians choice. The median number of infused CD34-positive cells was 3.98 × 106/kg (range, 1.36–26.87). The median post-transplant days of neutrophil recovery and platelet were 11 days (range, 9–20) and 12 days (range, 7–53), respectively. Common grade 3/4 non-hematological treatment-related toxicities within the first 100 days after transplant were nausea (24%), vomit (4%), stomatitis (15%), and diarrhea (9%). Infections included febrile neutropenia (85%), sepsis (15%) which contained catheter-related bacteremia (n=4) and pneumonia (n=2). Other less common severe toxicities were acute renal impairment (n=3), and pleural effusion (n=1). No sever cardiac, neurologic toxicity, or veno-occlusive disease of the liver was observed in any of the patients. Late-onset adverse effects during follow-up period includes chronic renal impairment (n= 5), therapy-related myelodysplastic syndrome (refractory anemia) (n=1, 120 months), and prostate cancer (n=1, 83 months). Serious late cardiac or pulmonary impairment was not diagnosed in this cohort. Median follow-up duration of 42 patients surviving at the time of the analysis was 52 months (range, 1–159). Relapse or disease progression after PBSCT was documented in 21 cases (range, 0–81 months after PBSCT). No therapy related mortality (TRM) associated with PBSCT was observed, and all the 13 deaths were due to disease relapse/progression. The 5-year estimated overall survival (OS) and progression-free survival (PFS) were 70.6% (95% CI, 54.0–82.1)% and 57.0% (95% CI, 39.5–71.2)% for all patients, respectively. In a univariate analysis for factors affecting OS and PFS, no significant factors associated with unfavorable OS and PFS was found including age at transplant, gender, history of rituximab use, histology, disease status at transplant, and previous BM involvement. Conclusion We conclude that the CEM regimen using MCNU would be a tolerable, effective conditioning regimen of autologous PBSCT for high-risk or relapsed de novo DLBCL or FL/DLBCL. Although no TRM was observed, relapse was the major cause of treatment failure. Late-onset complications in long-term survivors can occur, and should be carefully monitored. Disclosures: No relevant conflicts of interest to declare.

Abstract 1039 Background: The association between increase in body mass index (BMI) and development of acute promyelocytic leukemia (APL) has been reported. Interaction of APL cells with bone marrow adipocytes in vitro induces phosphorylation of STAT3 and MAPK, resulting in an anti-apoptotic effect in APL cells. However, the influence of obesity on the prognosis of APL is not well understood. Materials and Methods: To evaluate the effect of obesity on the prognosis of adult APL, we retrospectively analyzed 62 patients under the age of 65 with newly diagnosed APL. Patients who could not be administered intensified chemotherapy, because of either poor performance status or adverse events of chemotherapy, were excluded. Results: Median age of patients was 40 years (range, 14–63). Twenty-five were male and 37 were female. Median follow-up period was 646 days (range, 3–12,558). Median BMI was 22.7 (range, 16.0–33.0). Twenty-six patients diagnosed with APL between 1970 and 1992 had been treated with chemotherapy alone (chemotherapy group), and 36 patients diagnosed between 1995 and 2010 had been treated with all-trans retinoic acid (ATRA) ± chemotherapy (ATRA group). In both groups, the outcome was compared between high-BMI (BMI ≥ 24) and low-BMI (BMI < 24) patients. Eight out of 26 in the chemotherapy group and 16 out of 36 in the ATRA group were high-BMI patients. In the chemotherapy group, complete remission (CR) was obtained in 13 out of 18 (72.2%) low-BMI patients and in only 3 out of 8 (37.5%) high-BMI patients. By day 498, all 3 CR patients with high BMI had relapsed, whereas only 6 out of 13 (46.2%) low-BMI CR patients had relapsed. The 2-year relapse-free survival (RFS) rate, event-free survival (EFS) rate, and overall survival (OS) rate in high-BMI versus low-BMI patients were 0% v 50.5%, 0% v 61.1%, and 12.5% v 42.8%, respectively. Although the relapsed patients were salvaged with ATRA, arsenic trioxide, or allogeneic bone marrow transplantation, OS was significantly worse in high-BMI patients. On the contrary, in the ATRA group, prognosis between high-BMI and low-BMI patients was not statistically different. Rates of CR, relapse, 2-year RFS, EFS, and OS in high-BMI versus low-BMI patients were 87.5% v 94.7%, 35.7% v 38.9%, 73.3% v 46.2%, 55.0% v 52.5%, and 87.5% v 81.1%, respectively. The initial white blood cell (WBC) and platelet counts have been previously reported to be prognostic factors in APL; however, they were not associated with BMI in our study. A WBC count of >10 × 109/L and a platelet count of ≤40 × 109/L were observed in 4 (10.5%) and 28 (73.8%) out of 38 low-BMI, and 4 (16.5%) and 19 (79.2%) out of 24 high-BMI patients, respectively. Conclusions: Our results demonstrate that obesity at diagnosis is a poor prognostic factor in APL, especially in patients not administered ATRA. ATRA overcomes the anti-apoptotic effect of adipocytes for APL cells. Disclosures: No relevant conflicts of interest to declare.