Clinical significance of co-expression of MYC and BCL2 protein in aggressive B-cell lymphomas treated with a second line immunochemotherapy.

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.

Dose-intensified CHOP with rituximab (R-Double-CHOP) followed by consolidation high-dose chemotherapies for patients with advanced diffuse large B-cell lymphoma.

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.

Involvement of calpain-7 in epidermal growth factor receptor degradation via the endosomal sorting pathway.

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 (CAPN7(C290S) ) 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)-CAPN7(C290S) ] was mobilized to EGFR-positive endosomes upon epidermal growth factor stimulation in HeLa cells. Although mGFP-CAPN7(C290S) exhibited dominant-negative effects on EGFR degradation, a deletion mutant of MIT domains in mGFP-CAPN7(C290S) 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-CAPN7(C290S) . When IST1 was knocked down, mGFP-CAPN7(C290S) 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).

Safety and efficacy of high-dose cyclophosphamide, etoposide and ranimustine regimen followed by autologous peripheral blood stem cell transplant for patients with diffuse large B-cell lymphoma.

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.

Analysis of limited proteolytic activity of calpain-7 using non-physiological substrates in mammalian cells.

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.

Efficacy of a dose-intensified CHOP (Double-CHOP) regimen for peripheral T-cell lymphomas.

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, has 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. 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 median age of patients 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 the CR patients, 10 successfully tolerated a consolidated high-dose chemotherapy followed by ASCT and 7 received HDMTX. A single case of treatment-related mortality was recorded during the study. On a median 31-month follow-up, the estimated 3- or 5-year overall survival (OS) rates were 68 or 63%, respectively, while 3- or 5-year relapse-free survival (RFS) rates after CR were 60 or 43%, respectively. Although this study included elderly and excluded low-risk IPI and ALK+-ALCL patients, OS results were superiorly favourable, indicating the efficacy of this Double-CHOP regimen. However, an effective treatment strategy for refractory or relapsing patients needs to be validated and established.

Long-term follow-up of localized, primary gastric diffuse large B-cell lymphoma treated with rituximab and CHOP.

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.

An effective salvage treatment using ifosfamide, etoposide, cytarabine, dexamethasone, and rituximab (R-IVAD) for patients with relapsed or refractory aggressive B-cell lymphoma.

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.