Masaru Nakagawa

Induced Differentiation of Human Myeloid Leukemia Cells into M2 Macrophages by Combined Treatment with Retinoic Acid and 1α,25-Dihydroxyvitamin D3

Retinoids and 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) induce differentiation of myeloid leukemia cells into granulocyte and macrophage lineages, respectively. All-trans retinoic acid (ATRA), which is effective in the treatment of acute promyelocytic leukemia, can induce differentiation of other types of myeloid leukemia cells, and combined treatment with retinoid and 1,25(OH)2D3 effectively enhances the differentiation of leukemia cells into macrophage-like cells. Recent work has classified macrophages into M1 and M2 types. In this study, we investigated the effect of combined treatment with retinoid and 1,25(OH)2D3 on differentiation of myeloid leukemia THP-1 and HL60 cells. 9-cis Retinoic acid (9cRA) plus 1,25(OH)2D3 inhibited proliferation of THP-1 and HL60 cells and increased myeloid differentiation markers including nitroblue tetrazolium reducing activity and expression of CD14 and CD11b. ATRA and the synthetic retinoic acid receptor agonist Am80 exhibited similar effects in combination with 1,25(OH)2D3 but less effectively than 9cRA, while the retinoid X receptor agonist HX630 was not effective. 9cRA plus 1,25(OH)2D3 effectively increased expression of M2 macrophage marker genes, such as CD163, ARG1 and IL10, increased surface CD163 expression, and induced interleukin-10 secretion in myeloid leukemia cells, while 9cRA alone had weaker effects on these phenotypes and 1,25(OH)2D3 was not effective. Taken together, our results demonstrate selective induction of M2 macrophage markers in human myeloid leukemia cells by combined treatment with 9cRA and 1,25(OH)2D3.

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

Abstract 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.

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.

Abstract 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.

Negative impact of concurrent overexpression of MYC and BCL2 in patients with advanced diffuse large B-cell lymphoma treated with dose-intensified immunochemotherapy.

Abstract Co-expression of MYC and BCL2 proteins in diffuse large B-cell lymphoma (DLBCL), or ‘double-expressor lymphoma’ (DEL), results in poor patient prognosis, but the significance of DEL when aggressive treatments are applied remains uncertain. We performed a retrospective analysis of 40 patients with de novo DLBCL, who were categorized as being at high/high-intermediate risk according to the age-adjusted International Prognostic Index. Patients underwent an R-Double-CHOP regimen, a dose-intensified immunochemotherapy with or without consolidative high-dose chemotherapy followed by autologous stem cell transplantation. According to immunohistochemical analysis, 10 (25%) patients were categorized as having DEL, showing positivity for MYC (≥40%) and BCL2 (≥50%). The 3 year progression-free survival and overall survival of the DEL group were significantly worse compared with those of the non-DEL group (30% vs. 63%, p = 0.019 and 40% vs. 82%, p = 0.006, respectively). These results suggest that advanced DEL may need discrete treatment strategies.

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.

Combined treatment with benzo[ a ]pyrene and 1α,25-dihydroxyvitamin D 3 induces expression of plasminogen activator inhibitor 1 in monocyte/macrophage-derived cells

ABSTRACT Benzo[a]pyrene (BaP) is an environmental pollutant found in cigarette smoke and is implicated as a causative agent of tobacco‐related diseases, such as arteriosclerosis. In contrast, vitamin D signaling, which is principally mediated by conversion of vitamin D to the active form, 1&agr;,25‐dihydroxyvitamin D3 [1,25(OH)2D3], decreases cardiovascular disease risk. However, combined treatment with BaP and 1,25(OH)2D3 enhances BaP toxicity, including BaP‐DNA adduct formation. We further investigated the cross‐talk between BaP and 1,25(OH)2D3 signaling pathways, and found that combined treatment with these compounds induces mRNA and protein expression of plasminogen activator inhibitor 1 (PAI‐1) in monocyte/macrophage‐derived THP‐1 and U937 cells. Protein synthesis inhibitor treatment did not inhibit induction of the PAI‐1 gene (SERPINE1) in these cells. BaP plus 1,25(OH)2D3 induced differentiation markers, inhibited cellular proliferation, and induced apoptosis and oxidative stress in these cells. Reactive oxygen species scavenger treatment suppressed apoptosis but not SERPINE1 induction in cells treated with BaP plus 1,25(OH)2D3. Thus, combined treatment with BaP and 1,25(OH)2D3 induced SERPINE1 mRNA expression in these cells through a mechanism that does not require de novo protein synthesis or reactive oxygen species production. These findings suggest that induction of the proinflammatory factor PAI‐1 plays a role in BaP toxicity. Interestingly, PAI‐1 knockdown decreased expression of the cell surface antigen CD14, a monocytic differentiation marker, in THP‐1 cells treated with BaP plus 1,25(OH)2D3. PAI‐1 induction may also be related to a function of monocytes/macrophages in response to xenobiotic and vitamin D signaling. HIGHLIGHTSCombined benzo[a]pyrene (BaP) and 1,25(OH)2D3 treatment induces PAI‐1 in macrophages.BaP plus 1,25(OH)2D3 induces differentiation and apoptotic markers.Apoptosis by BaP plus 1,25(OH)2D3 is suppressed by a ROS scavenger.PAI‐1 knockdown suppresses differentiation induced by BaP plus 1,25(OH)2D3.PAI‐1 in macrophages may be related to cellular function or BaP‐induced diseases.

Enhanced perforin expression associated with dasatinib therapy in natural killer cells

We investigated the effects of dasatinib on natural killer (NK) cell-induced signaling protein and perforin expression as well as plasma cytokine levels by analyzing blood samples from patients with well-controlled chronic myeloid leukemia receiving tyrosine kinase inhibitor (TKI) therapy. Perforin expression and phosphorylation of signal transducer and activator of transcription (STAT) 1, STAT3, Janus kinase (JAK) 1, and JAK2 in NK cells were evaluated by flow cytometry, and the levels of plasma cytokines, including interferon (IFN)-γ and interleukin (IL)-2, were determined by enzyme-linked immunosorbent assays in 40 patients (dasatinib, n = 23; imatinib, n = 11; and nilotinib, n = 6). Perforin levels in NK cells were higher in dasatinib-treated patients before TKI treatment; phospho (p)-STAT1 levels were closely correlated with pJAK1 and perforin levels, and pSTAT3 levels were correlated with pJAK2 and perforin levels. The correlation between pJAK1 and pSTAT1 was apparent in dasatinib-treated patients but not in other TKI-treated patients, and the correlation between pJAK2 and pSTAT3 was apparent in patients treated with other TKIs. Constitutive expression of IFN-γ was higher in patients treated with dasatinib or with other TKIs than in those who were in treatment-free remission (TFR). In contrast, constitutive expression of IL-2 was lower in patients treated with other TKIs than in those treated with dasatinib or in those who were in TFR. These results provided insights into the effects of dasatinib on JAK/STAT signaling in NK cells in vivo and the mechanisms underlying NK cell activation induced by dasatinib therapy.

Tamibarotene maintenance improved relapse-free survival of acute promyelocytic leukemia: a final result of prospective, randomized, JALSG-APL204 study

Between April 2004 and December 2010, we conducted a prospective randomized controlled study comparing tamibarotene with all-trans retinoic acid (ATRA) in the maintenance therapy of newly diagnosed acute promyelocytic leukemia (APL), and here report the final results of this study with a median follow-up of 7.3 years. Of 344 eligible patients who had received ATRA and chemotherapy, 319 (93%) achieved complete remission (CR). After completion of three courses of consolidation chemotherapy, 269 patients in molecular remission underwent maintenance randomization, 135 to ATRA (45 mg/m2 daily), and 134 to tamibarotene (6 mg/m2 daily) for 14 days every 3 months for 2 years. The primary endpoint was relapse-free survival (RFS). The 7-year RFS was 84% in the ATRA arm and 93% in the tamibarotene arm (p = 0.027, HR = 0.44, 95% CI, 0.21 to 0.93). The difference was prominent in high-risk patients with initial leukocytes ≥ 10.0 × 109/L (62% vs. 89%; p = 0.034). Tamibarotene was significantly superior to ATRA by decreasing relapse in high-risk patients. Overall survival after randomization did not differ (96% vs. 97%; p = 0.520). Secondary hematopoietic disorders developed in nine patients, secondary malignancies in 11, and grade 3 or more late cardiac comorbidities in three. These late complications did not differ between the two arms.

Clinical effect of immunophenotyping on the prognosis of multiple myeloma patients treated with bortezomib

In the present study, the effect of immunophenotyping on the prognoses of patients with multiple myeloma (MM) treated with bortezomib plus dexamethasone was investigated. The study involved 46 patients with MM, and analyzed the prognostic significance of the expression of cluster of differentiation (CD)45, CD56 and mature plasma cell (MPC)-1, and other factors including the International Staging System (ISS) stage, age, gender, the immunoglobulin subtype and the treatment line number prior to bortezomib treatment. Although CD56 and MPC-1 expression did not appear to affect the time to next treatment (TNT) or overall survival rate (OS), the univariate analysis determined that CD45 positivity was an adverse prognostic factor for TNT and OS, and that being male was significantly associated with inferior TNT and OS. Multivariate analyses determined that CD45 expression was prognostically significant for TNT and OS. In conclusion, CD45 positivity is an adverse prognostic factor in MM patients treated with bortezomib. The data from the present study demonstrate the clinical importance of classifying MM cells immunophenotypically to determine the prognoses of patients.