Masakazu Higuchi

Prognostic impact of immunohistochemical biomarkers in diffuse large B-cell lymphoma in the rituximab era.

We evaluated the usefulness of prognostic markers in patients with diffuse large B‐cell lymphoma (DLBCL) treated with cyclophosphamide, vincristine, doxorubicin, and prednisolone (CHOP) ± rituximab (R‐CHOP) in Japan. We studied 730 patients with DLBCL; 451 received CHOP and 279 R‐CHOP. We analyzed biopsy samples immunohistochemically for markers of germinal center B cells (CD10, Bcl‐6), postgerminal center B cells (Multiple myeloma‐1), and apoptosis (Bcl‐2). The median follow‐up period for surviving patients was 56.4 months for the CHOP group and 25.2 months for the R‐CHOP group. DLBCL were categorized as germinal center B (GCB) subtype (352/730; 48.2%) or non‐GCB subtype (378/730; 51.8%). In the CHOP group, the high expression of CD10 (P = 0.022) or Bcl‐6 (P = 0.021), or GCB subtype (P = 0.05) was associated with better overall survival, whereas the high expression of Bcl‐2 (P = 0.001) or MUM1 (P = 0.011), or non‐GCB subtype (P = 0.05) was associated with worse overall survival. In the R‐CHOP group, however, these biomarkers except Bcl‐6 were not significant prognostic factors. The patients with non‐GCB subtype showed improved survival in the R‐CHOP group (P = 0.756). The International Prognostic Index was a useful clinical marker of survival in the CHOP group (P < 0.001) and also in the R‐CHOP group (P < 0.001). Results of improved survival with rituximab addition indicate that the relevance of previously recognized prognostic factors should be re‐evaluated. (Cancer Sci 2009; 100: 1842–1847)

Monitoring of minimal residual disease (MRD) is useful to predict prognosis of adult patients with Ph-negative ALL: results of a prospective study (ALL MRD2002 Study)

BackgroundAllogeneic hematopoietic stem cell transplantation (HSCT) for patients with Philadelphia chromosome (Ph)-negative acute lymphoblastic leukemia (ALL) in first complete remission (CR1) is much more intensive than multi-agent combined chemotherapy, although allogeneic HSCT is associated with increased morbidity and mortality when compared with such chemotherapy. Minimal residual disease (MRD) status has been proven to be a strong prognostic factor for adult patients with Ph-negative ALL.MethodsWe investigated whether MRD status in adult patients with ALL is useful to decide clinical indications for allogeneic HSCT. We prospectively monitored MRD after induction and consolidation therapy in adult patients with Ph-negative ALL.ResultsOf 110 adult ALL patients enrolled between July 2002 and August 2008, 101 were eligible, including 59 Ph-negative patients. MRD status was assessed in 43 patients by the detection of major rearrangements in TCR and Ig and the presence of chimeric mRNA. Thirty-nine patients achieved CR1, and their probabilities of 3-year overall survival and disease-free survival (DFS) were 74% and 56%, respectively. Patients who were MRD-negative after induction therapy (nu2009=u200926) had a significantly better 3-year DFS compared with those who were MRD-positive (nu2009=u200913; 69% vs. 31%, pu2009=u20090.004). All of 3 patients who were MRD-positive following consolidation chemotherapy and did not undergo allogeneic HSCT, relapsed and died within 3 years after CR.ConclusionsThese results indicate that MRD monitoring is useful for determining the clinical indications for allogeneic HSCT in the treatment of ALL in CR1.

Rituximab in combination with CHOP chemotherapy for the treatment of diffuse large B cell lymphoma in Japan: a retrospective analysis of 1,057 cases from Kyushu Lymphoma Study Group

We performed a retrospective analysis of patients with diffuse large B cell lymphoma treated with rituximab plus CHOP (cyclophosphamide, adriamycin, vincristine and prednisone) as a first-line therapy at 22 hospitals of the Kyushu Lymphoma Study Group. During the period 1996–2005, 1,057 patients (aged 22–90xa0years) were analyzed. Of these, 678 were treated with CHOP, and 379 were treated with rituximab plus CHOP (R-CHOP). The complete response rate was 59.9% in the CHOP group and 67.0% in the R-CHOP group (Pxa0<xa00.001). Three-year progression-free survival (PFS) and overall survival (OS) rates were significantly higher in the R-CHOP group than in the CHOP group (61.3 vs. 45.6% for PFS, Pxa0<xa00.001; 68.3 vs. 54.5% for OS, Pxa0<xa00.001). The International Prognostic Index was a good prognostic marker for both groups; a survival benefit of rituximab addition was found for each risk subgroup and also for both age groups (≤60 and >60xa0years). Among 345 patients who received localized radiation therapy, the adding rituximab to CHOP attenuated the survival difference between CHOP and R-CHOP groups (Pxa0=xa00.104), compared with no radiation group (Pxa0<xa00.001). Results of this large-scale, multicenter study confirm that rituximab plus CHOP provided a greater survival benefit than CHOP alone.

Demonstration of reversed flow in segmental branches of the portal vein with hand-held color Doppler ultrasonography after hematopoietic stem cell transplantation

Summary:Hepatic veno-occlusive disease (VOD) is a severe complication of hematopoietic stem cell transplantation (SCT). When monitored with hand-held color Doppler ultrasonography during day −7 to +35 around SCT, reversed blood flow in the segmental branches of the portal vein was detected in nine of 56 patients who had undergone SCT. Three of nine patients had clinical evidence of VOD, but six patients did not fulfill the criteria for diagnosis of VOD initially. Two patients progressed to clinical VOD at a later date and the reversed portal flow disappeared with or without treatment for VOD in the other four patients. Monitoring for reversed portal flow with color Doppler ultrasonography may be a useful tool for the early diagnosis of VOD, and may improve prognosis by allowing early initiation of treatment.

Bortezomib, doxorubicin and intermediate-dose dexamethasone (iPAD) therapy for relapsed or refractory multiple myeloma: a multicenter phase 2 study

We previously conducted a phase 1 study of bortezomib, doxorubicin and intermediate-dose dexamethasone (iPAD) therapy and determined the optimal dose of bortezomib to be 1.0xa0mg/m2. We then conducted a multicenter phase 2 study in patients with relapsed or refractory myeloma. Bortezomib 1.0xa0mg/m2 was administered intravenously on days 1, 4, 8 and 11, in combination with intravenous doxorubicin 9xa0mg/m2 on days 1–4, and dexamethasone 20xa0mg orally on days 1–2, 4–5, 8–9 and 11–12 at a 3-week interval for six cycles. The primary endpoint of this study was the complete remission (CR) rate, and the secondary endpoints were progression-free survival (PFS), overall survival (OS) and toxicity. Twenty-seven patients, median age of 63, were enrolled. An overall response rate was 89xa0% with CR rate of 30xa0%. The median PFS time was 12.1xa0months, and the median OS time was not reached. One patient died of pneumonia. Although the incidence of hematological toxicities was high, these were transient and manageable. The most common non-hematological toxicity was sensory neuropathy; grade 3 toxicity was observed in six patients (22xa0%) and treatment was discontinued in four. We conclude that iPAD therapy is feasible, and shows efficacy by inducing high response rates and long response duration.

A phase I study of bortezomib in combination with doxorubicin and intermediate-dose dexamethasone (iPAD therapy) for relapsed or refractory multiple myeloma

Bortezomib and doxorubicin have synergistic activity against myeloma cells in vitro. We underwent a dose finding study of bortezomib in combination with a fixed dose of doxorubicin and intermediate-dose dexamethasone (iPAD therapy) in patients with relapsed or refractory myeloma. Bortezomib was administered on days 1, 4, 8 and 11 at a dose of 1.0 and 1.3xa0mg/m2 in cohorts 1 and 2, respectively. Doxorubicin 9xa0mg/m2 was given by rapid intravenous infusion on days 1–4, and dexamethasone 20xa0mg on days 1–2, 4–5, 8–9 and 11–12. Treatment was repeated at a 3-week interval and the dose-limiting toxicity (DLT), defined as grade 4 hematological toxicity lasting more than 5xa0days and/or grade 3 or higher non-hematological toxicity, was evaluated. In cohort 1, 2 of 6 patients developed DLTs including grade 4 hyponatremia and grade 3 infection with appropriate neutrophil counts. No DLT was observed in the remaining 4 patients, indicating this dose was tolerable. In cohort 2, 3 of 5 patients developed DLTs including grade 4 thrombocytopenia lasting more than 5xa0days, grade 3 hepatic transaminase elevation and grade 3 ileus, indicating this dose was intolerable. It is concluded that bortezomib at the dose of 1.0xa0mg/m2 is recommended in combination with doxorubicin and intermediate-dose dexamethasone.

A spindle cell variant of diffuse large B‐cell lymphoma is characterized by T‐cell/myofibrohistio‐rich stromal alterations: analysis of 10 cases and a review of the literature

Spindle‐shaped diffuse large B‐cell lymphoma (Sp‐DLBCL) has been recognized as a rare morphologic variant of DLBCL. However, the biological processes that contribute to the specific features of Sp‐DLBCL remain poorly understood. In this study, a combined immunophenotypic and genetic analysis was performed in 10 Sp‐DLBCL. First, we investigated several unique markers for anaplasia (CD30, ALK, CD68, and EBER‐ISH), mesenchyma (SMA, desmin, and vimentin), and B‐cell differentiation (CD10, BCL6, and MUM1). We also performed conventional cytogenetic and fluorescence in situ hybridization studies to look for common chromosomal break points (BCL2, BCL6, and MYC). We found that most Sp‐DLBCLs were germinal center B cell‐like and that none had any other specific phenotypes or any karyotypic abnormalities. Instead, T cells, CD68‐positive macrophages and SMA‐positive myofibroblasts were significantly increased in Sp‐DLBCL when compared with conventional GCB origin DLBCL cases (n = 10) (P = 0.012, P < 0.001, and P < 0.0001, respectively). To further characterize Sp‐DLBCL, we next compared the expression of fibroblast growth factor 2 (FGF2) and transforming growth factor‐β1 (TGFβ1) between the two types of DLBCL. Finally, we confirmed that the number of FGF2‐ and TGFβ1‐positive stromal cells was markedly increased in Sp‐DLBCL and that the difference between these and conventional GCB origin DLBCLs was significant (P < 0.0001 and P = 0.0017, respectively). Thus, T‐cell/myofibrohistio‐rich stromal alterations in Sp‐DLBCL, especially those mediated by TGFβ1 and FGF2, may play a role in the transition of lymphoma cells into those with spindle‐shaped features.

Reconstitution of HLA-A*2402-Restricted Cytomegalovirus-Specific T-Cells Following Stem Cell Transplantation

Cytomegalovirus (CMV)-specific immune reconstitution early after stem cell transplantation (SCT) was evaluated prospectively by detecting CD8+ T-cells, which recognize the peptide QYDPVAALF in the context of HLA-A*2402. Fifteen allogeneic SCT recipients were included in the study. All recipients and donors were seropositive for CMV and had the HLA-A*2402 allele. CMV-specific T-cells were detected as early as 1 month after transplantation, and their numbers increased to peak levels 2 to 5 months after transplantation. The numbers of CMV-specific T-cells in patients who developed grade II to IV acute graft-versus-host disease (GVHD) and received corticosteroids for acute GVHD were low in the early period after allogeneic SCT. There was a trend toward earlier reconstitution of CMV-specific CD8+ T-cells in allogeneic peripheral blood SCT (PBSCT) patients than in allogeneic bone marrow transplantation patients. The contribution of T-cells in the graft to the recovery of CMV-specific immune responses was also suggested by the finding that the reconstitution of CMV-specific CD8+ T-cells was delayed in CD34-selected autologous PBSCT compared with unpurged autologous PBSCT. The reconstitution of CMV-specific CD8+ T-cells was delayed in patients with CMV disease or recurrent CMV reactivation. These observations suggest that the detection of CMV-specific T-cells with an HLA-peptide tetramer is useful to assess immune reconstitution against CMV and to identify patients at risk for CMV disease or recurrent CMV reactivation after SCT.

Calreticulin mutation does not contribute to disease progression in essential thrombocythemia by inhibiting phagocytosis

Somatic mutations of calreticulin (CALR) have been observed in many cases of essential thrombocythemia (ET) or primary myelofibrosis that harbor non-mutated Janus kinase 2 (JAK2). CALR mainly localizes within the endoplasmic reticulum lumen, but a small fraction of the total CALR pool is distributed over the cell surface. Cell surface CALR is known to transduce prophagocytic eat me signals to macrophages and acts as one of the important regulators for macrophage engulfment. In this study, we attempted to clarify whether mutant CALR may affect the threshold for macrophage engulfment and play an integral role in the pathogenesis of CALR-mutated ET. First, we compared the surface expression levels of CALR on hematopoietic stem and progenitor cells (HSPCs) and mature blood cells in patients with myeloproliferative neoplasms and found that the surface expression of mutant CALR did not change. Next, we compared the threshold for macrophage phagocytosis of each HSPC fraction and mature blood cells and found no significant change in the efficiency of macrophage engulfment. Our data suggest that CALR mutation does not affect sensitivity to phagocytosis by macrophages. Finally, we analyzed the phosphorylation statuses of molecules downstream of JAK2 at each HSPC level in patients with ET and found that CALR mutations activated the JAK-STAT pathway in a manner similar to that associated with JAK2 mutations. These results indicate that mutant CALR causes myeloproliferation because of the activation of JAK-STAT pathway and not by the inhibition of phagocytosis, which is similar to the myeloproliferation caused by JAK2 V617F mutation.

Factors prognostic of eligibility for allogeneic HCT among older patients with AML-CR1 and adverse- or intermediate-risk cytogenetics

The introduction of reduced-intensity conditioning (RIC) regimens has made possible allogeneic hematopoietic cell transplantation (allo-HCT) in older patients with acute myeloid leukemia (AML). However, the optimal timing of allo-HCT in these patients and its relative risks and benefits when compared with chemotherapies have not been determined. This retrospective study by the Fukuoka Blood and Marrow Transplant Group compared RIC allo-HSCT with non-transplant therapies, the choice based on donor availability, in AML patients in their first complete remission (CR1). The prognostic value of various patient characteristics and disease-specific variables were investigated in 299 patients aged ≥60xa0years with AML in CR1. Among the 107 patients aged 60–65xa0years, 54 of whom received allo-HCT and 53 of whom continued chemotherapies; allo-HCT, adverse-risk group, and hematopoietic cell transplantation-comorbidity index were significant predictors of survival outcomes. Among 192 patients aged ≥66xa0years deemed ineligible for allo-HCT, relapse and Karnofsky performance status after induction therapy were significant predictors of survival outcomes. Findings from this study may facilitate a new standard of care for older AML patients in CR1 who are considered candidates for allo-HCT.