Toshiki Uchida

Multicenter Phase II Study of Bendamustine Plus Rituximab in Patients With Relapsed or Refractory Diffuse Large B-Cell Lymphoma

PURPOSE Effective and less aggressive therapies are required for patients with relapsed or refractory diffuse large B-cell lymphoma (DLBCL) who are not eligible for or have undergone autologous stem-cell transplantation (ASCT). The present phase II study assessed the efficacy and safety of bendamustine plus rituximab (BR) in this population. PATIENTS AND METHODS Patients with relapsed or refractory DLBCL treated with one to three prior chemotherapy regimens received rituximab 375 mg/m(2) intravenous (IV) infusion on day 1 and bendamustine 120 mg/m(2) by IV infusion on days 2 and 3 of each 21-day cycle for up to six cycles. The primary end point was overall response rate (ORR), and the secondary end points were complete response (CR) rate, progression-free survival (PFS), and safety. RESULTS Sixty-three patients were enrolled, and 59 received BR. The median age was 67 years (range, 36 to 75 years), and 62.7% of patients were 65 years of age or older. Fifty-seven patients (96.6%) were previously treated with rituximab-containing chemotherapy. The ORR was 62.7% (95% CI, 49.1% to 75.0%), with a CR rate of 37.3% (95% CI, 25.0% to 50.9%). The ORRs were comparable between patients ≥ 65 years of age and less than 65 years (62.2% and 63.6%, respectively). The median PFS was 6.7 months (95% CI, 3.6 to 13.7 months). The most frequently observed grade 3 or 4 adverse events were hematologic: lymphopenia (78.0%), neutropenia (76.3%), leukopenia (72.9%), CD4 lymphopenia (66.1%), and thrombocytopenia (22.0%). CONCLUSION BR is a promising salvage regimen for patients with relapsed or refractory DLBCL after rituximab-containing chemotherapy, warranting further investigation.

Methylation status of the p15INK4B gene in hematopoietic progenitors and peripheral blood cells in myelodysplastic syndromes

We previously reported that the hypermethylation of the p15INK4B gene promoter was frequently observed in myelodysplastic syndromes (MDS), and that it may be associated with disease progression. An unanswered question is whether p15INK4B gene methylation is restricted to undifferentiated blastic cells, or whether differentiated cells such as granulocytes or erythrocytes of MDS origin also harbor this epigenetic alteration. In this study, we analyzed the methylation status of the p15INK4B gene in MDS by the methylation-specific PCR (MSP) method, which is more sensitive than Southern blotting. The bone marrow mononuclear cells (BM-MNCs) of 23 MDS patients were analyzed, and six of them showed p15INK4B methylation. Progenitor assay with methylcellulose medium was also performed in all patients. In two of the six patients with p15INK4B-methylated BM-MNCs, erythroid and/or non-erythroid colonies formed were subjected to molecular analysis. Colonies with and without p15INK4B methylation were detected in both patients. Furthermore, X-chromosome inactivation (XCI) pattern of each colony was simultaneously determined by MSP-based human androgen receptor gene analysis (HUMARA-MSP), and all p15INK4B-methylated colonies showed the same XCI pattern, which was dominant among the colonies, while p15INK4B-unmethylated colonies showed both patterns of XCI, in each of the two patients. We then examined the methylation status of the p15INK4B gene of granulocyte (PB-PMN) fractions from 10 patients with available peripheral blood cells. In all four patients with p15INK4B-methylated BM-MNCs, their PB-PMNs showed p15INK4B methylation. These results suggest that p15INK4Bmethylation in hematopoietic cells in MDS patients is restricted to the MDS clone but not necessarily to blast cells.

Short-term methotrexate could reduce early immune reactions and improve outcomes in umbilical cord blood transplantation for adults

Post transplant immune disorders are problematic in cord blood transplantation (CBT) for adult patients, and optimal prophylaxis has not been established. We investigated whether intensive graft-versus-host disease (GVHD) prophylaxis using short-term methotrexate (MTX) has a prognostic impact on CBT. Post-CBT immune reactions were classified according to time course as pre-engraftment immune reaction (PIR), engraftment syndrome (ES) or acute GVHD. Between March 2001 and November 2005, a total of 77 patients underwent CBT at eight transplantation centers. Median age was 48 years (range, 18–69 years). Preparative regimens comprised myeloablative (n=31) or reduced-intensity (n=46). Acute GVHD prophylaxis included cyclosporine alone (n=23), tacrolimus alone (n=12), cyclosporine plus MTX (n=17), tacrolimus plus short-term MTX (n=23) or cyclosporine plus methylprednisolone (n=2). Cumulative incidences of PIR, ES and grade II–IV GVHD were 36, 12 and 23%, respectively. Short-term MTX exerted significant favorable effects on post-CBT immune reactions (hazard ratio, 0.55; 95% confidence interval (95% CI), 0.31–0.98; P=0.04) in multivariate analysis. Overall survival rates for patients with and without short-term MTX at day 180 were 59% (95% CI, 42–73%) and 16% (95% CI, 6.6–30%) (P=0.0001), respectively. Short-term MTX could offer one optimal regimen to reduce immune reactions and improve outcomes in CBT.

Hypermethylation of p16INK4A gene promoter during the progression of plasma cell dyscrasia.

Recent studies have indicated a close relationship between inactivation of tumor suppressor genes (TSGs) and disease progression. The genes encoding the cyclin-dependent kinase inhibitors p16INK4A and p15INK4B are potent TSGs, and correlations between their inactivation and disease progression have also been reported in various malignancies. In this study, we analyzed the methylation status of p16INK4Aand p15INK4B gene promoters in plasma cell dyscrasias (PCDs) by methylation-specific PCR (MSP). In analyses using DNAs extracted from bone marrow mononuclear cells (BM-MNCs), patients with multiple myeloma (MM) showed frequent hypermethylation of the p16INK4A gene (15/37, 41%), whereas p15INK4B gene methylation was not so frequent (5/37, 14%). Many patients whose BM-MNC showed dense methylation of the p16INK4A gene had extramedullary plasmacytoma (extra-PC), and all available extra-PC samples showed alterations of the p16INK4A gene (4; dense methylation, 1; homozygous deletion). In contrast to MM, hypermethylation of the p16INK4A gene was significantly infrequent in indolent PCDs (2/22, 9%, P = 0.0055). The infrequency in indolent PCDs was also confirmed by analyses using DNAs extracted from BM smears (1/29, 3%). It is possible that hypermethylation of the p16INK4A gene promoter contributes to progression to aggressive MM from indolent PCD, especially to extra-PC development.

Multicenter phase II study of bendamustine for relapsed or refractory indolent B‐cell non‐Hodgkin lymphoma and mantle cell lymphoma

Bendamustine is a unique cytotoxic agent that has demonstrated efficacy in the treatment of indolent B‐cell non‐Hodgkin lymphomas (B‐NHLs). In this multicenter phase II trial, the efficacy and safety of bendamustine were evaluated in Japanese patients with relapsed or refractory indolent B‐NHL or mantle‐cell lymphoma (MCL). Patients received bendamustine (120 mg/m2) on days 1–2 of a 21‐day cycle, for up to six cycles. The primary endpoint was the overall response rate (ORR) as assessed by an extramural committee according to International Workshop Response Criteria (IWRC). Secondary endpoints included complete response (CR) rate, ORR according to Revised Response Criteria (revised RC), progression‐free survival (PFS), and safety. Fifty‐eight patients with indolent B‐NHL and 11 with MCL were enrolled. By IWRC, bendamustine produced an ORR of 91% (95% confidence interval [CI], 82–97%; 90% and 100% in patients with indolent B‐NHL and MCL, respectively), with a CR rate of 67% (95% CI, 54–78%). ORR and CR rates according to revised RC were 93% (95% CI, 84–98%) and 57% (95% CI, 44–68%), respectively. After a median follow‐up of 12.6 months, median PFS had not been reached. Estimated PFS rates at 1 year were 70% and 90% among indolent B‐NHL and MCL patients, respectively. Bendamustine was generally well tolerated. Reversible myelosuppression, including grade 3/4 leukopenia (65%) and neutropenia (72%), was the most clinically significant toxicity observed. Common non‐hematologic toxicities included mild gastrointestinal events and fatigue. These results demonstrate the high efficacy and tolerability of single‐agent bendamustine in relapsed patients with indolent B‐NHL or MCL histologies. (ClinicalTrials.gov ID: NCT00612183). (Cancer Sci 2010;)

Mutational analysis of the PTEN/MMAC1 gene in non-Hodgkin’s lymphoma

The PTEN/MMAC1 gene at 10q23.3, which has dual specific phosphatase activity, is a novel tumor suppressor gene candidate. Various kinds of tumors have mutations in this gene, including glioblastoma, endometrial carcinoma and prostate cancer. We examined 29 cases of primary non-Hodgkin’s lymphoma (NHL) for mutations in the PTEN/MMAC1 gene. One case of diffuse large B cell lymphoma had an 11 bp deletion, but the remaining 28 cases showed no mutations in the genome. Two of these 28 cases showed missense mutations in the PTEN/MMAC1 transcripts, but no alterations in the genomic DNA. These mRNA missense variants are similar to PTEN/MMAC1 transcript aberrations which have been reported in patients with breast cancer. These findings suggest that alterations in the PTEN/MMAC1 gene play a role in the pathogenesis of NHL.

Monitoring of Hepatitis B Virus (HBV) DNA and Risk of HBV Reactivation in B-Cell Lymphoma: A Prospective Observational Study

BACKGROUND There is no standard management of reactivation of hepatitis B virus (HBV) infection in HBV-resolved patients without hepatitis B surface antigen (HBsAg), but with antibodies against hepatitis B core antigen and/or antibodies against HBsAg (anti-HBs). METHODS We conducted a prospective observational study to evaluate the occurrence of HBV reactivation by serial monthly monitoring of HBV DNA and to establish preemptive therapy guided by this monitoring in B-cell non-Hodgkin lymphoma (B-NHL) treated with rituximab plus corticosteroid-containing chemotherapy (R-steroid-chemo). The primary endpoint was the incidence of HBV reactivation defined as quantifiable HBV DNA levels of ≥ 11 IU/mL. RESULTS With a median HBV DNA follow-up of 562 days, HBV reactivation was observed in 21 of the 269 analyzed patients. The incidence of HBV reactivation at 1.5 years was 8.3% (95% confidence interval, 5.5-12.4). No hepatitis due to HBV reactivation was observed in patients who received antiviral treatment when HBV DNA levels were between 11 and 432 IU/mL. An anti-HBs titer of <10 mIU/mL and detectable HBV DNA remaining below the level of quantification at baseline were independent risk factors for HBV reactivation (hazard ratio, 20.6 and 56.2, respectively; P < .001). Even in 6 patients with a rapid increase of HBV due to mutations, the monthly HBV DNA monitoring was effective at preventing HBV-related hepatitis. CONCLUSIONS Monthly monitoring of HBV DNA is useful for preventing HBV reactivation-related hepatitis among B-NHL patients with resolved HBV infection following R-steroid-chemo (UMIN000001299).

Phase I study of inotuzumab ozogamicin (CMC-544) in Japanese patients with follicular lymphoma pretreated with rituximab-based therapy

Inotuzumab ozogamicin (CMC‐544), an antibody‐targeted chemotherapeutic agent composed of an anti‐CD22 antibody conjugated to calicheamicin, a potent cytotoxic antibiotic, specifically targets the CD22 antigen present in >90% of B‐lymphoid malignancies, rendering it useful for treating patients with B‐cell non‐Hodgkin lymphoma (B‐NHL). This phase I study evaluated the safety, tolerability, efficacy, and pharmacokinetics of inotuzumab ozogamicin in Japanese patients. Eligible patients had relapsed or refractory CD22‐positive B‐NHL without major organ dysfunction. Inotuzumab ozogamicin was administered intravenously once every 28 days (dose escalation: 1.3 and 1.8 mg/m2). All 13 patients had follicular lymphoma, were previously treated with ≥1 rituximab‐alone or rituximab‐containing chemotherapy, and were enrolled into two dose cohorts (1.3 mg/m2, three patients; 1.8 mg/m2, 10 patients). No patient had dose‐limiting toxicities, and the maximum tolerated dose, previously determined in non‐Japanese patients (1.8 mg/m2), was confirmed. Drug‐related adverse events (AEs) included thrombocytopenia (100%), leukopenia (92%), lymphopenia (85%), neutropenia (85%), elevated AST (85%), anorexia (85%), and nausea (77%). Grade 3/4 drug‐related AEs in ≥15% patients were thrombocytopenia (54%), lymphopenia (31%), neutropenia (31%), and leukopenia (15%). The AUC and Cmax of inotuzumab ozogamicin increased dose‐dependently with pharmacokinetic profiles similar to non‐Japanese. Seven patients had complete response (CR, 54%) including unconfirmed CR, four patients had partial response (31%), and two patients had stable disease (15%). The overall response rate was 85% (11/13). Inotuzumab ozogamicin was well tolerated at doses up to 1.8 mg/m2 and showed preliminary evidence of activity in relapsed or refractory follicular lymphoma pretreated with rituximab‐containing therapy, warranting further investigations. This trial was registered in ClinicalTrials.gov (NCT00717925). (Cancer Sci 2010)

Clonality analysis by methylation-specific PCR for the human androgen-receptor gene (HUMARA-MSP)

The human androgen-receptor gene (HUMARA) has been used for analysis of X chromosome inactivation (XCI) pattern because of a polymorphic short tandem repeat (STR) near the 5′-promoter region correlated with XCI. We introduce a novel method to analyze XCI pattern, named HUMARA methylation-specific PCR (HUMARA-MSP) assay, which analyzes methylation status of the HUMARA gene by bisulfite modification instead of a methylation-sensitive restriction enzyme. Although the original MSP method shows whether there is a methylated band or not, our HUMARA-MSP method identifies the patterns of methylated and unmethylated bands. Because this method identifies either unmethylated or methylated alleles in each PCR tube and shows opposite band patterns dependent on methylation status, we can assess the XCI pattern independently twice. This method can avoid false results by incomplete enzyme digestion and incomplete bisulfite modification will not affect the results. Extremely small quantities of samples, such as hematopoietic colonies, were also available for HUMARA-MSP assay. Because DNA modified by sodium bisulfite is also available for assessment of methylation status of other genes by setting specific primers for them, we performed the simultaneous assessment of clonality and aberrant hypermethylation of p15INK4B gene in myelodysplastic syndromes. These simultaneous assessments were easily possible and provided much information despite requiring only a small volume of DNA. The HUMARA-MSP assay may facilitate the analyses for pathogenesis of hematological disorders because of its simplicity, sensitivity and wide applicability.

Truncated c-Myb expression in the human leukemia cell line TK-6.

The c-MYB proto-oncogene encodes a transcription factor which plays an important role in hematopoiesis. We detected truncated c-MYB mRNA (2.0 kb) and c-Myb protein (55 kDa) in the TK-6 cell line, which was established from a patient with chronic myelogenous leukemia in T cell blast crisis. Mutated c-MYB cDNA clone (WTK-1) was isolated from a TK-6 cell cDNA library and sequenced in its entirety. Compared with the wild-type human c-MYB sequence, the WTK-1 sequence diverged at the 3′ ends of exons 9. A termination codon was present as the second codon downstream from the point of divergence and was followed by a previously unknown rearranged sequence. The conceptual protein encoded by WTK-1 (MybTK-6) comprises 402 amino acids and lacks the negative regulatory domain of the normal c-Myb, reminiscent of the activated form of Myb protein. Luciferase reporter assay in NIH3T3 cells showed that the expression vector encoding MybTK-6 stimulated Myb-regulated mim-1 promoter more effectively than that encoding wild-type human c-Myb, suggesting that MybTK-6 is functional as a transcription factor, and thus as a potential transforming protein. Southern blot and mutant allele-specific polymerase chain reaction analyses showed that the same rearrangement of the c-MYB gene in TK-6 was present in late, but not in early, specimens obtained from the patient, indicating that this mutation had been acquired during disease progression.