Shigeru Kusumoto

Reactivation of hepatitis B virus following systemic chemotherapy for malignant lymphoma.

Reactivation of hepatitis B virus (HBV) has been reported not only in HBsAg-positive patients undergoing systemic chemotherapy, but also in a proportion of HBsAg-negative patients with HBc antibody and/or HBs antibody. Recently, rituximab-plus-steroid combination chemotherapy (R-CHOP, etc.) has been identified as a risk factor for HBV reactivation in HBsAg-negative patients with malignant lymphoma. Prophylaxis with antiviral drugs is essential for preventing HBV reactivation in HBsAg-positive patients, but there is little evidence on which to base the choice of drug or appropriate duration of prophylaxis. There are also few clinical data on HBsAg-negative patients and no established standard of care for such patients with HBV reactivation. Based on the limited number of previous reports, preemptive therapy, guided by serial HBV-DNA monitoring, is a reasonable strategy to prevent HBV reactivation in HBsAg-negative patients. However, clinical evidence alone is insufficient for determining optimal frequency of HBV-DNA monitoring during and after chemotherapy, or for determining when to stop preemptive therapy for HBV reactivation. Thus, well-designed clinical trials should be carried out to investigate the efficacy and safety of such preemptive therapy. Additionally, assessment of viral factors such as HBV genotypes and gene mutations may assist in the development of strategies to prevent the occurrence of severe hepatitis. In this review, we summarize the characteristics of HBV reactivation after systemic chemotherapy including rituximab, and propose a management strategy for malignant lymphoma patients suffering from HBV reactivation.

Defucosylated anti-CCR4 monoclonal antibody exercises potent ADCC-mediated antitumor effect in the novel tumor-bearing humanized NOD/Shi-scid, IL-2Rγnull mouse model

PurposeThere are no suitable small animal models to evaluate human antibody-dependent cellular cytotoxicity (ADCC) in vivo, due to species incompatibilities. Thus, the first aim of this study was to establish a human tumor-bearing mouse model in which human immune cells can engraft and mediate ADCC, but where the endogenous mouse immune cells cannot mediate ADCC. The second aim was to evaluate ADCC mediated in these humanized mice by the defucosylated anti-CC chemokine receptor 4 (CCR4) monoclonal antibody (mAb) which we have developed and which is now in phase I clinical trials.Experimental designNOD/Shi-scid, IL-2Rγnull (NOG) mice were the recipients of human immune cells, and CCR4-expressing Hodgkin lymphoma (HL) and cutaneous T-cell lymphoma (CTCL) cell lines were used as target tumors.ResultsHumanized mice have been established using NOG mice. The chimeric defucosylated anti-CCR4 mAb KM2760 showed potent antitumor activity mediated by robust ADCC in these humanized mice bearing the HL or CTCL cell lines. KM2760 significantly increased the number of tumor-infiltrating CD56-positive NK cells which mediate ADCC, and reduced the number of tumor-infiltrating FOXP3-positive regulatory T (Treg) cells in HL-bearing humanized mice.ConclusionsAnti-CCR4 mAb could be an ideal treatment modality for many different cancers, not only to directly kill CCR4-expressing tumor cells, but also to overcome the suppressive effect of Treg cells on the host immune response to tumor cells. In addition, using our humanized mice, we can perform the appropriate preclinical evaluation of many types of antibody based immunotherapy.

Bortezomib-resistant myeloma cell lines: a role for mutated PSMB5 in preventing the accumulation of unfolded proteins and fatal ER stress

Bortezomib is an effective agent for treating multiple myeloma (MM). To investigate the underlying mechanisms associated with acquired resistance to this agent, we established two bortezomib-resistant MM cell lines, KMS-11/BTZ and OPM-2/BTZ, the 50% inhibitory concentration values of which were respectively 24.7- and 16.6-fold higher than their parental cell lines. No activation of caspase and BH3-only proteins such as Noxa was noted in bortezomib-resistant cells after exposure to the drug. The accumulation of polyubiquitinated proteins was reduced in bortezomib-resistant cells compared with the parental cells, associated with avoidance of catastrophic ER stress as assessed by downregulation of CHOP expression. These resistant MM cells have a unique point mutation, G322A, in the gene encoding the proteasome β5 subunit (PSMB5), likely resulting in conformational changes to the bortezomib-binding pocket of this subunit. KMS-11 parental cells transfected to express mutated PSMB5 also showed reduced bortezomib-induced apoptosis compared with those expressing wild-type PSMB5 or the parental cells. Expression of mutated PSMB5 was associated with the prevention of the accumulation of unfolded proteins. Thus, a fraction of MM cells may acquire bortezomib resistance by suppressing apoptotic signals through the inhibition of unfolded protein accumulation and subsequent excessive ER stress by a mutation of the PSMB5 gene.

Hepatic toxicity and prognosis in hepatitis C virus–infected patients with diffuse large B-cell lymphoma treated with rituximab-containing chemotherapy regimens: a Japanese multicenter analysis

The influence of hepatitis C virus (HCV) infection on prognosis and hepatic toxicity in patients with diffuse large B-cell lymphoma in the rituximab era is unclear. Thus, we analyzed 553 patients, 131 of whom were HCV-positive and 422 of whom were HCV-negative, with DLBCL treated with rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (RCHOP)-like chemotherapy. Survival outcomes and hepatic toxicity were compared according to HCV infection. The median follow-up was 31 and 32 months for patients who were HCV-positive and HCV-negative, respectively. HCV infection was not a significant risk factor for prognosis (3-year progression-free survival, 69% vs 77%, P = .22; overall survival, 75% vs 84%, P = .07). Of 131 patients who were HCV-positive, 36 (27%) had severe hepatic toxicity (grade 3-4), compared with 13 of 422 (3%) patients who were HCV-negative. Multivariate analysis revealed that HCV infection was a significant risk factor for severe hepatic toxicity (hazard ratio: 14.72; 95% confidence interval, 6.37-34.03; P < .001). An exploratory analysis revealed that pretreatment transaminase was predictive of severe hepatic toxicity. HCV-RNA levels significantly increased during immunochemotherapy (P = .006). These results suggest that careful monitoring of hepatic function and viral load is indicated during immunochemotherapy for HCV-positive patients.

Identification of Toyocamycin, an agent cytotoxic for multiple myeloma cells, as a potent inhibitor of ER stress-induced XBP1 mRNA splicing

The IRE1α-XBP1 pathway, a key component of the endoplasmic reticulum (ER) stress response, is considered to be a critical regulator for survival of multiple myeloma (MM) cells. Therefore, the availability of small-molecule inhibitors targeting this pathway would offer a new chemotherapeutic strategy for MM. Here, we screened small-molecule inhibitors of ER stress-induced XBP1 activation, and identified toyocamycin from a culture broth of an Actinomycete strain. Toyocamycin was shown to suppress thapsigargin-, tunicamycin- and 2-deoxyglucose-induced XBP1 mRNA splicing in HeLa cells without affecting activating transcription factor 6 (ATF6) and PKR-like ER kinase (PERK) activation. Furthermore, although toyocamycin was unable to inhibit IRE1α phosphorylation, it prevented IRE1α-induced XBP1 mRNA cleavage in vitro. Thus, toyocamycin is an inhibitor of IRE1α-induced XBP1 mRNA cleavage. Toyocamycin inhibited not only ER stress-induced but also constitutive activation of XBP1 expression in MM lines as well as primary samples from patients. It showed synergistic effects with bortezomib, and induced apoptosis of MM cells including bortezomib-resistant cells at nanomolar levels in a dose-dependent manner. It also inhibited growth of xenografts in an in vivo model of human MM. Taken together, our results suggest toyocamycin as a lead compound for developing anti-MM therapy and XBP1 as an appropriate molecular target for anti-MM therapy.

Reactivation of hepatitis B virus following rituximab-plus-steroid combination chemotherapy

Reactivation of hepatitis B virus (HBV) has been reported as a fatal complication following systemic chemotherapy or other immunosuppressive therapy. The risk of HBV reactivation differs according to both the patient’s HBV infection status prior to systemic chemotherapy and the degree of immunosuppression due to chemotherapy. For establishing an optimal strategy for hepatitis prevention and treatment, it is necessary to understand the characteristics, the clinical course and the risk factors for HBV reactivation and to recognize the difference between hepatitis B surface antigen (HBsAg)-positive and -negative patients with HBV reactivation. Among the important viral risk factors, HBV-DNA level and HBV-related serum markers have been reported to be associated with HBV reactivation in addition to cccDNA, genotypes and gene mutations. Rituximab-plus-steroid combination chemotherapy has recently been identified as a host risk factor for HBV reactivation in hepatitis B core antibody (anti-HBc)-positive and/or hepatitis B surface antibody (anti-HBs) positive—but nonetheless HBsAg-negative—lymphoma patients. For these patients with resolved hepatitis B, preemptive therapy guided by serial HBV-DNA monitoring is a reasonable strategy to enable early diagnosis of HBV reactivation and initiation of antiviral therapy. In this review, we summarize the characteristics of HBV reactivation following rituximab-plus-steroid combination chemotherapy, mainly in HBsAg-negative lymphoma patients, and propose a strategy for managing HBV reactivation.

Stevens–Johnson Syndrome associated with mogamulizumab treatment of adult T-cell leukemia / lymphoma

We report an adult T‐cell leukemia/lymphoma patient suffering from Stevens–Johnson Syndrome (SJS) during mogamulizumab (humanized anti‐CCR4 monoclonal antibody) treatment. There was a durable significant reduction of the CD4+CD25highFOXP3+ regulatory T (Treg) cell subset in the patients PBMC, and the affected inflamed skin almost completely lacked FOXP3‐positive cells. This implies an association between reduction of the Treg subset by mogamulizimab and occurrence of SJS. The present case should contribute not only to our understanding of human pathology resulting from therapeutic depletion of Treg cells, but also alert us to the possibility of immune‐related severe adverse events such as SJS when using mogamulizumab. We are currently conducting a clinical trial of mogamulizumab for CCR4‐negative solid cancers (UMIN000010050), specifically aiming to deplete Treg cells.

Defucosylated Anti-CCR4 Monoclonal Antibody Exerts Potent ADCC against Primary ATLL Cells Mediated by Autologous Human Immune Cells in NOD/Shi-scid, IL-2Rγnull Mice In Vivo

There is a lack of suitable small animal models to evaluate human Ab-dependent cellular cytotoxicity (ADCC) in vivo, because of the species incompatibility between humans and animals or due to nonspecific allogeneic immune reactions. To overcome these problems, we established a human tumor-bearing mouse model, using NOD/Shi-scid, IL-2Rγnull (NOG) mice as recipients, in which autologous human immune cells are engrafted and mediate ADCC but in which endogenous murine cells are unable to mediate ADCC. In the present study, we used NOG mice bearing primary adult T cell leukemia/lymphoma (ATLL) cells and a therapeutic chimeric anti-CCR4 mAb, the Fc region of which is defucosylated to enhance ADCC. We report significant antitumor activity in vivo associated with robust ADCC mediated by autologous effector cells from the same patients. The present study is the first to report a mouse model in which a potent antitumor effect of the therapeutic mAb against primary tumor cells is mediated by autologous human immune cells. Human autologous ADCC in mice in vivo was confirmed by the depletion of human immune cells before ATLL PBMC inoculation. In addition, NOG mice bearing primary ATLL cells presented features identical with patients with ATLL. In conclusion, this approach makes it possible to model the human immune system active in Ab-based immunotherapy in vivo, and thus to perform more appropriate preclinical evaluations of novel therapeutic mAb. Furthermore, the potent ADCC mediated by defucosylated anti-CCR4 mAb, observed here in vivo in humanized mice, will be exploited in clinical trials in the near future.

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).

Bortezomib-induced apoptosis in mature T-cell lymphoma cells partially depends on upregulation of Noxa and functional repression of Mcl-1

Bortezomib, a proteasome inhibitor that was originally developed as an inhibitor of nuclear factor‐κB pathways, is currently used for the treatment of multiple myeloma (MM) and mantle cell lymphoma (MCL). The mechanisms of action of this antitumor agent have been studied by several investigators. Here, we explore the underlying mechanisms of bortezomib‐induced apoptosis in cutaneous T‐cell lymphoma (CTCL) and adult T‐cell leukemia/lymphoma (ATLL) at the level of mitochondrial membrane injury. In all cell lines including (KMS‐12‐PE [MM], HUT78 [CTCL], ATN1 [ATLL], and MT4 [ATLL]), antiapoptotic factors such as c‐Flip and XIAP were downregulated after exposure to bortezomib, probably via inhibition of nuclear factor‐κB signaling. In addition, among the members of the BH3‐only family, upregulation of Noxa was consistently seen at both the transcriptional and protein levels in a p53‐independent manner after exposure to bortezomib. Repression of Noxa by small interfering RNA partially rescued CTCL and ATLL cells from bortezomib‐induced apoptosis. Immunoprecipitation assays indicated time‐dependent binding of Noxa and Mcl‐1 in all cell types, suggesting that functional repression of Mcl‐1 led to the loss of mitochondrial outer membrane potential. Similar results were also obtained in primary tumor cells from patients with ATLL. Taken together, we conclude that bortezomib‐induced apoptosis in ATLL and CTCL cells at least partly depends on the upregulation of Noxa and functional repression of Mcl‐1, as is also the case in MM and malignant melanoma. (Cancer Sci 2009; 100: 341–348)