Yukiya Yamamoto

BCOR as a novel fusion partner of retinoic acid receptor alpha in a t(X;17)(p11;q12) variant of acute promyelocytic leukemia

The majority of acute promyelocytic leukemia (APL) cases are characterized by the presence of a promyelocytic leukemia-retinoic acid receptor alpha(RARA) fusion gene. In a small subset, RARA is fused to a different partner, usually involved in regulating cell growth and differentiation. Here, we identified a novel RARA fusion transcript, BCOR-RARA, in a t(X;17)(p11;q12) variant of APL with unique morphologic features, including rectangular and round cytoplasmic inclusion bodies. Although the patient was clinically responsive to all-trans retinoic acid, several relapses occurred with standard chemotherapy and all-trans retinoic acid. BCOR is a transcriptional corepressor through the proto-oncoprotein, BCL6, recruiting histone deacetylases and polycomb repressive complex 1 components. BCOR-RARA was found to possess common features with other RARA fusion proteins. These included: (1) the same break point in RARA cDNA; (2) self-association; (3) retinoid X receptor alpha is necessary for BCOR-RARA to associate with the RARA responsive element; (4) action in a dominant-negative manner on RARA transcriptional activation; and (5) aberrant subcellular relocalization. It should be noted that there was no intact BCOR found in the 45,-Y,t(X;17)(p11;q12) APL cells because they featured only a rearranged X chromosome. These results highlight essential features of pathogenesis in APL in more detail. BCOR appears to be involved not only in human congenital diseases, but also in a human cancer.

Clarifying the impact of polycomb complex component disruption in human cancers.

The dysregulation of proper transcriptional control is a major cause of developmental diseases and cancers. Polycomb proteins form chromatin-modifying complexes that transcriptionally silence genome regions in higher eukaryotes. The BCL6 corepressor (BCOR) complex comprises ring finger protein 1B (RNF2/RING1B), polycomb group ring finger 1 (PCGF1), and lysine-specific demethylase 2B (KDM2B) and is uniquely recruited to nonmethylated CpG islands, where it removes histone H3K36me2 and induces repressive histone H2A monoubiquitylation. Germline BCOR mutations have been detected in patients with oculofaciocardiodental and Lenz microphthalmia syndromes, which are inherited conditions. Recently, several variants of BCOR and BCOR-like 1 (BCORL1) chimeric fusion transcripts were reported in human cancers, including acute promyelocytic leukemia, bone sarcoma, and hepatocellular carcinoma. In addition, massively parallel sequencing has identified inactivating somatic BCOR and BCORL1 mutations in patients with acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), chronic myelomonocytic leukemia, medulloblastoma, and retinoblastoma. More importantly, patients with AML and MDS with BCOR mutations exhibit poor prognosis. This perspective highlights the detection of BCOR mutations and fusion transcripts of BCOR and BCORL1 and discusses their importance for diagnosing cancer subtypes and estimating the treatment responses of patients. Furthermore, this perspective proposes the need for additional functional studies to clarify the oncogenic mechanism by which BCOR and BCORL1 are disrupted in cancers, and how this may lead to the development of novel therapeutics. Mol Cancer Res; 12(4); 479–84. ©2014 AACR.

In vitro anticancer activity of methyl caffeate isolated from Solanum torvum Swartz. fruit.

The present study was undertaken to investigate the anticancer activity of methyl caffeate isolated from Solanum torvum Swartz. fruit and to explore the molecular mechanisms of action in MCF-7 cells. Cytotoxic properties of hexane, ethyl acetate and methanol extracts were carried out against MCF-7 cells using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay. Ethyl acetate extract showed good cytototoxic activities compared to hexane and methanol extracts. Methyl caffeate was isolated from the ethyl acetate extract using column chromatography. Cytotoxic properties of methyl caffeate was investigated against MCF-7, A549, COLO320, HepG-2 and Vero cells. The compound showed potent cytotoxic properties against MCF-7 cells compared to A549, COLO320 and HepG-2 cells. Methyl caffeate significantly reduced cell proliferation and increased formation of fragmented DNA and apoptotic body in MCF-7 cells. Bcl-2, Bax, Bid, p53, caspase-3, PARP and cytochrome c release were detected by western blot analysis. The activities of caspases-3 and PARP gradually increased after the addition of isolated compound. Bcl-2 protein was down regulated; Bid and Bax were up regulated after the treatment with methyl caffeate. Molecular docking studies showed that the compound bound stably to the active sites of poly (ADP-ribose) polymerase-1 (PARP1), B cell CLL/lymphoma-2 (BCL-2), E3 ubiquitin-protein ligase (MDM2) and tubulin. The results strongly suggested that methyl caffeate induced apoptosis in MCF-7 cells via caspase activation through cytochrome c release from mitochondria.

Construction and molecular characterization of a T-cell receptor-like antibody and CAR-T cells specific for minor histocompatibility antigen HA-1H

The genetic transfer of T-cell receptors (TCRs) directed toward target antigens into T lymphocytes has been used to generate antitumor T cells efficiently without the need for the in vitro induction and expansion of T cells with cognate specificity. Alternatively, T cells have been gene-modified with a TCR-like antibody or chimeric antigen receptor (CAR). We show that immunization of HLA-A2 transgenic mice with tetramerized recombinant HLA-A2 incorporating HA-1 H minor histocompatibility antigen (mHag) peptides and β2-microglobulin (HA-1 H/HLA-A2) generate highly specific antibodies. One single-chain variable region moiety (scFv) antibody, #131, demonstrated high affinity (KD=14.9 nM) for the HA-1 H/HLA-A2 complex. Primary human T cells transduced with #131 scFV coupled to CD28 transmembrane and CD3ζ domains were stained with HA-1 H/HLA-A2 tetramers slightly more intensely than a cytotoxic T lymphocyte (CTL) clone specific for endogenously HLA-A2- and HA-1 H-positive cells. Although #131 scFv CAR-T cells required >100-fold higher antigen density to exert cytotoxicity compared with the cognate CTL clone, they could produce inflammatory cytokines against cells expressing HLA-A2 and HA-1 H transgenes. These data implicate that T cells with high-affinity antigen receptors reduce the ability to lyse targets with low-density peptide/MHC complexes (~100 per cell), while they could respond at cytokine production level.

Differences in outcome for consecutive patients with diffuse large B-cell lymphoma before and after the advent of rituximab: a single-center experience

Abstract The beneficial effect of rituximab for first-line treatment of diffuse large B-cell lymphoma (DLBCL) has been demonstrated by several randomized controlled trials. To clarify whether results for selected patient populations also apply to unselected patients, we analyzed long-term outcomes for all the 277 consecutive adults diagnosed with de novo DLBCL in a single center between 1998 and 2008. The study population included 147 and 130 patients diagnosed before (Cohort A) and after the advent of rituximab (Cohort B). Progression-free survival (PFS) was significantly better for Cohort B than for Cohort A (P = 0.005). For patients age 60 or younger, PFS did not differ significantly between Cohort A and Cohort B (P = 0.329), but for patients over 60, Cohort B showed superior PFS (P = 0.002). Patients with high or high-intermediate risk according to the International Prognostic Index score showed less improvement in PFS than did those with low or low-intermediate risk primarily because of still unfavorable outcomes of patients with poor performance status. These results indicate that the advent of rituximab has significantly improved outcome for unselected patients with DLBCL, and that improvement was greater for older patients. Further investigations are warranted in the hope of improving outcomes for younger patients with DLBCL.

Severe hepatitis associated with varicella zoster virus infection in a patient with diffuse large B cell lymphoma treated with rituximab-CHOP chemotherapy.

Severe disseminated varicella zoster virus (VZV) infection rarely occurs in patients who are not recipients of hematopoietic stem cell transplantation. This report concerns severe disseminated VZV infection in a diffuse large B cell lymphoma (DLBCL) patient treated with rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisolone (R-CHOP). The patient was an 82-year-old male with DLBCL who had a history of type II diabetes mellitus. He incurred VZV infection with severe hepatitis and disseminated intravascular coagulopathy after three courses of R-CHOP. When the VZV infection occurred, anti-VZV IgG was not detected and lymphopenia was observed. We initiated treatment with acyclovir, immunoglobulin, and thrombomodulin alpha, and rescued this patient. We suggest that the use of chemotherapy for immune-suppressed elderly lymphoma patients may involve the risk of severe VZV infection.

The prognostic significance of EBV DNA load and EBER status in diagnostic specimens from diffuse large B‐cell lymphoma patients

Epstein–Barr virus (EBV)‐encoded small RNA in situ hybridization (EBER‐ISH) is a widely accepted method to evaluate EBV involvement in diffuse large B‐cell lymphoma (DLBCL), although little is known regarding associations between EBV DNA load and the EBER status and whether EBV DNA load data provide additional clinical information. In this study, we quantified EBV DNA load in diagnostic specimens from DLBCL patients diagnosed at our hospital to evaluate clinical implications of EBV DNA load in diagnostic specimens as contrasted with EBER‐ISH. Among 140 DLBCL patients without underlying immunodeficiency, 51 were evaluable for both EBER and EBV DNA load, 83 for EBER only and one for EBV DNA load only. The median EBV DNA load was 708 copies/µg. Although EBV DNA load was significantly higher for EBER‐positive patients than for EBER‐negative patients (p < 0.001), EBV DNA was detected in up to 72% of EBER‐negative patients. Progression‐free survival and overall survival were significantly worse for patients with EBV DNA load above 700 copies/µg than for those with EBV DNA load below 700 copies/µg (p = 0.009 and p = 0.003); they were also significantly worse for EBER‐positive patients than for EBER‐negative patients (p < 0.001 and p = 0.001). Even among EBER‐negative patients, higher EBV DNA load conferred worse progression‐free survival and overall survival (p = 0.041 and p = 0.013). These findings indicate that EBV DNA load in diagnostic specimens is not a simple surrogate for the EBER status and may be a potential biomarker associated with EBV involvement and prognosis in DLBCL. Copyright

Prognostic significance of Epstein–Barr virus DNA detection in pretreatment serum in diffuse large B‐cell lymphoma

It is still a matter of debate whether detection of Epstein–Barr virus (EBV) DNA in pretreatment serum has clinical implications for diffuse large B‐cell lymphoma. For this study, we measured EBV DNA load in pretreatment serum from 127 diffuse large B‐cell lymphoma patients without any underlying immunodeficiency to evaluate its effects on clinical manifestations and prognosis. Anthracycline‐based chemotherapy in combination with rituximab was given as initial therapy for 119 patients (94%). Epstein–Barr virus DNA was detected in 15 patients (12%), who were older (P = 0.005) and tended to be at a more advanced disease stage (P = 0.053). They showed significantly worse progression‐free survival (PFS) and overall survival (OS) than other patients (P < 0.001 each). This effect remained significant (P = 0.004 and P = 0.027, respectively) after adjustment for age, lactate dehydrogenase, performance status, stage, and extranodal sites. The status of EBV‐encoded small RNA in situ hybridization was known for 123 patients; 6 of 8 positive patients (75%) and 9 of 115 negative patients (8%) had detectable EBV DNA in pretreatment serum. While patients positive for EBV‐encoded small RNA had significantly worse PFS and OS than negative patients (P = 0.001 and P = 0.029, respectively), EBV DNA detection in pretreatment serum was associated with poorer PFS and OS even for the 115 patients negative for EBV‐encoded small RNA (P < 0.001 each). These findings suggest that EBV DNA detection in pretreatment serum may have an adverse prognostic impact for patients with diffuse large B‐cell lymphoma.

A varicella outbreak in B-cell lymphoma patients receiving rituximab-containing chemotherapy

Varicella, characterized by a vesicular rash, occurs primarily in young children. Although older individuals can also be affected or vaccinated, outbreaks among adults are rare. We investigated a small outbreak of varicella in B-cell lymphoma patients for elucidation of risk factor of the disease. We experienced four cases of varicella after an index herpes zoster case. All varicella cases were confirmed varicella zoster virus (VZV) infection by PCR. All varicella cases occurred in diffuse large B-cell lymphoma patients receiving rituximab-containing chemotherapy. On the other hand, only three of the 18 non-varicella patients in the same room were receiving rituximab-containing chemotherapy (P = 0.005). All varicella patients had detectable serum anti-varicella zoster virus IgG antibodies before chemotherapy. Even in the presence of neutralizing antibodies to the virus, lymphoma patients treated with rituximab-containing chemotherapy can possibly become re-infected with varicella. These findings suggest that zoster patients should be strictly isolated in hematology and oncology ward, and prophylactic acyclovir should be considered for such patients when exposed to zoster/varicella.

Functionally deregulated AML1/RUNX1 cooperates with BCR-ABL to induce a blastic phase-like phenotype of chronic myelogenous leukemia in mice

Patients in the chronic phase (CP) of chronic myelogenous leukemia (CML) have been treated successfully following the advent of ABL kinase inhibitors, but once they progress to the blast crisis (BC) phase the prognosis becomes dismal. Although mechanisms underlying the progression are largely unknown, recent studies revealed the presence of alterations of key molecules for hematopoiesis, such as AML1/RUNX1. Our analysis of 13 BC cases revealed that three cases had AML1 mutations and the transcript levels of wild-type (wt.) AML1 were elevated in BC compared with CP. Functional analysis of representative AML1 mutants using mouse hematopoietic cells revealed the possible contribution of some, but not all, mutants for the BC-phenotype. Specifically, K83Q and R139G, but neither R80C nor D171N mutants, conferred upon BCR-ABL-expressing cells a growth advantage over BCR-ABL-alone control cells in cytokine-free culture, and the cells thus grown killed mice upon intravenous transfer. Unexpectedly, wt.AML1 behaved similarly to K83Q and R139G mutants. In a bone marrow transplantation assay, K83Q and wt.AML1s induced the emergence of blast-like cells. The overall findings suggest the roles of altered functions of AML1 imposed by some, but not all, mutants, and the elevated expression of wt.AML1 for the disease progression of CML.