Yoko Inaguma

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.

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.

Induction of HLA‐B*40:02‐restricted T cells possessing cytotoxic and suppressive functions against haematopoietic progenitor cells from a patient with severe aplastic anaemia

Acquired aplastic anaemia (AA) is a syndrome characterized by bone marrow (BM) hypoplasia and pancytopenia. Autoimmunity to haematopoietic stem/progenitor cells (HSPCs) has been considered as a possible cause (Young et al, 2006). We recently described AA patients possessing clonal/oligoclonal haematopoiesis who had specifically lost either human leucocyte antigen (HLA) haplotype containing the HLA-A*02:06 or -B*40:02 (Katagiri et al, 2011). We surmized that selective pressure against HSPCs by specific cytotoxic T lymphocytes (CTLs) via these HLA molecules led to an escape of HSPCs without the causative HLA haplotype. We sought to determine whether HLA-B*40:02-restricted CTLs specific for HSPCs were present in patient peripheral blood or BM, and if so, how the CTLs induced BM failure as well as clonal haematopoiesis in AA patients who improved with immunosuppressive therapy (IST). Method details are provided in Data S1. This case report describes a 14-year-old male diagnosed with severe AA. Because he had no suitable donor, he received antithymocyte globulin and ciclosporin (CsA) and subsequently achieved remission (Fig 1A). His AA recurred twice but was successfully treated with CsA, indicating his haematopoiesis was CsA-dependent. He possessed a risk allele of HLA-B*40:02. His blood cells lacked one HLA haplotype containing HLA-B*40:02 (Figure S1) due to acquired copy number-neutral loss of heterozygosity of the 6p arms (6pLOH) (data not shown). To isolate HLA-B*40:02-restricted CTLs that could have triggered his BM failure (time point 4 in Fig 1A), we chose K562/B*40:02 as alternative antigen-presenting cells in place of the patient’s rare BM HSPCs because K562 has characteristics of HSPCs (Lozzio et al, 1981). The T cell line generated lysed K562/B*40:02 more intensely than K562/A*02:01, but did not lyse the non-haematopoietic cell line 293T/B*40:02 (Fig 1B). Of 84 putative clones after CTL cloning, clone A6 (CTL-A6) was selected for further analyses as a representative clone (Fig 1C). CTL-A6 did not recognize immortalized B and T lymphocytes expressing HLA-B*40:02 (Fig 1D), suggesting that mature lymphocytes might not express the target antigen. We also examined whether CTL-A6 recognized HLA-B*40:02-transduced cell lines derived from various haematological malignancies, but none of them was lysed by CTL-A6 (Fig 1E), indicating that CTL-A6 recognized antigen expressed only in K562 among the cell lines tested and might have been involved in the development of AA in the patient. Finally, we questioned whether reactivity of CTL-A6 to K562/B*40:02 would be altered with differentiation of K562. Phorbol 12-myristate 13-acetate (PMA) treatment (Witt et al, 2000) completely abrogated reactivity of CTL-A6 against K562/B*40:02 (Fig 1F) while reactivity of CTL-EH6 (Torikai et al, 2007) against simultaneously treated K562/A*02:01 expressing HLA-A*02:01-restricted minor histocompatibility antigen (mHag) HA-1H (den Haan et al, 1998) was retained (Fig 1G). Butyrate treatment produced a similar trend, but to a lesser extent (Colamonici et al, 1986). Morphological changes were apparent but their viability remained unchanged (Figure S2A). Flow cytometry revealed slight upregulation of HLA class I expression and diminished glycophorin A expression by PMA (Figure S2B). These data suggest that CTL-A6 recognized an antigen expressed in HSPC with limited maturation. We then examined the effects of CTL-A6 on HSPC growth using CD34 cells from normal BM. We identified BM from a HLA-A*02:01, B*40:02 healthy donor that was positive for HLA-B*40:02-restricted mHag encoded by SLC1A5 (Kamei et al, 2009), but negative for HA-1H. CTL-3B6 (Kamei et al, 2009) and CTL-EH6, corresponding to these mHags, were used as positive and negative controls, respectively (Fig 2A). While CD34 cells cultured with CTL-EH6 allowed expansion of immature and differentiated cells, those with CTL-3B6 showed no growth (Fig 2B). When CD34 cells were cocultured with CTL-A6, either partial growth inhibition (Experiment 1 with erythropoietin, left) or no inhibition (Experiment 2 without erythropoietin, right) was observed. After 14 d coculture in Experiment 2, cells cocultured with CTL-EH6 contained mature myeloid cells and some less mature myeloid cells (Fig 2C, upper left); the majority of those with CTL-A6 were mature myeloid cells, including some with degraded morphology (upper right); cells in the CTL-EH6 coculture contained CD13-, CD14or CD33-antigen high fractions (Fig 2C, lower left), whereas cells in the CTL-A6 coculture contained weakly positive and negative cells for CD13, CD14 or CD33; one-third of the latter contained CD8 cells most probably corresponding to residual CTL-A6 (lower right). All types of colony-forming units (CFU) were diminished by two to threefold, with the exception of mixed lineage CFU (CFU-GEMM), which showed greater than fourfold decrease, in the coculture with CTL-A6 compared with those with CTL-EH6 (Fig 2D). Altogether, correspondence

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

In vitro antiproliferative activity of 2,3-dihydroxy-9,10-anthraquinone induced apoptosis against COLO320 cells through cytochrome c release caspase mediated pathway with PI3K/AKT and COX-2 inhibition.

The present study investigated the anticancer activity of 2,3-dihydroxy-9,10-anthraquinone against different cancer cells such as MCF-7, COLO320, HepG-2, Skov-3, MOLM-14, NB-4, CEM, K562, Jurkat, HL-60, U937, IM-9 and Vero. 2,3-dihydroxy-9,10-anthraquinone showed good antiproliferative activity against COLO320 cells when compared to other tested cells. The cytotoxicity results showed 79.8% activity at the dose of 2.07 μM with IC50 value of 0.13 μM at 24 h in COLO320 cells. So we chose COLO320 cells for further anticancer studies. mRNA expression was confirmed by qPCR analysis using SYBR green method. Treatment with 2,3-dihydroxy-9,10-anthraquinone was found to trigger intrinsic apoptotic pathway as indicated by down regulation of Bcl-2, Bcl-xl; up regulation of Bim, Bax, Bad; release of cytochrome c and pro-caspases cleaving to caspases. Furthermore, 2,3-dihydroxy-9,10-anthraquinone stopped at G0/G1 phase with modulation in protein levels of cyclins. On the other hand PI3K/AKT signaling plays an important role in cell metabolism. We found that 2,3-dihydroxy-9,10-anthraquinone inhibits PI3K/AKT activity after treatment. Also, COX-2 enzyme plays a major role in colorectal cancer. Our results showed that the treatment significantly reduced COX-2 enzyme in COLO320 cells. These results indicated antiproliferative activity of 2,3-dihydroxy-9,10-anthraquinone involving apoptotic pathways, mitochondrial functions, cell cycle checkpoint and controlling the over expression genes during the colorectal cancer. Molecular docking studies showed that the compound bound stably to the active sites of Bcl-2, COX-2, PI3K and AKT. This is the first report of anticancer mechanism involving 2,3-dihydroxy-9,10-anthraquinone in COLO320 cells. The present results might provide helpful suggestions for the design of antitumor drugs toward colorectal cancer treatment.

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.

Reappraisal of Epstein–Barr virus (EBV) in diffuse large B-cell lymphoma (DLBCL): comparative analysis between EBV-positive and EBV-negative DLBCL with EBV-positive bystander cells

Epstein–Barr virus (EBV)‐positive diffuse large B‐cell lymphoma (DLBCL) not otherwise specified is defined as monoclonal EBV+ B‐cell proliferation affecting patients without any known immunosuppression. Non‐neoplastic EBV+ cells proliferating in or adjacent to EBV− DLBCL were reported recently, but their clinical significance is unclear. Thus, the aim of this study was to investigate the prognostic impact of EBV+ cells in DLBCL.