Masako Yokoo

Retrospective and prospective studies of hepatitis B virus reactivation in malignant lymphoma with occult HBV carrier

BACKGROUND In surface antigen of hepatitis B virus (HBsAg)-positive carrier for anticancer treatment of malignant lymphoma, it is well recognized that reactivation of hepatitis B virus (HBV) occasionally occurs. However, there have been only a few studies of HBV reactivation in serum HBsAg-negative and hepatitis B core antigen (HBcAb)-positive occult HBV carriers. We looked at both retrospective and prospective studies to determine the prevalence, clinical course and risk factor of HBV reactivation during chemotherapy in lymphoma patients. PATIENTS AND METHODS Forty-eight of 127 (37.8%) lymphoma patients were HBsAg negative and HBcAb positive, and 24 of these patients were then given liver function tests and HBsAg tests monthly and serum HBV DNA every 3 months. RESULTS HBV reactivation was observed in two patients (4.1%) who had received intensive chemotherapy including steroid and rituximab. Immediate administration of entecavir therapy after elevation of HBV DNA level was conducted, and this resulted in reduction of it and improvement of liver function test. CONCLUSIONS Rituximab plus steroid-containing regimens may increase the risk of HBV reactivation in HBsAg-negative and HBcAb-positive lymphoma patients. More ambitious prospective studies are required to establish clinically useful or cost-effective follow-up methods for control of HBV reactivation in lymphoma patients with occult HBV infection.

Induction of p53-mediated transcription and apoptosis by exportin-1 (XPO1) inhibition in mantle cell lymphoma.

The nuclear transporter exportin‐1 (XPO1) is highly expressed in mantle cell lymphoma (MCL) cells, and is believed to be associated with the pathogenesis of this disease. XPO1‐selective inhibitors of nuclear export (SINE) compounds have been shown to induce apoptosis in MCL cells. Given that p53 is a cargo protein of XPO1, we sought to determine the significance of p53 activation through XPO1 inhibition in SINE‐induced apoptosis of MCL cells. We investigated the prognostic impact of XPO1 expression in MCL cells using Oncomine analysis. The significance of p53 mutational/functional status on sensitivity to XPO1 inhibition in cell models and primary MCL samples, and the functional role of p53‐mediated apoptosis signaling, were also examined. Increased XPO1 expression was associated with poor prognosis in MCL patients. The XPO1 inhibitor KPT‐185 induced apoptosis in MCL cells through p53‐dependent and ‐independent mechanisms, and p53 status was a critical determinant of its apoptosis induction. The KPT‐185‐induced, p53‐mediated apoptosis in the MCL cells occurred in a transcription‐dependent manner. Exportin‐1 appears to influence patient survival in MCL, and the SINE XPO1 antagonist KPT‐185 effectively activates p53‐mediated transcription and apoptosis, which would provide a novel strategy for the therapy of MCL.

2-Hydroxypropyl-β-Cyclodextrin Acts as a Novel Anticancer Agent.

2-Hydroxypropyl-β-cyclodextrin (HP-β-CyD) is a cyclic oligosaccharide that is widely used as an enabling excipient in pharmaceutical formulations, but also as a cholesterol modifier. HP-β-CyD has recently been approved for the treatment of Niemann-Pick Type C disease, a lysosomal lipid storage disorder, and is used in clinical practice. Since cholesterol accumulation and/or dysregulated cholesterol metabolism has been described in various malignancies, including leukemia, we hypothesized that HP-β-CyD itself might have anticancer effects. This study provides evidence that HP-β-CyD inhibits leukemic cell proliferation at physiologically available doses. First, we identified the potency of HP-β-CyD in vitro against various leukemic cell lines derived from acute myeloid leukemia (AML), acute lymphoblastic leukemia and chronic myeloid leukemia (CML). HP-β-CyD treatment reduced intracellular cholesterol resulting in significant leukemic cell growth inhibition through G2/M cell-cycle arrest and apoptosis. Intraperitoneal injection of HP-β-CyD significantly improved survival in leukemia mouse models. Importantly, HP-β-CyD also showed anticancer effects against CML cells expressing a T315I BCR-ABL mutation (that confers resistance to most ABL tyrosine kinase inhibitors), and hypoxia-adapted CML cells that have characteristics of leukemic stem cells. In addition, colony forming ability of human primary AML and CML cells was inhibited by HP-β-CyD. Systemic administration of HP-β-CyD to mice had no significant adverse effects. These data suggest that HP-β-CyD is a promising anticancer agent regardless of disease or cellular characteristics.

Correlation between Plasma DNA and Tumor Status in an Animal Model

Overcoming metastasis is one of the most important issues with lung cancer. Since metastasis arises through complex steps, a suitable animal model is indispensable for investigation of metastasis. To establish an animal model reflecting human metastatic lung cancers, we used NOD/SCID/Jak3null (NOJ) mice, which exhibit deficiencies in NK cell activity, macrophage and dendritic cell function, and complement activation, as well as T and B cell deficiencies. After screening twenty human lung cancer cell lines through expression patterns of E-cadherin and vimentin according to epithelial mesenchymal transition features, an H1975 cell line carrying EGFR mutations, L858R and T790M, was selected for investigation. Inoculation of the cells into the dorsal flanks caused systemic metastases after one month in lymph nodes, liver, lung, and peritoneum, suggesting that metastases occurred both lymphogenically and hematogenously. We confirmed the existence of H1975 cells in metastatic lesions by detection of T790M and L858R using the mutation-biased PCR and quenching probe (MBP-QP) system previously established in our laboratory. In addition, tumor-derived plasma DNA could be detected using the MBP-QP method. The amount of tumor-derived DNA was associated with tumor volume, whereas an unrelated large amount of tumor-derived DNA was circulating in the presence of metastasis. We present a novel animal model with systemic metastasis with human lung cancer cells. The amount of tumor derived DNA would be related with tumor volume and tumor progression such as metastasis.

Non-germinal cell phenotype and bcl-2 expression in primary adrenal diffuse large B-cell lymphoma

Primary adrenal lymphoma is a rare subtype observed in only 0.83% of all non-Hodgkin lymphoma [1], although the adrenal gland may be involved in up to 25% of NHL cases, by autopsy [2]. The immunophenotype of primary adrenal lymphoma is usually B-cell, with a few T-cell types reported [1,3 – 6]. About 60% of primary adrenal lymphoma patients have bilateral involvement and two-thirds show adrenal insufficiency. Previous reports found that most primary adrenal lymphoma patients belonged to high-risk group by IPI classification, associated with poor prognosis with only 4 months over all survival [1]. It was recently shown that diffuse large B-cell lymphoma (DLBCL) can be divided into three prognostically important subgroups: germinal center B-cell-like (GCB)/DLBCL, activated B-cell-like DLBCL, and type 3, based on gene expression profiles using a cDNA microarray [7,8]. Germinal center B-cell-like DLBCL has a better prognosis than either activated B-cell or type 3. Hans et al. proposed that the immunohistochemical expression pattern of CD10, Bcl-6 and MUM-1 could classify cases of DLBCL into GCB and non-germinal center B-cell type (non-GCB), and predict survival similar to the cDNA microarray [9]. The majority of primary adrenal lymphoma cases are diffuse large B-cell lymphoma, but no precise immunohistochemical examination has been reported. We examined the phenotype of primary adrenal diffuse large B-cell lymphoma (PA-DLBCL) using immunohistochemistry for CD10, Bcl-6, MUM-1 and bcl-2. We found four cases (1.0%) of primary adrenal lymphoma among 390 cases of NHL diagnosed at Saga University Hospital from 1997 to 2006. PAL was diagnosed when the extra nodal site was the only adrenal gland mass, or when the bulk of disease was confined to site of adrenal gland [10] and tissue sample could be obtained only from adrenal mass. The four cases included three men and one woman from 48 to 78 years old, and CT or MRI showed bilateral adrenal masses in all cases. These tumors appeared to be complex masses with variable density in CT scan, no apparent hemorrhage or fibrotic change, and detectable normal adrenal glands. Adrenal insufficiency was present in Case 2 and 4, and bulky mass (410 cm) was observed in Case 2 and 4. Only Case 3 was stage IE, the other cases being stage IV. Case 1 showed B-symptom (fever), a high level of lactate dehydrogenase (LDH) was observed in Case 1, 2 and 4, and a high level of soluble IL-2 receptor (sIL-2R) were present in all cases. International prognostic index (IPI) was high or high-intermediate in three of these cases. The definitive diagnostic procedures for histopathological diagnosis were resection (Case 1 and 4) and ultrasound scan guided needle biopsy (Case 2 and 3). Hematoxylin and eosin staining showed that all cases demonstrated diffuse proliferation of large lymphoid cells [Figure 1(a)]. Case 2 included pleomorphic features, with most of these cells noncleaved/centroblastic morphology; Case 2 and 4 had fibrosis in various degrees; and Case 4 had

Comparative Study of the Anti-leukemic Effects of Imatinib Mesylate, Glivec™ Tablet and Its Generic Formulation, OHK9511

Long-term treatment with imatinib mesylate (IM) allows patients with chronic myeloid leukemia (CML) to live a near-normal lifespan. However, the fact that tyrosine kinase inhibitors, including IM, are extremely expensive is a major cause of poor adherence, resulting in disease relapse or drug resistance. Therefore, physicians are encouraged to prescribe generic drugs to reduce the financial burden of medical expenses. In Japan, only generic drugs that have a basic chemical structure and pharmacokinetic data that are the same as those of the original drug are approved. However, it is not mandatory to demonstrate that generic drugs have adequate biological effects. This is one of the reasons why Japanese hematologists do not often use generic IM. The aim of the present study was to compare the anti-leukemic effects of Glivec™ (a commercial IM) and its generic formulation, OHK9511. The IC50 values of OHK9511 and Glivec™ were comparable, and both induced similar levels of apoptosis in several CML cell lines. Furthermore, the overall survival of OHK9511-treated mice transplanted with BCR-ABL-positive cells was similar to that of mice treated with Glivec™. Although the experiments performed herein were basic, the results suggest that physicians should consider using generic IM.

Harmonization of quantitative BCR-ABL measurements using the secondary reference material anchored to the WHO primary standards.

Sir, The oncogenic BCR-ABL fusion gene is a hallmark of chronic myeloid leukemia (CML), and tyrosine kinase inhibitors (TKIs) specifically disable the constitutive activity of the BCR-ABL kinase [1–4]. Responses to TKI treatment were defined using the practice guidelines issued by the National Comprehensive Cancer Network (NCCN) and the European Leukemia Net (ELN), which recommend measuring BCR-ABL gene expression relative to that of a control gene in bone marrow or peripheral blood samples [5–7]. The International Randomized Study of Interferon and STI571 (IRIS) demonstrated that a 3-log reduction of BCR-ABL mRNA compared with a standardized transcript baseline level of diagnostic CML is a desirable therapeutic response [1, 8]. Because of an extensive variation in the BCR-ABL mRNA levels reported by individual laboratories [9, 10], efforts have been made to standardize the reporting of real-time quantitative polymerase chain reaction (RQ-PCR) results using the International Scale (IS) percentage. The IS percentage (% BCR-ABL IS) does not require a baseline value at diagnosis, and it exhibits better correlation with the complete cytogenetic response (CCyR) than the log reduction method [11]. A sample exchange system with reference laboratories has been shown to be effective in comparing the results from different laboratories. However, the validation procedure is a lengthy and costly and requires continuous revalidation [12–14]. Therefore, now, the effort is ongoing to distribute IS value predefined reference materials. The World Health Organization International Genetic Reference Panel for the quantitation of BCR-ABL1 mRNA (World Health Organization document, World Health Organization/BS/09.2106) has been distributed to manufacturers to generate secondary reference materials [13]. Currently, several manufacturers are attempting commercial production of secondary materials. It was recently reported that a synthetic Armored RNA Quant IS Calibrator Panel (ARQ IS; Asuragen, Inc., Austin, TX, USA) provides accurate BCRABL IS values [14]. In this study, we aimed to utilize commercially produced secondary reference materials to standardize BCR-ABL measurements. We calibrated our laboratory-developed (local) RQ-PCR values using synthetic ARQ IS reference panels with a sensitivity of 0.0033% of the IS. Peripheral blood samples were collected from 38 CML patients who achieved a complete hematological response (defined as normalization of blood cell counts with no immature cells, <5% basophils and no palpable spleen) [15] receiving imatinib, nilotinib, dasatinib, or interferon therapy at the Saga University Hospital in Japan between February and April 2013. The diagnosis of CML was determined through the detection of the Philadelphia chromosome using cytogenetic evaluation and/or BCR-ABL transcription using RQ-PCR. Patients provided written informed consent, and the study was approved by the local research ethics committee. Total leukocyte RNA was extracted using a QIAamp RNA Blood Mini Kit (Qiagen, Hilden, Germany), and Transcriptor Universal cDNA Master reverse transcriptase (Roche Diagnostics, Mannheim, Germany) was used for cDNA synthesis. cDNA was amplified by 55 cycles of RTPCR using the LightCycler 2.0 (Roche Diagnostics) and LightCycler TaqMan Master in accordance with the manufacturer’s instructions. ABL was used as the control gene. The primers and the probes used were as follows: BCR-ABL; forward primer, 50-TGACCAACTCGTGTGTGA AACTC-30; reverse primer, 50-CACTCAGACCCTGAGGCT CAA-30; probe, 50-CCCTTCAGCGGCCAGTAGCATCTGA -30, ABL; forward primer, 50-CGAAGGGAGGGTGTACCAT TA-30; reverse primer, 50-CAACTCGGCCAGGGTGTT-30; probe, 50-CTTCTGATGGCAAGCTCTACGTCTCCTCC-30. The sequences were obtained from GenBank Accession No. X02596 for BCR and No. X16416 for ABL. The probe contained the fluorescent reporter dye 6-carboxyfluorescein (FAM) at the 50-end and the fluorescent quencher dye Black Hole Quencher (BHQ) at the 30-end. The results were reported as a BCR-ABL/ABL ratio (%). An in vitro transcribed RNA of the BCR-ABL gene from the K562 cell line was used to determine the lower

Successful reduced-intensity umbilical cord blood transplant for fulminant hemophagocytic syndrome in an adult with pre-existing rheumatoid arthritis and autoimmune hemolytic anemia

Hemophagocytic syndrome (HPS) is a potentially fatal syndrome of dysregulated immune activation characterized by severe clinical manifestations such as pancytopenia, high fever and hepatosplenomega...

Abstract 3093: Gene regulation induced by constitutive expression of HIF-1α in transgenic mice

Hypoxia-inducible factor-1α (HIF-1α) has a central role on regulation of various genes under hypoxic condition linked to regulation of angiogenesis, metabolism and tumor development. Expression of HIF-1α was reported in many cancers associated with tumor progression, invasion and metastasis. We reported that constitutive expression of HIF-1α in transgenic mice induced tumors in lymphoid, lung and breast tissues in last this meeting. The incidence of tumors in transgenic mice was up to 90% until 18 months after birth. To clarify the molecular mechanism of lymphomagenesis in the transgenic mice, lymphocyte subsets, gene expression and mitogenic responsiveness on lymphoid tissues were analyzed. Although population of T and B cell subset in spleen, maturation pattern of T cells in thymus, or cell growth rate after treatment with various mitogens were not changed by HIF-1α overexpression, the lymphocytes from transgernic mice showed longer survival than those from wild mice. The lymphocytes also showed resistance for genotoxic stimuli like topoisomerase inhibitors. We next compared gene expression profile on lymphocyte from transgenic mice and wild mice by cDNA microarray and RT-PCR. The expression of over a hundred of genes including HIF-1α itself was altered on T and B cells in transgenic mice. Among them the gene associated with cell survival and cell transformation-related genes were further analyzed by validation study. In this paper, we present the results concerning the role of HIF-1α in lymphoma occurrence, and will discuss the contribution of the transcription factor in human cancer development. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3093. doi:10.1158/1538-7445.AM2011-3093

Successful treatment of post-transplant relapsed adult T cell leukemia after cord blood transplantation with low-dose, short-term lenalidomide

Adult T cell leukemia (ATL) is a peripheral T cell malignancy associated with human T cell leukemia virus type I infection. ATL is incurable with conventional chemotherapies, and allogeneic stem ce...