Yujin Kobayashi

Haplotype-Based Case-Control Study Revealing an Association between the Adrenomedullin Gene and Proteinuria in Subjects with Essential Hypertension

Adrenomedullin (AM) has various physiological actions on the cardiovascular system, including vasodilatation, diuresis, natriuresis, inhibition of aldosterone secretion, and increases of the cardiac output, all of which cause hypotension. Since AM plays a role in the pathophysiology of vascular diseases, genes controlling AM might be involved in the development and etiology of essential hypertension (EH). However, there have been few studies examining the relationship between the AM gene and hypertension. The aims of this study were to genotype some of the genetic markers for the human AM gene in Japanese subjects, and via a haplotype-based case-control study, assess the association between and the AM gene and EH or its risk factors, such as hyperlipidemia, renal damage, and proteinuria. We genotyped 205 EH patients and 210 age-matched normotensive (NT) individuals for two single nucleotide polymorphisms of rs4399321, rs7944706 and a microsatellite polymorphism located approximately 5,400 base pairs downstream of the 3′ end of the human AM gene. The overall distribution in each variant and haplotype did not significantly differ between the two groups. However, after dividing the groups into those subjects with and without proteinuria, the haplotype analysis revealed a positive association. In conclusion, a possible mutation linked to the haplotype may indicate a genetic predisposition for proteinuria in EH.

Identification and analysis of an early diagnostic marker for malignant melanoma: ZAR1 intra-genic differential methylation

BACKGROUND Epigenetic changes such as aberrant DNA methylation and histone modification have been shown to play an important role in the tumorigenesis of malignant melanoma. OBJECTIVE To identify novel tumor-specific differentially methylated regions (DMRs) in human malignant melanoma. METHODS The aberrant methylation at 14 candidate human genomic regions identified through a mouse model study with quantitative DNA methylation analysis using the Sequenom MassARRAY system was performed. RESULTS The CpG island Exon 1 region of the Zygote arrest 1 (ZAR1) gene, which is responsible for oocyte-to-embryo transition, showed frequent aberrant methylation of 28 out of 30 (93%) melanoma surgical specimens, 16 of 17 (94%) melanoma cell lines, 0% of 4 normal human epidermal melanocyte (NHEM) cell lines, 0% of 10 melanocytic nevi and 100% of 51 various cancer cell lines. According to the real-time RT-PCR, the ZAR1 gene was overexpressed in part of the hypermethylated cell lines, while its low expression with bivalent histone methylation status was seen in unmethylated cell lines. CONCLUSION Our findings suggest that the ZAR1 intra-genic differentially methylated region would be a useful tumor marker for malignant melanoma and may be other type of cancers. The involvement of ZAR1 in the carcinogenesis of melanoma, still remains unclear, although we have examined tumorigenic capacities by exogenous full-length ZAR1 over-expression and siRNA knock-down experiments.

Positive impact of chronic graft-versus-host disease on the outcome of patients with de novo myelodysplastic syndrome after allogeneic hematopoietic cell transplantation: a single-center analysis of 115 patients.

To evaluate the impact of graft‐versus‐host disease (GVHD) and prognostic factors for patients with myelodysplastic syndrome (MDS) after allogeneic hematopoietic cell transplantation (allo‐HCT), we retrospectively reviewed 115 patients with MDS or acute myeloid leukemia with multilineage dysplasia (AML‐MLD) after allo‐HCT at our center. Eighty one patients received reduced‐intensity conditioning (RIC) regimens, whereas 34 received myeloablative conditioning regimens. Although the RIC group was significantly older and included more patients with poor cytogenetic risk, no difference in 4‐yr overall survival (OS) was seen between the two groups. In a multivariate analysis, covariates associated with a worse OS were the French‐American‐British stage of refractory anemia excess blasts in transformation/AML‐MLD at peak, poor cytogenetic risk, bone marrow blasts of 20% or higher at HCT and the absence of chronic GVHD (cGVHD). By using semi‐landmark analyses, we found that the presence of cGVHD significantly improved OS in high‐risk patients or the RIC group. However, there was no difference in OS between those with and without cGVHD among low‐risk MDS patients. These findings suggest that the graft‐versus‐leukemia effect may be more beneficial in high‐risk patients who do not receive intensive preparative regimens.

Clinical significance of co-expression of MYC and BCL2 protein in aggressive B-cell lymphomas treated with a second line immunochemotherapy.

Abstract The clinical significance of concurrent expression of MYC and BCL2 protein, known as “double-expressor lymphoma” (DEL), among patients with relapsed or refractory aggressive B-cell lymphomas, remains unclear. A retrospective analysis was performed of 38 patients treated with a salvage treatment consisting of rituximab, ifosfamide, etoposide, cytarabine and dexamethasone followed by consolidative high-dose chemotherapies. A total of 17 cases (45%) were categorized as DEL using immunohistochemical assay with a cut-off value of positivity of 40% for MYC and 50% for BCL2, respectively. DEL was associated with a lower overall response rate (35% vs 71%, p = 0.0481), worse 2-year progression-free survival (9% vs 67%, p = 0.001) and overall survival (35% vs 71%, p = 0.037). This analysis suggests that DEL is common among patients with relapsed/refractory aggressive B-cell lymphomas and that such patients require novel treatment strategies.

Identification of a novel E-box binding pyrrole-imidazole polyamide inhibiting MYC-driven cell proliferation

The MYC transcription factor plays a crucial role in the regulation of cell cycle progression, apoptosis, angiogenesis, and cellular transformation. Due to its oncogenic activities and overexpression in a majority of human cancers, it is an interesting target for novel drug therapies. MYC binding to the E‐box (5′‐CACGTGT‐3′) sequence at gene promoters contributes to more than 4000 MYC‐dependent transcripts. Owing to its importance in MYC regulation, we designed a novel sequence‐specific DNA‐binding pyrrole–imidazole (PI) polyamide, Myc‐5, that recognizes the E‐box consensus sequence. Bioinformatics analysis revealed that the Myc‐5 binding sequence appeared in 5′‐ MYC binding E‐box sequences at the eIF4G1, CCND1, and CDK4 gene promoters. Furthermore, ChIP coupled with detection by quantitative PCR indicated that Myc‐5 has the ability to inhibit MYC binding at the target gene promoters and thus cause downregulation at the mRNA level and protein expression of its target genes in human Burkitts lymphoma model cell line, P493.6, carrying an inducible MYC repression system and the K562 (human chronic myelogenous leukemia) cell line. Single i.v. injection of Myc‐5 at 7.5 mg/kg dose caused significant tumor growth inhibition in a MYC‐dependent tumor xenograft model without evidence of toxicity. We report here a compelling rationale for the identification of a PI polyamide that inhibits a part of E‐box‐mediated MYC downstream gene expression and is a model for showing that phenotype‐associated MYC downstream gene targets consequently inhibit MYC‐dependent tumor growth.

Safety and efficacy of high-dose cyclophosphamide, etoposide and ranimustine regimen followed by autologous peripheral blood stem cell transplant for patients with diffuse large B-cell lymphoma.

Abstract We retrospectively evaluated the safety and efficacy of high-dose chemotherapy consisting of cyclophosphamide, etoposide and ranimustine (CEM) with autologous peripheral blood stem cell transplant (PBSCT) in 55 adult patients with relapsed or high-risk de novo diffuse large B-cell lymphoma (DLBCL) or DLBCL associated with follicular lymphoma. This included 36 patients in the upfront setting in their first complete remission. The median follow-up of 42 patients surviving at the time of the analysis was 52 months (range 1–159). Relapse or disease progression after PBSCT was a frequent cause of death, but no therapy-related mortality associated with PBSCT was observed. The 5-year overall survival and progression-free survival were 70.6% (95% confidence interval [CI], 54.0–82.1) and 57.0% (95% CI, 39.5–71.2), respectively. Chronic renal impairment, therapy-related myelodysplastic syndrome and prostate cancer were the major late complications. The CEM regimen is a tolerable, effective conditioning regimen for autologous PBSCT for DLBCL, with no therapy-related mortality observed.

Negative impact of concurrent overexpression of MYC and BCL2 in patients with advanced diffuse large B-cell lymphoma treated with dose-intensified immunochemotherapy.

Abstract Co-expression of MYC and BCL2 proteins in diffuse large B-cell lymphoma (DLBCL), or ‘double-expressor lymphoma’ (DEL), results in poor patient prognosis, but the significance of DEL when aggressive treatments are applied remains uncertain. We performed a retrospective analysis of 40 patients with de novo DLBCL, who were categorized as being at high/high-intermediate risk according to the age-adjusted International Prognostic Index. Patients underwent an R-Double-CHOP regimen, a dose-intensified immunochemotherapy with or without consolidative high-dose chemotherapy followed by autologous stem cell transplantation. According to immunohistochemical analysis, 10 (25%) patients were categorized as having DEL, showing positivity for MYC (≥40%) and BCL2 (≥50%). The 3 year progression-free survival and overall survival of the DEL group were significantly worse compared with those of the non-DEL group (30% vs. 63%, p = 0.019 and 40% vs. 82%, p = 0.006, respectively). These results suggest that advanced DEL may need discrete treatment strategies.

Successful treatment of secondary Helicobacter pylori eradication for chronic immune thrombocytopenic purpura

Eradication of Helicobacter pylori (H. pylori) is one of the useful treatments of H. pylori-positive chronic immune thrombocytopenic purpura (ITP) [1, 2], and considered as a first-line treatment in Japan [3]. However, there are few reports concerning the efficacy of secondary eradication of residual H. pylori for chronic ITP. Here, we report a case of successful secondary eradication of residual H. pylori for chronic ITP. A 78-year-old man was referred to our hospital because of progressive thrombocytopenia. Initial investigations revealed normal hemoglobin (13.0 g/dl) and leukocyte count (3.5 10/l) and differentiation, but decreased platelet count to 51 10/l. Based on the clinical manifestation, laboratory tests and findings of bone marrow aspiration and biopsy, the patient was diagnosed as having chronic ITP. Because H. pylori infection was present by detecting anti-H.pylori IgG antibody, H. pylori eradication was selected as an initial treatment for ITP with amoxicillin, clarithromycin and proton pomp inhibitor (PPI). The patient had no gastrointestinal manifestation that associated with H. pylori infection or eradication. Two weeks after eradication, the platelet count increased to 88 10/l. However, H. pylori remained positive by the urea breath test. Six months after the first eradication, ITP was exacerbated with the decreased platelet count to 10 10/l, and the patient suffered from macroscopic hematuria. Although immunosuppressive therapy with prednisolone was recommended for the patient as a next therapeutic option, the patient declined any treatment other than secondary eradication of H. pylori. Therefore, after obtaining informed consent from the patient, we decided to treat the patient of secondary eradication with amoxicillin, metronidazole and PPI [4]. No serious adverse effect of this treatment was observed. Three months after the secondary eradication, the platelet count increased to 125 10/l without bleeding, and H. pylori had returned negative by the urea breath test. The platelet count remained normal 6 months after the second eradication. Eradication of H. pylori is now considered one of the first-line treatments for H. pylori-positive ITP, especially in Japan [3], and metronidazole was recommended as second-line therapy after failure of first-line eradication therapy in the Japanese guideline [4]. The duration of treatment will be shorter and less costly than immunosuppressive agents such as prednisolone, or trombopoietin receptor antagonists, and therefore less toxic in terms of long-term adverse effects. Although little is known about the efficacy of secondary eradication of residual H. pylori for ITP, it would be a useful treatment. Further investigation will be required.

Efficacy of a dose-intensified CHOP (Double-CHOP) regimen for peripheral T-cell lymphomas.

Peripheral T-cell lymphomas (PTCLs) are a rare and heterogeneous group of non-Hodgkin lymphomas, often resulting in poor prognoses. The CHOP chemotherapy regimen, which includes cyclophosphamide, doxorubicin, vincristine and prednisone, has been used previously to treat other types of lymphomas. Here, we examined the efficacy and safety of a dose-intensified CHOP regimen (Double-CHOP), which was followed by autologous stem-cell transplantation (ASCT) or high-dose methotrexate (HDMTX), in PTCL patients. Twenty-eight PTCL patients, who received 3 courses of Double-CHOP at our institution, were retrospectively studied from 1996 to 2012. Patients with anaplastic lymphoma kinase-positive anaplastic large-cell lymphoma (ALK+-ALCL) were excluded from this study. The median age of patients was 58 years (range: 17-69). They had low-intermediate (n=11), high-intermediate (n=10) or high (n=7) risk according to the International Prognostic Index (IPI). The overall complete remission (CR) rate following Double-CHOP treatment was 68%. Of the CR patients, 10 successfully tolerated a consolidated high-dose chemotherapy followed by ASCT and 7 received HDMTX. A single case of treatment-related mortality was recorded during the study. On a median 31-month follow-up, the estimated 3- or 5-year overall survival (OS) rates were 68 or 63%, respectively, while 3- or 5-year relapse-free survival (RFS) rates after CR were 60 or 43%, respectively. Although this study included elderly and excluded low-risk IPI and ALK+-ALCL patients, OS results were superiorly favourable, indicating the efficacy of this Double-CHOP regimen. However, an effective treatment strategy for refractory or relapsing patients needs to be validated and established.

Copper deficiency anaemia

A 74-year-old man was admitted to the hospital with progressive leucopenia and anaemia. He had undergone total gastrectomy for gastric carcinoma 31 years previously and ileocaecal resection for colon carcinoma 2 years previously. Laboratory tests showed a haemoglobin concentration of 70 g/l, a white cell count of 1 75 9 10/l (neutrophils, 0 595 9 10/l) and a platelet count of 138 9 10/l. Bone marrow aspiration showed a left shift in granulocyte and erythrocyte maturation, and cytoplasmic vacuolization of erythroid and myeloid precursors (top left, top right). Ring sideroblasts were not found, but other abnormal sideroblasts were present (bottom left, bottom right). Both serum copper (0 9 lmol/l, normal range, 10 4–20 5) and caeruloplasmin (24 mg/l, normal range, 210–370) were decreased, and 24-h urinary excretion of copper was below the detection limit. We diagnosed the patient with copper deficiency anaemia. Copper deficiency is an under-recognized cause of reversible anaemia and leucopenia, particularly neutropenia, and is often misdiagnosed as a myelodysplastic syndrome.