BRAF V600 mutations and pathological features in Japanese melanoma patients.

Ultraviolet radiation is a risk factor for BRAF V600 mutations frequently found in melanomas that cause constitutive BRAF activation. Primary sites of melanoma and the frequency of BRAF mutations might differ between races. Melanoma is rare in Japan (1500-2000 cases/year compared with 132 000/year worldwide) and the frequency and distribution of BRAF V600 mutations are unknown. We aimed to investigate the frequency of BRAF V600 mutations in a cohort of Japanese patients with melanoma and determine the relationship between mutations and clinical/pathologic features. DNA was extracted from 80 formalin-fixed, paraffin-embedded tumours from individuals diagnosed with melanoma. BRAF V600 mutations were detected using the Cobas 4800 System with z480 Analyzer and Cobas 4800 BRAF V600 Mutation Test reagents. BRAF V600 mutations were detected in 41.8% of tested tumours, with an invalid rate of 1.3%. The mutation rate was more than 60% in patients aged less than 60 years and more than 36% in patients with stage III/IV disease. No sex difference in the mutation rate was observed. BRAF V600 mutations were detected in 18.8% of acral lentiginous melanomas (ALMs), 64.7% of superficial spreading melanomas, 50.0% of lentigo maligna melanomas and 20.0% of nodular melanomas. Although the mutation rate was low in ALMs, 36.4% were mutation positive at stage III/IV compared with 9.5% at stage I/II. This study confirmed associations among BRAF V600 mutations, pathological features and subtypes of melanoma. BRAF V600 mutations were more frequent in late-stage ALMs than in early-stage ALMs. Superficial spreading melanomas had similar mutation rates at all stages. These insights suggest improved treatment predictions for stage III/IV melanoma patients.

TLX controls angiogenesis through interaction with the von Hippel-Lindau protein.

TLX is known as the orphan nuclear receptor indispensable for maintaining neural stem cells in adult neurogenesis. We report here that neuroblastoma cell lines express high levels of TLX, which further increase in hypoxia to enhance the angiogenic capacity of these cells. The proangiogenetic activity of TLX appears to be induced by its direct binding to the von Hippel-Lindau protein (pVHL), which stabilizes TLX. In turn, TLX competes with hydroxylated hypoxia-inducible factor (HIF-α) for binding to pVHL, which contributes to the stabilization of HIF-2α in neuroblastoma during normoxia. Upon hypoxia, TLX increases in the nucleus where it binds in close proximity of the HIF-response element on the VEGF-promoter chromatin, and, together with HIF-2α, recruits RNA polymerase II to induce VEGF expression. Conversely, depletion of TLX by shRNA decreases the expression of HIF-2α and VEGF as well as the growth-promoting and colony-forming capacity of the neuroblastoma cell lines IMR-32 and SH-SY5Y. On the contrary, silencing HIF-2α will slightly increase TLX, suggesting that TLX acts to maintain a hypoxic environment when HIF-2α is decreasing. Our results demonstrate TLX to play a key role in controlling angiogenesis by regulating HIF-2α. TLX and pVHL might counterbalance each other in important fate decisions such as self-renewal and differentiation, as well as angiogenesis and anti-angiogenesis.

Personalized treatment in the eradication therapy for Helicobacter pylori.

Helicobacter pylori (HP) is known to be a causative bacterium of gastritis and peptic ulcers. The combination treatment consisting of a proton pump inhibitor (PPI), amoxicillin and clarithromycin (CAM) is widely used in eradication therapy, but the eradication fails in some patients. The main causes are CAM resistance of HP and individual variability in PPI metabolism related to the activity of the cytochrome P450 2C19 (CYP2C19) enzyme. In this study, we examined the usefulness of the prediction of the pharmacotherapeutic efficacy using a newly developed analysis system for HP CAM resistance and CYP2C19 genotypes. After obtaining the informed consent from 45 subjects with HP-positive peptic ulcers, biopsy specimens of the gastric mucosa were obtained by endoscopy. HP DNA extracted from the gastric mucosa was examined by the SELMAP-PCR method, the direct sequencing method or the single-nucleotide primer extension (SNuPE) method. HP detection rates by culture and the SELMAP-PCR method were 71% and 100%, respectively. Among 32 cultured HP, CAM resistance was confirmed in 6 samples by the in vitro drug susceptibility test. CAM-resistant gene mutations were also examined by the SELMAP-PCR method using 32 DNAs from cultured HP and the results were consistent with the drug susceptibility test. Among 22 patients, the eradication rate was 77%. Among 4 patients with CAM resistance determined by both the in vitro drug susceptibility test and the SNuPE method, eradication was successful in one intermediate metabolizer (IM), but not in three extensive metabolizers (EMs). Patients were divided into three groups according to their CYP2C19 phenotype: EMs, IMs and poor metabolizers (PMs). The eradication rates for 6 EMs, 12 IMs and 4 PMs were 33.3%, 91.7% and 100%, respectively. Based on these results, the information on CAM resistance in HP and CYP2C19 phenotypes in carriers could predict the pharmacotherapeutic efficacy and probability of eradication. It can then be possible to vary the dosing or to select another drug by the prediction of the pharmacotherapeutic efficacy.

Neurotrophic receptor tyrosine kinase B induces c-fos-associated cell survival.

The neurotrophic receptor tyrosine kinase B (TrkB) is a cell surface receptor for brain-derived neurotrophic factor (BDNF) with kinase activity. This protein is expressed in various tumors and thought to participate in various cellular processes. In this study, we established 293T cells stably expressing human TrkB to elucidate its intracellular functions. Using this cell system, we examined the biological roles of TrkB and its downstream target molecules. The TrkB expressing cells showed an increased survival rate through increased c-fos mRNA expression by BDNF, which were completely suppressed by TrkB inhibitor. Moreover, the combination of inhibitors of mitogen-activated protein kinase (MEK) and phosphatidylinositol 3-kinase (PI3K) partially reduced both the cell survival rate and c-fos mRNA expression, whereas monotreatment of these reagents could not affect cell survival nor c-fos mRNA expression. These results suggested that TrkB could play a role in c-fos-associated cell survival through both MEK and PI3K pathway. It is conceivable that activation of TrkB has a significant impact on tumorigenesis and metastasis.