Atsuko Ashida

NADPH oxidase 4 contributes to transformation phenotype of melanoma cells by regulating G2-M cell cycle progression.

Generation of reactive oxygen species (ROS) has been implicated in carcinogenic development of melanoma, but the underlying molecular mechanism has not been fully elucidated. We studied the expression and function of the superoxide-generating NADPH oxidase (Nox)4 in human melanoma cells. Nox4 was up-regulated in 13 of 20 melanoma cell lines tested. Silencing of Nox4 expression in melanoma MM-BP cells by small interfering RNAs decreased ROS production and thereby inhibited anchorage-independent cell growth and tumorigenecity in nude mice. Consistently, a general Nox inhibitor, diphenylene iodonium, and antioxidants vitamine E and pyrrolidine dithiocarbamate blocked cell proliferation of MM-BP cells. Flow cytometric analysis indicated that Nox4 small interfering RNAs and diphenylene iodonium induced G(2)-M cell cycle arrest, which was also observed with another melanoma cell line, 928mel. This was accompanied by induction of the Tyr-15 phosphorylated, inactive form of cyclin-dependent kinase 1 (a hallmark of G(2)-M checkpoint) and hyperphosphorylation of cdc25c leading to its increased binding to 14-3-3 proteins. Ectopic expression of catalase, a scavenger of ROS, also caused accumulation of cells in G(2)-M phase. Immunohistochemistry revealed that expression of Nox4 was detected in 31.0% of 13 melanoma patients samples, suggesting the association of Nox4 expression with some steps of melanoma development. The findings suggest that Nox4-generated ROS are required for transformation phenotype of melanoma cells and contribute to melanoma growth through regulation of G(2)-M cell cycle progression.

Pathological activation of KIT in metastatic tumors of acral and mucosal melanomas

Recent studies showed KIT gene aberrations in a substantial number of melanomas on acral skin and mucosa, suggesting the therapeutic benefit of tyrosine kinase inhibitors, such as imatinib. We therefore examined the expression and mutations of KIT in 4 primary and 24 metastatic acral and mucosal melanomas. Immunohistochemistry revealed moderate or strong KIT protein expression in 13 (48%) tumors. Sequence analysis revealed K642E and D820Y mutations in two metastases. Amplification of KIT was identified by real‐time PCR in 4 tumors, including one that had K642E. Western blot analysis showed phosphorylation of the KIT receptor in 8 (62%) of 13 cryopreserved samples, indicating the frequent pathological activation of the receptor in vivo. Phosphorylation of KIT protein was detected in 2 tumors harboring KIT mutations, as well as in one tumor with KIT gene amplification. Furthermore, 5 tumors without detectable KIT gene aberrations showed phosphorylation of the KIT receptor. Expression of stem cell factor (SCF) in melanoma cells as well as stromal cells suggests SCF/KIT autocrine and paracrine activation in these tumors. Finally, we found significant growth suppressive effects of sunitinib in two acral melanoma cell lines; one harboring the D820Y mutation and one showing SCF‐dependent KIT activation. These results show pathological activation of KIT in a substantial number of metastatic tumors of acral and mucosal melanomas, and suggest a potential therapeutic benefit of sunitinib for these melanomas.

Interferon-β therapy for malignant melanoma: the dose is crucial for inhibition of proliferation and induction of apoptosis of melanoma cells

We investigated the anti-tumor effect of human interferon-β (HuIFN-β) against malignant melanoma. In vitro study revealed that HuIFN-β not only inhibited proliferation of melanoma cells (seven cell lines: MM-AN, MM-BP, MM-LH, MM-RU, PM-WK, RPM-EP, RPM-MC) but also induced apoptosis in a dose dependent fashion, though the sensitivity to HuIFN-β was different among cell lines. In addition, we administered HuIFN-β into cutaneous metastatic lesions of melanoma and evaluated clinical and histopathological effects. Although the size of the metastatic cutaneous lesion did not change by the intralesional injection of HuIFN-β, histopathological examination revealed apoptotic changes of melanoma cells along with dense lymphohistiocytic infiltration. The present study confirmed direct and indirect inhibitory effects of HuIFN-β on human melanoma cells and suggests that local higher concentration of HuIFN-β is needed to eradicate melanoma lesions.

Clinical characteristics associated with BRAF, NRAS and KIT mutations in Japanese melanoma patients

BACKGROUND The importance of the genetic background of melanoma cells to the individual susceptibility to treatment has become apparent. In Caucasians, BRAF mutations are frequently detected in lesions on the skin of younger patients compared to NRAS and KIT mutations. However, clinical and pathological characteristics associated with BRAF, NRAS and KIT mutations have not been fully evaluated in East Asians. OBJECTIVE To clarify clinical and pathological characteristics associated with BRAF, NRAS and KIT mutations in Japanese melanoma patients. METHODS Clinical data were retrospectively collected from 11 hospitals in Japan. BRAF, NRAS and KIT mutations were evaluated with polymerase chain reaction and Sanger sequencing. The relationships between these gene mutations and pathological and clinical findings were analyzed. RESULTS The number of cases examined was 171 (primary: 135, metastases: 11, paired: 25), and all were Japanese patients. The detection rates of BRAF, NRAS and KIT mutations were 30.4%, 12.3% and 12.9%, respectively. Compared with the wild type, the presence of BRAF mutations was significantly associated with younger age (median, 50.0 years vs. 70.0 years, p<0.001). BRAF mutation was frequently detected in the lesions of the scalp (80%; 4/5), trunk (72.0%; 18/25), extremities (56.7%; 17/30) and neck (44.4%; 4/9), and the least prevalent were the face (22.2%; 2/9), nail (12.5%; 3/24), palm or sole (8.9%; 4/45) and mucosa (0%). NRAS mutations were prevalent in the face (33.3%) and palm or sole (20.0%), and the median age of these patients was 70.5 years. A KIT mutation was observed in the nail apparatus (25%), palm or sole (15.6%) and mucosa (18.2%). The median age of the patients with a KIT mutation was 63.0 years. Heterogeneity of mutations between primary and metastatic lesions was detected in six of 25 cases (24%). Solar elastosis was identified in 12 of 71 cases (15.3%), among which four cases harbored BRAF(V600E) (2 cases), BRAF(V600K), NRAS(Q61K) or NRAS(Q61L), respectively. CONCLUSION Some clinical characteristics associated with BRAF, NRAS and KIT mutations were observed in Japanese patients, and we observed both similarities to and differences from those of Caucasians. Our findings could provide useful information in efforts to clarify the tumor genesis of malignant melanomas.

Clinical course of drug‐induced hypersensitivity syndrome treated without systemic corticosteroids

Background /aim  Drug‐induced hypersensitivity syndrome (DIHS) is a severe reaction to drugs which characteristically occurs after a long latency period. In addition, human herpes virus 6 (HHV‐6) reactivation is a characteristic finding in DIHS, which has been known to be related to disease severity. Because DIHS has generally been treated by systemic corticosteroids, the natural clinical course is not clear.

Coexpression of EpCAM, CD44 variant isoforms and claudin-7 in anaplastic thyroid carcinoma.

Background Anaplastic thyroid cancer is considered to be one of the most aggressive human malignancies, and the mean survival time after diagnosis is approximately six months, regardless of treatments. This study aimed to examine how EpCAM and its related molecules are involved in the characteristics of anaplastic thyroid carcinoma. Methodology/Principal Findings Two differentiated thyroid cancer cell lines (TPC-1 and FTC-133), and two anaplastic thyroid cancer cell lines (FRO, ACT-1) were analyzed for expression of CD44 standard isoform (CD44s), CD44 variant isoforms, and EpCAM, and human aldehyde dehydrogenase-1 (ALDH1) enzymatic activity using flow cytometry. CD44s expression was higher in TPC-1 and FTC-133 than in the FRO and ACT-1, whereas ALDH1 activities were higher in FRO and ACT-1 than in TPC-1 and FTC-133. An inverse correlation between CD44s expression and ALDH1 activity was observed in all thyroid cancer cell lines. As for the expressions of CD44 variant isoforms, ACT-1 showed higher and FRO showed moderate CD44v6 expressions, whereas either TPC-1 or FTC-133 showed negative CD44v6 expression. EpCAM expressions in FRO and ACT-1 were higher than those in TPC-1 and FTC-133, and EpCAM expressions inversely correlated with those of CD44s. A positive correlation was observed between EpCAM expression and ALDH1 activity in thyroid cancer cell lines. In the RT-PCR analysis, the expression levels of EpCAM, caludin-7 and ALDH1 in FRO and ATC-1 cells were significantly higher than those in TPC-1 and FTC-133 cells. In clinical specimens of thyroid cancers, nuclear expression of EpCAM and high expression of CD44v6 were detected significantly more frequently in anaplastic carcinomas. Conclusions/Significance Our study suggests the possibility that EpCAM, together with CD44v6 and claudin-7 as well as ALDH1, may be involved in the development of the aggressive phenotype of anaplastic thyroid carcinoma. Our findings may suggest a novel therapeutic strategy for treatment of anaplastic thyroid carcinoma.

Establishment of a novel melanoma cell line SMYM-PRGP showing cytogenetic and biological characteristics of the radial growth phase of acral melanomas

We established a novel melanoma cell line, SMYM‐PRGP, which was non‐tumorigenic in vivo, from an acral melanoma in radial growth phase under a low‐oxygen environment. SMYM‐PRGP was wild‐type for known mutation sites in the BRAF and NRAS genes, and showed focal amplification of the human telomerase reverse transcriptase and cyclin D1 genes as well as the fibroblast growth factor‐3 and fibroblast growth factor‐4 genes. Neither mutation nor copy number loss of the CDKN2A gene was observed. The p16INK4A protein was expressed at a level equal to that in normal melanocytes. Among the various melanocyte growth factors added to the culture of SMYM‐PRGP cells, endothelin‐1 was the strongest growth stimulator, the effect of which was significantly augmented by the addition of calcium chloride. The growth stimulatory effect of endothelin‐1 was shown to be mediated via the endothelin B receptor. The protein level of cyclin D1 in SMYM‐PRGP cells was approximately 10 times higher than that in normal melanocytes. Although the stimulation with endothelin‐1 plus calcium chloride increased cyclin D1 protein levels after 4–6 h, the level of phosphorylated retinoblastoma protein did not increase, suggesting that overexpression of cyclin D1 protein may have little effect on cell cycle progression but rather act as a pro‐survival factor. SMYM‐PRGP is an excellent tool for investigating the development and progression of acral melanoma. (Cancer Sci 2007; 98: 958–963)

Melanoma with BRAF Mutation in Circulating Cell-free DNA despite no Mutation in the Primary Lesion: A Case Report.

Substantial advances have been made recently in the treatment of metastatic melanoma, with the advent of therapies such as BRAF inhibitors (iBRAF), which have been shown to be effective in the treatment of BRAF mutation-positive melanoma (1). The frequency of BRAF mutations is 50–60% in Caucasians and 20–30% in Asian populations (1–3). Mutation is usually determined by Sanger sequencing and high-sensitivity PCR-based methods, such as the Cobas 4800 BRAF Mutation Test (Roche Molecular Diagnostics, Basel, Switzerland), using primary or metastatic lesions. However, the accuracy of these tests is grossly influenced by the quality and quantity of the DNA extracted from the tissues. Thus, BRAF mutation can be missed in cases of contamination, widespread necrosis, or when only small quantities of DNA are available. We describe here a case of advancedstage melanoma with a BRAFV600E mutation. Although the BRAFV600E mutation was not detected in the DNA of the primary lesion, it was detected in circulating cell-free DNA (cfDNA) in peripheral blood. cfDNA includes DNA from apoptotic and necrotic cancer cells, it harbours the genetic alterations present in the tumour (4, 5), and is useful for evaluating tumour features in patients with advanced cancer.

Circulating Tumour DNA for Monitoring Treatment Response to Anti-PD-1 Immunotherapy in Melanoma Patients

Anti-programmed cell death-1 (anti-PD-1) antibody shows high therapeutic efficacy in patients with advanced melanoma. However, assessment of its therapeutic activity can be challenging because of tumour enlargement associated with intratumoural inflammation. Because circulating tumour DNA (ctDNA) correlates with tumour burden, we assessed the value of ctDNA levels as an indicator of tumour changes. Quantification of ctDNA (BRAFmutant or NRASmutant) levels by droplet digital PCR in 5 patients with BRAF or NRAS mutant melanoma during the treatment course showed dynamic changes corresponding to radiological and clinical alterations. In 3 cases in which the anti-PD-1 antibody was effective, ctDNA levels decreased within 2-4 weeks after treatment initiation. In 2 cases in which the anti-PD-1 antibody was ineffective, ctDNA levels did not decrease after treatment initiation. ctDNA could be a useful biomarker to predict early response to treatment in patients with advanced melanoma treated with anti-PD-1 immunotherapy.

Low p53 positivity in verrucous skin lesion in diabetic neuropathy occurring on the dorsum of the foot

Typically, the skin findings will improve with remission of malignancy but may subsequently worsen with recurrence of the primary neoplasm; however, when patients exhibit MAN eruption, the malignancy is usually already in the progressive stage, and it may therefore be difficult to achieve complete resolution of MAN. Although there is a predilection for flexural areas, eruptions may become widespread and generalized in cases of MAN, especially in severe cases. The unique feature of the present case is not only that the eruptions became generalized but also that the major skin folds of the abdomen and preaxillar regions were spared, producing the deck-chair sign. The deck-chair sign defines a peculiar clinical pattern observed in patients with PEO, characterized by selective sparing of major skin folds and flexures. Although the deck-chair sign was originally considered a specific feature of PEO, this sign has also been observed in certain other skin diseases. However, the deck-chair sign has never been reported in association with MAN in the English literature so far. PEO is essentially a clinical diagnosis and its etiology is unclear. Therefore, the reason skin creases is spared and its clinical significance is also obscure. The present case provides another example suggesting that the deck-chair sign is not a specific feature of PEO but rather a pattern of expression in several inflammatory dermatoses. However, the precise pathogenesis and clinical significance of the deck-chair sign remains to be elucidated.