Kazutoshi Harada

Mechanisms underlying the dysfunction of melanocytes in vitiligo epidermis: role of SCF/KIT protein interactions and the downstream effector, MITF-M.

Little is known about the mechanisms involved in the dysfunction of melanocytes in vitiligo epidermis. It is hypothesized that some cytokine/receptor interactions may be affected, resulting in dysfunction and/or loss of melanocytes. This study has compared the expression of endothelin (ET)‐1, the ET‐1 receptor (ETBR), granulocyte macrophage colony stimulating factor (GM‐CSF), stem cell factor (SCF), the SCF receptor (KIT protein), tyrosinase, and S100α between lesional and non‐lesional vitiligo epidermis. Analysis by reverse transcription‐polymerase chain reaction (RT‐PCR) and by western blotting for ET‐1 and SCF unexpectedly demonstrated up‐regulated expression of these cytokines in lesional vitiligo epidermis. Immunohistochemistry with antibodies to melanocyte markers revealed that at the edge of the lesional epidermis, melanocytes remain and express tyrosinase, S100α and ETBR, but not KIT protein or melanocyte‐specific microphthalmia‐associated transcription factor (MITF‐M). Quantitation of the staining revealed a slight or moderate decrease in the number of S100α, tyrosinase, and ETBR‐positive cells at the edge of the lesional epidermis. In contrast, the number of cells expressing KIT protein was markedly decreased at the edge of the lesional epidermis compared with the non‐lesional epidermis. At the centre of the lesional epidermis, there was complete loss of melanocytes expressing KIT protein, S100α, ETBR, and/or tyrosinase. Western blotting revealed down‐regulated expression of c‐kit and MITF‐M proteins at the edge of the lesional epidermis in vitiligo. These findings suggest that reduction in the expression of KIT protein by melanocytes and its downstream effectors, including MITF‐M, may be associated with the dysfunction and/or loss of melanocytes in vitiligo epidermis. Copyright

Mutation analysis of BRAF and KIT in circulating melanoma cells at the single cell level

Background:The availability of molecular-targeted therapies for the treatment of melanoma has emphasised the need to identify mutations in target genes such as BRAF and KIT. Circulating tumour cells (CTC) are present in the peripheral blood of a significant proportion of cancer patients.Methods:High molecular weight melanoma-associated antigen (HMW-MAA) was used to isolate melanoma cells from peripheral blood as it is selectively expressed at high levels on melanomas. The HMW-MAA-positive cells were isolated using immunomagnetic beads. After removing CD45+ cells, CTC were identified by staining with MART-1- and gp100-specific antibodies (HMW-MAA+, CD45−, MART-1/gp100+). Single, isolated CTC were then subjected to BRAF and KIT mutational analysis.Results:CTC (HMW-MAA+, CD45−, MART-1/gp100+) were isolated from the blood of 11 patients and BRAF and KIT were sequenced in nine and four patients, respectively. The BRAF sequences identified in the CTC were inconsistent with those identified in autologous melanoma tumours in three patients and the KIT sequences were inconsistent in three patients. In addition, polyclonal BRAF mutations were identified in one patient and concomitant mutations in BRAF and KIT were identified in another patient.Conclusion:Melanoma cells show clonal heterogeneity. Therefore, CTC genotyping may be crucial for successful molecular-targeted therapy.

Melanoma Cells Control Antimelanoma CTL Responses via Interaction between TIGIT and CD155 in the Effector Phase

Recently, T-cell immunoreceptor with Ig and ITIM domains (TIGIT) was reported as a candidate for novel immune checkpoints. However, the impact of TIGIT on melanoma-specific cytotoxic T lymphocytes in the effector phase remains unclear. In this study, we demonstrated that melanoma cells control antimelanoma cytotoxic T lymphocyte responses via the TIGIT-CD155 interaction in the effector phase. TIGIT is an inhibitory receptor expressed on T cells, and CD155 is one of the cognate ligands expressed on the tumor cells or antigen-presenting cells. First, we confirmed that CD155 was constitutively expressed on melanoma cells. We then demonstrated that CD155 on melanoma cells suppressed cytokine release from melanoma-specific cytotoxic T lymphocytes via interaction with TIGIT. Overexpression of CD155 enhanced and its downregulation attenuated the suppressive effect. This suggested that antimelanoma cytotoxic T lymphocyte responses are controlled not only by an imbalance in CD226 (an activating molecule that binds to CD155) and TIGIT expression on T cells but also by the expression levels of CD155 on melanoma cells. In addition, the co-blockade of TIGIT and PD-1 signals synergistically elicited a response of tumor-infiltrating lymphocytes on autologous melanoma cells. These results suggest that the CD155-TIGIT interaction should be blocked for enhancement of antimelanoma immune responses.

Overexpression of GRIM-19 in cancer cells suppresses STAT3-mediated signal transduction and cancer growth.

Constitutive activation of signal transducer and activator of transcription 3 (STAT3) is common in many human and murine cancer cells, and its activation leads to cellular transformation. STAT3 pathway inhibitors have been reported to suppress cancer growth. To investigate the antitumor effects of inhibiting the STAT3-mediated signaling cascade in the cancer microenvironment, using a molecular-targeting approach, we focused on the gene associated with retinoid-IFN-induced mortality 19 (GRIM-19). GRIM-19 has been reported to interact physically with STAT3 and inhibit STAT3-dependent signal transduction. We used the nona-arginine (R9)-protein transduction domain (R9-PTD) as a protein carrier to induce high levels of GRIM-19 expression in vitro and in vivo. We generated an R9-PTD–containing GRIM-19 fusion protein (rR9-GRIM19) and successfully induced overexpression in the cytoplasm of cancer cells. Analysis of the expression of downstream molecules of STAT3 confirmed that in vitro rR9-GRIM19 treatment of constitutively activated STAT3 (STAT3c) cancer cells significantly reduced STAT3-dependent transcription. Moreover, intratumoral injections of rR9-GRIM19 in STAT3c cancer-bearing mice significantly suppressed tumor growth. These results suggest that intratumoral injections of rR9-GRIM19 have potential as a novel anticancer therapy in STAT3c cancer due to their ability to inhibit STAT3-mediated signal transduction without major systemic side effects. Mol Cancer Ther; 9(8); 2333–43. ©2010 AACR.

Malassezia species and their associated skin diseases

Malassezia spp. are lipophilic fungi that occur on all skin surfaces of humans and animals as commensal and pathogenic organisms. In the 2000s, several new species were added to the Malassezia genus by Japanese researchers. The genus Malassezia now includes 14 species of basidiomycetous yeast. Culture‐independent molecular analysis clearly demonstrated that the DNA of Malassezia spp. was predominantly detected in core body and arm sites, suggesting that they are the dominant fungal flora of the human body. Malassezia spp. have been implicated in skin diseases including pityriasis versicolor (PV), Malassezia folliculitis (MF), seborrheic dermatitis (SD) and atopic dermatitis (AD). While Malassezia spp. are directly responsible for the infectious diseases, PV and MF, they act as an exacerbating factor in AD and SD. The fatty acids generated by Malassezia lipase can induce inflammation of the skin, resulting in development of SD. Patch and serum immunoglobulin E tests revealed that AD patients were hypersensitive to Malassezia. However, these findings only partially elucidated the mechanism by which Malassezia spp. induce inflammation in the skin; understanding of the pathogenetic role of Malassezia spp. in SD or AD remains incomplete. In this article, the latest findings of Malassezia research are reviewed with special attention to skin diseases.

CD271 on Melanoma Cell Is an IFN-γ-Inducible Immunosuppressive Factor that Mediates Downregulation of Melanoma Antigens

IFN-γ released from cytotoxic T lymphocytes (CTLs) during the effector phase is essential for rejecting bulky melanoma tumors. In contrast, IFN-γ is known to induce certain immunosuppressive factors in tumor cells such as programmed cell death 1 ligand 1 (PD-L1). In this study, we have identified candidates for IFN-γ-inducible CTL-suppressive factors in melanoma cells using complementary DNA microarray analysis, and CD271/p75/neurotrophin receptor (NTR) was one of the candidate genes. Recently, CD271 was identified as a marker of the cancer stem cell-like population in human melanoma tissues. In this study, we showed that overexpression of CD271 on melanoma cells suppressed the in vitro activation of melanoma-specific CTLs. This suppression was mediated by CD271 ligation with activated CTL-derived nerve growth factor and the subsequent downregulation of melanoma antigens. Moreover, we found that the expression levels of PD-L1 on melanoma cells correlated with those of CD271, and they additively suppressed the activation of melanoma-specific CTLs. To the best of our knowledge, the role of overexpression of CD271 in an anti-melanoma T-cell response has been unreported.

A case of lymphoepithelioma-like carcinoma of the skin associated with Epstein-Barr virus infection.

Lymphoepithelioma-like carcinoma of the skin (LELCS) is a rare cutaneous neoplasm with histopathologic similarities to nasopharyngeal carcinoma. The association of nasopharyngeal carcinoma with Epstein-Barr virus (EBV) is well documented. EBV has also been reported to be associated with LELC in only four sites (the stomach, salivary glands, lung, and thymus), but not in the skin. We report herein a case of EBV-positive LELCS. An 82-year-old female presented with a red nodule on the right cheek. Histologically, the entire dermis was occupied by atypical tumor cell nests with dense lymphocytic infiltration. Neoplastic cells were strongly positive for cytokeratin 14 but were negative for cytokeratins 19 and 20. EBV genomes in neoplastic cells were detected by polymerase chain reaction analysis and in situ hybridization for EBV-encoded RNA, suggesting an association with EBV.

A Case of Metastatic Extramammary Paget’s Disease Responding to Trastuzumab plus Paclitaxel Combination Therapy

There is no effective treatment for advanced extramammary Paget’s disease (EMPD). The human epidermal growth factor receptor 2 (HER2) protein is often overexpressed in EMPD. Trastuzumab is a humanized monoclonal antibody against HER2 used in the treatment of breast cancers in which HER2 is overexpressed. We report a case of advanced EMPD in which trastuzumab and paclitaxel combination therapy was effective. The patient was a70-year-old Japanese woman who presented with EMPD on the vulva and multiple metastatic lymph nodes. Immunohistochemical staining revealed strong HER2 protein expression in the primary tumor and metastatic lymph nodes. The patient received trastuzumab and paclitaxel. After 4 courses of this regimen, the mass on the vulva and the metastatic lymph nodes regressed. Our findings may imply that trastuzumab plus paclitaxel combination therapy is useful for the treatment of advanced EMPD overexpressing HER2.

A case of endocrine mucin-producing sweat gland carcinoma

Endocrine mucin‐producing sweat gland carcinoma (EMPSGC), which is an uncommon sweat gland tumor with a predilection for the eyelids, is morphologically analogous to solid papillary carcinoma of the breast. We report the case of a 55‐year‐old man with a subcutaneous tumor of the upper cheek. The pathological findings for this patient were compatible with those of reported cases of EMPSGC, and p63 staining revealed partial microinvasion into the dermis. On the basis of these findings, the patient was diagnosed with EMPSGC. It is reported that EMPSGC is a precursor of invasive mucinous carcinoma of the skin. Therefore, this patient was treated and followed up as if he had mucinous carcinoma of the skin. To the best of our knowledge, this is the first report of such a case from Japan.

Case of acute anterior uveitis and Vogt-Koyanagi-Harada syndrome-like eruptions induced by nivolumab in a melanoma patient.

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