Minoru Takata

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.

Polyclonality of BRAF mutations in primary melanoma and the selection of mutant alleles during progression

Background:Oncogenic BRAF mutation had been considered to be a founder event in the formation of melanocytic tumours; however, we recently argued against this notion by showing marked polyclonality of BRAF mutations in acquired melanocytic nevi (Lin et al, J Natl Cancer Inst., 2009; 101:1423–7). Here, we tested whether similar heterogeneity of BRAF mutations exists in primary melanomas.Methods:We isolated and sequenced single melanoma cells from five primary melanoma tissues using antibodies against human high-molecular-weight melanoma-associated antigen. We also examined 10 primary melanomas by the sensitive Mutector assay detecting the BRAFV600E mutation, as well as by cloning and sequencing of separated alleles. Furthermore, we estimated the frequency of BRAF mutant alleles in paired samples of primary tumour and recurrence or metastasis in three patients.Results:Single-cell mutation analyses revealed that four of five primary melanomas contained both BRAF-wild-type and BRAF-mutant tumour cells. Tumour heterogeneity in terms of BRAF mutations was also shown in 8 of 10 primary melanomas. Selection of BRAF mutant alleles during progression was demonstrated in all the three patients.Conclusion:Acquisition of a BRAF mutation is not a founder event, but may be one of the multiple clonal events in melanoma development, which is selected for during the progression.

Genetic and epigenetic alterations in the differential diagnosis of malignant melanoma and spitzoid lesion.

Background  The histopathological differentiation of malignant melanoma and Spitz naevus often presents diagnostic problems.

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.

A New Melanoma Antigen Fatty Acid-Binding Protein 7, Involved in Proliferation and Invasion, Is a Potential Target for Immunotherapy and Molecular Target Therapy

The identification of molecules that are preferentially expressed in melanoma cells and involved in their malignant phenotypes is important for understanding melanoma biology and the development of new diagnostic and therapeutic methods. By comparing the expression profile of a melanoma cell line with those of various normal tissues using GeneChip and by confirming the actual expression of the selected genes by reverse transcription-PCR and Northern and Western blot analyses, fatty acid-binding protein 7 (FABP7), which is frequently expressed in melanomas, was identified. Immunohistochemical examination revealed that FABP7 was expressed in 11 of 15 melanoma tissues. By down-regulating the FABP7 expression with FABP7-specific small interfering RNAs, in vitro cell proliferation and Matrigel invasion were suppressed in two of six melanoma cell lines. Overexpression of FABP7 in a FABP7-negative embryonic kidney cell line 293T by transfecting with the FABP7 cDNA resulted in enhanced cell proliferation and Matrigel invasion, indicating that FABP7 plays a role in the malignant phenotype of some melanoma cell lines. IgG antibodies specific for the phage or bacterial recombinant FABP7 protein were detected in 14 of 25 (56%) or in 8 of 31 (26%) sera from melanoma patients, respectively, but not in sera from healthy individuals, indicating that FABP7 is an immunogenic antigen in melanoma patients. These results showed that FABP7 is frequently expressed in melanoma, may be involved in cell proliferation and invasion, and may be a potential target for development of diagnostic and therapeutic methods.

Molecular pathogenesis of malignant melanoma: a different perspective from the studies of melanocytic nevus and acral melanoma

The Clark model for melanoma progression emphasizes a series of histopathological changes beginning from benign melanocytic nevus to melanoma via dysplastic nevus. Several models of the genetic basis of melanoma development and progression are based on this Clark’s multi‐step model, and predict that the acquisition of a BRAF mutation can be a founder event in melanocytic neoplasia. However, our recent investigations have challenged this view, showing the polyclonality of BRAF mutations in melanocytic nevi. Furthermore, it is suggested that many melanomas, including acral and mucosal melanomas, arise de novo, not from melanocytic nevus. While mutations of the BRAF gene are frequent in melanomas on non‐chronic sun damaged skin which are prevalent in Caucasians, acral and mucosal melanomas harbor mutations of the KIT gene as well as the amplifications of cyclin D1 or cyclin‐dependent kinase 4 gene. Amplifications of the cyclin D1 gene are detected in normal‐looking ‘field melanocytes’, which represent a latent progression phase of acral melanoma that precedes the stage of atypical melanocyte proliferation in the epidermis. Based on these observations, we propose an alternative genetic progression model for melanoma.

Dermoscopy of pigmented lesions on mucocutaneous junction and mucous membrane.

Background  The dermoscopic features of pigmented lesions on the mucocutaneous junction and mucous membrane are different from those on hairy skin. Differentiation between benign lesions and malignant melanomas of these sites is often difficult.

Eccrine porocarcinoma: Clinical and pathological studies of 12 cases

Twelve cases of eccrine porocarcinoma have been reported at our facility in the past 10 years. All of them were Japanese; half had lymph node metastases; and one‐third died of this disease. Lymph node metastasis was correlated with pathological lymphovascular invasion. Death was correlated with a pathological growth pattern and clinical lymph node metastasis. Sentinel lymph node biopsy was performed usefully in two patients.

Early cancers of the skin: clinical, histopathological, and molecular characteristics.

Because skin lesions are visible and easily accessible, skin cancers provide us with an excellent in vivo model to study the development of cancers. Cutaneous malignant melanoma and squamous cell carcinoma (SCC) both arise from the epidermis and have an initial progression stage in which proliferation of the neoplastic cells is confined to the epidermis. This stage is called melanoma in situ or SCC in situ. Molecular analyses of melanoma in situ and of solar keratosis, a prototype of early SCC in situ, show that loss of p16INK4a/p14ARF and dysfunction of p53 play a critical role, respectively. Furthermore, there seems to be potential precursor cells to these in situ lesions, which are not discernible with conventional hematoxylin and eosin-stained sections. The precursor cells have minimal but critical genetic alterations, such as cyclin D1 amplification and p53 mutation, and can be identified using fluorescent in situ hybridization and immunostaining with p53 antibodies, respectively. These precursor cells may be defective in repair response to DNA damage, and would have proliferative or survival advantages over their normal neighboring counterparts in the presence of growth factor stimulation or genotoxic events, such as ultraviolet irradiation. Such precursor clones may be induced at a rather young age, and their number and size increase with accumulating carcinogenic stimuli. If these lesions acquire additional mutations, they could progress to clinically visible lesions of in situ carcinoma. Precise molecular analyses of early stages of skin cancers may have a strong impact on our understanding of in vivo development of cancers in other human organs.

Molecular diagnosis of basal cell carcinoma and other basaloid cell neoplasms of the skin by the quantification of Gli1 transcript levels

Background:  Distinguishing basal cell carcinoma (BCC) from other benign and malignant skin tumors is sometimes a difficult task for the pathologists. Because the activation of hedgehog signals and the up‐regulation of its critical transcriptional factor Gli1 are well documented in BCC, a molecular technique measuring Gli1 transcripts may aide the diagnosis.