Contextual role of E2F1 in suppression of melanoma cell motility and invasiveness.

The general transcription factor E2F1 reportedly functions in a protumorigenic manner in several cancer models. We show that the genetic context of cancer cells influence E2F1's role to impede the protumorigenic role. Thirty to fifty percent of melanoma patients carry mutant BRAF with about 90% of mutant BRAF melanomas being V600E mutation. Tissue microarrays from melanoma patients were used to establish an association between E2F1 and BRAFV600E . We show for the first time that low E2F1 levels in BRAFV600E melanomas are associated with lymph node metastasis. Genetic manipulation of E2F1 in BRAFV600E and BRAFwt cells were used to determine its role in malignant melanoma progression by examining effects on migration and invasion. E2F1-mediated negative regulation of myosin light chain kinase (MYLK) increased migration and invasion in BRAFV600E cells by phosphorylating myosin light chain and increased stress fiber formation. We show that E2F1 inhibits extracellular signal-regulated kinase (ERK) activation in BRAFV600E cells and provide evidence for a negative feedback loop between E2F1 and ERK in these cells. This study shows for the first time that E2F1 has a cancer protective role in oncogenic BRAF-activated melanoma cells and that loss of E2F1 can allow disease progression through a novel mechanism of E2F1-mediated MYLK regulation. This study has implications for oncogenic BRAF-activated tumors and resistance to targeted oncogenic BRAF therapy.

Desmosomal defects in acantholytic squamous cell carcinomas.

BACKGROUND: Acantholytic squamous cell carcinoma (Acantholytic SCC) are epithelial tumors characterized by a loss of cell adhesion between neoplastic keratinocytes. The mechanism underlying loss of cell-cell adhesion in these tumors is not understood. METHODS: A retrospective analysis of acantholytic SCC (n = 17) and conventional SCC (n = 16, controls not showing acantholysis) was conducted using a set of desmosomal and adherens junction protein antibodies. Immunofluorescence microscopy was used to identify tumors with loss of adhesion protein expression. RESULTS: The vast majority of acantholytic SCC (89%) showed focal loss of at least one desmosomal cell adhesion protein. Most interestingly, 65% of these tumors lost expression of two or more desmosomal proteins. CONCLUSIONS: Loss of cell adhesion in acantholytic SCC is most likely linked to the focal loss of desmosomal protein expression, thus providing potential mechanistic insight into the patho-mechanism underlying this malignancy.

HLA antigen expression in melanocytic lesions: is acquisition of HLA antigen expression a biomarker of atypical (dysplastic) melanocytes?

BACKGROUND: Although criteria are established for the histologic diagnosis of atypical nevi (AN), consensus about the criteria in the diagnosis of and in the definition of AN is limited. Moreover, intraobserver and interobserver differences in the application of these criteria for the diagnosis of AN have been observed. OBJECTIVE: We sought to determine the usefulness of HLA antigen expression as a biomarker of AN. METHODS: The immunoperoxidase reaction was used to mark common nevi and AN with HLA class I heavy chain-, ß2microglobulin (ß2m)-, and HLA class II ß chain-specific monoclonal antibodies. RESULTS: HLA class I heavy chain, ß2m, and HLA class II ß chain were expressed in 5 (8.6%) of the 58 common nevi and in 46 (∼72%) of the 64 atypical melanocytic lesions. Among common lesions, only halo nevi expressed HLA class I heavy chain, ß2m, and HLA class II ß chain. The level of HLA class I heavy chain ß2m and of HLA class II ß chain expression correlated with the degree of cytologic atypia and architectural disorder. LIMITATIONS: The number of lesions tested and the subjective nature of the analysis of immunohistochemical staining of tissue sections are both limitations. CONCLUSIONS: The data presented suggest that HLA antigen expression is an objective biomarker that correlates well with the degree of cytologic atypia in AN and may: (1) be useful to distinguish common nevi from AN, and (2) represent a more objective measure to determine which AN should be excised.

Loss of Desmocollin 3 in skin tumor development and progression.

Desmocollin 3 (DSC3) is a desmosomal cadherin that is required for maintaining cell adhesion in the epidermis as demonstrated by the intra-epidermal blistering observed in Dsc3 null skin. Recently, it has been suggested that deregulated expression of DSC3 occurs in certain human tumor types. It is not clear whether DSC3 plays a role in the development or progression of cancers arising in stratified epithelia such as the epidermis. To address this issue, we generated a mouse model in which Dsc3 expression is ablated in K-Ras oncogene-induced skin tumors. Our results demonstrate that loss of Dsc3 leads to an increase in K-Ras-induced skin tumors. We hypothesize that acantholysis-induced epidermal hyperplasia in the Dsc3 null epidermis facilitates Ras-induced tumor development. Further, we demonstrate that spontaneous loss of DSC3 expression is a common occurrence during human and mouse skin tumor progression. This loss occurs in tumor cells invading the dermis. Interestingly, other desmosomal proteins are still expressed in tumor cells that lack DSC3, suggesting a specific function of DSC3 loss in tumor progression. While loss of DSC3 on the skin surface leads to epidermal blistering, it does not appear to induce loss of cell-cell adhesion in tumor cells invading the dermis, most likely due to a protection of these cells within the dermis from mechanical stress. We thus hypothesize that DSC3 can contribute to the progression of tumors both by cell adhesion-dependent (skin surface) and likely by cell adhesion-independent (invading tumor cells) mechanisms.

Imiquimod-induced vitiligo after treatment of nodular basal cell carcinoma.

An 84-year-old male presented with recurrent nodular infiltrative basal cell carcinoma on the left shoulder. The patient was treated with curettage followed by the application of topical imiquimod 5% cream five times a week. The patient discontinued imiquimod after a total of 18 applications because of local inflammation. Depigmentation was noted in the treated area 11 months after the initiation of treatment with imiquimod. The depigmented area did not resolve 14 months after treatment initiation. Histologic examination of the depigmented area established the absence of melanin using Fontana-Masson stain and the absence of melanocytes using S-100 and Melan A stains. The adjacent unaffected skin showed a normal number of melanocytes and melanin pigment. To our knowledge, this is the first biopsy-proven case of vitiligo in an imiquimod-treated area.

A mediator of Rho-dependent invasion moonlights as a methionine salvage enzyme.

RhoA controls changes in cell morphology and invasion associated with cancer phenotypes. Cell lines derived from melanoma tumors at varying stages revealed that RhoA is selectively activated in cells of metastatic origin. We describe a functional proteomics strategy to identify proteins regulated by RhoA and report a previously uncharacterized human protein, named "mediator of RhoA-dependent invasion (MRDI)," that is induced in metastatic cells by constitutive RhoA activation and promotes cell invasion. In human melanomas, MRDI localization correlated with stage, showing nuclear localization in nevi and early stage tumors and cytoplasmic localization with plasma membrane accentuation in late stage tumors. Consistent with its role in promoting cell invasion, MRDI localized to cell protrusions and leading edge membranes in cultured cells and was required for cell motility, tyrosine phosphorylation of focal adhesion kinase, and modulation of actin stress fibers. Unexpectedly MRDI had enzymatic function as an isomerase that converts the S-adenosylmethionine catabolite 5-methylribose 1-phosphate into 5-methylribulose 1-phosphate. The enzymatic function of MRDI was required for methionine salvage from S-adenosylmethionine but distinct from its function in cell invasion. Thus, mechanisms used by signal transduction pathways to control cell movement have evolved from proteins with ancient function in amino acid metabolism.

Tattoo pigment interpreted as lymph node metastasis in a case of subungual melanoma.

We present a patient with subungual melanoma of the thumb who, during radioisotope-guided selective sentinel lymphadenectomy, was found to have black, hard lymph nodes at multiple axillary node levels. This finding was interpreted intraoperatively as clinical evidence of metastasis and a formal axillary dissection was carried out. Pathological examination of excised nodes failed to demonstrate metastasis but instead showed collections of tattoo pigment.

Human papillomavirus infection and ultraviolet light exposure as epidermoid inclusion cyst risk factors in a patient with epidermodysplasia verruciformis?

Epidermoid inclusion cysts are common lesions with unclear etiology. We sought to examine evidence for human papillomavirus (HPV) infection and ultraviolet light (UV) exposure as risk factors in the formation of epidermoid inclusion cysts. We performed HPV typing of biopsied cysts with polymerase chain reaction for a patient with darkly-pigmented skin, epidermodysplasia verruciformis (EV) and more than 250 photodistributed cysts. HPV types 8 and 6 DNA was demonstrated within biopsy specimens of 3 cysts. In one biopsy specimen, abnormal keratinocytes bridging the follicular infundibulum were seen. We concluded that UV exposure and HPV viral infection may be risk factors for the formation of epidermoid inclusion cysts.

Gastrointestinal melanoma or clear cell sarcoma? Molecular evaluation of 7 cases previously diagnosed as malignant melanoma.

Clear cell sarcoma (CCS) is a rare tumor classically associated with the tendons and aponeuroses of distal extremities of young adults. CCS and malignant melanoma (MM) share immunohistochemical profiles and ultrastructural features, but classic CCS has characteristic morphology with low mitotic activity and minimal pleomorphism. Occasional cases show pleomorphism, high mitotic index, and/or melanin pigmentation, making CCS indistinguishable from MM based on morphology. However, CCS is genetically distinct owing to its consistent association with a t(12;22)(q13;q12) chromosomal translocation, leading to the formation of the EWS/ATF1 fusion transcript. This translocation has never been documented in cutaneous melanoma, and thus is regarded as specific for CCS. Recent evidence suggests that primary "malignant melanomas" in unusual anatomic sites, most notably the gastrointestinal (GI) tract, may be CCS. This is supported by 11 cases of primary GI CCS with the t(12;22) translocation. We used reverse-transcription polymerase chain reaction and fluorescence in situ hybridization to examine whether a proportion of cases diagnosed as MM of the GI tract in patients without a history of cutaneous MM actually represent primary GI CCS. In total, we examined 7 cases: Four with no prior history of MM, 2 with histories of cutaneous MM, and 1 with an anal MM. All 4 cases for which there was no history of cutaneous/mucosal MM harbored the EWS/ATF1 fusion transcript. We report the largest series of GI CCS and have shown that molecular studies may be warranted in cases that otherwise seem to represent MM of unusual primary locations.

Platelet-derived growth factor receptor alpha mutational status and immunohistochemical expression in Merkel cell carcinoma: implications for treatment with imatinib mesylate.

BACKGROUND: It is not known if Merkel cell carcinomas (MCCs) show mutations in the platelet-derived growth factor receptor alpha (PDGFRA) gene similar to a subset of gastrointestinal stromal cell tumors. The purpose of this study was to analyze MCCs for mutations in the PDGFRA gene as well as to analyze those MCCs exhibiting a possible mutation in the PDGFRA gene for immunohistochemical expression of PDGFRA. METHODS: We extracted tumor DNA from nine MCCs, performed polymerase chain reaction amplification of PDGFRA exons 10, 12, 14 and 18, and directly sequenced those gene products for mutations. In addition, we evaluated for PDGFRA immunostaining in three MCCs showing a possible PDGFRA gene mutation. RESULTS: Three out of nine (33.3%) MCCs showed an identical novel single heterozygous base change in exon 10 of the PDGFRA gene leading to an amino acid substitution at codon 478. In addition, all three (100%) of those MCCs expressed PDGFRA. CONCLUSIONS: Although it is unknown whether the base change described above represents a true mutation or a single nucleotide polymorphism, the fact that this change was absent in our control specimens suggests that this mutation may be oncogenic in nature and may make imatinib mesylate a possible therapeutic option in MCC.

Pustulo-ovoid bodies of Milian in granular cell tumors.

BACKGROUND: Granular cell tumors (GCTs) are benign neural tumors with a distinct histologic appearance on light microscopy, characterized by eosinophilic cytoplasmic granules. Pustulo-ovoid bodies of Milian (POB) are larger granules surrounded by a clear halo. There have been no histologic studies to document their prevalence in GCT. METHODS: We examined the sections of 47 cases of GCT stained with hematoxylin and eosin to determine the frequency of POB within this tumor. POB were measured per 10 high-power fields (HPFs) and were divided into the following categories: less than 10 POB per 10 HPFs, 10-30 per 10 HPFs, 30-50 per 10 HPFs and greater than 50 per 10 HPFs. RESULTS: POB were present in 100% of the specimens examined. Eleven cases (23%) had between 1 and 9 POBs per 10 HPFs, twelve cases (26%) had between 10 and 29 POBs per HPFs, five cases (11%) had between 30 and 50 POBs per HPFs and nineteen cases (40%) had greater than 50 POBs per HPFs. When grouped according to clinical characteristics, there was an even distribution of POB by age, sex and site of the tumor. CONCLUSIONS: In our series, POB were present in varying numbers in all the tumors studied. They appear to represent the heterogeneity of the lysosomes, giving the appearance of large granules that have partially detached from the adjacent cytoplasm. POB are an easily recognizable component of GCTs.

Merkel cell carcinoma: evaluation of KIT (CD117) expression and failure to demonstrate activating mutations in the C-KIT proto-oncogene - implications for treatment with imatinib mesylate.

BACKGROUND: Merkel cell carcinomas (MCCs) express the tyrosine kinase receptor KIT. However, it is not known if MCCs have activating mutations in KIT that would make them responsive to treatment with imatinib mesylate. The purpose of this article is to describe the KIT immunohistological staining pattern (CD117) of MCCs and analyze those MCCs for mutations in areas of KIT where mutations are found in gastrointestinal stromal cell tumors. METHODS: We evaluated KIT immunostaining in nine MCCs from nine patients. In addition, we extracted DNA from the same MCCs, performed PCR amplification of C-KIT exons 9, 11, 13, and 17, and sequenced those gene products for mutations. RESULTS: Eight of nine (88.8%) MCCs expressed KIT. No mutations were found. CONCLUSIONS: The majority of MCCs express KIT but do not contain activating mutations in exons 9, 11, 13, or 17 of KIT. Imatinib mesylate is unlikely to provide effective therapy in MCC unless activating mutations in other areas of KIT or activating mutations in other related genes can be detected.

Granulomatous reaction to titanium alloy: an unusual reaction to ear piercing.

A 68-year-old man presented with 4 firm, flesh-colored, and slightly erythematous nodules located on the superior pole and lobule of each ear. Although reluctant to provide details, it was discovered he had pierced his ears approximately 10 years earlier, and the nodules developed at the sites of the piercings. Keloids were suggested clinically and the lesions were excised. Microscopic examination demonstrated epithelialized tracts surrounded by a granulomatous infiltrate of macrophages, lymphocytes, and plasma cells. Closer examination revealed minute brown-black particles within macrophages. Dark-field microscopy confirmed the metallic nature of the particles. Environmental scanning electron microscopy with energy dispersive spectroscopy revealed the particles to be composed of titanium, aluminum, and vanadium. It would appear that in rare circumstances titanium alloy used in body piercing may engender a granulomatous dermatitis. The rarity of such a response to titanium alloy is discussed and the literature appraised.

Kindler syndrome: a new mutation and new diagnostic possibilities.

BACKGROUND: Kindler syndrome (KS) is a rare genetic disorder that is characterized by blistering in infancy, followed by the onset of poikiloderma and photosensitivity in childhood. The recently elucidated molecular pathogenesis involves mutations in KIND1, a gene encoding the protein kindlin-1, which is involved in the attachment of the actin cytoskeleton to the extracellular matrix in basal keratinocytes. OBSERVATIONS: We describe a child with the neonatal diagnosis of epidermolysis bullosa simplex who developed poikiloderma and skin fragility at 6 years of age. His skin showed diminished staining with anti-kindlin-1 antibody, and genetic analysis revealed that he was a compound heterozygote with a previously unreported mutation in KIND1. Ultrastructural clues to the diagnosis of KS were present in a biopsy specimen that was obtained when the patient was 10 months old, before he developed poikiloderma and photosensitivity. CONCLUSIONS: In this case, a combination of a known mutation (R271X) and a newly described mutation (1755delT) in the KIND1 gene produced loss of function in kindlin-1, leading to the clinical features of KS. Ultrastructural findings characteristic of KS were evident years before the onset of poikiloderma and sun sensitivity. In infancy, electron microscopy can enable early, accurate diagnosis of KS.

Expression of c-kit (CD117) in Spitz nevus and malignant melanoma.

BACKGROUND: CD117, the receptor for kit-ligand, which is a growth factor for melanocyte migration and proliferation, has shown differential staining in various benign and malignant melanocytic lesions. The purpose of this study is to compare CD117 immunohistological staining in Spitz nevus versus malignant melanoma, to determine whether CD117 can aid in the diagnosis of these two lesions. METHODS: CD-117 immunohistological staining was performed in 22 clinically and pathologically diagnosed pigmented lesions including 9 cases of Spitz nevus, 10 cases of primary MM and 3 cases of metastatic melanoma. RESULTS: There was no significant difference in CD117 staining in either epidermis or dermis between Spitz nevi and primary melanomas. However staining of metastatic melanomas is less than dermal staining of primary MM and Spitz nevus. CONCLUSIONS: CD117 is unlikely a useful diagnostic tool in differentiating Spitz nevus from primary MM. On the other hand, CD 117 may be useful in differentiating metastatic melanoma from primary melanoma in patients who had a history of melanoma and who present with new dermal lesions.