Cellular sheddases are induced by Merkel cell polyomavirus small tumour antigen to mediate cell dissociation and invasiveness.

Merkel cell carcinoma (MCC) is an aggressive skin cancer with a high propensity for recurrence and metastasis. Merkel cell polyomavirus (MCPyV) is recognised as the causative factor in the majority of MCC cases. The MCPyV small tumour antigen (ST) is considered to be the main viral transforming factor, however potential mechanisms linking ST expression to the highly metastatic nature of MCC are yet to be fully elucidated. Metastasis is a complex process, with several discrete steps required for the formation of secondary tumour sites. One essential trait that underpins the ability of cancer cells to metastasise is how they interact with adjoining tumour cells and the surrounding extracellular matrix. Here we demonstrate that MCPyV ST expression disrupts the integrity of cell-cell junctions, thereby enhancing cell dissociation and implicate the cellular sheddases, A disintegrin and metalloproteinase (ADAM) 10 and 17 proteins in this process. Inhibition of ADAM 10 and 17 activity reduced MCPyV ST-induced cell dissociation and motility, attributing their function as critical to the MCPyV-induced metastatic processes. Consistent with these data, we confirm that ADAM 10 and 17 are upregulated in MCPyV-positive primary MCC tumours. These novel findings implicate cellular sheddases as key host cell factors contributing to virus-mediated cellular transformation and metastasis. Notably, ADAM protein expression may be a novel biomarker of MCC prognosis and given the current interest in cellular sheddase inhibitors for cancer therapeutics, it highlights ADAM 10 and 17 activity as a novel opportunity for targeted interventions for disseminated MCC.

Increased expression of EZH2 in Merkel cell carcinoma is associated with disease progression and poorer prognosis.

Enhancer of zeste homolog 2 (EZH2) is a histone methyltransferase that affects tumorigenesis by epigenetic gene silencing. Merkel cell carcinoma (MCC) is a rare cutaneous neuroendocrine carcinoma that has a high risk of disease progression with nodal and distant metastases. Here, we evaluated EZH2 expression by immunohistochemistry in a cohort of 85 MCC tumors (29 primary tumors, 41 lymph node metastases, 13 in-transit metastases, and 2 distant metastases) with clinical follow-up. We show strong/moderate EZH2 expression in 54% of tumors. Importantly, weak expression of EZH2 in the primary tumor, but not nodal metastases, correlated with improved prognosis compared to moderate/strong EZH2 expression (5-year MCC-specific survival of 68% versus 22%, respectively, P=.024). In addition, EZH2 was expressed at higher levels in nodal metastases compared to primary tumors (P=.005). Our data demonstrate that EZH2 has prognostic value and may play an oncogenic role in MCC.

TERT promoter mutations and gene amplification: promoting TERT expression in Merkel cell carcinoma.

Telomerase activation through the induction of its catalytic component TERT is essential in carcinogenesis. The regulatory mechanism and clinical significance underlying cancer-specific TERT expression have been extensively investigated in various human malignancies, but little is known about these in Merkel cell carcinoma (MCC), an aggressive neuroendocrine skin tumor. Here we addressed these issues by determining TERT promoter mutations, gene amplification, mRNA expression and association with clinical variables in MCC. TERT mRNA was expressed in 6/6 MCC cell lines and 41 of 43 tumors derived from 35 MCC patients. Telomerase activity was detectable in all 6 cell lines and 11 tumors analyzed. TERT promoter mutations were identified in 1/6 cell lines and 4/35 (11.4%) MCC cases. The mutation exhibited UV signature and occurred in sun-exposed areas. Increased TERT gene copy numbers were observed in 1/6 cell lines and 11/14 (79%) tumors, and highly correlated with its mRNA expression (r = 0.7419, P = 0.0024). Shorter overall survival was significantly associated with higher TERT mRNA levels in MCC patients (P = 0.032). Collectively, TERT expression and telomerase activity is widespread in MCC, and may be attributable to TERT promoter mutations and gene amplification. Higher TERT expression predicts poor patient outcomes.

Chronic mTOR activation promotes cell survival in Merkel cell carcinoma.

Merkel cell carcinoma (MCC) is an aggressive skin cancer with rising incidence. In this study, we demonstrate that mTOR activation and suppressed autophagy is common in MCCs. mTOR inhibition in two primary human MCC cell lines induces autophagy and cell death that is independent of caspase activation but can be attenuated by autophagy inhibition. This is the first study to evaluate mTOR and autophagy in MCC. Our data suggests a potential role of autophagic cell death upon mTOR inhibition and thus uncovers a previously underappreciated role of mTOR signaling and cell survival, and merits further studies for potential therapeutic targets.

Expression of anaplastic lymphoma kinase in Merkel cell carcinomas.

This study examines the presence of anaplastic lymphoma kinase protein and anaplastic lymphoma kinase gene rearrangements in Merkel cell carcinomas. A total of 32 cases of Merkel cell carcinomas and 12 cases of small cell lung carcinomas were analyzed. Immunohistochemistry was performed using 3 different anaplastic lymphoma kinase antibody clones (D5F3, 5A4, and anaplastic lymphoma kinase 1). Tumors were divided into high (intensity score 2-3+ in ≥25% of the tumor cells) and low expressors (all other positive expression patterns). Anaplastic lymphoma kinase reactivity in Merkel cell carcinoma was observed in 93.8% (30/32) with clone D5F3, 87.5% (28/32) with clone 5A4, and 12.5% (4/32) with clone anaplastic lymphoma kinase 1. One small cell lung carcinoma (1/12; 8.3%) showed anaplastic lymphoma kinase low expression with clone D5F3. Anaplastic lymphoma kinase high expression was observed in 81.3% (26/32) of the Merkel cell carcinomas with clone D5F3, 71.9% (23/32) with clone 5A4, and none with clone anaplastic lymphoma kinase 1. The specificity of anaplastic lymphoma kinase expression in Merkel cell carcinoma versus small cell lung carcinoma was 91.7% with clone D5F3 and 100% with the clones 5A4 and anaplastic lymphoma kinase 1. Interphase fluorescence in situ hybridization with the anaplastic lymphoma kinase dual-color, break-apart rearrangement probe was performed on 10 randomly selected Merkel cell carcinoma anaplastic lymphoma kinase high expressors. No rearrangement or other cytogenetic aberration of the anaplastic lymphoma kinase gene locus was identified. In conclusion, the anaplastic lymphoma kinase protein was detected with high frequency in Merkel cell carcinomas and was useful in distinguishing Merkel cell carcinoma from small cell lung carcinoma. No correlation with anaplastic lymphoma kinase rearrangement was found. Our findings could have important therapeutic consequences for patients, but the role of anaplastic lymphoma kinase in the pathogenesis of Merkel cell carcinoma needs to be further elucidated.

Activation of the PI3K/AKT pathway in Merkel cell carcinoma.

Merkel cell carcinoma (MCC) is a highly aggressive skin cancer with an increasing incidence. The understanding of the molecular carcinogenesis of MCC is limited. Here, we scrutinized the PI3K/AKT pathway, one of the major pathways activated in human cancer, in MCC. Immunohistochemical analysis of 41 tumor tissues and 9 MCC cell lines revealed high levels of AKT phosphorylation at threonine 308 in 88% of samples. Notably, the AKT phosphorylation was not correlated with the presence or absence of the Merkel cell polyoma virus (MCV). Accordingly, knock-down of the large and small T antigen by shRNA in MCV positive MCC cells did not affect phosphorylation of AKT. We also analyzed 46 MCC samples for activating PIK3CA and AKT1 mutations. Oncogenic PIK3CA mutations were found in 2/46 (4%) MCCs whereas mutations in exon 4 of AKT1 were absent. MCC cell lines demonstrated a high sensitivity towards the PI3K inhibitor LY-294002. This finding together with our observation that the PI3K/AKT pathway is activated in the majority of human MCCs identifies PI3K/AKT as a potential new therapeutic target for MCC patients.

Significant correlation of peptidyl-prolyl isomerase overexpression in Merkel cell carcinoma with overall survival of patients.

BACKGROUND: An overexpression of PIN1, the peptidyl-prolyl cis-trans isomerase, might cause cell cycle arrest and growth inhibition by binding to the p53 protein, a process leading to p53 stabilization. The rationale of this retrospective analysis was to evaluate the expression pattern of PIN1 in Merkel cell carcinomas (MCCs) and its suitability as a prognostic factor. METHODS: Samples of 27 MCCs were immunhistochemically stained for PIN1 expression and correlated with overall and disease-free survival of patients. RESULTS: All samples expressed PIN1. We showed a significantly better overall survival in patients with an overexpression of PIN1 than in patients with a weak PIN1 expression (p = .031), but expression was not significant for disease-free survival (p = .821). The 5-year overall survival rate was 14.4% in patients with weak and 50.9% in patients with overexpression of PIN1. CONCLUSIONS: PIN1 seems to be a prognostic factor for a better overall survival rate of patients with MCC.

Limb preservation with isolated limb infusion for locally advanced nonmelanoma cutaneous and soft-tissue malignant neoplasms.

OBJECTIVE: To demonstrate the efficacy of isolated limb infusion (ILI) in limb preservation for patients with locally advanced soft-tissue sarcomas and nonmelanoma cutaneous malignant neoplasms. BACKGROUND: Locally advanced nonmelanoma cutaneous and soft-tissue malignant neoplasms, including soft-tissue sarcomas of the extremities, can pose significant treatment challenges. We report our experience, including responses and limb preservation rates, using ILI in cutaneous and soft-tissue malignant neoplasms. METHODS: We identified 22 patients with cutaneous and soft-tissue malignant neoplasms who underwent 26 ILIs with melphalan and dactinomycin from January 1, 2004, through December 31, 2009, from 5 institutions. Outcome measures included limb preservation and in-field response rates. Regional toxic effects were measured using the Wieberdink scale and serum creatinine phosphokinase levels. RESULTS: The median age was 70 years (range, 19-92 years), and 12 patients (55%) were women. Fourteen patients (64%) had sarcomas, 7 (32%) had Merkel cell carcinoma, and 1 (5%) had squamous cell carcinoma. The median length of stay was 5.5 days (interquartile range, 4-8 days). Twenty-five of the 26 ILIs (96%) resulted in Wieberdink grade III or less toxicity, and 1 patient (4%) developed grade IV toxicity. The median serum creatinine phosphokinase level was 127 U/L for upper extremity ILIs and 93 U/L for lower extremity ILIs. Nineteen of 22 patients (86%) underwent successful limb preservation. The 3-month in-field response rate was 79% (21% complete and 58% partial), and the median follow-up was 8.6 months (range, 1-63 months). Five patients underwent resection of disease after an ILI, of whom 80% are disease free at a median of 8.6 months. CONCLUSIONS: Isolated limb infusion provides an attractive alternative therapy for regional disease control and limb preservation in patients with limb-threatening cutaneous and soft-tissue malignant neoplasms. Short-term response rates appear encouraging, yet durability of response is unknown.

Merkel cell carcinoma: lack of KIT positivity and implications for the use of imatinib mesylate.

The large variability (7% to 100%) in previously reported rates of receptor tyrosine kinase KIT expression in Merkel cell carcinoma (MCC) may be owing to the use of heat-induced epitope retrieval. High frequency of reported KIT reactivity by immunohistochemistry (IHC) in part prompted the initiation of a phase 2 clinical trial of imatinib mesylate (Gleevec, Novartis Pharmaceuticals, East Hanover, NJ) for the treatment of advanced MCC. Our experience has been that a small number of MCCs (12.5%) are positive for KIT by IHC. We also found a higher rate of apparently KIT-positive MCCs (75%) using heat-induced epitope retrieval. Our anecdotal experience with the use of imatinib mesylate has been disappointing. As IHC detection of KIT expression does not correlate with the presence of KIT-activating mutations, protein expression as tested by IHC should not be used to determine if patients would respond to imatinib mesylate. Indeed, our review of the literature and the apparent lack of efficacy of imatinib mesylate for MCC in a recent phase 2 trial suggest a minor role for KIT signaling in MCC tumorigenesis.

Cyclooxygenase-2 expression in primary and metastatic Merkel cell carcinoma.

Cyclooxygenase-2 (COX-2) is involved in the development and progression of many tumors, and its inhibition has been shown to block tumor growth. This study examined COX-2 expression in primary and metastatic Merkel cell carcinoma (MCC). Formalin-fixed paraffin-embedded tissues from 26 primary MCCs and 7 lymph node metastases were stained immunohistochemically with a monoclonal antibody directed against COX-2, and the percentage and intensity of staining were analyzed semiquantitatively. Immunopositivity for COX-2 was found in 20 primary tumors (77%), and was diffuse in 16 of them (80%). Staining intensity was strong in 5 tumors (19%), moderate in 6 (23%), and weak in 9 (35%). Five metastases (71%) showed similar staining. Prominent mitotic activity was associated with more diffuse COX-2 immunopositivity. No association was found between COX-2 expression and outcome. This study confirms that most MCCs express COX-2 and shows that COX-2 expression is related to one parameter of aggressive behavior--a high mitotic rate--but not to any others. The possibility of treating MCC with COX-2 inhibitors should be considered.

Reactivity with TdT in Merkel cell carcinoma: a potential diagnostic pitfall.

Merkel cell carcinoma (MCC) is a high-grade neuroendocrine carcinoma of skin characterized by cells with a "blastic" appearance, scant cytoplasm, and fine, evenly distributed chromatin. Terminal deoxynucleotidyl transferase (TdT) is a DNA polymerase present in thymic T cells, lymphoblastic lymphoma/leukemia, and some cases of acute myeloid leukemia. After observing TdT immunoreactivity in a case of MCC, we analyzed 26 tumors by immunohistochemical analysis to determine their spectrum of reactivity with TdT and identified TdT in 19 (73%) of 26 MCCs. Staining intensity was variable but was often moderate to strong and present in a significant percentage of cells. Because MCC has cytomorphologic features similar to those of lymphoblastic lymphoma and may manifest as metastatic disease, reactivity with TdT in MCC could represent a diagnostic pitfall in the differential diagnosis with lymphoblastic lymphoma, particularly because the latter may lack CD45 and/or CD20, yet both neoplasms may express PAX-5, a B-cell-associated marker.

Activation of the MAP kinase pathway induces apoptosis in the Merkel cell carcinoma cell line UISO.

Merkel cell carcinoma (MCC) is a rare but highly aggressive tumor of the skin. Recently, we have shown that MCC cells in situ are characterized by a complete absence of mitogen-activated protein kinase (MAPK) pathway signaling, which is preserved in the MCC cell line UISO. Here we present data suggesting that silencing of the MAPK pathway is essential for the survival of MCC cells. Activation of the MAPK pathway could be achieved by inducing a regulatable form of the c-Raf-1 kinase domain in UISO cells. Consequently, MAPK signaling led to morphological changes, loss of actin stress fibers, and induction of apoptosis, which could be prevented by the MAP kinase kinase-specific inhibitor U0126. Hence, despite the fact that activation of the MAPK pathway contributes to oncogenesis in many cancers, it seems to be a negative selection factor for MCC cells. Since ERK phosphorylation was also inducible by the Raf-activating pharmacological agent ZM336372, these results provide new perspectives for potential therapeutics for this highly aggressive tumor.

Cyclooxygenase-2 expression in primary Merkel cell carcinoma.

BACKGROUND: Merkel cell carcinoma (MCC) is a highly malignant neuroendocrine skin tumor. The typical course of MCC is rapid progression of the primary tumor and metastatic dissemination to the regional lymph nodes. Thus far, no biological, prognostic marker has been established for this aggressive neoplasm. Cyclooxygenase-2 (Cox-2) is undetectable in most normal tissues, but it is induced in various cell types by inflammation and carcinogenesis. Although the expression and function of Cox-2 have been studied extensively in several carcinomas, little is known about Cox-2 expression in neuroendocrine carcinomas. The aim of the present report was to study Cox-2 expression in MCC and find out whether this expression correlates with outcome. METHODS: Immunohistochemical analysis for Cox-2 was performed on 22 primary MCC samples. RESULTS: Almost 70% of the samples showed positive staining. Protein expression of Cox-2 was sparse and low in intensity. We found a tendency for enhanced Cox-2 expression in tumors located in sun-exposed areas. Cox-2 expression had no significant statistical correlation with clinical parameters. CONCLUSIONS: MCC expresses Cox-2 in low levels, and the expression did not prove to be a prognostic factor. Furthermore, the low expression suggests that the primary treatment option for MCC is not therapeutic inhibition of Cox-2.

Alterations of the SDHD gene locus in midgut carcinoids, Merkel cell carcinomas, pheochromocytomas, and abdominal paragangliomas.

Several types of endocrine tumors show frequent somatic deletions of the distal part of chromosome arm 11q, where the tumor-suppressor gene SDHD (succinate-ubiquinone oxidoreductase subunit D), constitutionally mutated in paragangliomas of the head and neck, is located. In this study, we screened 18 midgut carcinoids, 7 Merkel cell carcinomas, 46 adrenal pheochromocytomas (37 sporadic and 9 familial), and 7 abdominal paragangliomas for loss of heterozygosity (LOH) and/or mutations at the SDHD gene locus. LOH was detected in 5 out of 8 (62%) informative midgut carcinoids, in 9 out of 30 (30%) sporadic pheochromocytomas, in none of the familial pheochromocytomas (0%), and in 1 out of 6 (17%) abdominal paragangliomas. No sequence variants were detected in the pheochromocytomas or paragangliomas. However, two constitutional putative missense mutations, H50R and G12S, were detected in two midgut carcinoids, which were both associated with LOH of the other allele. The same sequence variants were also detected in two Merkel cell carcinomas. In addition, the S68S polymorphism was found to coexist with the G12S sequence variant in both cases. In conclusion, we show that alterations of the SDHD gene seem to be involved in the tumorigenesis of both midgut carcinoids and Merkel cell carcinomas.

Telomerase activity of Merkel cell carcinomas and Merkel cell carcinoma-derived cell cultures.

Merkel cell carcinomas are rare malignant tumors of the skin, which are predominantly observed in elderly patients (mean age 65-70 years). It is believed but not yet proven that these tumors are derived from the Merkel cells of the epidermis and hair follicles. The Merkel cells themselves probably originate from an asymmetric cell division of basal keratinocytes and the resulting differentiated Merkel cells have presumably, at least in humans, lost their growth potential. The capability of indefinite cell division in germ line cells and in the great majority of malignant tumors as well as an increased growth potential in certain somatic cells (such as basal cells of renewable tissues) is correlated with cellular telomerase activity, which is absent in differentiated somatic cells. In this study the telomerase activity in cryostat sections of frozen Merkel cell tumor biopsies and in in vitro cultivated Merkel cell carcinoma cells was analyzed. We detected telomerase activity in four tumors and three of four cell cultures. These results show that despite their pronounced neuroendocrine differentiation and their occurrence in patients of advanced age, Merkel cell carcinomas possess telomerase activity similar to that of common carcinoma types.