The diagnostic utility of Merkel cell polyomavirus immunohistochemistry in a fine needle aspirate of metastatic Merkel cell carcinoma of unknown primary to the pancreas

Merkel cell carcinoma (MCC) is an aggressive skin tumor with a high tendency for metastases. We report a case of MCC initially presenting as axillary and pancreatic metastases. A 33‐year‐old HIV‐positive Hispanic male presented with a history of a rapidly growing axillary mass. A needle core biopsy demonstrated an epithelioid neoplasm composed of small to medium‐sized cells with high nuclear‐cytoplasmic ratio, nuclear molding, and frequent mitotic figures. A subsequent PET scan revealed a 1.5 cm FDG avid mass in the pancreas. Endoscopic ultrasound‐guided FNA of the pancreatic mass showed neoplastic cells with similar morphology to those of the axillary mass. The tumor cells were positive with pancytokeratin AE1/AE3, CK20, CD56, synatophysin, chromogranin, and Merkel cell polyomavirus (MCPyV). This case of MCC most likely originated from a resolved primary skin lesion drained by the involved axillary lymph node with subsequent metastases to the pancreas and distant lymph nodes.     

Association of Merkel cell polyomavirus infection with morphologic differences in Merkel cell carcinoma

Recently, it has been shown that approximately 80% of Merkel cell carcinomas harbor a novel polyomavirus named Merkel cell polyomavirus, thought to be a carcinogenic agent. However, it is not fully elucidated whether Merkel cell carcinomas differ with regard to the presence or absence of Merkel cell polyomavirus. To address this, we investigated morphologic differences between Merkel cell polyomavirus-positive and -negative Merkel cell carcinomas by morphometry. Using polymerase chain reaction and real-time quantitative polymerase chain reaction, Merkel cell polyomavirus was detected in 20 (77%) of 26 Merkel cell carcinoma cases, including 4 Merkel cell carcinomas combined with squamous cell carcinomas. Interestingly, Merkel cell polyomavirus was detected only in ordinary (pure) Merkel cell carcinomas; none of the 4 combined Merkel cell carcinomas + squamous cell carcinomas was positive for Merkel cell polyomavirus (P = .001). Morphometric analyses revealed that Merkel cell polyomavirus-negative Merkel cell carcinomas had more irregular nuclei (P < .001) and more abundant cytoplasm (P = .001) than Merkel cell polyomavirus-positive Merkel cell carcinomas, which had uniform round nuclei and scant cytoplasm. Reliability of the morphometry was confirmed using intraobserver and interobserver reliability tests. These results demonstrated statistically significant differences in tumor cell morphology between Merkel cell polyomavirus-positive and -negative Merkel cell carcinomas and reconfirmed the absence of Merkel cell polyomavirus in combined tumors. Furthermore, the results strongly suggest fundamental biological differences between Merkel cell polyomavirus-positive and -negative Merkel cell carcinomas, supporting that Merkel cell polyomavirus plays an important role in the pathogenesis of Merkel cell polyomavirus-positive Merkel cell carcinoma.     

Detection of Merkel cell polyomavirus in Merkel cell carcinoma and Kaposi's sarcoma

Merkel cell carcinoma is a rare malignancy that sometimes occurs in the skin of elderly people. Recently, a new human polyomavirus, Merkel cell polyomavirus (MCPyV) was identified in Merkel cell carcinoma. In the present study, MCPyV‐DNA was detected in 6 of 11 (55%) cases of Merkel cell carcinoma by nested PCR and real‐time PCR. Histologically, MCPyV‐positive cases showed round and vesicular nuclei with a fine granular chromatin and small nucleoli, whereas MCPyV‐negative cases showed polygonal nuclei with diffusely distributed chromatin. Real‐time PCR analysis to detect the MCPyV gene revealed that viral copy numbers ranged 0.04–0.43 per cell in cases of Merkel cell carcinoma. MCPyV was also detected in 3 of 49 (6.1%) cases of Kaposi's sarcoma (KS), but not in 192 DNA samples of other diseases including 142 autopsy samples from 20 immunodeficient patients. The MCPyV copy number in KS was lower than that in Merkel cell carcinoma. PCR successfully amplified a full‐length MCPyV genome from a case of KS. Sequence analysis revealed that the MCPyV isolated from KS had 98% homology to the previously reported MCPyV genomes. These data suggest that the prevalence of MCPyV is low in Japan, and is at least partly associated with the pathogenesis of Merkel cell carcinoma. J. Med. Virol. 81:1951–1958, 2009. © 2009 Wiley‐Liss, Inc.     

Nuclear localization of Merkel cell polyomavirus large T antigen in Merkel cell carcinoma

To clarify whether mutations in the large T gene encoded by Merkel cell polyomavirus affect the expression and function of large T antigen in Merkel cell carcinoma cases, we investigated the expression of large T antigen in vitro and in vivo. Immunohistochemistry using a rabbit polyclonal antibody revealed that large T antigen was expressed in the nuclei of Merkel cell carcinoma cells with Merkel cell polyomavirus infection. Deletion mutant analyses identified an Arg-Lys-Arg-Lys sequence (amino acids 277-280) as a nuclear localization signal in large T antigen. Sequence analyses revealed that there were no mutations in the nuclear localization signal in any of the eleven Merkel cell polyomavirus strains examined. Furthermore, stop codons were not observed in the upstream of the nuclear localization signal in any of the Merkel cell carcinoma cases examined. These data suggest that the nuclear localization signal is highly conserved and functional in Merkel cell carcinoma cases.     

High load of Merkel cell polyomavirus DNA detected in the normal skin of Japanese patients with Merkel cell carcinoma

BackgroundAlthough Merkel cell polyomavirus (MCPyV) has the potential to cause Merkel cell carcinoma (MCC), it is also found in the normal skin of healthy individuals. However, the mechanism for transformation of MCPyV to an oncogenic form is unknown.ObjectivesTo investigate the levels of MCPyV infection in the normal skin patients with MCC compared with those in a control cohort.Study designWe studied a total of six Japanese patients with cutaneous MCC. Sun-exposed and sun-unexposed skin swabs were obtained and analyzed for MCPyV loads using quantitative real-time polymerase chain reaction.ResultsAt first, we found a patient with MCC carrying an extremely high load of MCPyV DNA in normal skin. This unique case prompted us to further explore the levels of MCPyV as skin microbiota in patients with MCC. We showed that MCPyV DNA levels were significantly higher in swabs obtained from normal skin samples of six patients with MCC compared with those from 30 age-matched healthy individuals and 19 patients with other cutaneous cancers. Whereas MCPyV strains obtained from the normal skin of patients with MCC had gene sequences without structural alterations, sequences of the tumor-derived strains showed truncating mutations or deletions.ConclusionsAlthough the number of patients with MCC studied was small, our findings suggest that MCC may occur with a background of high MCPyV load in the skin, and are expected to stimulate further studies on whether such skin virome levels could be one of predictive markers for the development of MCC.     

The spectrum of Merkel cell polyomavirus expression in Merkel cell carcinoma, in a variety of cutaneous neoplasms, and in neuroendocrine carcinomas from different anatomical sites

Most Merkel cell carcinomas display pure neuroendocrine differentiation (pure Merkel cell carcinoma), whereas a minority show combined neuroendocrine and nonneuroendocrine elements (combined Merkel cell carcinoma). Recent identification of Merkel cell polyomavirus DNA and Merkel cell polyomavirus large T antigen expression in a proportion of Merkel cell carcinomas has suggested viral-induced oncogenesis. To date, Merkel cell polyomavirus immunohistochemistry has shown an absence of viral large T antigen expression in combined Merkel cell carcinoma as well as select non-Merkel cell carcinoma cutaneous lesions and visceral neuroendocrine tumors. In our series, we aimed to further characterize the frequency and pattern of Merkel cell polyomavirus large T antigen expression by CM2B4 immunohistochemistry in primary and metastatic Merkel cell carcinoma (pure Merkel cell carcinoma and combined Merkel cell carcinoma) and various non-Merkel cell carcinoma lesions from patients with Merkel cell carcinoma, patients without Merkel cell carcinoma, and individuals with altered immune function. Merkel cell polyomavirus large T antigen was detected in 17 (63%) of 27 pure Merkel cell carcinomas and absent in all 15 (0%) combined Merkel cell carcinomas. Furthermore, complete concordance (100%) of Merkel cell polyomavirus large T antigen expression was observed in 10 cases of primary Merkel cell carcinoma and subsequent tumor metastases. We also evaluated 70 non-Merkel cell carcinoma lesions including 15 cases each of pulmonary and gastrointestinal neuroendocrine tumors. All 70 non-Merkel cell carcinoma lesions were negative for Merkel cell polyomavirus by CM2B4 immunohistochemistry, irrespective of any known Merkel cell carcinoma diagnosis and immune status. In summary, our identification of Merkel cell polyomavirus large T antigen expression in a subset of Merkel cell carcinoma and lack of findings in combined Merkel cell carcinomas and non-Merkel cell carcinoma lesions concur with earlier findings and implicate Merkel cell polyomavirus-independent pathogenesis in these cases. Overall, CM2B4 immunohistochemistry appears to be a specific method for Merkel cell polyomavirus detection and has the potential to play an important role in the diagnosis and classification of Merkel cell carcinoma in the future.     

Recent advances in the biology of Merkel cell carcinoma

Recent outstanding research has rapidly revealed new aspects of the biology, etiology, and clinicopathology of Merkel cell carcinoma, a rare but highly aggressive neuroendocrine skin malignancy that affects the elderly and immunosuppressed patients. Molecular biological studies, especially the discovery of Merkel cell polyomavirus, have shed new light on the pathogenesis of the disease. Increasing evidence strongly suggests that this virus is causally related to the development of Merkel cell carcinoma. On the other hand, many studies have also indicated that a subset (approximately 20%) of Merkel cell carcinomas are not likely to be associated with the virus. Tumors with and without the virus have been shown to be significantly different in prognosis, oncogene expression, and histologic appearance, suggesting that they have different etiologies. Moreover, studies on the histopathology, immunohistochemistry, and cytogenetics have revealed several biological factors that are related to the clinical behavior and prognosis of the disease. This review summarizes the advances in the molecular biology of Merkel cell carcinoma based on recent study results. Although the exact molecular pathway of the pathogenesis of Merkel cell carcinoma remains unclear, further understanding of the pathophysiology of this tumor is expected to result in novel therapeutic approaches for management of the disease and contribute to better patient outcomes.     

Detection of Merkel cell polyomavirus and human papillomavirus DNA in porocarcinoma

BackgroundIncreasing evidences support the role of Merkel cell polyomavirus (MCPyV) and human papillomavirus (HPV) in non-cutaneous and cutaneous tumours. Porocarcinoma is a rare malignant neoplasm that arises from the intraepidermal ductal portion of the eccrine sweat glands. The aetiology of porocarcinoma is largely unknown and no systematic studies have been done to investigate the implication of infectious agents in the pathogenesis of this tumour.ObjectivesTo investigate the possible association between MCPyV and/or HPV infection and porocarcinoma.Study designForty-four formalin-fixed paraffin-embedded (FFPE) porocarcinomas (40 primary and 4 metastatic) and 10 healthy skin specimens (controls), were analysed for the presence of MCPyV and HPV DNA using molecular detection methods.ResultsMCPyV DNA was found in 27/40 (68%) primary porocarcinomas and in 3/10 (30%) controls (Fisher exact test: p < 0.04). No significant difference in viral load was observed between tumours and healthy skin. Moreover, 2/40 primary porocarcinomas tested positive for high-risk HPV16. Cutaneous beta-HPV infection was detected in 16/40 (40%) porocarcinomas and in 6/10 (60%) controls. No particular beta-HPV types were significantly associated with tumour or with healthy skin. Two out of 4 metastatic biopsies were MCPyV DNA positive. All metastatic samples had mixed infections with cutaneous HPV types.ConclusionsThis study demonstrated a significantly high prevalence of MCPyV and the presence of a broad spectrum of HPV types in porocarcinoma and provided the first available data about viral infections in this tumour. To understand the role, if any, of viral infections in the pathogenesis of porocarcinoma further studies are needed.     

Merkel cell carcinoma: a review and update on current concepts

Merkel cell carcinoma (MCC) is a malignant, cutaneous neuroendocrine tumour of the elderly with an increasing worldwide incidence. Clinical presentation is generally characterized by a rapidly-evolving dermal tumour on sun-exposed skin of the head, neck or extremities. Histologically, there are sheets and cords of uniform, small cells with hyperchromatic nuclei and multiple small nucleoli. Mitoses and apoptotic bodies are widespread and lymphovascular involvement is commonly present. Aggressive surgical treatment of localized primary lesions followed by radiotherapy remains the mainstay of treatment. Lymph node metastases, local recurrences, and widespread dissemination are commonly seen. The 10-year survival rates for MCC are 71%, 48%, and 20% for localized, regional, and distant disease, respectively. Merkel cell polyomavirus (MCV) has been implicated as a contributing factor in the pathogenesis of MCC with approximately 80% of tumours showing positivity for the virus. This review provides an up-to-date overview of the clinicopathologic features, current knowledge of MCV, and recent advances in diagnosis, prognostication, and management of MCC.     

MicroRNA dysregulations in Merkel cell carcinoma: Molecular mechanisms and clinical applications

Merkel cell carcinoma (MCC) is an aggressive skin malignancy with two distinct etiologies. The first, which accounts for the highest proportion, is caused by Merkel cell polyomavirus (MCPyV), a DNA tumor virus. A second, UV-induced, MCC form has also been identified. Few MCC diagnostic, prognostic, and therapeutic options are available. MicroRNAs (miRNAs) are small noncoding RNA molecules, which play a key role in regulating various physiologic cellular functions including cell cycling, proliferation, differentiation, and apoptosis. Numerous miRNAs are dysregulated in cancer, by acting as either tumor suppressors or oncomiRs. The aim of this review is to collect, summarize, and discuss recent findings on miRNAs whose dysregulation has been assumed to play a role in MCC. The potential clinical application of miRNAs as diagnostic and prognostic biomarkers in MCC is also described. In the future, miRNAs will potentially gain clinical significance for the improvement of MCC diagnostic, prognostic, and therapeutic options.     

An update on diagnostic features of Merkel cell carcinoma

Merkel cell carcinoma (MCC) is an aggressive primary cutaneous neuroendocrine carcinoma that predominantly affects the elderly and immunosuppressed. Although rare, the incidence of MCC is increasing. Evidence suggests that the pathogenesis of MCC is related to Merkel cell polyomavirus infection or ultraviolet mutagenesis. Clinically, MCC typically presents as an asymptomatic violaceous papule on the head and neck. Histologically MCC is a small round blue cell tumor that must be differentiated from other small cell tumors, therefore immunohistochemistry is necessary for diagnosis of MCC. However, recent evidence suggests that some standard diagnostic markers may be less reliable in virus-negative MCC. MCC may develop local and distant metastases, with poor prognosis for advanced disease. Immunotherapy has recently been shown to be effective for many advanced cases. Here, we review diagnostic features of MCC including potential diagnostic and prognostic differences between virus-positive and virus-negative tumors.     

Immunohistochemistry for Merkel cell polyomavirus is highly specific but not sensitive for the diagnosis of Merkel cell carcinoma in the Australian population

Recent studies have demonstrated a high frequency of detection of Merkel cell polyomavirus in Merkel cell carcinoma. However, most of these studies are from European or North American centers that have relatively low sun exposure and may have a higher incidence of virus-driven oncogenesis compared with the highly sun-exposed but predominantly fair-skinned Australian population. We performed immunohistochemistry for Merkel cell polyomavirus on 104 cases of Merkel cell carcinoma and 74 cases of noncutaneous small cell–undifferentiated carcinoma from 3 major Australian centers. Nineteen (18.3%) cases of Merkel cell carcinoma showed positive staining for Merkel cell polyomavirus versus 1 (1.3%) of small cell–undifferentiated carcinoma. All 15 cases (14.3%) of Merkel cell carcinoma with areas of mixed squamous differentiation showed negative staining. We found positive staining in only 3 (7.7%) of 39 Merkel cell carcinoma from the head and neck (the most sun-exposed area) versus 16 (24.6%) of 65 of tumors from other sites (P < .05). Our findings support the concept of a Merkel cell polyomavirus–driven and a non-Merkel cell polyomavirus–driven (primarily sun-dependent) pathway in Merkel cell carcinoma carcinogenesis, with the latter being significantly more frequent in Australia and in mixed squamous–Merkel cell carcinoma (which is also more frequent in Australia). Although immunohistochemistry for Merkel cell polyomavirus seems to be highly specific in all populations, the low incidence of Merkel cell polyomavirus–positive Merkel cell carcinoma in a highly sun-exposed population limits its diagnostic utility in this setting.     

Human Merkel cell polyomavirus: virological background and clinical implications

The Merkel cell polyomavirus (MCPyV), identified in humans in 2008, is associated with a relatively rare but aggressive neuroendocrine skin cancer, the Merkel cell carcinoma (MCC). MCC incidence is increasing due to the advancing age of the population, the increase in damaging sun exposure and in the number of immunocompromised individuals. MCPyV must be considered as the etiological agent of MCC and thus is the first example of a human oncogenic polyomavirus. MCPyV infection is common, and seroprevalence studies indicate that widespread exposure begins early in life. The majority of adults have anti‐MCPyV antibodies and there is a growing body of evidence that healthy human skin harbors resident or transient MCPyV suggesting that MCPyV infection persists throughout life. However, the mode of transmission, the host cells, and the latency characteristics of this virus remain to be elucidated. In addition, it is still not clear whether MCPyV is associated with diseases or lesions other than Merkel cell carcinoma. The etiologic role of MCPyV in MCC opens up opportunities to improve the understanding of this cancer and to potentially improve its treatment.