Characterisation of four merkel cell carcinoma adherent cell lines

We have previously described the establishment of a number of cell lines from Merkel cell carcinoma (MCC), also known as small cell cancer of the skin or neuroendocrine carcinoma of the skin. These cells, all of which grew as suspension cultures, were found to resemble small cell lung cancer (SCLC) lines types 1, 2 and 3 by their morphology and growth characteristics. We now report 4 more MCC cell lines which resemble the SCLC type 4 cell lines in that they grow as adherent monolayers. These MCC lines would belong to the variant subgroup as they no longer express most neuroendocrine markers, grow at low cell density and have population doubling times of 1–5 days in contrast to the MCC suspension lines which have doubling times of 6–12 days. MCC 14/1 and MCC 14/2 were established from the same metastatic node and would appear to represent 2 clones of the tumour which differ in morphology, histochemical markers and DNA content. We present details of the morphology, DNA content and immunohistochemistry of these 4 lines and com-pare their growth patterns with those of SCLC and MCC lines which grow in suspension. © 1995 Wiley-Liss, Inc.     

Merkel cell carcinoma

Merkel cell carcinoma (MCC) is a rare, aggressive cutaneous cancer that predominately affects elderly Caucasians with fair skin and has a propensity for local recurrence and regional lymph node metastases. A variety of terms have been used to describe this tumor, including trabecular cell carcinoma, neuroendocrine or primary small cell carcinoma of the skin, and anaplastic cancer of the skin. Although the skin lesion is most commonly found on sun-exposed areas of the head and neck or extremities, it can occur on the trunk, genitalia, and perianal region. The median age is 69 years, but it may occur earlier and more frequently in immunosuppressed patients. Patients with MCC frequently present with a nonspecific erythematous or violaceous firm nodule or small plaque that may be surrounded by small satellite tumors. MCC usually arises in the dermis and extends into the subcutis. It may be difficult to accurately diagnose MCC by light microscopy alone and ancillary techniques, including electron microscopy and immunohistochemistry, may be necessary to make a definitive diagnosis. Management of MCC is dependent on stage of the disease and is hampered by its rarity and lack of randomized trials. Nonetheless, for localized disease most guidelines include wide local excision of the primary tumor either alone or with radiation therapy. Sentinel lymph node biopsy can be helpful in staging and prognosis, but its benefit in survival remains to be seen. Systemic chemotherapy, akin to regimens for small cell carcinoma of the lung, may be considered as an adjuvant following surgery or to treat locoregional or distant disease. The prognosis of MCC is variable. Some patients with localized disease have an indolent course and are well controlled with local excision alone. On the other hand, many tumors are aggressive and have a tendency for locoregional recurrence and distant metastases. Such patients have a grim prognosis with a median survival of 9 months. Successful outcomes are most often seen in patients with early diagnosis and complete excision.     

Merkel cell carcinomas

Merkel cell carcinoma (MCC) is a rare and extremely aggressive skin cancer that arises from primary neural cells. It presents most commonly in the elderly and immunocompromised patients. Pathologically, MCC should be distinguished from extrapulmonary small cell lung cancer or metastatic small cell lung cancer or a small cell variant of melanoma. The prognosis is based largely on the stage of disease at the time of presentation. Therapeutic options for MCC include wide resection with or without adjuvant radiotherapy or chemotherapy. Novel therapies based on the understanding of the molecular aspects of MCC are currently being explored.     

The Merkel cell carcinoma challenge

Merkel cell carcinoma (MCC) is a highly aggressive neuroendocrine carcinoma of the skin that occurs primarily in elderly or immunocompromised patients. For this report, the authors reviewed the diagnostic challenges associated with MCC encountered on their fine‐needle aspiration (FNA) service and also conducted an in‐depth review of the literature on MCC. A computer search for patients who were diagnosed with MCC by FNA at the authors' institution from 2006 to 2010 was conducted, and 5 patients were selected for cytologic and immunochemical analyses based on their varied and diagnostically challenging clinical presentations. The 5 selected patients had clinical findings commonly associated with MCC, including advanced age (4 of the 5 patients were ages 75‐85 years) and a history of previous malignancies (3 of the 5 patients had a history of previous malignancy), and 1 patient was diagnosed with a concomitant low‐grade lymphoma. The patients and their disease illustrated the protean clinical presentation of MCC and the clinical and cytologic challenges associated with this neoplasm. The current findings indicate the need for cytopathologists to be aware of the deceptive presentation of this neoplasm and its cytologic and immunochemical features to correctly diagnose this insidious neoplasm. Cancer (Cancer Cytopathol) 2013;121:179–188. © 2012 American Cancer Society.     

Frequent allelic loss at 10q23 but low incidence of PTEN mutations in Merkel cell carcinoma

Merkel cell carcinoma (MCC) is a rare, highly metastatic skin tumor of neuroectodermal origin. The disease shares clinical and histopathological features with small cell lung carcinoma (SCLC). The genetic mechanisms underlying the development and tumor progression of MCC are poorly understood. We recently showed by comparative genomic hybridization (CGH) that the pattern of chromosomal abnormalities in MCC resembles that of SCLC. One of the most frequently observed losses involved the entire chromosome 10 or partial loss of the chromosome 10 long arm (33% of examined MCC cases). The PTEN tumor-suppressor gene has been mapped to 10q23.3 and was shown to be mutated in a variety of human cancers including SCLC. Germline PTEN mutations have been observed in familial predisposing cancer syndromes including Cowden disease. Interestingly, an association between Cowden syndrome and Merkel cell carcinoma has been reported. To study the possible role of PTEN in MCC oncogenesis, loss of heterozygosity (LOH) analysis for the 10q23 region was performed on 26 MCC tumor samples from 23 MCC patients. The PTEN locus was deleted in 9 of 21 (43%) informative MCC tumor samples [7 of 18 (39%) MCC patients]. Despite this high frequency of LOH at 10q23, mutation and homozygous deletion screening of the PTEN gene revealed only one tumor with a nonsense mutation and a second with a homozygous deletion of exon 9. These data suggest that either alternative mechanisms lead to inactivation of the PTEN gene or that other tumor-suppressor genes at chromosome 10 are implicated in the development of MCC.     

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.     

皮肤Ｍｅｒｋｅｌ细胞癌的研究进展

Ｍｅｒｋｅｌ细胞癌（ＭＣＣ）是一种好发于皮肤的少见恶性神经内分泌瘤,预后较差,易局部复发和转移。ＭＣＣ的临床诊断可参考“ＡＥＩＯＵ”五元音法,但必须由组织病理学确诊。ＭＣＣ在高度紫外线暴露的地区较常见,且与免疫抑制密切相关。ＭＣＣ可能与Ｍｅｒｋｅｌ细胞多瘤病毒（ＭＣＰｙＶ）相关。目前ＭＣＣ尚无成熟的治疗方案,但首选手术治疗并常常辅助放疗或化疗。     

Detection of Merkel cell virus and correlation with histologic presence of Merkel cell carcinoma in sentinel lymph nodes

Background:

Adjuvant treatment can dramatically improve the survival of patients with metastatic Merkel cell carcinoma (MCC), making early, accurate detection of nodal disease critical. The purpose of this study was to correlate Merkel cell virus (MCV) detection with histopathologic disease in sentinel lymph nodes (SLNs) of MCC.

Methods:

Merkel cell carcinoma cases with SLN (n=25) were compared with negative controls (n=27). Viral load was obtained by quantitative polymerase chain reaction (PCR) for regions VP1 and LT3 of MCV. Histopathologic disease and viral load were correlated.

Results:

Merkel cell virus was detected in 16 out of 17 (94%) of primary MCC (mean viral load (MVL)=1.44 copies per genome). Viral load in the negative controls was <0.01 copies per genome. Merkel cell carcinoma was present in 5 out of 25 (20%) SLN by histopathology, and MCV was detected in 11 out of 25 (44%) MCC SLN (MVL=1.68 copies per genome). In all, 15 out of 25 (60%) SLN showed correlation between histologic and MCV results. In all, 2 out of 25 (8%) samples were histopathologically positive and PCR negative. Of note, 8 out of 25 (32%) samples had detectable MCV without microscopic disease.

Conclusion:

Patients with positive SLN for MCV even if negative by histopathology were identified. The application of molecular techniques to detect subhistologic disease in SLN of MCC patients may identify a subset of patients who would benefit from adjuvant nodal treatment.     

Clinical emergence of neurometastatic merkel cell carcinoma: a surgical case series and literature review

Merkel cell carcinoma (MCC) is a rare cutaneous neuroendocrine neoplasm of possible viral origin and is known for its aggressive behavior. The incidence of MCC has increased in the last 15 years. Merkel cell carcinoma has the potential to metastasize, but rarely involves the central nervous system. Herein, we report three consecutive surgical cases of MCC presenting at a single institution within 1 year. We used intracavitary BCNU wafers (Gliadel^®) in two cases. Pathological features, including CK20 positivity, consistent with MCC, were present in all cases. We found 33 published cases of MCC with CNS involvement. We suggest that the incidence of neurometastatic MCC may be increasing, parallel to the increasing incidence of primary MCC. We propose a role for intracavitary BCNU wafers in the treatment of intra-axial neurometastatic MCC.     

Merkel cell carcinoma in a renal transplant patient

Merkel cell carcinoma (MCC) is an aggressive neuroendocrine skin cancer which seems to be common in transplant recipients. We describe the case of a renal transplant patient who developed a MCC on the right glutaeus eight years after transplantation.     

Clinical added-value of 18FDG PET in neuroendocrine-merkel cell carcinoma

Merkel cell carcinoma (MCC) is a rare and highly malignant skin cancer with neuroendocrine differentiation. We studied the potential value of 18FDG PET in the management of MCC. Eleven patients with MCC were examined by 18FDG PET and PET-CT for staging purpose (n=4) or for detection of recurrence (n=7). Qualitative and quantitative interpretation of PET studies was performed routinely. 18FDG PET observations were compared to clinical and radiological findings. In 6 patients, PET findings were also compared to histology. In 7 patients, the 18FDG tumor uptake was compared to the MCC proliferative activity expressed by the Ki-67 index. 18FDG PET was contributive in 10/11 MCC patients. In 7 patients, 18FDG PET detected focal lesions or a disseminated stage of the disease including dermal, nodal and visceral metastases. In 3 patients, a normal 18FDG PET confirmed complete remission of disease. Most MCC patients exhibited highly 18FDG-avid sites suggestive of increased glucose metabolism. This imaging pattern was related to a high proliferative activity (Ki-67 index >50%). In 1 patient with a weakly proliferative nodal MCC (Ki-67<10%), a false negative result was yielded by metabolic imaging. In 4/11 patients, 18FDG PET revealed an unsuspected second neoplasm in addition to MCC. It is concluded that whole-body 18FDG PET may be useful in the management of MCC patients. However, a normal 18FDG PET aspect cannot rule out MCC with low proliferative activity.     

Antibodies to merkel cell polyomavirus T antigen oncoproteins reflect tumor burden in merkel cell carcinoma patients

Merkel cell polyomavirus (MCPyV) is a common infectious agent that is likely involved in the etiology of most Merkel cell carcinomas (MCC). Serum antibodies recognizing the MCPyV capsid protein VP1 are detectable at high titer in nearly all MCC patients and remain stable over time. Although antibodies to the viral capsid indicate prior MCPyV infection, they provide limited clinical insight into MCC because they are also detected in more than half of the general population. We investigated whether antibodies recognizing MCPyV large and small tumor-associated antigens (T-Ag) would be more specifically associated with MCC. Among 530 population control subjects, these antibodies were present in only 0.9% and were of low titer. In contrast, among 205 MCC cases, 40.5% had serum IgG antibodies that recognize a portion of T-Ag shared between small and large T-Ags. Among cases, titers of T-Ag antibodies fell rapidly (～8-fold per year) in patients whose cancer did not recur, whereas they rose rapidly in those with progressive disease. Importantly, in several patients who developed metastases, the rise in T-Ag titer preceded clinical detection of disease spread. These results suggest that antibodies recognizing T-Ag are relatively specifically associated with MCC, do not effectively protect against disease progression, and may serve as a clinically useful indicator of disease status.     

PD-L1 blockade with avelumab: A new paradigm for treating Merkel cell carcinoma

Merkel cell carcinoma (MCC) is a rare and aggressive neuroendocrine skin cancer. Until recently, no durable treatment options were available for patients with advanced disease. As an immunogenic cancer, MCC was hypothesized to be a candidate for PD-L1/PD-1 targeted therapy. On March 23, 2017 the US Food and Drug Administration granted accelerated approval for avelumab, an anti-PD-L1 monoclonal antibody, for the treatment of metastatic MCC on the basis of the JAVELIN Merkel 200 trial. Here we examine the results and implications of this pivotal study, published in Lancet Oncology by Kaufman et al., as well as current developments in the use of immune-checkpoint therapies for treating patients with MCC.     

Gene-expression profiling reveals distinct expression patterns for Classic versus Variant Merkel cell phenotypes and new classifier genes to distinguish Merkel cell from small-cell lung carcinoma

Merkel cell carcinoma (MCC) is a rare aggressive skin tumor which shares histopathological and genetic features with small-cell lung carcinoma (SCLC), both are of neuroendocrine origin. Comparable to SCLC, MCC cell lines are classified into two different biochemical subgroups designated as 'Classic' and 'Variant'. With the aim to identify typical gene-expression signatures associated with these phenotypically different MCC cell lines subgroups and to search for differentially expressed genes between MCC and SCLC, we used cDNA arrays to profile 10 MCC cell lines and four SCLC cell lines. Using significance analysis of microarrays, we defined a set of 76 differentially expressed genes that allowed unequivocal identification of Classic and Variant MCC subgroups. We assume that the differential expression levels of some of these genes reflect, analogous to SCLC, the different biological and clinical properties of Classic and Variant MCC phenotypes. Therefore, they may serve as useful prognostic markers and potential targets for the development of new therapeutic interventions specific for each subgroup. Moreover, our analysis identified 17 powerful classifier genes capable of discriminating MCC from SCLC. Real-time quantitative RT-PCR analysis of these genes on 26 additional MCC and SCLC samples confirmed their diagnostic classification potential, opening opportunities for new investigations into these aggressive cancers.     

Molecular and immune targets for Merkel cell carcinoma therapy and prevention

Merkel cell carcinoma (MCC) is a rare neuroendocrine carcinoma of the skin, for which the exact mechanisms of carcinogenesis remain unknown. Therapeutic options for this highly aggressive malignancy have historically been limited in both their initial response and response durability. Recent improvements in our understanding of MCC tumor biology have expanded therapeutic options for these patients, namely through the use of immunotherapies such as immune checkpoint inhibitors. Further elucidation of the tumor mutational landscape has identified molecular targets for therapies, which have demonstrated success in other cancer types. In this review, we discuss both current and investigational immune and molecular targets of therapy for MCC.     

Merkel cell carcinoma after chronic lymphocytic leukemia: case report and literature review

Increasing evidence supports an association of Merkel cell carcinoma (MCC) with immunodeficiency and neoplasia, and the management and outcome of these patients requires study. This report describes a 72-year-old man with newly diagnosed chronic lymphocytic leukemia (CLL) who developed MCC of his right upper extremity and died of bone marrow metastases at 8 months. In the five previously reported cases of MCC after CLL, a shorter time interval between the diagnosis of CLL and the onset of MCC was associated with a better prognosis. In contrast, in this case the near simultaneous onset of CLL and MCC was followed by a rapid, lethal outcome.     

Merkel cell carcinoma,chronic lymphocytic leukemia and other lymphoproliferative disorders: an old bond with possible new viral ties

Merkel cell carcinoma (MCC) is a rare and aggressive skin tumor. The link between tumorigenesis and immunosuppression is well known and the increased prevalence of MCC in human immunodeficiency virus carriers and organ transplant recipients and in patients with hemato-oncological neoplasias is now well recognized over the past decade. In this respect, chronic lymphocytic leukemia (CLL) seems to be the most frequent neoplasia associated with the development of MCC. Very recently, a newly described virus, the Merkel cell polyomavirus, was found in ∼80% of MCC tumor samples and is in fact the first member of the polyomavirus family to be associated with human tumors. The virus appears to play a role in the pathogenesis of MCC and may constitute the missing link between immunosuppression and the development of MCC. This review summarizes the current knowledge relating to MCC and its pathogenesis, stressing the link with hematologic neoplasias in general and to CLL in particular. We describe the permissive immunologic environment, which enables the virus-containing tumor cells to survive and proliferate in disorders like CLL. More studies are still needed to confirm this appealing theory in a more convincing manner.     

Transcriptomic analyses reveal three distinct molecular subgroups of Merkel cell carcinoma with differing prognoses

Merkel cell carcinoma (MCC) is a cutaneous neuroendocrine malignancy with a poor prognosis and an unknown cell of origin. Proffered cells of origin include epithelial stem cells of the hair follicle or interfollicular epidermis, dermal stem cells and pro/pre- or pre-B cells. MCC has also been proposed to have more than one cell of origin and indeed to represent more than one type of carcinoma, currently grouped together due to phenotypic similarities. We explored the heterogeneous nature of MCC by studying the most variably expressed genes with the goal of identifying gene expression patterns that are either clinically relevant or have implications regarding the cell(s) of origin. We performed RNA sequencing on primary tumor samples from 102 patients and identified the top 200 most variably expressed genes. These genes and the tumor samples were hierarchically clustered based on their expression. The functions of three gene clusters exhibiting clearly divergent expression between samples were studied by cross-referencing the lists of genes with online databases. High expression of a gene cluster related to embryonic developmental processes and low expression of a gene cluster related to neuroendocrine processes distinguished Merkel cell polyomavirus (MCPyV)-negative tumors from MCPyV-positive tumors. Furthermore, two prognostically relevant subgroups of MCPyV-positive MCC were identified based on dichotomic expression of genes related to epidermal structures and processes. We identified three distinct molecular subgroups of MCC with prognostic relevance. We propose that the dichotomic expression of epidermis-related genes might reflect both an epidermal and a nonepidermal origin for MCPyV-positive MCC.     

Combined effect of aloe-emodin and chemotherapeutic agents on the proliferation of an adherent variant cell line of Merkel cell carcinoma

Merkel cell carcinoma (MCC) has only limited sensitivity to chemotherapeutic agents. The aim of the study was to determine if members of the anthraquinone family could be used as adjuncts to increase the growth inhibiting effect of anticancer agents in MCC. An adherent variant of MCC was derived from a previously established MCC cell line suspension. Cells were characterized by immunocytochemical methods using specific antibodies against epithelial (low molecular weight cytokeratins and cytokeratin 20) and neuroendocrine (neuron-specific enolase, neurofilament protein, chromogranin A and synaptophysin) antigens. Emodin and aloe-emodin, members of the anthraquinone family, inhibited proliferation of the adherent MCC cells, with a slight advantage of aloe-emodin over emodin. Aloin had no effect on cell proliferation. The chemotherapeutic agents, cis-platinol (abiplastin), doxorubicin (adriablastin), and 5-fluorouracil, and the tyrosine kinase inhibitor STI 571, all independently inhibited the proliferation of adherent MCC cells. The addition of aloe-emodin potentiated their inhibitory effect, especially when low concentrations of the anticancer compounds were used. The antiproliferative action of STI 571 may be associated with the presence of anti-c-kit antibodies. The combined use of anticancer agents, especially at low concentrations, and aloe-emodin may be considered a preferable means for treating MCC.