Detection of wildtype Merkel cell polyomavirus genomic sequence and VP1 transcription in a subset of Merkel cell carcinoma.

AIMS: Merkel cell carcinoma (MCC) is frequently caused by the Merkel cell polyomavirus (MCPyV). Characteristic for these virus-positive (VP) MCC is MCPyV integration into the host genome and truncation of the viral oncogene Large T antigen (LT), with full-length LT expression considered as incompatible with MCC growth. Genetic analysis of a VP-MCC/trichoblastoma combined tumour demonstrated that virus-driven MCC can arise from an epithelial cell. Here we describe two further cases of VP-MCC combined with an adnexal tumour, i.e. one trichoblastoma and one poroma. METHODS AND RESULTS: Whole-genome sequencing of MCC/trichoblastoma again provided evidence of a trichoblastoma-derived MCC. Although an MCC-typical LT-truncating mutation was detected, we could not determine an integration site and we additionally detected a wildtype sequence encoding full-length LT. Similarly, Sanger sequencing of the combined MCC/poroma revealed coding sequences for both truncated and full-length LT. Moreover, in situ RNA hybridization demonstrated expression of a late region mRNA encoding the viral capsid protein VP1 in both combined as well as in a few cases of pure MCC. CONCLUSION: The data presented here suggest the presence of wildtype MCPyV genomes and VP1 transcription in a subset of MCC.

Distinct regulations driving YAP1 expression loss in poroma, porocarcinoma and RB1-deficient skin carcinoma.

AIMS: Recently, YAP1 fusion genes have been demonstrated in eccrine poroma and porocarcinoma, and the diagnostic use of YAP1 immunohistochemistry has been highlighted in this setting. In other organs, loss of YAP1 expression can reflect YAP1 rearrangement or transcriptional repression, notably through RB1 inactivation. In this context, our objective was to re-evaluate the performance of YAP1 immunohistochemistry for the diagnosis of poroma and porocarcinoma. METHODS AND RESULTS: The expression of the C-terminal part of the YAP1 protein was evaluated by immunohistochemistry in 543 cutaneous epithelial tumours, including 27 poromas, 14 porocarcinomas and 502 other cutaneous tumours. Tumours that showed a lack of expression of YAP1 were further investigated for Rb by immunohistochemistry and for fusion transcripts by real-time PCR (YAP1::MAML2 and YAP1::NUTM1). The absence of YAP1 expression was observed in 24 cases of poroma (89%), 10 porocarcinoma (72%), 162 Merkel cell carcinoma (98%), 14 squamous cell carcinoma (SCC) (15%), one trichoblastoma and one sebaceoma. Fusions of YAP1 were detected in only 16 cases of poroma (n = 66%), 10 porocarcinoma (71%) all lacking YAP1 expression, and in one sebaceoma. The loss of Rb expression was detected in all cases except one of YAP1-deficient SCC (n = 14), such tumours showing significant morphological overlap with porocarcinoma. In-vitro experiments in HaCat cells showed that RB1 knockdown resulted in repression of YAP1 protein expression. CONCLUSION: In addition to gene fusion, we report that transcriptional repression of YAP1 can be observed in skin tumours with RB1 inactivation, including MCC and a subset of SCC.

Recurrent FOXK1::GRHL and GPS2::GRHL fusions in trichogerminoma.

We report 21 cases of trichogerminoma harbouring previously undescribed FOXK1::GRHL1/2 or GPS2::GRHL1/2/3 in-frame fusion transcripts. Microscopic examination of a preliminary set of five cases revealed well-delimitated tumours located in the dermis with frequent extension to the subcutaneous tissue. Tumours presented a massive and nodular architecture and consisted of a proliferation of basaloid cells. A biphasic pattern sometime resulting in tumour cell nests ('cell balls') was present. Immunohistochemistry demonstrated the expression of cytokeratins (CKs) 15, 17, and PHLDA1. In addition, numerous CK20-positive Merkel cells were detected. RNA sequencing (RNA-seq) revealed a FOXK1::GRHL1 chimeric transcript in three cases and a FOXK1::GRHL2 fusion in two cases. In a second series for validation (n = 88), FOXK1::GRHL1/2 fusion transcripts were detected by RT-qPCR or FISH in an additional 12 trichogerminomas and not in any other follicular tumour entities or basal cell carcinoma cases (n = 66). Additional RNA-seq analysis in trichogerminoma cases without detected FOXK1::GRHL1/2 rearrangements revealed GPS2::GRHL1 fusion transcripts in two cases, GPS2::GRHL2 in one case, and GPS2::GRHL3 fusion transcript in one case. Therefore, our study strongly suggests that GRHL1/2/3 gene rearrangements might represent the oncogenic driver in trichogerminoma, a subset of follicular tumours characterized by immature features and numerous Merkel cells. © 2022 The Pathological Society of Great Britain and Ireland.

Effect of decalcification protocols on immunohistochemistry and molecular analyses of bone samples.

Diagnosis of osteocartilaginous pathologies depends on morphological examination and immunohistochemical and molecular biology analyses. Decalcification is required before tissue processing, but available protocols often lead to altered proteins and nucleic acids, and thus compromise the diagnosis. The objective of this study was to compare the effect of different methods of decalcification on histomolecular analyses required for diagnosis and to recommend an optimal protocol for processing these samples in routine practice. We prospectively submitted 35 tissue samples to different decalcification procedures with hydrochloric acid, formic acid, and EDTA, in short, overnight and long cycles for 1 to >10 cycles. Preservation of protein integrity was examined by immunohistochemistry, and quality of nucleic acids was estimated after extraction (DNA and RNA concentrations, 260/280 ratios, PCR cycle thresholds), analysis of DNA mutations (high-resolution melting) or amplifications (PCR, in situ hybridization), and detection of fusion transcripts (RT-PCR, in situ hybridization). Hydrochloric acid- and long-term formic acid-based decalcification induced false-negative results on immunohistochemistry and molecular analysis. EDTA and short-term formic acid-based decalcification (<5 cycles of 6 h each) did not alter antigenicity and allowed for detection of gene mutations, amplifications or even fusion transcripts. EDTA showed superiority for in situ hybridization techniques. According to these results and our institutional experience, we propose recommendations for decalcification of bone samples, from biopsies to surgical specimens.

Diagnostic accuracy of a panel of immunohistochemical and molecular markers to distinguish Merkel cell carcinoma from other neuroendocrine carcinomas.

Merkel cell carcinoma is a rare neuroendocrine carcinoma of the skin mostly induced by Merkel cell polyomavirus integration. Cytokeratin 20 (CK20) positivity is currently used to distinguish Merkel cell carcinomas from other neuroendocrine carcinomas. However, this distinction may be challenging in CK20-negative cases and in cases without a primary skin tumor. The objectives of this study were first to evaluate the diagnostic accuracy of previously described markers for the diagnosis of Merkel cell carcinoma and second to validate these markers in the setting of difficult-to-diagnose Merkel cell carcinoma variants. In a preliminary set (n = 30), we assessed optimal immunohistochemical patterns (CK20, thyroid transcription factor 1 [TTF-1], atonal homolog 1 [ATOH1], neurofilament [NF], special AT-rich sequence-binding protein 2 [SATB2], paired box protein 5, terminal desoxynucleotidyl transferase, CD99, mucin 1, and Merkel cell polyomavirus-large T antigen) and Merkel cell polyomavirus load thresholds (real-time PCR). The diagnostic accuracy of each marker was then assessed in a validation set of 103 Merkel cell carcinomas (9 CK20-negative cases and 15 cases without a primary skin tumor) and 70 extracutaneous neuroendocrine carcinoma cases. The most discriminant markers for a diagnosis of Merkel cell carcinoma were SATB2, NF expression, and Merkel cell polyomavirus DNA detection (positive likelihood ratios: 36.6, 44.4, and 28.2, respectively). Regarding Merkel cell carcinoma variants, cases without a primary skin tumor retained a similar immunohistochemical  profile and CK20-negative tumors displayed a different profile (decrease frequency of NF and SATB2 expression), but Merkel cell polyomavirus DNA remained detected (78% of cases by qPCR). Moreover, 8/9 (89%) CK20-negative Merkel cell carcinoma cases but only 3/61 (5%) CK20-negative extracutaneous neuroendocrine cases were positive for at least one of these markers. In conclusion, detection of SATB2 and NF expression and Merkel cell polyomavirus DNA helps distinguish between Merkel cell carcinoma classical and variant cases and extracutaneous neuroendocrine carcinomas.

Morphologic and immunophenotypical features distinguishing Merkel cell polyomavirus-positive and negative Merkel cell carcinoma.

In 2008, Feng et al. identified Merkel cell polyomavirus integration as the primary oncogenic event in ~80% of Merkel cell carcinoma cases. The remaining virus-negative Merkel cell carcinoma cases associated with a high mutational load are most likely caused by UV radiation. The current study aimed to compare the morphological and immunohistochemical features of 80 virus-positive and 21 virus-negative Merkel cell carcinoma cases. Microscopic evaluation revealed that elongated nuclei-similar to the spindle-shape variant of small cell lung cancer-were less frequent in Merkel cell polyomavirus-positive Merkel cell carcinoma compared to the virus-negative subset (p = 0.005). Moreover, virus-negative cases more frequently displayed a "large-cell neuroendocrine carcinoma" phenotype with larger cell size (p = 0.0026), abundant cytoplasm (p = 4×10-7) and prominent nucleoli (p = 0.002). Analysis of immunohistochemical data revealed frequent positivity for thyroid transcription factor 1 and cytokeratin 7, either absence or overexpression of p53, as well as frequent lack of neurofilament expression in virus-negative cases. By contrast, cytokeratin 8, 18 and 20 and a CD99 with a dot pattern as well as high EMA expression were identified as characteristic features of virus-positive Merkel cell carcinoma. In particular, the CD99 dot-like expression pattern was strongly associated with presence of the Merkel cell polyomavirus in Merkel cell carcinoma (sensitivity = 81%, specificity = 90%, positive likelihood ratio = 8.08). To conclude, virus-positive and -negative Merkel cell carcinoma are characterized by distinct morphological and immunohistochemical features, which implies a significant difference in tumor biology and behavior. Importantly, we identified the CD99 staining pattern as a marker indicating the virus status of this skin cancer.

Interest of variations in microRNA-152 and -122 in a series of hepatocellular carcinomas related to hepatitis C virus infection.

AIM: Hepatocellular carcinoma (HCC) is a common outcome of chronic hepatitis C virus (HCV) infection and constitutes the main burden of this disease. The molecular mechanisms underlying the development of HCC are multiple and might involve certain microRNA (miR). As discordant results have been reported concerning the detection of expression of miR-152 and miR-122 in HCC, our aim was to measure the levels of both miRs in serum and liver samples. METHODS: We analyzed miR-152 and miR-122 expression by reverse transcription-quantitative polymerase chain reaction in a serum cohort from 14 HCV-infected patients who developed HCC, 20 HCV+ patients without HCC, and 19 control patients. We also studied miR-152 and miR-122 in an independent tissue cohort from 11 normal livers, and from paired HCC and non-tumor adjacent livers of 11 HCV-infected patients and 12 non-infected patients. RESULTS: In serum samples, higher levels of miR-122 were found in non-HCC HCV+ compared to HCC HCV+ and control groups, whereas miR-152 was detectable in a lower range in HCC HCV+ compared to non-HCC HCV+ and control groups. We found higher signals for miR-122 and miR-152 in non-tumor liver and HCC tissues compared to control tissues. Hepatocellular carcinoma etiology had no detectable influence on miR-122 expression, whereas miR-152 was increased in HCV+ tissue samples. CONCLUSIONS: Detection of low values of circulating miR-152 is a potentially interesting marker of hepatocarcinogenesis in HCV+ patients, in contrast to miR-122, which varies according to hepatocyte damage.

Measurement of Telomere Length in Colorectal Cancers for Improved Molecular Diagnosis.

All tumors have in common to reactivate a telomere maintenance mechanism to allow for unlimited proliferation. On the other hand, genetic instability found in some tumors can result from the loss of telomeres. Here, we measured telomere length in colorectal cancers (CRCs) using TRF (Telomere Restriction Fragment) analysis. Telomeric DNA content was also quantified as the ratio of total telomeric (TTAGGG) sequences over that of the invariable Alu sequences. In most of the 125 CRCs analyzed, there was a significant diminution in telomere length compared with that in control healthy tissue. Only 34 tumors exhibited no telomere erosion and, in some cases, a slight telomere lengthening. Telomere length did not correlate with age, gender, tumor stage, tumor localization or stage of tumor differentiation. In addition, while telomere length did not correlate with the presence of a mutation in BRAF (V-raf murine sarcoma viral oncogene homolog B), PIK3CA (phosphatidylinositol 3-kinase catalytic subunit), or MSI status, it was significantly associated with the occurrence of a mutation in KRAS. Interestingly, we found that the shorter the telomeres in healthy tissue of a patient, the larger an increase in telomere length in the tumor. Our study points to the existence of two types of CRCs based on telomere length and reveals that telomere length in healthy tissue might influence telomere maintenance mechanisms in the tumor.

Intratumoral distribution of EGFR mutations and copy number in metastatic lung cancer, what impact on the initial molecular diagnosis?

BACKGROUND: Activating epidermal growth factor receptor (EGFR) mutations characterize a subgroup of non-small-cell lung cancer that benefit from first line EGFR tyrosine kinase inhibitors (EGFR-TKI). However, the existence of polyclonal cell populations may hinder personalized-medicine strategies as patients' screening often depends upon a single tumor-biopsy sample. The purpose of this study is to clarify and to validate in clinical testing conditions the accuracy of EGFR genotyping using different tumor sites and various types of samples (transthoracic, surgical or endoscopic biopsies and cytology specimens). METHODS: We conducted a retrospective review of 357 consecutive patients addressed for EGFR mutation screening in accordance with the directive of the European Medicines Agency (stage IV NSCLC). Fifty-seven samples were EGFR mutated and 40 had adequate tumor specimens for analysis on multiple spatially separated sites. Ten wild type samples were also analyzed. A total of 153 and 39 tumor fragments, from mutated and non-mutated cases respectively, were generated to analyze tumor heterogeneity or primary-metastatic discordances. After histological review of all fragments, EGFR genotyping was assessed using the routine diagnostic tools: fragment analysis for insertions and deletions and allele specific TaqMan probes for point mutations. EGFR copy number (CN) was evaluated by qPCR using TaqMan probes. RESULTS: The identification of EGFR mutations was independent of localization within primary tumor, of specimen type and consistent between primary and metastases. At the opposite, for half of the samples, tumor loci showed different EGFR copy number that may affect mutation detection cut-off. CONCLUSIONS: This is the largest series reporting multiple EGFR testing in Caucasians. It validates the accuracy of EGFR mutation screening from single tumor-biopsy samples before first line EGFR-TKI. The unpredictable variability in EGFR CN and therefore in EGFR wild type/mutant allelic ratio justifies the implementation of sensitive methods to identify patients with EGFR mutated tumors.

BRCA1/2 Pathogenic Variants Are Not Common in Merkel Cell Carcinoma: Comprehensive Molecular Study of 30 Cases and Meta-Analysis of the Literature.

Merkel cell carcinoma (MCC) is a rare and aggressive cutaneous neuroendocrine cancer. Management of advanced MCC is mainly based on immune-checkpoint inhibitors. The high failure rate warrants an investigation of new therapeutic targets. The recent identification of BRCA1 or BRCA2 (BRCA1/2) mutations in some MCC raises the issue of the use of poly-(ADP-Ribose)-polymerase inhibitors in selected advanced cases. The main objective of our study is to determine the accurate frequency of BRCA1/2 pathogenic variants. We studied a series of 30 MCC and performed a meta-analysis of BRCA1/2 variants of published cases in the literature. In our series, we detected only one BRCA2 pathogenic variant. The low frequency of BRCA1/2 pathogenic variants in our series of MCC (3%) was confirmed by the meta-analysis of BRCA1/2 variants in the literature. Among the 915 MCC from 13 published series studied for molecular alterations of BRCA1/2, only 12 BRCA1/2 pathogenic mutations were identified (1-2% of MCC), whereas many other BRCA1/2 variants were variants of unknown significance or benign. BRCA1/2 pathogenic variants are uncommon in MCC. However, in BRCA-mutated MCC, poly-(ADP-Ribose)-polymerase inhibitors might be a valuable therapeutic option requiring validation by clinical trials.

Digital Papillary Adenocarcinoma in Nonacral Skin: Clinicopathologic and Genetic Characterization of 5 Cases.

Digital papillary adenocarcinoma (DPA) is a rare sweat gland neoplasm that has exceptionally been reported outside acral locations. Recently, human papillomavirus 42 was identified as the main oncogenic driver of DPA. Herein, we report 5 tumors arising in extra-acral locations predominantly in the female anogenital skin. Four patients were female and 1 patient was male. The mean age at the diagnosis time was 65 years (range: 55 to 82 y). Tumors were located on the vulva (n=3), perianal area (n=1), and forearm (n=1). Histologically, all tumors were lobular and mainly solid and composed of sheets of cells with rare focal papillae and frequent glandular structures in a "back-to-back" pattern and lined by atypical basophilic cells. Immunohistochemistry showed diffuse positivity for SOX10. Epithelial membrane antigen and carcinoembryonic antigen highlighted the luminal cells and staining for p63 and p40 revealed a consistent and continuous myoepithelial component around glandular structures. Follow-up was available in 3 cases (mean duration: 12 mo [range: 8 to 16 mo]). One patient developed local recurrence and 1 experienced regional lymph node metastases. HPV Capture Next-generation sequencing revealed the presence of the HPV42 genome in all samples. Viral reads distributions were compatible in the 5 cases with an episomal nature of the viral genome, with a recurrent deletion in the E1 and/or E2 open reading frames. In conclusion, this study demonstrates that digital DPA may rarely present in nonacral locations mainly in the female anogenital area, usually with a more solid pattern as compared with those cases presenting on the digits and it is also associated with HPV42.

Sweat Gland Tumors Arising on Acral Sites: A Molecular Survey.

Recurrent oncogenic drivers have been identified in a variety of sweat gland tumors. Recently, integration of human papillomavirus type 42 (HPV42) has been reported in digital papillary adenocarcinoma (DPA). The main objectives of the present study were (i) to provide an overview of the prevalence of previously identified oncogenic drivers in acral sweat gland tumors and (ii) to genetically characterize tumors in which no recurrent genetic alteration has been identified yet. Cases of acral sweat gland tumors were identified from the database of the French network CARADERM. After histologic review, the presence of previously identified genetic alterations was investigated in the entire cohort (n=79) using a combination of immunohistochemistry and targeted DNA and RNA sequencing. Tumor entities with no recurrent genetic alterations were submitted to whole-transcriptome sequencing. CRTC1::MAML2 fusion was identified in cases of hidradenoma and hidradenocarcinoma (n=9/12 and n=9/12). A p.V600E mutation of BRAF was observed in all cases of tubular adenoma (n=4). YAP1:MAML2 and YAP1::NUTM1 fusions were observed in poroid tumors (n=15/25). ETV6::NTRK3 and TRPS1::PLAG1 fusion transcripts were identified in secretory carcinoma (n=1/1) and cutaneous mixed tumors (n=3/4), respectively. The HPV42 genome was detected in most cases of DPA (n=10/11) and in 1 adnexal adenocarcinoma not otherwise specified. Finally, whole-transcriptome analysis revealed BRD3::NUTM1 or NSD3::NUTM1 fusions in 2 cases of NUT adnexal carcinoma and NCOA4::RET and CCDC6::RET fusion transcripts in 2 cystadenoma/hidrocystoma-like tumors. Our study confirms distinctive cytogenetic abnormalities in a wide number of acral adnexal neoplasms and supports the use of molecular analysis as a valuable aid in the diagnosis of these rare and often difficult to diagnose group of neoplasms.

Distinct Regulation of EZH2 and its Repressive H3K27me3 Mark in Polyomavirus-Positive and -Negative Merkel Cell Carcinoma.

Merkel cell carcinoma (MCC) is an aggressive skin cancer for which Merkel cell polyomavirus integration and expression of viral oncogenes small T and Large T have been identified as major oncogenic determinants. Recently, a component of the PRC2 complex, the histone methyltransferase enhancer of zeste homolog 2 (EZH2) that induces H3K27 trimethylation as a repressive mark has been proposed as a potential therapeutic target in MCC. Because divergent results have been reported for the levels of EZH2 and trimethylation of lysine 27 on histone 3, we analyzed these factors in a large MCC cohort to identify the molecular determinants of EZH2 activity in MCC and to establish MCC cell lines' sensitivity to EZH2 inhibitors. Immunohistochemical expression of EZH2 was observed in 92% of MCC tumors (156 of 170), with higher expression levels in virus-positive than virus-negative tumors (P = 0.026). For the latter, we showed overexpression of EZHIP, a negative regulator of the PRC2 complex. In vitro, ectopic expression of the large T antigen in fibroblasts led to the induction of EZH2 expression, whereas the knockdown of T antigens in MCC cell lines resulted in decreased EZH2 expression. EZH2 inhibition led to selective cytotoxicity on virus-positive MCC cell lines. This study highlights the distinct mechanisms of EZH2 induction between virus-negative and -positive MCC.

EGFR-Mutant Non-Small-Cell Lung Cancer at Surgical Stages: What Is the Place for Tyrosine Kinase Inhibitors?

The ADAURA trial has been significant for the perception of EGFR tyrosine kinase inhibitors (TKIs) as a tool for early stage non-small-cell lung cancer (NSCLC). It produced such great insight that the main TKI, Osimertinib, was rapidly integrated into international guidelines for adjuvant use. However, EGFR-mutant NSCLC is a complex entity and has various targeting drugs, and the benefits for patients might not be as clear as they seem. We reviewed trials and meta-analyses considering TKI adjuvant and neoadjuvant use. We also explored the influence of mutation variability and financial evaluations. We found that TKIs often show disease-free survival (DFS) benefits, yet studies have struggled to improve the overall survival (OS); however, the results from the literature might be confusing because of variability in the stages and mutations. The safety profiles and adverse events are acceptable, but costs remain high and accessibility might not be optimal. TKIs are promising drugs that could allow for tailored treatment designs.

Investigation of the RB1-SOX2 axis constitutes a tool for viral status determination and diagnosis in Merkel cell carcinoma.

MCC (Merkel cell carcinoma) is an aggressive neuroendocrine cutaneous neoplasm. Integration of the Merkel cell polyomavirus (MCPyV) is observed in about 80% of the cases, while the remaining 20% are related to UV exposure. Both MCPyV-positive and -negative MCCs-albeit by different mechanisms-are associated with RB1 inactivation leading to overexpression of SOX2, a major contributor to MCC biology. Moreover, although controversial, loss of RB1 expression seems to be restricted to MCPyV-negative cases.The aim of the present study was to assess the performances of RB1 loss and SOX2 expression detected by immunohistochemistry to determine MCPyV status and to diagnose MCC, respectively.Overall, 196 MCC tumors, 233 non-neuroendocrine skin neoplasms and 70 extra-cutaneous neuroendocrine carcinomas (NEC) were included. SOX2 and RB1 expressions were assessed by immunohistochemistry in a tissue micro-array. Diagnostic performances were determined using the likelihood ratio (LHR).RB1 expression loss was evidenced in 27% of the MCC cases, 12% of non-neuroendocrine skin tumors and 63% of extra-cutaneous NEC. Importantly, among MCC cases, RB1 loss was detected in all MCPyV(-) MCCs, while MCPyV( +) cases were consistently RB1-positive (p < 0.001). SOX2 diffuse expression was observed in 92% of the MCC cases and almost never observed in non-neuroendocrine skin epithelial neoplasms (2%, p < 0.0001, LHR + = 59). Furthermore, SOX2 diffuse staining was more frequently observed in MCCs than in extra-cutaneous NECs (30%, p < 0.001, LHR + = 3.1).These results confirm RB1 as a robust predictor of MCC viral status and further suggest SOX2 to be a relevant diagnostic marker of MCC.

Merkel Cell Polyomavirus‒Negative Merkel Cell Carcinoma Originating from In Situ Squamous Cell Carcinoma: A Keratinocytic Tumor with Neuroendocrine Differentiation.

Although virus-negative Merkel cell carcinoma (MCC) is characterized by a high frequency of UV-induced mutations, the expression of two viral oncoproteins is regarded as a key mechanism driving Merkel cell polyomavirus‒positive MCC. The cells in which these molecular events initiate MCC oncogenesis have yet not been identified for both MCC subsets. A considerable proportion of virus-negative MCC is found in association with squamous cell carcinoma (SCC), suggesting (i) coincidental collision, (ii) one providing a niche for the other, or (iii) one evolving from the other. Whole-exome sequencing of four combined tumors consisting of SCC in situ and Merkel cell polyomavirus‒negative MCC showed many mutations shared between SCC and MCC in all cases, indicating a common ancestry and thereby a keratinocytic origin of these MCCs. Moreover, analyses of the combined cases as well as of pure SCC and MCC suggest that RB1 inactivation in SCC facilitates MCC development and that epigenetic changes may contribute to the SCC/MCC transition.