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 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.

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.

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.

MiR-31-3p do not predict anti-EGFR efficacy in first-line therapy of RAS wild-type metastatic right-sided colon cancer.

BACKGROUND: Low miR-31-3p expression was identified as predictive of anti-EGFR efficacy in RAS-wt mCRC. Primary tumor side was also proposed as a predictive factor of anti-EGFR benefit. This retrospective multicentric study evaluated the predictive role of miR-31-3p in right-sided RAS-wt mCRC patients treated with first-line CT+anti-EGFR or CT+bevacizumab (Beva). METHODS: Seventy-two right-sided RAS-wt mCRC patients treated in first-line with CT+anti-EGFR (n = 43) or Beva (n = 29) were included. Overall survival (OS), progression-free survival (PFS) and response rate (RR) were analyzed and stratified according to tumor miR-31-3p expression level and targeted therapy (TT). RESULTS: BRAF V600E mutation was more frequent in high vs low miR-31-3p expressers (60.6% vs 15.4%, P < 0.001). PFS was significantly longer with CT+Beva than with CT+anti-EGFR (13 vs 7 months; P = 0.024). Among low miR-31-3p expressers, PFS, OS and RR were not significantly different between the two groups, while in high miR-31-3p expressers, only PFS was longer in the CT+Beva group (11 vs 6 months; P = 0.03). In patients treated with CT+anti-EGFR, low miR-31-3p expressers had a significantly longer OS (20 vs 13 months; P = 0.02) than high miR-31-3p expressers. ORR was not significantly different between the two groups of treatment, in both low and high miR-31-3p expressers. MiR-31-3p expression status was statistically correlated between primary tumors and corresponding metastases. CONCLUSION: In this study, miR-31-3p couldn't identify a subgroup of patients with right-sided RAS-wt mCRC who might benefit from anti-EGFR and suggest that Beva is the TT of choice in first-line treatment of these patients.

Antibody-Drug Conjugates as an Emerging Therapy in Oncodermatology.

Antibody-drug conjugates (ADCs) are an emerging class of therapeutics, with twelve FDA- and EMA-approved drugs for hematological and solid cancers. Such drugs consist in a monoclonal antibody linked to a cytotoxic agent, allowing a specific cytotoxicity to tumor cells. In recent years, tremendous progress has been observed in therapeutic approaches for advanced skin cancer patients. In this regard, targeted therapies (e.g., kinase inhibitors) or immune checkpoint-blocking antibodies outperformed conventional chemotherapy, with proven benefit to survival. Nevertheless, primary and acquired resistances as well as adverse events remain limitations of these therapies. Therefore, ADCs appear as an emerging therapeutic option in oncodermatology. After providing an overview of ADC design and development, the goal of this article is to review the potential ADC indications in the field of oncodermatology.

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.

Voltage-Gated Sodium Channel NaV1.5 Controls NHE-1-Dependent Invasive Properties in Colon Cancer Cells.

Colorectal cancer (CRC) is the second leading cause of death worldwide, with 0.9 million deaths per year. The metastatic stage of the disease is identified in about 20% of cases at the first diagnosis and is associated with low patient-survival rates. Voltage-gated sodium channels (NaV) are abnormally overexpressed in several carcinomas including CRC and are strongly associated with the metastatic behavior of cancer cells. Acidification of the extracellular space by Na+/H+ exchangers (NHE) contributes to extracellular matrix degradation and cell invasiveness. In this study, we assessed the expression levels of pore-forming α-subunits of NaV channels and NHE exchangers in tumor and adjacent non-malignant tissues from colorectal cancer patients, CRC cell lines and primary tumor cells. In all cases, SCN5A (gene encoding for NaV1.5) was overexpressed and positively correlated with cancer stage and poor survival prognosis for patients. In addition, we identified an anatomical differential expression of SCN5A and SLC9A1 (gene encoding for NHE-1) being particularly relevant for tumors that originated on the sigmoid colon epithelium. The functional activity of NaV1.5 channels was characterized in CRC cell lines and the primary cells of colon tumors obtained using tumor explant methodologies. Furthermore, we assessed the performance of two new small-molecule NaV1.5 inhibitors on the reduction of sodium currents, as well as showed that silencing SCN5A and SLC9A1 substantially reduced the 2D invasive capabilities of cancer cells. Thus, our findings show that both NaV1.5 and NHE-1 represent two promising targetable membrane proteins against the metastatic progression of CRC.

Kallikrein-related peptidase 5 contributes to the remodeling and repair of bronchial epithelium.

Inflammation, oxidative stress, and protease/protease inhibitor imbalance with excessive production of proteases are factors associated with pathogenesis of the chronic obstructive pulmonary disease (COPD). In this study, we report that kallikrein-related peptidase 5 (KLK5) is a crucial protease involved in extracellular matrix (ECM) remodeling and bronchial epithelial repair after injury. First, we showed that KLK5 degrades the basal layer formed by culture of primary bronchial epithelial cells from COPD or non-COPD patients. Also, exogenous KLK5 acted differently on BEAS-2B cells already engaged in epithelial-to-mesenchymal transition (EMT) or on 16HBE 14o- cells harboring epithelial characteristics. Indeed, by inducing EMT, KLK5 reduced BEAS-2B cell adherence to the ECM. This effect, neutralized by tissue factor pathway inhibitor 2, a kunitz-type serine protease inhibitor, was due to a direct proteolytic activity of KLK5 on E-cadherin, ß-catenin, fibronectin, and α5ß1 integrin. Thus, KLK5 may strengthen EMT mechanisms and promote the migration of cells by activating the mitogen-activated protein kinase signaling pathway required for this function. In contrast, knockdown of endogenous KLK5 in 16HBE14o- cells, accelerated wound healing repair after injury, and exogenous KLK5 addition delayed the closure repair. These data suggest that among proteases, KLK5 could play a critical role in airway remodeling events associated with COPD during exposure of the pulmonary epithelium to inhaled irritants or smoking and the inflammation process.

Two repeated motifs enriched within some enhancers and origins of replication are bound by SETMAR isoforms in human colon cells.

Setmar is a gene specific to simian genomes. The function(s) of its isoforms are poorly understood and their existence in healthy tissues remains to be validated. Here we profiled SETMAR expression and its genome-wide binding landscape in colon tissue. We found isoforms V3 and V6 in healthy and tumour colon tissues as well as incell lines. In two colorectal cell lines SETMAR binds to several thousand Hsmar1 and MADE1 terminal ends, transposons mostly located in non-genic regions of active chromatin including in enhancers. It also binds to a 12-bp motifs similar to an inner motif in Hsmar1 and MADE1 terminal ends. This motif is interspersed throughout the genome and is enriched in GC-rich regions as well as in CpG islands that contain constitutive replication origins. It is also found in enhancers other than those associated with Hsmar1 and MADE1. The role of SETMAR in the expression of genes, DNA replication and in DNA repair are discussed.

Head-to-Head Comparison of 18F-DOPA PET/CT and 68Ga-DOTANOC PET/CT in Patients With Midgut Neuroendocrine Tumors.

PURPOSE: The aim of this study was to compare retrospectively 18F-DOPA PET/CT versus 68Ga-DOTANOC PET/CT in a group of patients affected by midgut NET. PATIENTS AND METHODS: Patients with histologically proven grade 1 or grade 2 midgut NET were explored after injection of 150 MBq of 68Ga-DOTANOC and 210 MBq of 18F-DOPA. The PET/CTs were analyzed visually and semiquantitatively at the patient level, regional level (7 defined regions), and lesion level (maximum of 5 lesions/organ). The criterion standard was determined on the basis of histology and imaging follow-up. RESULTS: Thirty patients (17 males and 13 females; median age, 63.5 years [37-82 years]) were included. Both PET/CTs were negative in 3 patients and positive in 25 patients. PET/CTs were discordant in 2 patients, with 18F-DOPA positive and 68Ga-DOTANOC negative. 18F-DOPA PET/CT detected more involved regions and more metastatic lesions than 68Ga-DOTANOC PET/CT in 6 (20%) and 10 (33.3%) patients, respectively. Of the 81 confirmed affected regions, 77 (95%) were detected by 18F-DOPA PET/CT and 71 (87.7%) by 68Ga-DOTANOC PET/CT (P < 0.0001). 18F-DOPA PET/CT detected significantly more lesions (211/221) than 68Ga-DOTANOC PET/CT (195/221), corresponding to a sensitivity of 95.5% and 88.2%, respectively (P < 0.0001). Tumor-to-background ratios were more favorable in liver for 18F-DOPA than for 68Ga-DOTANOC. Interestingly, a correlation was found between 18F-DOPA SUVmax and tumor burden and especially with the number of regions involved by the disease (P = 0.019). CONCLUSIONS: 18F-DOPA PET/CT is superior to 68Ga-DOTANOC PET/CT for the detection of lesions, and when available, this tracer may be recommended as the first-line examination for an accurate staging of midgut NET.

Specific Genomic Alterations in High-Grade Pulmonary Neuroendocrine Tumours with Carcinoid Morphology.

INTRODUCTION: High-grade lung neuroendocrine tumours with carcinoid morphology have been recently reported; they may represent the thoracic counterparts of grade 3 digestive neuroendocrine tumours. We aimed to study their genetic landscape including analysis of tumoral heterogeneity. METHODS: Eleven patients with high-grade (>20% Ki-67 and/or >10 mitoses) lung neuroendocrine tumours with a carcinoid morphology were included. We analysed copy number variations, somatic mutations, and protein expression in 16 tumour samples (2 samples were available for 5 patients allowing us to study spatial and temporal heterogeneity). RESULTS: Genomic patterns were heterogeneous ranging from "quiet" to tetraploid, heavily rearranged genomes. Oncogene mutations were rare and most genetic alterations targeted tumour suppressor genes. Chromosomes 11 (7/11), 3 (6/11), 13 (4/11), and 6-17 (3/11) were the most frequently lost. Altered tumour suppressor genes were common to both carcinoids and neuroendocrine carcinomas, involving different pathways including chromatin remodelling (KMT2A, ARID1A, SETD2, SMARCA2, BAP1, PBRM1, KAT6A), DNA repair (MEN1, POLQ, ATR, MLH1, ATM), cell cycle (RB1, TP53, CDKN2A), cell adhesion (LATS2, CTNNB1, GSK3B) and metabolism (VHL). Comparative spatial/temporal analyses confirmed that these tumours emerged from clones of lower aggressivity but revealed that they were genetically heterogeneous accumulating "neuroendocrine carcinoma-like" genetic alterations through progression such as TP53/RB1 alterations. CONCLUSION: These data confirm the importance of chromatin remodelling genes in pulmonary carcinoids and highlight the potential role of TP53 and RB1 to drive the transformation in more aggressive high-grade tumours.