Unlicensed origin DNA melting by MCV and SV40 polyomavirus LT proteins is independent of ATP-dependent helicase activity.

Cellular eukaryotic replication initiation helicases are first loaded as head-to-head double hexamers on double-stranded (ds) DNA origins and then initiate S-phase DNA melting during licensed (once per cell cycle) replication. Merkel cell polyomavirus (MCV) large T (LT) helicase oncoprotein similarly binds and melts its own 98-bp origin but replicates multiple times in a single cell cycle. To examine the actions of this unlicensed viral helicase, we quantitated multimerization of MCV LT molecules as they assembled on MCV DNA origins using real-time single-molecule microscopy. MCV LT formed highly stable double hexamers having 17-fold longer mean lifetime (τ, >1,500 s) on DNA than single hexamers. Unexpectedly, partial MCV LT assembly without double-hexamer formation was sufficient to melt origin dsDNA as measured by RAD51, RPA70, or S1 nuclease cobinding. DNA melting also occurred with truncated MCV LT proteins lacking the helicase domain, but was lost from a protein without the multimerization domain that could bind only as a monomer to DNA. SV40 polyomavirus LT also multimerized to the MCV origin without forming a functional hexamer but still melted origin DNA. MCV origin melting did not require ATP hydrolysis and occurred for both MCV and SV40 LT proteins using the nonhydrolyzable ATP analog, adenylyl-imidodiphosphate (AMP-PNP). LT double hexamers formed in AMP-PNP, and melted DNA, consistent with direct LT hexamer assembly around single-stranded (ss) DNA without the energy-dependent dsDNA-to-ssDNA melting and remodeling steps used by cellular helicases. These results indicate that LT multimerization rather than helicase activity is required for origin DNA melting during unlicensed virus replication.

Membrane-bound Merkel cell polyomavirus middle T protein constitutively activates PLCγ1 signaling through Src-family kinases.

Merkel cell polyomavirus (MCV or MCPyV) is an alphapolyomavirus causing human Merkel cell carcinoma and encodes four tumor (T) antigen proteins: large T (LT), small tumor (sT), 57 kT, and middle T (MT)/alternate LT open reading frame proteins. We show that MCV MT is generated as multiple isoforms through internal methionine translational initiation that insert into membrane lipid rafts. The membrane-localized MCV MT oligomerizes and promiscuously binds to lipid raft-associated Src family kinases (SFKs). MCV MT-SFK interaction is mediated by a Src homology (SH) 3 recognition motif as determined by surface plasmon resonance, coimmunoprecipitation, and bimolecular fluorescence complementation assays. SFK recruitment by MT leads to tyrosine phosphorylation at a SH2 recognition motif (pMTY114), allowing interaction with phospholipase C gamma 1 (PLCγ1). The secondary recruitment of PLCγ1 to the SFK-MT membrane complex promotes PLCγ1 tyrosine phosphorylation on Y783 and activates the NF-κB inflammatory signaling pathway. Mutations at either the MCV MT SH2 or SH3 recognition sites abrogate PLCγ1-dependent activation of NF-κB signaling and increase viral replication after MCV genome transfection into 293 cells. These findings reveal a conserved viral targeting of the SFK-PLCγ1 pathway by both MCV and murine polyomavirus (MuPyV) MT proteins. The molecular steps in how SFK-PLCγ1 activation is achieved, however, differ between these two viruses.

The immunogenetics of viral antigen response is associated with subtype-specific glioma risk and survival.

Glioma is a highly fatal cancer with prognostically significant molecular subtypes and few known risk factors. Multiple studies have implicated infections in glioma susceptibility, but evidence remains inconsistent. Genetic variants in the human leukocyte antigen (HLA) region modulate host response to infection and have been linked to glioma risk. In this study, we leveraged genetic predictors of antibody response to 12 viral antigens to investigate the relationship with glioma risk and survival. Genetic reactivity scores (GRSs) for each antigen were derived from genome-wide-significant (p < 5 × 10-8) variants associated with immunoglobulin G antibody response in the UK Biobank cohort. We conducted parallel analyses of glioma risk and survival for each GRS and HLA alleles imputed at two-field resolution by using data from 3,418 glioma-affected individuals subtyped by somatic mutations and 8,156 controls. Genetic reactivity scores to Epstein-Barr virus (EBV) ZEBRA and EBNA antigens and Merkel cell polyomavirus (MCV) VP1 antigen were associated with glioma risk and survival (Bonferroni-corrected p < 0.01). GRSZEBRA and GRSMCV were associated in opposite directions with risk of IDH wild-type gliomas (ORZEBRA = 0.91, p = 0.0099/ORMCV = 1.11, p = 0.0054). GRSEBNA was associated with both increased risk for IDH mutated gliomas (OR = 1.09, p = 0.040) and improved survival (HR = 0.86, p = 0.010). HLA-DQA1∗03:01 was significantly associated with decreased risk of glioma overall (OR = 0.85, p = 3.96 × 10-4) after multiple testing adjustment. This systematic investigation of the role of genetic determinants of viral antigen reactivity in glioma risk and survival provides insight into complex immunogenomic mechanisms of glioma pathogenesis. These results may inform applications of antiviral-based therapies in glioma treatment.

Merkel Cell Polyomavirus-Pathophysiology and Treatment in the Era of Gene-Targeted Therapies.

Merkel cell polyomavirus (MCPyV) is a significant contributor to the development of Merkel cell carcinoma (MCC), an aggressive skin cancer with high recurrence and a low survival rate. In fact, it is the deadliest skin cancer. The precise routes of transmission for MCPyV-positive MCC remain unclear, but several factors may trigger its development. Conventional treatments for MCC are not highly effective, especially in patients with metastasis, with a clear need for new treatment options. Gene-targeted therapies hold great promise for the treatment of MCC, including the use of siRNA and CRISPR/Cas (C/Cas) but critically none have yet been translated into clinical trials. Validating this approach is the fact that several siRNA products are already FDA licenced, while C/Cas has entered clinical trial, albeit for conditions other than MCC. There are many challenges that must be overcome to move from preclinical research to the clinic. In this review, we provide a comprehensive summary of the current understanding of MCC, with a particular focus on MCPyV-positive MCC, and the status of gene-targeted therapies. Additionally, we discuss the major obstacles that impede MCC research and explore future prospects.

A Novel Artificial Intelligence-Based Parameterization Approach of the Stromal Landscape in Merkel Cell Carcinoma: A Multi-Institutional Study.

Tumor-stroma ratio (TSR) has been recognized as a valuable prognostic indicator in various solid tumors. This study aimed to examine the clinicopathologic relevance of TSR in Merkel cell carcinoma (MCC) using artificial intelligence (AI)-based parameterization of the stromal landscape and validate TSR scores generated by our AI model against those assessed by humans. One hundred twelve MCC cases with whole-slide images were collected from 4 different institutions. Whole-slide images were first partitioned into 128 × 128-pixel "mini-patches," then classified using a novel framework, termed pre-tumor and stroma (Pre-TOAST) and TOAST, whose output equaled the probability of the minipatch representing tumor cells rather than stroma. Hierarchical random samplings of 50 minipatches per region were performed throughout 50 regions per slide. TSR and tumor-stroma landscape (TSL) parameters were estimated using the maximum-likelihood algorithm. Receiver operating characteristic curves showed that the area under the curve value of Pre-TOAST in discriminating classes of interest including tumor cells, collagenous stroma, and lymphocytes from nonclasses of interest including hemorrhage, space, and necrosis was 1.00. The area under the curve value of TOAST in differentiating tumor cells from related stroma was 0.93. MCC stroma was categorized into TSR high (TSR ≥ 50%) and TSR low (TSR < 50%) using both AI- and human pathology-based methods. The AI-based TSR-high subgroup exhibited notably shorter metastasis-free survival (MFS) with a statistical significance of P = .029. Interestingly, pathologist-determined TSR subgroups lacked statistical significance in recurrence-free survival, MFS, and overall survival (P > .05). Density-based spatial clustering of applications with noise analysis identified the following 2 distinct TSL clusters: TSL1 and TSL2. TSL2 showed significantly shorter recurrence-free survival (P = .045) and markedly reduced MFS (P < .001) compared with TSL1. TSL classification appears to offer better prognostic discrimination than traditional TSR evaluation in MCC. TSL can be reliably calculated using an AI-based classification framework and predict various prognostic features of MCC.

The prognostic value of the Merkel cell polyomavirus serum antibody test: A dual institutional observational study.

BACKGROUND: Merkel cell carcinoma (MCC) is an aggressive cancer with often poor outcomes. Limited biomarkers exist for predicting clinical outcomes. The Merkel cell polyomavirus (MCPyV) serum antibody test (AMERK) has shown potential for indicating better recurrence-free survival in a single-institution study. The study aimed to evaluate the link between initial AMERK serostatus and survival. Secondary objectives included examining the relationship between initial AMERK titer levels and tumor burden. METHODS: A retrospective cohort study across two institutions analyzed patients tested with AMERK within 90 days of MCC diagnosis. Regression models assessed the association of survival outcomes with serostatus, considering various factors. The relationship between AMERK titer and tumor burden indicators was evaluated using ANOVA. Significance testing was exploratory, without a fixed significance level. RESULTS: Of 261 MCC patients tested, 49.4% were initially seropositive (titer ≥75). Multivariable analysis showed that seropositivity improved recurrence, event-free, overall, and MCC-specific survival rates. Strong associations were found between initial AMERK titer and clinical, tumor, and nodal stages, tumor size, and disease extent. Notably, improved survival with seropositivity was observed only in patients with localized disease at initial presentation. CONCLUSION: Circulating antibodies to MCPyV oncoproteins, as indicated by the AMERK test, are linked with better survival in MCC patients with localized disease at presentation. This could enhance patient risk profiling and treatment personalization. The study's retrospective nature and exploratory analysis are key limitations. PLAIN LANGUAGE SUMMARY: Merkel cell carcinoma (MCC) is a potentially aggressive skin cancer, and tools to predict patient outcomes are limited. A blood test called anti-Merkel cell panel (AMERK), which checks for specific antibodies related to this cancer, might give us some clues. In this study, we looked at 261 MCC patients who took the AMERK test within 90 days of diagnosis. We found that patients with an initial positive AMERK result tended to have better outcomes, especially if their cancer was in the early stages. However, it is important to note that this study has limitations, including using retrospective data and exploratory analyses.

Characterization of the Impact of Merkel Cell Polyomavirus-Induced Interferon Signaling on Viral Infection.

Merkel cell polyomavirus (MCPyV) has been associated with approximately 80% of Merkel cell carcinoma (MCC), an aggressive and increasingly incident skin cancer. The link between host innate immunity, viral load control, and carcinogenesis has been established but poorly characterized. We previously established the importance of the STING and NF-κB pathways in the host innate immune response to viral infection. In this study, we further discovered that MCPyV infection of human dermal fibroblasts (HDFs) induces the expression of type I and III interferons (IFNs), which in turn stimulate robust expression of IFN-stimulated genes (ISGs). Blocking type I IFN downstream signaling using an IFN-ß antibody, JAK inhibitors, and CRISPR knockout of the receptor dramatically repressed MCPyV infection-induced ISG expression but did not significantly restore viral replication activities. These findings suggest that IFN-mediated induction of ISGs in response to MCPyV infection is not crucial to viral control. Instead, we found that type I IFN exerts a more direct effect on MCPyV infection postentry by repressing early viral transcription. We further demonstrated that growth factors normally upregulated in wounded or UV-irradiated human skin can significantly stimulate MCPyV gene expression and replication. Together, these data suggest that in healthy individuals, host antiviral responses, such as IFN production induced by viral activity, may restrict viral propagation to reduce MCPyV burden. Meanwhile, growth factors induced by skin abrasion or UV irradiation may stimulate infected dermal fibroblasts to promote MCPyV propagation. A delicate balance of these mutually antagonizing factors provides a mechanism to support persistent MCPyV infection. IMPORTANCE Merkel cell carcinoma is an aggressive skin cancer that is particularly lethal to immunocompromised individuals. Though rare, MCC incidence has increased significantly in recent years. There are no lasting and effective treatments for metastatic disease, highlighting the need for additional treatment and prevention strategies. By investigating how the host innate immune system interfaces with Merkel cell polyomavirus, the etiological agent of most of these cancers, our studies identified key factors necessary for viral control, as well as conditions that support viral propagation. These studies provide new insights for understanding how the virus balances the effects of the host immune defenses and of growth factor stimulation to achieve persistent infection. Since virus-positive MCC requires the expression of viral oncogenes to survive, our observation that type I IFN can repress viral oncogene transcription indicates that these cytokines could be explored as a viable therapeutic option for treating patients with virus-positive MCC.

Exploring the effects of Merkel cell polyomavirus T antigens expression in REH and MCC13 cells by methylome and transcriptome profiling.

Merkel cell carcinoma (MCC) is a rare, aggressive skin cancer with a tripled incidence in the US and Europe over the past decade. Around 80% of MCC is linked to Merkel cell polyomavirus, but the cell of origin remains unknown. We stably introduced Merkel cell polyomavirus (MCPyV)-sT) and LT antigens to MCC13 and REH cell lines, analyzing DNA methylation and gene transcriptional regulation. Gene ontology analysis assessed MCPyV effects, and integrative analysis correlated gene expression and methylation. Expression patterns were compared with 15 previously sequenced primary MCCs. We found that MCPyV-LT induces DNA methylation changes in both cell lines, while MCPyV-sT only affected REH cells. Greater gene expression changes are observed in MCC13 cells, with upregulated genes associated with cellular components and downregulated genes related to biological processes. Integrative analysis of differentially expressed genes (DEG) and differentially methylated regions (DMR) of REH cell lines revealed that no genes were commonly methylated and differentially expressed. The study compared DEGs and DMG in MCC13 and REH cells to overlapping genes in MCPyV-positive cell lines (MKL1, MKL2, and WaGa), identifying hypomethylated genes in the gene body and hypermethylated genes at TSS1500. GO analysis of the two cell lines showed that MCPyV-TAs can downregulate genes in MHC-I pathways; this downregulation offers a target that can be used to create novel and efficient MCC immunotherapy approaches. Finally, it was confirmed that MCPyV-LT controls gene expression in MCC tissues using an integrative investigation of DNA methylation and gene expression.

Merkel cell polyomavirus pan-T antigen knockdown reduces cancer cell stemness and promotes neural differentiation independent of RB1.

Merkel cell carcinoma (MCC) is a highly aggressive skin cancer associated with integration of Merkel cell polyomavirus (MCPyV). MCPyV-encoded T-antigens (TAs) are pivotal for sustaining MCC's oncogenic phenotype, i.e., repression of TAs results in reactivation of the RB pathway and subsequent cell cycle arrest. However, the MCC cell line LoKe, characterized by a homozygous loss of the RB1 gene, exhibits uninterrupted cell cycle progression after shRNA-mediated TA repression. This unique feature allows an in-depth analysis of the effects of TAs beyond inhibition of the RB pathway, revealing the decrease in expression of stem cell-related genes upon panTA-knockdown. Analysis of gene regulatory networks identified members of the E2F family (E2F1, E2F8, TFDP1) as key transcriptional regulators that maintain stem cell properties in TA-expressing MCC cells. Furthermore, minichromosome maintenance (MCM) genes, which encodes DNA-binding licensing proteins essential for stem cell maintenance, were suppressed upon panTA-knockdown. The decline in stemness occurred simultaneously with neural differentiation, marked by the increased expression of neurogenesis-related genes such as neurexins, BTG2, and MYT1L. This upregulation can be attributed to heightened activity of PBX1 and BPTF, crucial regulators of neurogenesis pathways. The observations in LoKe were confirmed in an additional MCPyV-positive MCC cell line in which RB1 was silenced before panTA-knockdown. Moreover, spatially resolved transcriptomics demonstrated reduced TA expression in situ in a part of a MCC tumor characterized by neural differentiation. In summary, TAs are critical for maintaining stemness of MCC cells and suppressing neural differentiation, irrespective of their impact on the RB-signaling pathway.

Human papillomavirus and Merkel cell polyomavirus in Korean patients with nonsmall cell lung cancer: Evaluation and genetic variability of the noncoding control region.

Human papillomavirus (HPV) is an important causative factor of cervical cancer and is associated with nonsmall cell lung cancer (NSCLC). Merkel cell polyomavirus (MCPyV) is a rare and highly fatal cutaneous virus that can cause Merkel cell carcinoma (MCC). Although coinfection with oncogenic HPV and MCPyV may increase cancer risk, a definitive etiological link has not been established. Recently, genomic variation and genetic diversity in the MCPyV noncoding control region (NCCR) among ethnic groups has been reported. The current study aimed to provide accurate prevalence information on HPV and MCPyV infection/coinfection in NSCLC patients and to evaluate and confirm Korean MCPyV NCCR variant genotypes and sequences. DNA from 150 NSCLC tissues and 150 adjacent control tissues was assessed via polymerase chain reaction (PCR) targeting regions of the large T antigen (LT-ag), viral capsid protein 1 (VP1), and NCCR. MCPyV was detected in 22.7% (34 of 150) of NSCLC tissues and 8.0% (12 of 150) of adjacent tissues from Korean patients. The incidence rates of HPV with and without MCPyV were 26.5% (nine of 34) and 12.9% (15 of 116). The MCPyV NCCR genotype prevalence in Korean patients was 21.3% (32 of 150) for subtype I and 6% (nine of 150) for subtype IIc. Subtype I, a predominant East Asian strain containing 25 bp tandem repeats, was most common in the MCPyV NCCR data set. Our results confirm that coinfection with other tumor-associated viruses is not associated with NSCLC. Although the role of NCCR rearrangements in MCPyV infection remains unknown, future studies are warranted to determine the associations of MCPyV NCCR sequence rearrangements with specific diseases.

Merkel Cell Polyomavirus Antibody in Tumor and Plasma Specimens in Patients with Merkel Cell Carcinoma.

BACKGROUND: Merkel cell carcinoma (MCC) is associated with Merkel cell polyomavirus (MCPyV) infection. MCPyV antibodies (MCPyV-Ab) in plasma correlate with survival, while MCPyV-Ab within the tumor has never been investigated. This study evaluated plasma MCPyV-Ab and tumor MCPyV-Ab titers to evaluate their role in outcomes and prognostication. METHODS: A single-institution, prospective database was retrospectively reviewed for patients diagnosed with MCC from 2014 to 2021. MCPyV-Ab plasma and tumor titers, as well as patient and treatment factors, were collected. Two-year overall survival (OS) and disease-free survival (DFS) were examined based on MCPyV-Ab presence in tumor. RESULTS: Forty patients were identified, with a median follow-up of 27.6 months. Patients were stratified into four groups based on the presence of MCPyV-Ab in plasma (P+, P-) and tumor (T+, T-). Most patients (60.0%) were P-/T-. Of the remaining patients, 22.5% were P+/T+, 12.5% were P-/T+, and 5.0% were P+/T-. Two-year DFS of the P-/T- group was 16.6 months, which was not different from the other groups (p = 0.79). Two-year OS of P-/T- was 18.3 months, and 2-year OS of P-/T+ was 28.1 months, which was similar between groups (p = 0.80). CONCLUSIONS: Most patients P+ for MCPyV had antibody-positive tumors (T+), and P- patients were also T-; however, there was a subset of patients where plasma and tumor antibody findings were incongruent. Patients with MCPyV-Ab in either plasma or tumor had a trend toward improved 2-year DFS and OS, but was limited by a small cohort. This study offers an exploratory investigation into the relationship between plasma and tumor antibodies to MCPyV on which to base future work.

The small tumor antigen of Merkel cell polyomavirus accomplishes cellular transformation by uniquely localizing to the nucleus despite the absence of a known nuclear localization signal.

BACKGROUND: Merkel Cell Carcinoma (MCC) is an aggressive skin cancer that is three times deadlier than melanoma. In 2008, it was found that 80% of MCC cases are caused by the genomic integration of a novel polyomavirus, Merkel Cell Polyomavirus (MCPyV), and the expression of its small and truncated large tumor antigens (ST and LT-t, respectively). MCPyV belongs to a family of human polyomaviruses; however, it is the only one with a clear association to cancer. METHODS: To investigate the role and mechanisms of various polyomavirus tumor antigens in cellular transformation, Rat-2 and 293A cells were transduced with pLENTI MCPyV LT-t, MCPyV ST, TSPyV ST, HPyV7 ST, or empty pLENTI and assessed through multiple transformation assays, and subcellular fractionations. One-way ANOVA tests were used to assess statistical significance. RESULTS: Soft agar, proliferation, doubling time, glucose uptake, and serum dependence assays confirmed ST to be the dominant transforming protein of MCPyV. Furthermore, it was found that MCPyV ST is uniquely transforming, as the ST antigens of other non-oncogenic human polyomaviruses such as Trichodysplasia Spinulosa-Associated Polyomavirus (TSPyV) and Human Polyomavirus 7 (HPyV7) were not transforming when similarly assessed. Identification of structural dissimilarities between transforming and non-transforming tumor antigens revealed that the uniquely transforming domain(s) of MCPyV ST are likely located within the structurally dissimilar loops of the MCPyV ST unique region. Of all known MCPyV ST cellular interactors, 62% are exclusively or transiently nuclear, suggesting that MCPyV ST localizes to the nucleus despite the absence of a canonical nuclear localization signal. Indeed, subcellular fractionations confirmed that MCPyV ST could achieve nuclear localization through a currently unknown, regulated mechanism independent of its small size, as HPyV7 and TSPyV ST proteins were incapable of nuclear translocation. Although nuclear localization was found to be important for several transforming properties of MCPyV ST, some properties were also performed by a cytoplasmic sequestered MCPyV ST, suggesting that MCPyV ST may perform different transforming functions in individual subcellular compartments. CONCLUSIONS: Together, these data further elucidate the unique differences between MCPyV ST and other polyomavirus ST proteins necessary to understand MCPyV as the only known human oncogenic polyomavirus.

Mapping of Human Polyomavirus in Renal Cell Carcinoma Tissues.

Worldwide, the incidence of renal cell carcinoma (RCC) is rising, accounting for approximately 2% of all cancer diagnoses and deaths. The etiology of RCC is still obscure. Here, we assessed the presence of HPyVs in paraffin-embedded tissue (FFPE) resected tissue from patients with RCC by using different molecular techniques. Fifty-five FFPE tissues from 11 RCC patients were included in this study. Consensus and HPyV-specific primers were used to screen for HPyVs. Both PCR approaches revealed that HPyV is frequently detected in the tissues of RCC kidney resections. A total of 78% (43/55) of the tissues tested were positive for at least one HPyV (i.e., MCPyV, HPyV6, HPyV7, BKPyV, JCPyV, or WUyV). Additionally, 25 tissues (45%) were positive for only one HPyV, 14 (25%) for two HPyVs, 3 (5%) for three HPyVs, and 1 one (1%) tissue specimen was positive for four HPyVs. Eleven (20%) RCC specimens were completely devoid of HPyV sequences. MCPyV was found in 24/55 RCC tissues, HPyV7 in 19, and HPyV6 in 8. The presence of MCPyV and HPyV6 was confirmed by specific FISH or RNA-ISH. In addition, we aimed to confirm HPyV gene expression by IHC. Our results strongly indicate that these HPyVs infect RCC and nontumor tissues, possibly indicating that kidney tissues serve as a reservoir for HPyV latency. Whether HPyVs possibly contribute to the etiopathogenesis of RCC remains to be elucidated.

Immunological evidence of an early seroconversion to oncogenic Merkel cell polyomavirus in healthy children and young adults.

Oncogenic Merkel cell polyomavirus (MCPyV) provokes a widespread and asymptomatic infection in humans. Herein, sera from healthy children and young adults (HC, n = 344) aged 0-20 years old were evaluated for anti-MCPyV immunoglobulin G (IgG) and IgM antibodies employing a recently developed immunoassay. Serum MCPyV IgG data from healthy subjects (HS, n = 510) and elderlies (ES, n = 226), aged 21-65/66-100 years old, from our previous studies, were included. The anti-MCPyV IgG and IgM rates in HC sera were 40.7% and 29.7%, respectively. A lower prevalence of anti-MCPyV IgGs was found in HC aged 0-5 years old (13%) compared to 6-10 (52.3%), 11-15 (60.5%) and 16-20 years old (61.6%) cohorts. Age-stratified HCs exhibited similar anti-MCPyV IgM rates (27.9%-32.9%). Serological profiles indicated that anti-MCPyV IgGs and IgMs had low optical densities (ODs) during the first years of life, while IgM ODs appeared to decrease throughout young adulthood. A lower anti-MCPyV IgGs rate was found in HC (40.7%) than HS (61.8%) and ES (63.7%). Upon the 5-years range age-stratification, a lower anti-MCPyV IgGs rate was found in the younger HC cohort aged 0-5 years old compared to the remaining older HC/HS/ES cohorts (52.3%-72%). The younger HC cohort exhibited the lowest anti-MCPyV IgG ODs than the older cohorts. Low anti-MCPyV IgMs rates and ODs were found in the 21-25 (17.5%) and 26-30 (7.7%) years old cohorts. Our data indicate that, upon an early-in-life seroconversion, the seropositivity for oncogenic MCPyV peaks in late childhood/young adulthood and remains at high prevalence and relatively stable throughout life.

Expression of Genes Associated With Epithelial-Mesenchymal Transition in Merkel Cell Polyomavirus-Negative Merkel Cell Carcinoma.

Two accepted possible pathways for Merkel cell carcinoma (MCC) pathogenesis include the clonal integration of the Merkel cell polyomavirus (MCPyV) into the neoplastic cells and by UV irradiation. We hypothesize that, in UV etiology, the expression of genes associated with epithelial-mesenchymal transition (EMT) would be higher in MCPyV-negative MCCs. We compared RNA expression in 16 MCPyV-negative with that in 14 MCPyV-positive MCCs in 30 patients using NanoString panel of 760 gene targets as an exploratory method. Subsequently, we confirmed the findings with a publicly available RNA sequencing data set. The NanoString method showed that 29 of 760 genes exhibited significant deregulation. Ten genes (CD44, COL6A3, COL11A1, CXCL8, INHBA, MMP1, NID2, SPP1, THBS1, and THY1) were part of the EMT pathway. The expression of CDH1/E-cadherin, a key EMT gene, and TWIST1, regulator gene of EMT, was higher in MCPyV-negative tumors. To further investigate the expression of EMT genes in MCPyV-negative MCCs, we analyzed publicly available RNA sequencing data of 111 primary MCCs. Differential expression and gene set enrichment analysis of 35 MCPyV-negative versus 76 MCPyV-positive MCCs demonstrated significantly higher expression of EMT-related genes and associated pathways such as Notch signaling, TGF-ß signaling, and Hedgehog signaling, and UV response pathway in MCPyV-negative MCCs. The significance of the EMT pathway in MCPyV-negative MCCs was confirmed independently by a coexpression module analysis. One of the modules (M3) was specifically activated in MCPyV-negative MCCs and showed significant enrichment for genes involved in EMT. A network analysis of module M3 revealed that CDH1/E-cadherin was among the most connected genes (hubs). E-cadherin and LEF1 immunostains demonstrated significantly more frequent expression in MCPvV-negative versus MCPyV-positive tumors (P < .0001). In summary, our study showed that the expression of EMT-associated genes is higher in MCPyV-negative MCC. Because EMT-related proteins can be targeted, the identification of EMT pathways in MCPyV-negative MCCs is of potential therapeutic relevance.

Ancient Human Genomes and Environmental DNA from the Cement Attaching 2,000-Year-Old Head Lice Nits.

Over the past few decades, there has been a growing demand for genome analysis of ancient human remains. Destructive sampling is increasingly difficult to obtain for ethical reasons, and standard methods of breaking the skull to access the petrous bone or sampling remaining teeth are often forbidden for curatorial reasons. However, most ancient humans carried head lice and their eggs abound in historical hair specimens. Here we show that host DNA is protected by the cement that glues head lice nits to the hair of ancient Argentinian mummies, 1,500-2,000 years old. The genetic affinities deciphered from genome-wide analyses of this DNA inform that this population migrated from north-west Amazonia to the Andes of central-west Argentina; a result confirmed using the mitochondria of the host lice. The cement preserves ancient environmental DNA of the skin, including the earliest recorded case of Merkel cell polyomavirus. We found that the percentage of human DNA obtained from nit cement equals human DNA obtained from the tooth, yield 2-fold compared with a petrous bone, and 4-fold to a bloodmeal of adult lice a millennium younger. In metric studies of sheaths, the length of the cement negatively correlates with the age of the specimens, whereas hair linear distance between nit and scalp informs about the environmental conditions at the time before death. Ectoparasitic lice sheaths can offer an alternative, nondestructive source of high-quality ancient DNA from a variety of host taxa where bones and teeth are not available and reveal complementary details of their history.

Distinct retinoic gene signatures discriminate Merkel cell polyomavirus-positive from -negative Merkel cell carcinoma cells.

Limited molecular knowledge of Merkel cell polyomavirus (MCPyV)-positive and -negative Merkel cell carcinoma (MCC) subsets (MCCP/MCCN) has prevented so far the identification of the MCC origin cell type and, therefore, the development of effective therapies. The retinoic gene signature was investigated in various MCCP, MCCN, and control fibroblast/epithelial cell lines to elucidate the heterogeneous nature of MCC. Hierarchical clustering and principal component analysis indicated that MCCP and MCCN cells were clusterizable from each other and control cells, according to their retinoic gene signature. MCCP versus MCCN differentially expressed genes (n = 43) were identified. Protein-protein interaction network indicated SOX2, ISL1, PAX6, FGF8, ASCL1, OLIG2, SHH, and GLI1 as upregulated hub genes and JAG1 and MYC as downregulated hub genes in MCCP compared to MCCN. Numerous MCCP-associated hub genes were DNA-binding/-transcription factors involved in neurological and Merkel cell development and stemness. Enrichment analyses indicated that MCCP versus MCCN differentially expressed genes predominantly encode for to DNA-binding/-transcription factors involved in development, stemness, invasiveness, and cancer. Our findings suggest the neuroendocrine origin of MCCP, by which neuronal precursor cells could undergo an MCPyV-driven transformation. These overarching results might open the way to novel retinoid-based MCC therapies.

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.

The Evolving Treatment Landscape of Merkel Cell Carcinoma.

OPINION STATEMENT: Merkel cell carcinoma (MCC) has a high risk of recurrence and requires unique treatment relative to other skin cancers. The patient population is generally older, with comorbidities. Multidisciplinary and personalized care is therefore paramount, based on patient preferences regarding risks and benefits. Positron emission tomography and computed tomography (PET-CT) is the most sensitive staging modality and reveals clinically occult disease in ~ 16% of patients. Discovery of occult disease spread markedly alters management. Newly diagnosed, localized disease is often managed with sentinel lymph node biopsy (SLNB), local excision, primary wound closure, and post-operative radiation therapy (PORT). In contrast, metastatic disease is usually treated systemically with an immune checkpoint inhibitor (ICI). However, one or more of these approaches may not be indicated. Criteria for such exceptions and alternative approaches will be discussed. Because MCC recurs in 40% of patients and early detection/treatment of advanced disease is advantageous, close surveillance is recommended. Given that over 90% of initial recurrences arise within 3 years, surveillance frequency can be rapidly decreased after this high-risk period. Patient-specific assessment of risk is important because recurrence risk varies widely (15 to > 80%: Merkelcell.org/recur) depending on baseline patient characteristics and time since treatment. Blood-based surveillance tests are now available (Merkel cell polyomavirus (MCPyV) antibodies and circulating tumor DNA (ctDNA)) with excellent sensitivity that can spare patients from contrast dye, radioactivity, and travel to a cancer imaging facility. If recurrent disease is locoregional, management with surgery and/or RT is typically indicated. ICIs are now the first line for systemic/advanced MCC, with objective response rates (ORRs) exceeding 50%. Cytotoxic chemotherapy is sometimes used for debulking disease or in patients who cannot tolerate ICI. ICI-refractory disease is the major problem faced by this field. Fortunately, numerous promising therapies are on the horizon to address this clinical need.

Identification and confirmation via in situ hybridization of Merkel cell polyomavirus in rare cases of posttransplant cutaneous T-cell lymphoma.

BACKGROUND: Viral infection is an oncogenic factor in many hematolymphoid malignancies. We sought to determine the diagnostic yield of aligning off-target reads incidentally obtained during targeted hematolymphoid next-generation sequencing to a large database of viral genomes to screen for viral sequences within tumor specimens. METHODS: Alignment of off-target reads to viral genomes was performed using magicBLAST. Localization of Merkel cell polyomavirus (MCPyV) RNA was confirmed by RNAScope in situ hybridization. Integration analysis was performed using Virus-Clip. RESULTS: Four cases of post-cardiac-transplant folliculotropic mycosis fungoides (fMF) and one case of peripheral T-cell lymphoma (PTCL) were positive in off-target reads for MCPyV DNA. Two of the four cases of posttransplant fMF and the case of PTCL showed localization of MCPyV RNA to malignant lymphocytes, whereas the remaining two cases of posttransplant fMF showed MCPyV RNA in keratinocytes. CONCLUSIONS: Our findings raise the question of whether MCPyV may play a role in rare cases of T-lymphoproliferative disorders, particularly in the skin and in the heavily immunosuppressed posttransplant setting.