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Background: Integration of human papillomavirus (HPV) DNA has been considered a late event in cervical carcinogenesis. However, integrated forms of HPV were recently detected in cancer precursor lesions using a new real time polymerase chain reaction (PCR) to detect the deletions at the 3362–3443 region of HPV16 E2 Objective: To study the frequency of HPV16 DNA integration in cervical lesions and compare the sensitivity of an additional upstream region of the E2 ORF (2962–3138) in detecting HPV integration. Methods: Using the TaqMan based PCR, HPV16 positive DNA samples were analysed in 164 cervical scrapings from women participating in a multicentre screening trial. Biopsy confirmation was available in 62 cases. Results: Primers targeting the 3362–3443 region detected the majority of E2 deletions. In only 23% of the samples was the E2 upstream region equal or better target than the 3362–3443 region. Mixed (episomal/integrated) pattern was the most prevalent physical state of HPV16, also present in PAP smears with normal morphology. Pure integrated form was most prevalent in HSIL and cancer lesions, but also detectable in low grade abnormalities (NSIL, ASC-US, LSIL). Women with only integrated HPV16 were almost 10 years older than those with episomal HPV16. Viral load of integrated HPV16 was related to cytological abnormality (p = 0.003) but not to histology. Conclusions: Integrated HPV16 is present in low grade cervical lesions, mostly mixed with the episomal form. Women with the pure integrated form of HPV16 are older than those with the other forms.

In host–pathogen arms races, increases in host resistance prompt counteradaptation by pathogens, but the nature of that counteradaptation is seldom directly observed outside of laboratory models. The best-documented field example is the coevolution of myxoma virus (MYXV) in European rabbits. To understand how MYXV in Australia has continued to evolve in wild rabbits under intense selection for genetic resistance to myxomatosis, we compared the phenotypes of the progenitor MYXV and viral isolates from the 1950s and the 1990s in laboratory rabbits with no resistance. Strikingly, and unlike their 1950s counterparts, most virus isolates from the 1990s induced a highly lethal immune collapse syndrome similar to septic shock. Thus, the next step in this canonical case of coevolution after a species jump has been further escalation by the virus in the face of widespread host resistance.

Merkel cell polyomavirus (MCV) contributes to approximately 80% of all Merkel cell carcinomas (MCCs), a highly aggressive neuroendocrine carcinoma of the skin. MCV-positive MCC expresses small T antigen (ST) and a truncated form of large T antigen (LT) and usually contains wild-type p53 (TP53) and RB (RB1). In contrast, virus-negative MCC contains inactivating mutations in TP53 and RB1. While the MCV-truncated LT can bind and inhibit RB, it does not bind p53. We report here that MCV LT binds to RB, leading to increased levels of ARF, an inhibitor of MDM2, and activation of p53. However, coexpression of ST reduced p53 activation. MCV ST recruits the MYC homologue MYCL (L-Myc) to the EP400 chromatin remodeler complex and transactivates specific target genes. We observed that depletion of EP400 in MCV-positive MCC cell lines led to increased p53 target gene expression. We suspected that the MCV ST–MYCL–EP400 complex could functionally inactivate p53, but the underlying mechanism was not known. Integrated ChIP and RNA-sequencing analysis following EP400 depletion identified MDM2 as well as CK1α, an activator of MDM4, as target genes of the ST–MYCL–EP400 complex. In addition, MCV-positive MCC cells expressed high levels of MDM4. Combining MDM2 inhibitors with lenalidomide targeting CK1α or an MDM4 inhibitor caused synergistic activation of p53, leading to an apoptotic response in MCV-positive MCC cells and MCC-derived xenografts in mice. These results support dual targeting of MDM2 and MDM4 in virus-positive MCC and other p53 wild-type tumors.

Over 15% of cancers worldwide are caused by viruses. Merkel cell polyomavirus (MCPyV) is the most recently discovered human oncovirus and is the only polyomavirus that drives malignant tumors in humans. Here, we show that MCPyV + Merkel cell carcinoma is defined by neuroendocrine-lineage core regulatory (CR) transcription factors (TFs) (ATOH1, INSM1, ISL1, LHX3, POU4F3, and SOX2) that were essential for tumor survival and that co-bound chromatin with the viral small T antigen at super enhancers. Moreover, MCPyV integration sites were enriched at these neuroendocrine super enhancers. We further discovered that the MCPyV noncoding control region contained a homeodomain binding motif absent in other polyomaviruses that bound ISL1 and LHX3 and depended on them for T antigen expression. To therapeutically target the CR factors, we used histone deacetylase (HDAC) inhibitors to collapse the chromatin architecture and induce topological blurring of superenhancer loops, abrogating core TF expression and halting tumor growth. To our knowledge, our study presents the first example of oncogenic cross-regulation between viral and human epigenomic circuitry to generate interlocking and essential transcriptional feedback circuits that explain why MCPyV causes neuroendocrine cancer and represent a tumor dependency that can be targeted therapeutically.

Background Merkel cell carcinoma (MCC) is a highly aggressive neuroendocrine carcinoma of the skin caused by either the integration of Merkel cell polyomavirus (MCPyV) and expression of viral T antigens or by ultraviolet-induced damage to the tumor genome from excessive sunlight exposure. An increasing number of deep sequencing studies of MCC have identified significant differences between the number and types of point mutations, copy number alterations, and structural variants between virus-positive and virus-negative tumors. However, it has been challenging to reliably distinguish between virus positive and UV damaged MCC. Methods In this study, we assembled a cohort of 71 MCC patients and performed deep sequencing with OncoPanel, a clinically implemented, next-generation sequencing assay targeting over 400 cancer-associated genes. To improve the accuracy and sensitivity for virus detection compared to traditional PCR and IHC methods, we developed a hybrid capture baitset against the entire MCPyV genome and software to detect integration sites and structure. Results Sequencing from this approach revealed distinct integration junctions in the tumor genome and generated assemblies that strongly support a model of microhomology-initiated hybrid, virus-host, circular DNA intermediate that promotes focal amplification of host and viral DNA. Using the clear delineation between virus-positive and virus-negative tumors from this method, we identified recurrent somatic alterations common across MCC and alterations specific to each class of tumor, associated with differences in overall survival. Finally, comparing the molecular and clinical data from these patients revealed a surprising association of immunosuppression with virus-negative MCC and significantly shortened overall survival. Conclusions These results demonstrate the value of high-confidence virus detection for identifying molecular mechanisms of UV and viral oncogenesis in MCC. Furthermore, integrating these data with clinical data revealed features that could impact patient outcome and improve our understanding of MCC risk factors.

Approximately 80% of Merkel cell carcinoma (MCC) cases are caused by Merkel cell polyomavirus (MCV), driven by its T antigen oncogene. Why MCV drives MCC, a skin cancer that displays the neuroendocrine Merkel cell phenotype, remains unclear. In this issue of the JCI , Miao et al. demonstrated that MCC tumor survival requires neuroendocrine-lineage transcription factors, which are recruited to superenhancers (SEs) with the viral small T antigen oncoprotein to promote the neuroendocrine Merkel cell lineage of the cancer. Surprisingly, SEs mapped near the MCV integration site in MCC, and two SE-associated neuroendocrine transcription factors drove viral T antigen gene expression. MCV oncogene and neuroendocrine transcriptional network interactions rendered this viral tumorigenesis dependent on the Merkel cell lineage. Together with reports from other groups, the findings explain why MCV-associated cancer is specifically linked to the Merkel cell phenotype and identify epigenetic strategies targeting of lineage-dependent oncogene circuitry to treat virus-positive MCC.

Merkel cell carcinoma (MCC) is an aggressive neuroendocrine skin cancer that frequently carries an integrated Merkel cell polyomavirus (MCPyV) genome and expresses viral transforming antigens (TAgs). MCC tumor cells also express signature genes detected in skin-resident, postmitotic Merkel cells, including atonal bHLH transcription factor 1 (ATOH1), which is required for Merkel cell development from epidermal progenitors. We now report the use of in vivo cellular reprogramming, using ATOH1, to drive MCC development from murine epidermis. We generated mice that conditionally expressed MCPyV TAgs and ATOH1 in epidermal cells, yielding microscopic collections of proliferating MCC-like cells arising from hair follicles. Immunostaining of these nascent tumors revealed p53 accumulation and apoptosis, and targeted deletion of transformation related protein 53 (Trp53) led to development of gross skin tumors with classic MCC histology and marker expression. Global transcriptome analysis confirmed the close similarity of mouse and human MCCs, and hierarchical clustering showed conserved upregulation of signature genes. Our data establish that expression of MCPyV TAgs in ATOH1-reprogrammed epidermal cells and their neuroendocrine progeny initiates hair follicle-derived MCC tumorigenesis in adult mice. Moreover, progression to full-blown MCC in this model requires loss of p53, mimicking the functional inhibition of p53 reported in human MCPyV-positive MCCs.

Merkel cell carcinoma (MCC) is a rare but lethal cancer with the highest case-by-case fatality rate among all skin cancers. Eighty percent of cancers are associated with the Merkel cell polyomavirus (MCPyV). Twenty percent of MCCs are virus negative. Recent epidemiological data suggest that there are important, clinically relevant differences between these two subtypes of MCC. Recent studies in cancer genomics, mouse genetics, and virology experiments have transformed our understanding of MCC pathophysiology. Importantly, dramatic differences in the genetics of these two MCC subtypes suggest fundamental differences in their pathophysiology. We review these recent works and find that they provocatively suggest that MCPyV-positive and MCPyV-negative MCCs arise from two different cells of origin: the MCPyV-negative MCC from epidermal keratinocytes and the MCPyV-positive MCC from dermal fibroblasts. If true, this would represent the first cancer that we are aware of that evolves from cells of origin from two distinct germ layers: MCPyV-negative MCCs from ectodermal keratinocytes and MCPyV-positive MCCs from mesodermal fibroblasts. Future epigenetic experiments may prove valuable in confirming these distinct lineages for these MCC subtypes, especially for the clinical importance the cell of origin has on MCC treatment and prevention.

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.

BK polyomavirus (BKPyV) is a ubiquitous human virus that establishes a persistent infection in renal tubular epithelial cells and mainly causes disease in kidney transplant recipients. The closely related simian polyomavirus SV40 is known to cause cytoplasmic vacuolization in simian kidney cells, possibly increasing progeny release and cell death. This study aimed to determine whether BKPyV causes cytoplasmic vacuolization in primary human renal proximal tubule epithelial cells (RPTECs) and to investigate its potential role in the replication cycle. Using a large infectious dose (MOI 100–1000), a fraction of RPTECs (10–72%) showed early-wave vacuolization from 3 hours post-infection (hpi), which was mainly reversed by 36 hpi. Independent of the infectious dose, late-wave vacuolization occurred around the timepoint of progeny release. BKPyV receptor binding and internalization were required, as neuraminidase pretreatment and preincubation or treatment with a BKPyV-specific neutralizing antibody prevented early or late-occurring vacuolization. Microscopy revealed that the vacuoles were enlarged acidic endo-/lysosomal structures (dextran, EEA1, Rab5, Rab7, LAMP1, and/or Lysoview positive) that contained membrane-bound BKPyV. Time-lapse microscopy and quantitative PCR revealed that cell death and progeny release preceded late-wave vacuolization, mainly affecting cells directly neighboring the lysed cells. Thus, vacuolization had little impact on cell death or progeny release. Addition of the V-ATPase inhibitor Bafilomycin A1 at 0 hpi blocked vacuolization and BKPyV replication, but addition at 2 hpi only blocked vacuolization, suggesting that continuous endosomal acidification and maturation is needed for vacuole formation, but not for BKPyV replication. Our study shows that a massive uptake of BKPyV in RPTECs induces transient enlargement of endo-/lysosomes and is an early event in the viral replication cycle. Vacuolization gives no clear benefit for BKPyV and is possibly the result of a transiently overloaded endocytic pathway. Focal vacuolization around lysed cells suggests that the spread of BKPyV is preferably local.