Probes for HPV

Up-to-date, several molecular markers of prognosis have been studied in Oral Squamous Cell Carcinoma (OSCC), but none entered in the clinical setting. Therapy of OSCC tumors mainly relies on surgery, radiotherapy and partially on chemotherapy; there is an urgent need for biomarkers able to better stratify OSCC patients' risk to address targeted therapeutic strategies. The role of immune response in the pathogenesis and biological behavior of OSCC has been investigated by several authors, and promising results have been obtained with immune checkpoint inhibitors. We already investigated the role of the immune modulator FK506-binding protein 51 (FKBP51), a FK506-binding immunophilin, in cutaneous melanoma biology, and its expression in several human solid tumors. In the present study, we aimed to assess the value of FKBP51 expression in OSCC tumor cells as a marker of outcome. We collected clinical data from 72 patients who underwent surgery for Squamous Cell Carcinoma (SCC) of the tongue, floor, lips and palate. FKBP51 expression was assessed by immunohistochemistry on paraffin-embedded tumor tissues. In addition, we evaluated the human papillomavirus (HPV) status of primary tumors by immunohistochemistry, viral subtyping and In Situ Hybridization (ISH) assay. We found that high FKBP51-expressing tumors characterized the OSCCs with the worst prognosis: the high immunohistochemical expression of FKBP51 associated with death occurring within five years from the diagnosis with a sensitivity of 88.46% and a specificity of 91.67%. The estimated positive predictive value of the test was 88.45% and negative predictive value 91.67%. We tested FKBP51 mRNA presence, by RT-PCR assay, in a selected series of OSCC tumors, and we found that mRNA correlated well to the protein expression and to the clinical outcome. Applying the Bayes formula, we estimated an 88% probability of dying within five years from the diagnosis of OSCC patients with a high FKBP51 immunohistochemical (IHC) test result (>51% of FKBP51 positive tumor cells). On the basis of our analysis, we propose tumor tissue expression of FKBP51 protein as a reliable prognostic marker for OSCC tumors.

Papillomaviruses exhibit species-specific tropism, thereby limiting understanding and research of several aspects of HPV infection and carcinogenesis. The discovery of a murine papillomavirus (MmuPV1) provides the opportunity to study papillomavirus infections in a tractable, in vivo laboratory model. MmuPV1 infects and causes disease in the cutaneous epithelium, as well as the mucosal epithelia of the oral cavity and anogenital tract. In this report, we describe a murine model of MmuPV1 infection and neoplastic disease in the female reproductive tracts of wild-type immunocompetent FVB mice. Low-grade dysplastic lesions developed in reproductive tracts of FVB mice infected with MmuPV1 for 4 months, and mice infected for 6 months developed significantly worse disease, including squamous cell carcinoma (SCC). We also tested the contribution of estrogen and/or UV radiation (UVR), two cofactors we previously identified as being involved in papillomavirus-mediated disease, to cervicovaginal disease. Similar to HPV16 transgenic mice, exogenous estrogen treatment induced high-grade precancerous lesions in the reproductive tracts of MmuPV1-infected mice by 4 months and together with MmuPV1 efficiently induced SCC by 6 months. UV radiation and exogenous estrogen cooperated to promote carcinogenesis in MmuPV1-infected mice. This murine infection model represents the first instance of de novo papillomavirus-mediated carcinogenesis in the female reproductive tract of wild-type mice resulting from active virus infection and is also the first report of the female hormone estrogen contributing to this process. This model will provide an additional platform for fundamental studies on papillomavirus infection, cervicovaginal disease, and the role of cellular cofactors during papillomavirus-induced carcinogenesis.IMPORTANCE Tractable and efficient models of papillomavirus-induced pathogenesis are limited due to the strict species-specific and tissue-specific tropism of these viruses. Here, we report a novel preclinical murine model of papillomavirus-induced cervicovaginal disease in wild-type, immunocompetent mice using the recently discovered murine papillomavirus, MmuPV1. In this model, MmuPV1 establishes persistent viral infections in the mucosal epithelia of the female reproductive tract, a necessary component needed to accurately mimic HPV-mediated neoplastic disease in humans. Persistent MmuPV1 infections were able to induce progressive neoplastic disease and carcinogenesis, either alone or in combination with previously identified cofactors of papillomavirus-induced disease. This new model will provide a much-needed platform for basic and translational studies on both papillomavirus infection and associated disease in immunocompetent mice.

Papillomaviruses (PV) are associated with benign mucosal and cutaneous epithelial proliferations. In dogs, PV-associated pigmented plaques and papillomas can undergo malignant transformation, but this is rare, and most cases of canine squamous cell carcinoma do not arise from PV-induced precursor lesions. We describe herein the progression of pigmented plaques to invasive and metastatic squamous cell carcinoma associated with 2 canine papillomaviruses (CPV) in 2 related Basenji dogs. Immunohistochemistry for PV antigen revealed strong nuclear immunoreactivity within keratinocytes from pigmented plaques from both dogs, consistent with a productive viral infection. Polymerase chain reaction (PCR) using degenerate primers for the L1 gene revealed PV DNA sequences from 2 different CPVs. In situ hybridization for CPV revealed strong hybridization signals within the pigmented plaques and neoplastic squamous epithelial cells from both dogs. We report here progression of PV-associated pigmented plaques to metastatic squamous cell carcinoma within 2 Basenji dogs associated with 2 different CPVs.

The diverse malignant, stromal, and immune cells in tumors affect growth, metastasis, and response to therapy. We profiled transcriptomes of ∼6,000 single cells from 18 head and neck squamous cell carcinoma (HNSCC) patients, including five matched pairs of primary tumors and lymph node metastases. Stromal and immune cells had consistent expression programs across patients. Conversely, malignant cells varied within and between tumors in their expression of signatures related to cell cycle, stress, hypoxia, epithelial differentiation, and partial epithelial-to-mesenchymal transition (p-EMT). Cells expressing the p-EMT program spatially localized to the leading edge of primary tumors. By integrating single-cell transcriptomes with bulk expression profiles for hundreds of tumors, we refined HNSCC subtypes by their malignant and stromal composition and established p-EMT as an independent predictor of nodal metastasis, grade, and adverse pathologic features. Our results provide insight into the HNSCC ecosystem and define stromal interactions and a p-EMT program associated with metastasis.