Probes for HPV

Abstract BACKGROUND: The majority of human papillomavirus (HPV)-associated oropharyngeal carcinomas are squamous cell carcinomas; however, there are rare reports of HPV-associated neuroendocrine carcinomas (HPV-NECs) in the upper aerodigestive tract. The aim of this study was to characterize the diagnostic features of fine-needle aspiration (FNA) cases of head and neck HPV-NEC. METHODS: Cytology cases of HPV-NEC were identified over a 3-year period from 2 institutions. Clinical, cytomorphologic, and ancillary test results were evaluated. RESULTS: Five FNA cases of HPV-NEC were identified from 4 patients with cervical lymph node metastases with primaries in the oropharynx (n = 2), nasopharynx (n = 1), and larynx (n = 1). Three cases showed mixed small cell and large cell neuroendocrine morphologies; 1 case was a small cell carcinoma, and the last case appeared as a large cell neuroendocrine carcinoma. All tumors were strongly positive for synaptophysin and p16 and negative for p63/p40. Two cases tested for INSM1 showed diffuse nuclear staining. HPV was confirmed by in situ hybridization in 4 cases, and HPV-18 was detected by polymerase chain reaction in the fifth case. Retinoblastoma (Rb) staining was moderate to weak (5/5), and p53 was weakly positive (5/5). CONCLUSIONS: Head and neck HPV-NEC is a rare, aggressive entity that can show mixed small and large cell features and p16 upregulation; p53 and Rb are variable with limited diagnostic utility. Because p16 positivity can be nonspecific, confirmatory HPV testing is required and may be helpful in determining the primary site for neuroendocrine carcinoma of an unknown primary. The accurate diagnosis of HPV-NEC is also important because of its worse prognosis in comparison with HPV-associated squamous cell carcinoma.

The prevalence and prognostic significance of high-risk human papillomavirus (HR-HPV) have been well-established in oropharyngeal squamous cell carcinoma (OPSCC), but not in hypopharyngeal squamous cell carcinoma (HPSCC) or laryngeal squamous cell carcinoma (LSCC). Moreover, HR-HPV infection in squamous cell carcinoma with multisite involvement has not been examined. To clarify these issues, we retrospectively collected 480 invasive tumors from 467 patients with HPSCC, LSCC, or OPSCC, and comprehensively analyzed the detailed tumor localization, transcriptionally active HR-HPV infection by messenger RNA in situ hybridization, and immunohistochemical staining for p16 and Rb. HR-HPV infection was observed in 115/480 tumors (24%). Human papillomavirus (HPV)-positive cases were closely related with p16 positivity and the partial loss pattern of Rb. HR-HPV was detected in 104 of 161 tumors (64.6%) in the pure OPSCC group and only 1 of 253 tumors (0.4%) in the pure HP/LSCC group; the positive case occurred in the vocal cords. In the multisite-involving combined-type squamous cell carcinoma group, HPV infection was observed in 10/40 (25%) cases, and the 10 HPV-positive cases had OPSCC extending to the larynx or hypopharynx. Among high T-stage (T3/T4) cases of pure OPSCC, HPV-positive cases showed a better prognosis (P=0.0144), whereas the HPV-positive combined OPSCC group did not show a better prognosis (P=0.9428), as compared with HPV-negative counterpart. The results suggest that HR-HPV infection in pure HPSCC and LSCC may be extremely rare. HR-HPV infection seems to be present in a substantial proportion of patients with combined OPSCC and HPSCC/LSCC, but it may not improve prognosis at such advanced disease stages. Confirmation of these points awaits future studies with larger cohorts.

Human papillomaviruses are DNA viruses that ubiquitously infect humans and have been associated with hyperproliferative lesions. The recently discovered mouse specific papillomavirus (MmuPV1) provides the opportunity to study papillomavirus infections in vivo in the context of a common laboratory mouse model (Mus musculus). To date, a major challenge in the field has been the lack of tools to identify, observe, and characterize individually the papillomavirus hosting cells and also trace the progeny of these cells over time. Here, we present the successful generation of an in vivo lineage-tracing model of MmuPV1-harboring cells and their progeny by means of genetic reporter activation. Following the validation of the system both in vitro and in vivo, we used it to provide a proof-of-concept of its utility. Using flow-cytometry analysis, we observed increased proliferation dynamics and decreased MHC-I cell surface expression in MmuPV1-treated tissues which could have implications in tissue regenerative capacity and ability to clear the virus. This model is a novel tool to study the biology of the MmuPV1 host-pathogen interactions.

The main objective of our study was to understand the impact of immune cell composition and the tumor-reactivity of tumor infiltrating lymphocytes (TIL) in HPV-positive (HPV+) and HPV-negative (HPV-) head and neck squamous cell carcinoma (HNSCC). TIL cultures were established from primary HNSCC tumors, the T cell subsets were phenotypically characterized using flow cytometry, and Interferon (IFN)-γ ELISA assay was used to determine TIL function. NanoString Immune Profiler was used to determine an immune signature by HPV-status, and multiplex immunohistochemistry (MIHC) was used to quantify immune cell distributions and their spatial relationships. Results showed that HPV+ and HPV- HNSCC had similar capacity to expand IFN-γ reactive TIL populations, and these TIL populations had similar characteristics. NanoString analysis revealed increased differential expression of genes related to B cell functions in HPV+ HNSCC, which were significant at a Benjamini-Yekutieli adjusted p-value of < 0.001. MIHC also displayed increased CD8+ T cell and CD19/CD20+ B cell densities in the tumor region of HPV+ HNSCC as opposed to HPV- HNSCC (p < 0.01). Increases in a combined metric of tumor B cell content and stromal plasma cell content was associated with increased progression-free survival in HPV- HNSCC patients treated with immune checkpoint inhibitor therapy (p = 0.03). In summary, TIL populations expanded from HPV+ and HPV- HNSCC displayed similar IFN-γ reactivity. However, we identified a strong B-cell signature present within HPV+ HNSCC, and higher B and plasma cell content associated with improved PFS in HPV- HNSCC patients treated with immune checkpoint inhibitors.