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The high number of marginal mandibular nerve (MMN) anatomical variants have a well-known clinical significance due to the risk of nerve injury in several surgical procedures. The aim of this study was to find and systematize the available anatomical data concerning this nerve. The PubMed and Scopus databases were investigated in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-analyses guidelines. All studies reporting extractable data on the origin, course, splitting, anastomosis and relationship of the MMN with the mandible or the facial vessels were included. We included 28 studies analyzing 1861 halves. The MMN had one (PP = 35% 95% CI:18-54%), two (PP =35% 95% CI:18-54%), three (PP = 18% 95% CI:0-35%), or four branches (PP = 2% 95% CI:0-8%). Anastomosis with the great auricular nerve, transverse cervical nerve, mental nerve, and other branches of the facial nerve were defined. The origin of the MMN in relation to the parotid and the mandible was variable. The MMN nearly always crossed the anterior facial vein laterally (PP = 38% 95% CI:9-72% if single, PP = 57% 95% CI:22-90% when multiple); its relation with other vessels was less constant. At least one branch of the MMN was found below the inferior border of the mandible (IBM), with a PP of 39% (95% CI:30-50%). The MMN has high anatomical variability and it is more often represented by one or two branches; its origin is frequently described at the parotid apex and above the IBM, although in its course at least one branch often runs below the IBM. Its most frequent anastomosis is with the buccal branch of the facial nerve. Clin. Anat., 33:739-750, 2020. © 2019 Wiley Periodicals, Inc.

Objective. Human Papillomavirus (HPV)-induced oropharyngeal squamous cell carcinoma (OPSCC) represents an increasing tumor entity with typical clinical and histopathological features, associated with a rising incidence and improved prognosis (1). Detection of HPV-DNA is the most widely used method to diagnose HPV infection in clinical samples. Commercially available diagnostic assays show variable sensitivity and specificity and no international standards have been proposed yet (2). The main purpose of this preliminary study was to evaluate the reliability of EasyPap Direct HPV DNA Test (Ulisse BioMed) on formalin-fixed paraffin-embedded samples (FFPE), despite this test has been designed to work in vaginal mucus, where preliminary data showed a sensitivity of 97%. The test is real-time PCR based, it does not require DNA extraction and it can perform high-risk HPV genotyping. Kit performances have been compared with a commercial kit which requires DNA extraction. Materials and Methods. This pilot study included 38 cases of OPSCC that were surgically treated in our institute. Tumor samples were fixed in buffered formalin, embedded in paraffin and stored at room temperature for 1-13 years (mean storage period 4 ± 4 years). In order to confirm histology and lesion grade, all samples were reviewed by hematoxylin-eosin staining. DNA extraction has been performed starting from FFPE samples through QIAamp® DNA FFPE Tissue Kit (Qiagen, Hilden, Germany) and HPV has been typed through Real Time PCR HPV sign® Pyro Mark System Q96 IDTM (CE-IVD Diatech Pharmacogentetics Iesi Italy). We found 16 cases positive for HPV16, 1 case positive for HPV35 and 21 HPV negative samples. In order to perform HPV detection through EasyPap Direct HPV DNA test, samples underwent deparaffinization with xylene and rehydration with ethanol. Genotyping data were compared to those obtained with Qiagen kit. Results. EasyPap Direct HPV DNA test identified 16 HPVpositive cases and 22 HPV negative cases. Genotyping data showed that 15 samples were HPV16 and 1 sample was HPV35 positive. Concordance analysis showed an agreement- coefficient Kappa Cohen K=0.95 IC95% [0.84-1.00] (excellent agreement, Fleiss classification). The sensibility and specificity of the EasyPap HPV DNA test was 94.1% (95%, confidence interval 80.1%-98.8%) and 100% (95%, confidence interval 88.6%-100%), respectively. The positive predictive value was 100% (95%, confidence interval 88.6%-100%) and the negative predictive value was 95.5% (95% confidence interval, 81.9%-99.3%). Conclusions. Hitherto it was well known that DNA extraction contributes to increase the sensitivity of PCR-based HPV test starting from FFPE tissue samples (3). Despite small sample size has been analyzed, preliminary data obtained through EasyPap Direct HPV DNA test successfully showed that high sensitivity and specificity can be achieved without DNA extraction, with a remarkable decrease of assay cost and time. These results lead us to perform large scale EasyPap testing.

OBJECTIVE. Human Papillomavirus (HPV)-related squamous cell carcinoma of the oropharynx (HPV-OPSCC) usually originates from the reticulated epithelium lining the crypts of Waldeyer’s ring. The increasing epidemiologic evidence of this type of tumors and the peculiar positive prognosis, suggested to introduce a dedicated staging system (1). The 8th edition of the American Joint Committe on Cancer (AJCC) staging manual presented a new classification to discriminate HPV-related cancers based on p16 over-expression, since it demonstrated to be an important biomarker (2). The detection of HPV-DNA could not be used as a defining factor due to its cost and lack of universal applicability. The main purpose of this preliminary study was to evaluate the concordance between the results of immunohistochemistry for p16 overexpression and the HPV-DNA detection performed with a commercial kit as control. MATERIALS AND METHODS. We retrospectively collected 32 cases of OPSCC diagnosed in 2016 at the Academic Hospital of Cattinara in Trieste, whose samples had been tested with Real Time PCR (Pyro Mark System Q96 IDTM - CE-IVD Diatech Pharmacogentetics Iesi Italy). The study was performed using tumor samples fixed in buffered formalin, embedded in paraffin and stored at room temperature for 6 to 18 months. All samples were selected by a pathologist based on sections stained with hematoxylin and eosin to confirm the adequacy of the tissue. The samples were tested for p16 by immunohistochemistry (CINtech® p16 Histology E6H4 ROCHE) and the positivity was assessed when nuclear expression was ≥+2/+3 intensity and ≥75% distribution according to the 8th edition of the AJCC staging manual. In the context of oropharyngeal cancer, the optimal cut-off for p16 overexpression is ≥+2/+3 intensity (+/- cytoplasmic staining) with ≥75% distribution. RESULTS. Seven cases were excluded because of lack of histological stored material for p16 immunohistochemical analysis (only cytological specimen). Among the remaining 25 cases, there were 5 HPV-positive and 20 HPV-negative tumors. The sensibility and specificity of the p16 immunostaining was equal to 100% (95%, confidence interval 83.4%-100%) and 100% (95%, confidence interval 83.4%-97.8%), respectively; the positive predictive value was 100% (95%, confidence interval 83.4%-100%) and the negative predictive value 100% (95% confidence interval, 83.4%-100%). CONCLUSIONS. In agreement with the literature, p16 overexpression demonstrated to be a reliable parameter to identify the cases of OPSCC in which performing an in-depth analysis by means the detection of HPV-DNA would be indicated. Indeed, it is widely known that the association of p16 immunostaining and HPV-DNA detection by quantitative PCR increases the prognostic discriminator (3). We intend to carry on this study adopting a prospective design increasing the sample size and the test accuracy.

Objectives/hypothesis The accurate diagnostic assessment of clinically relevant human papillomavirus (HPV) infections in patients with head and neck squamous cell carcinoma represents an urgent unmet medical need. The aim of this study was to determine feasibility, accuracy, and clinical significance of HPV16/18 E6 oncoprotein detection on cytological specimens from oropharyngeal squamous cell carcinoma (OPSCC) and neck lymph node metastasis of SCC from unknown primary tumor (CUP) via a protein immunochromatographic assay. Study design Cross-sectional study. Methods Cytological specimens from primary tumor and neck metastases were collected from 34 patients with OPSCC or CUP and applied to a lateral flow format test that detects HPV16 and HPV18 E6 oncoproteins. E6 oncoprotein positivity or negativity in these specimens was compared to the specimens' "HPV-driven" reference status, defined by presence of HPV-DNA in combination with p16INK4a overexpression and/or HPV E6 seropositivity. Results Eighteen of 29 OPSCC (62%) and three of five CUP (60%) were HPV-driven according to our reference method. The E6 oncoprotein lateral flow test had a sensitivity of 94% (95% CI: 70%-100%) and a specificity of 100% (95% CI: 66%-100%) on primary tumor, and a sensitivity of 88% (95% CI: 64%-99%) and a specificity of 100% (95% CI: 74%-100%) on neck metastases. Test agreement between the E6 lateral flow test and the clinical reference method, HPV-DNA plus p16INK4a was excellent, both for primary lesion and neck metastases. Conclusions We found the detection of HPV16/18 E6 oncoproteins to be a feasible, highly reliable, and low-invasive method to assess "HPV-driven" status in OPSCC and CUP. Level of evidence II Laryngoscope, 131:1042-1048, 2021.

Patients with neck squamous cell carcinomas of unknown primary tumour (NSCCUP) present with lymph node metastasis without evidence for a primary tumour. Most patients undergo an aggressive multimodal treatment, which induces severe, potentially unnecessary toxicity. Primary tumours of NSCCUP can be hidden in the oropharynx. Human papillomavirus (HPV) is causally involved in a subgroup of oropharyngeal squamous cell carcinomas (OPSCC) associated with early lymph node metastasis and good prognosis. Detection of markers for HPV transformation in NSCCUP could allow focussing on the oropharynx in primary tumour search and could be of value for choice and extent of treatment. In a retrospective multicentre study (Germany, Italy and Spain), we analysed metastatic lymph nodes from 180 NSCCUP patients for the presence of HPV DNA, HPV E6*I mRNA and cellular p16(INK4a) overexpression, a surrogate marker for HPV-induced transformation. HPV status, defined as positivity for viral mRNA with at least one additional marker, was correlated with clinical parameters and survival outcome. A substantial proportion (16%) of NSCCUP were HPV-driven, mainly by HPV16 (89%). HPV prevalence increased with-year of diagnosis from 9% during 1998-2004 to 23% during 2005-2014 (p = 0.007). HPV-driven NSCCUP had significantly better overall and progression-free survival rates (p < 0.008). Based on this survival benefit, it is contended that HPV RNA status should be included in NSCCUP diagnosis and in therapeutic decision-making. Deintensification of radiation in patients with HPV-driven NSCCUP, while concurrently concentrating on the oropharynx appears to be a promising therapeutic strategy, the efficacy of which should be assessed in prospective trials. To our knowledge, this is the largest study on HPV in NSCCUP. (C) 2017 Elsevier Ltd. All rights reserved.