Probes for INS

Given the comparable strains of high-risk human papillomavirus (HPV) present in a subset of Barrett's dysplasia and esophageal adenocarcinoma as in head and neck squamous cell carcinomas and the anatomical proximity of both lesions, we hypothesized that oral sex may increase the risk of Barrett's dysplasia/esophageal adenocarcinoma. Therefore, we compared the sexual behavior of patients with Barrett's dysplasia/esophageal adenocarcinoma and controls (hospital, reflux, and Barrett's metaplasia) to explore a plausible mechanism of viral transmission to the lower esophagus. A hospital-based case-control study involving 36 Barrett's dysplasia/esophageal adenocarcinoma subjects and 55 controls with known HPV DNA status and markers of transcriptional activity i.e p16INK4A and E6/E7 mRNA of the esophageal epithelium was conducted to evaluate differences in sexual history (if any). Barrett's dysplasia/esophageal adenocarcinoma patients were more likely than controls to be positive for HPV DNA (18 of 36, 50% vs. 6/55, 11%, p for trend <0.0001), be male (P = 0.001) and in a relationship (P = 0.02). Viral genotypes identified were HPV 16 (n = 14), 18 (n = 2), 11 (n = 1) and 6 (n = 1). HPV exposure conferred a significantly higher risk for Barrett's dysplasia/esophageal adenocarcinoma as compared with hospital/reflux/Barrett's metaplasia controls (OR = 6.8, 95% CI: 2.1-23.1, adjusted P = 0.002). On univariate analysis, ≥6 lifetime oral sex partners were significantly associated with dysplastic Barrett's esophagus and adenocarcinoma (OR, 4.0; 95% CI: 1.2-13.7, P = 0.046). After adjustment for confounders, HPV exposure and men with ≥2 lifetime sexual partners were at significant risk for Barrett's dysplasia/esophageal adenocarcinoma. If these initial findings can be confirmed in larger studies, it could lead to effective prevention strategies in combating some of the exponential increase in the incidence of esophageal adenocarcinoma in the West.

Human Papilloma Virus (HPV) can play a causative role in the development of sinonasal tract malignancies. In fact, HPV may be the most significant causative agent implicated in sinonasal tumorigenesis and is implicated in as many as 21% of sinonasal carcinomas. To date, there are no definitive, reliable and cost-effective, diagnostic tests approved by the FDA for the unequivocal determination of HPV status in head and neck cancers. We followed an exhaustive algorithm to correctly test HPV infection, including a sequential approach with p16INK4a IHC, viral DNA genotyping and in situ hybridization for E6/E7 mRNA. Here, we report a case of sinonasal carcinoma with discordant results using HPV test assays. The tumor we describe showed an irregular immunoreactivity for p16INK4a, and it tested positive for HPV DNA; nevertheless, it was negative for HR-HPV mRNA. We discuss the possible meaning of this discrepancy. It would be advisable to test HPV transcriptional status of sinonasal carcinoma on a diagnostic routine basis, not only by p16INK4a IHC assay, but also by HPV DNA genotyping and HR-HPV mRNA assessment.

Regardless of host, pathogenic mycobacteria of the Mycobacterium tuberculosiscomplex such as Mycobacterium bovis, induce a characteristic lesion known as agranuloma, tubercle or tuberculoid granuloma. Granulomas represent a distinct host response to chronic antigenic stimuli, such as foreign bodies, certain bacterial components, or persistent pathogens such as M. bovis. Granulomas are composed of specific cell types including epithelioid macrophages, lymphocytes and a morphologically distinctive cell type, the multinucleated giant cell. Multinucleated giant cells are formed by the fusion of multiple macrophages; however, their function remains unclear. In humans, giant cells in tuberculous granulomas have been shown to express various cytokines, chemokines and enzymes important to the formation and maintenance of the granuloma. The objective of this study was to quantitatively assess multinucleated giant cell cytokine expression in bovine tuberculoid granulomas; focusing on cytokines of suspected relevance to bovine tuberculosis. Using calves experimentally infected with M. bovis, in situ cytokine expression was quantitatively assessed using RNAScope^® for the following cytokines TNF-α, IFN-γ, TGF-β, IL-17A and IL-10. Multinucleated giant cells in bovine tuberculoid granulomas expressed all examined cytokines to varying degrees, with differential expression of TGF-β, IL-17A and IL-10 in giant cells from early versus late stage granulomas. There was a modest, positive correlation between the level of cytokine expression and cell size or number of nuclei. These results suggest that multinucleated giant cells are active participants within bovine tuberculoid granulomas, contributing to the cytokine milieu necessary to form and maintain granulomas.

The hallmark lesion of tuberculosis in humans and animals is the granuloma. The granuloma represents a distinct host cellular immune response composed of epithelioid macrophages, lymphocytes, and multinucleated giant cells, often surrounding a caseous necrotic core. Within the granuloma, host-pathogen interactions determine disease outcome. Factors within the granulomas such as cytokines and chemokines drive cell recruitment, activity, function and ultimately the success or failure of the host's ability to control infection. Hence, an understanding of the granuloma-level cytokine response is necessary to understand tuberculosis pathogenesis. In-situ cytokine expression patterns were measured using a novel in situ hybridization assay, known as RNAScope^® in granulomas of the lungs, tracheobronchial lymph nodes and caudal mediastinal lymph nodes of cattle experimentally infected with Mycobacterium bovis via aerosol exposure. In spite of microscopic morphological similarities, significant differences were seen between late stage granulomas of the lung compared to those of the tracheobronchial lymph nodes for IL-17A, IFN-γ, TGF-β, IL10 and IL-22 but not for TNF-α. Additionally, significant differences were noted between granulomas from two different thoracic lymph nodes that both receive afferent lymphatics from the lungs (i.e., tracheobronchial and caudal mediastinal lymph nodes) for TNF-α, IL-17A, IFN-γ, TGF-β and IL-10 but not for IL-22. These findings show that granuloma morphology alone is not a reliable indicator of granuloma function as granulomas of similar morphologies can have disparate cytokine expression patterns. Moreover, anatomically distinct lymph nodes (tracheobronchial vs caudal mediastinal) differ in cytokine expression patterns even when both receive afferent lymphatics from a lung containing tuberculoid granulomas. These findings show that selection of tissue and anatomic location are critical factors in assessing host immune response to M. bovis and should be considered carefully.

Abstract

Importance  
High-risk human papillomavirus (HPV) has been associated with Barrett dysplasia and esophageal adenocarcinoma. Nevertheless, the prognostic significance of esophageal tumor HPV status is unknown.

Objective  
To determine the association between HPV infection and related biomarkers in high-grade dysplasia or esophageal adenocarcinoma and survival.

Design, Setting, and Participants  
Retrospective case-control study. The hypothesis was that HPV-associated esophageal tumors would show a favorable prognosis (as in viral-positive head and neck cancers). Pretreatment biopsies were used for HPV DNA determination via polymerase chain reaction, in situ hybridization for E6 and E7 messenger RNA (mRNA), and immunohistochemistry for the proteins p16INK4A and p53. Sequencing of TP53 was also undertaken. The study took place at secondary and tertiary referral centers, with 151 patients assessed for eligibility and 9 excluded. The study period was from December 1, 2002, to November 28, 2017.

Main Outcomes and Measures  
Disease-free survival (DFS) and overall survival (OS).

Results  
Among 142 patients with high-grade dysplasia or esophageal adenocarcinoma (126 [88.7%] male; mean [SD] age, 66.0 [12.1] years; 142 [100%] white), 37 were HPV positive and 105 were HPV negative. Patients who were HPV positive mostly had high p16INK4A expression, low p53 expression, and wild-type TP53. There were more Tis, T1, and T2 tumors in HPV-positive patients compared with HPV-negative patients (75.7% vs 54.3%; difference, 21.4%; 95% CI, 4.6%-38.2%; P = .02). Mean DFS was superior in the HPV-positive group (40.3 vs 24.1 months; difference, 16.2 months; 95% CI, 5.7-26.8; P = .003) as was OS (43.7 vs 29.8 months; difference, 13.9 months; 95% CI, 3.6-24.3; P = .009). Recurrence or progression was reduced in the HPV-positive cohort (24.3% vs 58.1%; difference, −33.8%; 95% CI, −50.5% to −17.0%; P < .001) as was distant metastasis (8.1% vs 27.6%; difference, −19.5%; 95% CI, −31.8% to −7.2%; P = .02) and death from esophageal adenocarcinoma (13.5% vs 36.2%; difference, −22.7%; 95% CI, −37.0% to −8.3%; P = .01). Positive results for HPV and transcriptionally active virus were both associated with a superior DFS (hazard ratio [HR], 0.33; 95% CI, 0.16-0.67; P = .002 and HR, 0.44; 95% CI, 0.22-0.88; P = .02, respectively [log-rank test]). Positivity for E6 and E7 mRNA, high p16INK4Aexpression, and low p53 expression were not associated with improved DFS. On multivariate analysis, superior DFS was demonstrated for HPV (HR, 0.39; 95% CI, 0.18-0.85; P = .02), biologically active virus (HR, 0.36; 95% CI, 0.15-0.86; P = .02), E6 and E7 mRNA (HR, 0.36; 95% CI, 0.14-0.96; P = .04), and high p16 expression (HR, 0.49; 95% CI, 0.27-0.89; P = .02).

Conclusions and Relevance  
Barrett high-grade dysplasia and esophageal adenocarcinoma in patients who are positive for HPV are distinct biological entities with a favorable prognosis compared with viral-negative esophageal tumors. Confirmation of these findings in larger cohorts with more advanced disease could present an opportunity for treatment de-escalation in the hope of reducing toxic effects without deleteriously affecting survival.

Objective: To observe the clinicopathologic features of oropharyngeal squamous cell carcinoma associated with human papilloma virus (OPSCC-HPV) and discuss the role and value of different in situ hybridization (ISH) detection methods for HPV in pathologic diagnosis. Methods: Fifteen cases of OPSCC-HPV were collected from Department of Pathology, Beijing Tongren Hospital, Capital Medical University from January 2016 to August 2018. These cases were diagnosed in accordance with the WHO classification of head and neck tumors. The histopathologic features and the clinicopathologic data were retrospectively analyzed. Immunohistochemistry (two-step EnVision method) was done to evaluate the expression of p16, Ki-67 and p53. ISH was used to detect HPV DNA (6/11 and 16/18). RNAscope technology was used to evaluate the presence of HPV mRNAs (16 and 18). Results: The mean age for the 15 patients (8 males, 7 females) was 47 years (range from 30 to 69 years). OPSCC-HPV typically presentedat an advanced clinical stage, six patients had cervical lymphadenopathy (large and cystic), seven had tonsillar swelling, one had tumor at base of tongue, and one had odynophagia. Microscopically the tumors exhibited distinctive non-keratinizing squamous cell carcinoma morphology. Cervical nodal metastases were large and cystic, with thickening of lymph node capsules. OPSCC-HPV raised from crypt epithelium and extended beneath the tonsillar surface epithelial lining as nests and lobules, often with central necrosis. Tumor cells displayed a high N: C ratio, and high mitotic and apoptotic rates. Tumor nests are often embedded within lymphoid stroma, and may be infiltrated by lymphoid cells.Fifteen cases (15/15) were strongly positive for p16; Ki-67 index were 60%-90%; they were focally positive or negative for p53. Ten cases (10/10) were negative for HPV 6/11 DNA, and one case(1/10) was focally positive for HPV16/18 DNA. Eleven cases (11/11) were strongly positive for HPV16 mRNA, one case was focally positive for HPV18 mRNA. Conclusions: OPSCC-HPV is a pathologically and clinically distinct form of head and neck squamous cell carcinoma. OPSCC-HPV is associated with high-risk HPV (type 16) in all cases. Detection of high-risk HPV16 mRNA by RNAscope is of great significance in the final diagnosis and pathogen identification.