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.

Human papillomavirus (HPV) infection is associated with several genetic alterations including oncogene amplification, leading to increased aggression of tumors. Recently, a relationship between HPV infection and oncogene amplification has been reported, but this finding remains controversial. This study therefore investigated relationships between HPV infection and amplification of genes in the epidermal growth factor receptor (EGFR) signaling cascade in oral squamous cell carcinoma (OSCC). Extracted DNA from 142 formalin-fixed paraffin-embedded (FFPE) OSCC tissues was performed to investigate the copy number of EGFR, KRAS, c-myc and cyclin D1 genes using real-time polymerase chain reaction (RT-PCR) and compared with calibrators. A tissue microarray of OSCC tissues was used for detection of c-Myc expression and HPV infection by immunohistochemistry and HPV E6/E7 RNA in situ hybridization, respectively. HPV infection was also investigated using PCR and RT-PCR. Of the 142 OSCC samples, 81 (57%) were HPV-infected cases. The most frequently amplified gene was c-myc (55.6%), followed by cyclin D1 (26.1%), EGFR (23.9%) and KRAS (19.7%). Amplification of c-myc was significantly associated with levels of its protein product. EGFR amplification was also significantly associated with amplification of genes in the signaling cascade: KRAS (50.0%), c-myc (34.2%) and cyclin D1 (46.0%). Interestingly, HPV infection was significantly associated with amplification of both EGFR (76.5%) and cyclin D1 (73.0%). Only cyclin D1 amplification was significantly associated with severity of OSCC histopathology. HPV infection may play an important synergistic role in amplification of genes in the EGFR signaling cascade, leading to increased aggression in oral malignancies.

We have previously demonstrated that transcriptionally active high-risk HPV (hr-HPV) is strongly incriminated in Barrett's dysplasia (BD) and oesophageal adenocarcinoma (OAC) using mainly fresh frozen tissue. This study aimed to identify biomarkers of active HPV infection in Barrett's metaplasia, (BM)/BD/OAC by immunohistochemical staining (IHC) of formalin-fixed paraffin embedded (FFPE) tissue for aberrations of p53 and the retinoblastoma (pRb) pathway which are targets for the viral oncoproteins, E6/E7 respectively. Prospectively, BM(n=81)/BD(n=72)/OAC(n=65) FFPE specimens were subjected to IHC staining for pRb, p16INK4A , cyclin D1 , p53 and RNA in-situ hybridization (ISH) for E6/E7 transcripts. HPV DNA was determined via PCR in fresh frozen specimens. Viral load measurement (real-time PCR) and Next Generation Sequencing of TP53 was also performed. Of 218 patients, 56 were HPV DNA positive [HPV16 (n=42), 18 (n=13), 6 (n=1)]. Viral load was low. Transcriptionally active HPV (DNA+ /RNA+ ) was only found in the dysplastic and adenocarcinoma group (n=21). The majority of HPV DNA+ /RNA+ BD/OAC were characterized by p16INK4Ahigh (14/21, 66.7%), pRblow (15/21, 71.4%) and p53low (20/21, 95%) and was significantly different to controls [combination of HPV DNA- /RNA- (n=94) and HPV DNA+ /RNA- cohorts (n=22)]. p53low had the strongest association with DNA+ /RNA+ oesophageal lesions (OR=23.5, 95% CI=2.94-187.8, p=0.0029). Seventeen HPV DNA+ /RNA+BD/OAC identified as p53low, were sequenced and all but one exhibited wild-type status. pRblow /p53low provided the best balance of strength of association (OR=8.0, 95% CI=2.6-25.0, p=0.0003) and sensitivity (71.4%)/specificity (71.6%) for DNA+ /RNA+ BD/OAC. Active HPV involvement in BD/OAC is characterized by wild-type p53 and aberrations of the retinoblastoma protein pathway.

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.

Abstract

PURPOSE:

Clinical responses with programmed death (PD-1) receptor directed antibodies occur in about 20% of patients with advanced head and neck squamous cell cancer (HNSCCa). Viral neoantigens, such as the E6/E7 proteins of HPV16/18 are attractive targets for therapeutic immunization, and offer an immune activation strategy that may be complementary to PD-1 inhibition.

EXPERIMENTAL DESIGN:

We report Phase Ib/II safety, tolerability and immunogenicity results of immunotherapy with MEDI0457 (DNA immunotherapy targeting HPV16/18 E6/E7 with IL-12 encoding plasmids) delivered by electroporation with CELLECTRA^® constant current device. Twenty-two patients with locally advanced, p16+ HNSCCa received MEDI0457.

RESULTS:

MEDI0457 was associated with mild injection site reactions but no treatment related grade 3-5 adverse events (AEs). Eighteen of 21 evaluable patients showed elevated antigen specific T cell activity by IFNg ELISpot and persistent cellular responses surpassing 100 SFU/106 PBMC were noted out to one year. Induction of HPV-specific CD8+ T cells was observed. MEDI0457 shifted the CD8+/FoxP3+ ratio in 4/5 post-immunotherapy tumor samples and increased the number of perforin+ immune infiltrates in all five patients. One patient developed metastatic disease and was treated with anti-PD-1 therapy with a rapid and durable complete response. Flow cytometric analyses revealed induction of HPV16 specific PD-1+ CD8+ T cells that were not found prior to MEDI0547 (0% vs. 1.8%).

CONCLUSIONS:

These data demonstrate that MEDI0457 can generate durable HPV16/18 antigen-specific peripheral and tumor immune responses. This approach may be used as a complementary strategy to PD-1/PD-L1 inhibition in HPV-associated HNSCCa to improve therapeutic outcomes.

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.