Probes for HPV

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.

Abstract

BACKGROUND:

In situ hybridization for human papillomavirus (HPV) messenger RNA (HPV RNA ISH) recently was introduced as an ancillary tool in the diagnosis of cervical squamous intraepithelial lesions, and can aid in the distinction between low-grade squamous intraepithelial lesions (LSILs) versus reactive/negative biopsies. Prior work has shown that up to one-half of cases originally diagnosed as LSIL are reclassified as negative/reactive by expert consensus review of morphology, and negative HPV RNA ISH results most often correlate with an expert diagnosis of negative/reactive. Given that LSIL overdiagnoses on biopsy may result in the erroneous clinical impression that a cervical lesion has been sampled appropriately, the authors proposed that HPV RNA ISH can inform cytology-histology correlation for challenging LSIL biopsies.

METHODS:

A total of 92 cervical biopsies originally diagnosed as LSIL were reviewed by 3 gynecologic pathologists and reclassified based on consensus opinion of morphology. ISH was performed for high-risk and low-risk HPV E6/E7 mRNA. Prior/concurrent cytology results were collected.

RESULTS:

Based on expert consensus morphologic review, 49% of biopsies (45 of 92 biopsies) originally diagnosed as LSIL were reclassified as negative, 6.5% (6 of 92 biopsies) were reclassified as high-grade squamous intraepithelial lesion, and 44.5% (41 of 92 biopsies) were maintained as LSIL. The majority of LSIL biopsies reclassified as negative (80%; 36 of 45 biopsies) were HPV RNA negative, whereas 93% of LSIL biopsies (39 of 41 biopsies) and 100% of high-grade squamous intraepithelial lesion biopsies were HPV RNA positive.

CONCLUSIONS:

LSIL often is overdiagnosed by morphology on biopsy, potentially leading to the false impression that a lesion identified on cytology has been sampled. Performing RNA ISH on biopsies decreases histologic LSIL overdiagnosis, and potentially can prompt further sampling when there is cytology-histology discordance.

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.

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

BACKGROUND:

Human papillomavirus (HPV)-related squamous cell carcinoma (SqCC) of the oropharynx is an epidemiologically and clinically distinct form of SqCC that is associated with an improved prognosis. However, HPV-related small cell carcinoma of the oropharynx is a rare and newly described variant that is associated with aggressive clinical behavior and poor outcomes. To date, fewer than 2 dozen reports of this entity exist in the literature, and there is no discussion of cytopathologic features. This article reports 6 cases and discusses the salient cytomorphologic findings, ancillary studies, and challenges when this entity is encountered.

METHODS:

Anatomic pathology archives were searched to identify patients with a diagnosis of HPV-related small cell carcinoma of the oropharynx. Medical records were reviewed to document the following: age, sex, smoking status, other relevant clinical history, primary location, treatment, and clinical outcome. Both p16 and high-risk HPV in situ hybridization (ISH) studies were positive in at least 1 specimen from each patient. The pathologic diagnoses, cytomorphologic characteristics, immunocytochemical stains, and HPV ISH studies were reviewed and recorded for all available cases.

RESULTS:

Six patients with 11 cytopathology specimens of HPV-related small cell carcinoma of the oropharynx were identified. The mean age was 61.3 years, and all patients died with widely metastatic disease (mean, 23 months; range, 12-48 months). Mixed small cell carcinoma and SqCC components were present in half of the cases.

CONCLUSIONS:

The identification of a small cell component can be reliably performed with cytology preparations and is crucial because this (and not the HPV status) determines the prognosis.

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.

Although 2014 World Health Organization criteria require unequivocal glandular and squamous differentiation for a diagnosis of cervical adenosquamous carcinoma, in practice, adenosquamous carcinoma diagnoses are often made in tumors that lack unequivocal squamous and/or glandular differentiation. Considering the ambiguous etiologic, morphological, and clinical features and outcomes associated with adenosquamous carcinomas, we sought to redefine these tumors. We reviewed slides from 59 initially diagnosed adenosquamous carcinomas (including glassy cell carcinoma and related lesions) to confirm an adenosquamous carcinoma diagnosis only in the presence of unequivocal malignant glandular and squamous differentiation. Select cases underwent immunohistochemical profiling as well as human papillomavirus (HPV) testing by in situ hybridization. Of the 59 cases originally classified as adenosquamous carcinomas, 34 retained their adenosquamous carcinoma diagnosis, 9 were reclassified as pure invasive stratified mucin-producing carcinomas, 10 as invasive stratified mucin-producing carcinomas with other components (such as HPV-associated mucinous, usual-type, or adenosquamous carcinomas), and 4 as HPV-associated usual or mucinous adenocarcinomas with benign-appearing squamous metaplasia. Two glassy cell carcinomas were reclassified as poorly differentiated usual-type carcinomas based on morphology and immunophenotype. There were significant immunophenotypic differences between adenosquamous carcinomas and pure invasive stratified mucin-producing carcinomas with regard to HPV (p < 0.0001), PAX8 (p = 0.038; more in adenosquamous carcinoma), p40 (p < 0.0001; more in adenosquamous carcinoma), p63 (p = 0.0018; more in adenosquamous carcinoma) and MUC6 (p  < 0.0001; less in adenosquamous carcinoma), HNF-1beta (p = 0.0023), vimentin (p = 0.0003), p53 (p = 0.0004), and CK7 (p = 0.0002) expression. Survival outcomes were similar between all groups. Adenosquamous carcinomas should be diagnosed only in the presence of unequivocal malignant glandular and squamous differentiation. The two putative glassy cell carcinomas studied did not meet our criteria for adenosquamous carcinoma, and categorizing them as such should be reconsidered.

Abstract

Background

Laryngeal squamous cell carcinoma (LSCC) is strongly associated with tobacco use, but recent reports suggest an increasing incidence of LSCC in patients without traditional risk factors, suggesting an alternative etiology of tumorigenesis. The purpose of this study is to characterize this non-smoking population and to compare immunohistochemical markers in tumor specimens from non-smokers and smokers with LSCC.

Methods

A retrospective chart review of patients with LSCC at Johns Hopkins Hospital (JHH) was performed. A tissue microarray (TMA) was constructed with tumor specimen from non-smokers with stage and age-matched smokers and stained for a variety of immunologic and molecular targets.

Results

In the JHH cohort of 521 patients, 12% (n = 63) were non-smokers. Non-smokers were more likely to be <45 years old at time of diagnosis (OR 4.13, p = 0.001) and to have glottic tumors (OR 2.46, p = 0.003). The TMA was comprised of tumors from 34 patients (14 non-smokers, 20 smokers). Only 2 patients (6%) were human-papillomavirus (HPV) positive by high-risk RNA in situ hybridization (ISH). There was no correlation between smoking status and p16 (p = 0.36), HPV-ISH positivity (p = 0.79), phosphatase and tensin homolog (PTEN, p = 0.91), p53 (p = 0.14), or programmed death-ligand 1 (PD-L1, p = 0.27) expression.

Conclusions

Non-smokers with LSCC are more likely to be younger at the time of diagnosis and have glottic tumors than smokers with LSCC. In TMA analysis of stage and age-matched specimens from smoker and non-smokers with LSCC, the pattern of expression for common molecular and immunologic markers is similar. Further, HPV does not appear to be a major causative etiology of LSCC in either smokers or non-smokers in our cohort of patients.

The International Endocervical Adenocarcinoma Criteria and Classification was developed to separate endocervical adenocarcinomas (ECAs) into 2 main categories on the basis of morphology such as human papilloma virus-associated (HPVA) and non-human papilloma virus-associated adenocarcinomas. We aimed to improve the diagnostic accuracy of International Endocervical Adenocarcinoma Criteria and Classification by performing a comprehensive immunohistochemical evaluation and constructing objective immunohistochemical-based algorithms for the classification of these tumors. Tissue microarrays were constructed from 297 of 409 cases used to develop the original classification. Immunostains included p16, p53, estrogen receptor (ER), progesterone receptor, androgen receptor, Vimentin, CK7, CK20, HER2, HIK1083, MUC6, CA-IX, SATB2, HNF-1beta, napsin A, PAX8, CDX2, GATA3, p63, p40, and TTF-1. High-risk human papilloma virus (HR-HPV) was detected by in situ hybridization (ISH) using probes against E6 and E7 mRNA expressed in 18 different virus types. Vimentin, ER, and progesterone receptor were expressed in a significant minority of ECAs, mostly HPVAs, limiting their use in differential diagnosis of endometrioid carcinoma when unaccompanied by HPV-ISH or p16. HR-HPV ISH had superior sensitivity, specificity, and negative and positive predictive values compared with p16, as published previously. HNF-1beta did not have the anticipated discriminatory power for clear cell carcinoma, nor did MUC6 or CA-IX for gastric-type carcinoma. HNF-1beta and napsin A were variably expressed in clear cell carcinoma, with HNF-1beta demonstrating less specificity, as it was ubiquitously expressed in gastric-type carcinoma and in the majority of HPV-associated mucinous (predominantly intestinal-type and invasive ECA resembling stratified mucin-producing intraepithelial lesion [iSMILE]) and usual-type carcinomas. HIK1083 was expressed in nearly half of gastric-type carcinomas, but not in the vast majority of other subtypes. GATA3 was positive in 10% of usual-type adenocarcinomas and in single examples of other subtypes. Rare gastric-type and HPVA mucinous carcinomas displayed HER2 overexpression. Androgen receptor was positive in 6% of usual-type adenocarcinomas. Aberrant p53 expression was found in only 3.6% of usual-type HPVA carcinomas, but it was more prevalent in mucinous (intestinal type and iSMILE) HPVAs and non-human papilloma virus-associates (particularly in gastric-type carcinoma, >50% of cases). The following diagnostic classification algorithms were developed with the above data. Carcinomas without overt cytoplasmic mucin (endometrioid, usual-type endocervical, clear cell, and mesonephric carcinomas) can be subclassified using HR-HPV ISH, ER, and GATA3, whereas carcinomas with easily appreciated cytoplasmic mucin (endometrioid carcinoma with mucinous features, HPVA mucinous, and gastric-type carcinomas) can be subclassified with HR-HPV ISH and ER.