Probes for HPV

Human papillomavirus (HPV) infection modulates several host cytokines contributing to cancer development. Oncostatin M (OSM), an IL-6 family cytokine, acts to promote cell senescence and inhibit growth. Its dysregulation promotes cell survival, cell proliferation and metastasis in various malignancies. The effect of HPV on OSM dysregulation has not been investigated. To elucidate this, immunohistochemistry was used on formalin-fixed, paraffin-embedded oral squamous cell carcinoma (OSCC) tissues: HPV-positive (50) and HPV-negative (50) cases. Immortalized human cervical keratinocytes expressing HPV16E6 (HCK1T, Tet-On system) were used to demonstrate the role of HPV16E6 in OSM expression. In addition, a vector containing HPV16E6/E7 was transiently transfected into oral cancer cell lines. Cell viability, cell-cycle progression and cell migration were evaluated using flow cytometry and a wound healing assay, respectively. The results showed various intensities of OSM expression in OSCC. Interestingly, the median percentages of strongly stained cells were significantly higher in HPV-positive OSCCs than in HPV-negative OSCCs. To explore the role of HPV oncoproteins on OSM expression, the expression of HPV16E6 in the HCK1T Tet-On condition was induced by doxycycline and HPV16E6 was found to significantly upregulate levels of OSM mRNA and protein, with concomitant upregulation of c-Myc. In addition, the levels of OSM mRNA and protein in E6/E7 transiently transfected oral cancer cells also gradually increased in a time-dependent manner and these transfected cells showed greater viability and higher migration rates and cell-cycle progression than controls. This result demonstrates that HPV16 oncoproteins upregulate OSM and play an important role to promote OSCC development.

Abstract
Background:

Although alcohol and tobacco use are known risk factors for development of squamous cell carcinoma in the head and neck, human papillomavirus (HPV) has been increasingly associated with this group of cancers. We describe the case of a married couple who presented with HPV-positive oropharynx squamous cell carcinoma within two months of each other.

Methods:

Tumor biopsies were positive for p16 and high-risk HPV in both patients. Sanger sequencing showed a nearly identical HPV16 strain in both patients. Both patients received chemoradiation, and one  patient also underwent transoral robotic tongue base resection with bilateral neck dissection.

Results:

Both patients showed no evidence of recurrent disease on follow-up PET imaging.

Conclusions:

New head and neck symptoms should be promptly evaluated in the partner of a patient with known HPV-positive oropharynx cancer. This case expands the limited current literature on concurrent presentation of HPV-positive oropharynx squamous cell carcinoma in couples. 

Our aim was to examine the clinicopathological features of squamous cell carcinoma (SCC) of the oral cavity and oropharynx in a group of young patients who were dignosed during a 15-year period (2000-2014). Patients' clinical details, risk factors, and survival were obtained from medical records. Formalin-fixed, paraffin-embedded, tissue was tested for high-risk human papillomavirus (HPV). The results were compared with those of a matching group of older patients. We identified 91 patients who were younger than 45 years old, and the 50 youngest patients were studied in detail. The male:female ratio was 2:1, with more tumours located in the oral cavity than in the oropharynx (35 compared with 15). HPV-related SCC was restricted to the oropharynx. When matched for site, stage and HPV status, five-year overall survival was similar in young and matched older patients (log-rank test, p=0.515). Our findings suggest that young patients with oral SCC have a disease profile similar to that of older patients with the condition. It is plausible that prognostic information generally available for oral cancers is applicable to young patients with the disease.

Detection of human papilloma virus (HPV)-related head and neck squamous cell carcinoma (HNSCC) is important, as HPV-associated HNSCCs respond better to therapy. The RNAscope HPV-test is a novel RNA in situ hybridization (ISH) technique which strongly stains transcripts of E6 and E7 mRNA in formalin-fixed, paraffin-embedded tissue, with the potential to replace the indirect immunohistochemical (IHC) marker for p16 protein. A direct clinical comparison between p16 IHC and an automated RNA ISH using 18 probes has not been established. Samples from 27 formalin-fixed, paraffin-embedded HNSCC cases from the Emory University Hospital archives were stained using 18 individual RNA ISH probes for high-risk HPV (RNAscope 2.5 LS Probe ) on a Leica autostainer (Buffalo Grove, IL) and were compared with p16 IHC. Two pathologists reviewed and reached a consensus on all interpretations. The RNAscope technique was positive in 89% (24/27) and the p16 IHC was positive in 78% (21/27). The RNAscope was negative in 11.1% of samples (3/27) and the p16 IHC-negative in 22.2% (6/27). The RNA ISH detected 100% of the p16-positive IHC-stained slides and had a concordance of 88.9% (24/27). This easy to interpret automated staining method for 18 high-risk HPV genotypes is a feasible replacement for the indirect p16 IHC method.

Persistent high-risk human papillomavirus (HPV) infection is strongly associated with development of high-grade cervical intraepithelial neoplasia or cancer (CIN3+). In single type infections serial type-specific viral-load measurements predict the natural history of the infection. In infections with multiple HPV-types, the individual type-specific viral-load profile could distinguish progressing HPV-infections from regressing infections. A case-cohort natural history study was established using samples from untreated women with multiple HPV-infections who developed CIN3+ (n=57) or cleared infections (n=88). Enriched cell pellet from liquid based cytology samples were subjected to a clinically validated real-time qPCR-assay (18 HPV-types). Using serial type-specific viral-load measurements (≥3) we calculated HPV-specific slopes and coefficient of determination (R2 ) by linear regression. For each woman slopes and R2 were used to calculate which HPV-induced processes were ongoing (progression, regression, serial transient, transient). In transient infections with multiple HPV-types, each single HPV-type generated similar increasing (0.27copies/cell/day) and decreasing (-0.27copies/cell/day) viral-load slopes. In CIN3+ at least one of the HPV-types had a clonal progressive course (R2 ≥0.85;0.0025copies/cell/day). In selected CIN3+ cases (n=6) immunostaining detecting type-specific HPV 16,31,33,58 and 67 RNA showed an even staining in clonal populations (CIN3+), whereas in transient virion-producing infections the RNA-staining was less in the basal layer compared to the upper layer where cells were ready to desquamate and release newly-formed virions. RNA-hybridization patterns matched the calculated ongoing processes measured by R2 and slope in serial type-specific viral-load measurements preceding the biopsy. In women with multiple HPV-types, serial type-specific viral-load measurements predict the natural history of the different HPV-types, and elucidates HPV-genotype attribution. 

Abstract

Background

Accurate identification of HPV-driven oropharyngeal cancer (OPC) is a major issue and none of the current diagnostic approaches is ideal. An in situ hybridization (ISH) assay that detects high-risk HPV E6/E7 mRNA, called the RNAscope HPV-test, has been recently developed. Studies have suggested that this assay may become a standard to define HPV-status.

Methods

To further assess this test, we compared its performance against the strategies that are used in routine clinical practice: p16 immunohistochemistry (IHC) as a single test and algorithms combining p16-IHC with HPV-DNA identification by PCR (algorithm-1) or ISH (algorithm-2).

Results

105 OPC specimens were analyzed. The prevalence of HPV-positive samples varied considerably: 67% for p16-IHC, 54% for algorithm-1, 61% for algorithm-2 and 59% for the RNAscope HPV-test. Discrepancies between the RNAscope HPV-test and p16-IHC, algorithm-1 and 2 were noted in respectively 13.3%, 13.1%, and 8.6%.

The 4 diagnostic strategies were able to identify 2 groups with different prognosis according to HPV-status, as expected. However, the greater survival differential was observed with the RNAscope HPV-test [HR: 0.19, 95% confidence interval (CI), 0.07–0.51, p = 0.001] closely followed by algorithm-1 (HR: 0.23, 95% CI, 0.08–0.66, p = 0.006) and algorithm-2 (HR: 0.26, 95% CI, 0.1–0.65, p = 0.004). In contrast, a weaker association was found when p16-IHC was used as a single test (HR: 0.33, 95% CI, 0.13–0.81, p = 0.02).

Conclusions

Our findings suggest that the RNAscope HPV-test and p16-based algorithms perform better that p16 alone to identify OPC that are truly driven by HPV-infection. The RNAscope HPV-test has the advantage of being a single test.

Abstract

BACKGROUND:

There is increasing evidence that high-risk human papillomavirus plays significant role in oropharyngeal cancer; however, there is lack of knowledge on the interplay between the virus and its downstream related molecules and their possible prognostic values. The objectives of the study are to better understand the interplay of the HR-HPV and its associated downstream molecules and to evaluate potential biomarkers for patient outcomes.

METHODS:

We conducted a retrospective study with available formalin-fixed, paraffin-embedded tissue from 244 oropharyngeal cancer patients that received curative radiotherapy or concurrent chemoradiotherapy from 2000 to 2008. In addition to chart review, we performed HPV DNA and RNA in situ hybridization and immunohistochemistry for p53, the retinoblastoma protein, p16, and cyclin D1 analysis. Cox-proportional hazard and Kaplan-Meier survival analysis were used to determine the prognostic markers for clinical outcomes.

RESULTS:

Patients averaged 57.3±9.4 year-old and were mostly males (76.2%) and ever-smokers (76.2%). All patients received curative radiotherapy and 44.3% received concurrent chemoradiotherapy. We detected the human papillomavirus in 77.9% of study patients. Ever-smokers, more advanced tumor stage, and receiving radiotherapy only had poorer 5-year overall survival, disease-specific survival, and loco-regional recurrence. Cases with positive human papillomavirus and p53 overexpression had poorer disease-specific survival. Cases without human papillomavirus, but cyclin D1 overexpression, was associated with poorer 5-year overall survival.

CONCLUSIONS:

Our data suggests that additional p53 and cyclin D1 testing may benefit oropharyngeal cancer patients with known human papillomavirus status.

Human Papillomavirus (HPV) is known as causative for squamous cell carcinoma (SCC) of the oropharynx, but is also not infrequently found in carcinomas of the sinonasal tract. Recently, a subset of these carcinomas was recognized to harbour HPV33 and have a significant morphological overlap with adenoid cystic carcinoma (ACC), a rare and aggressive carcinoma originating in the minor salivary glands. Termed HPV-related carcinoma with ACC-like features, only 9 cases have been reported. To clarify the occurrence of these tumours we screened a large material for presence of HPV-related ACC-like carcinoma. The identified tumours were characterized immunohistochemically and with fluorescence in situ hybridization and clinicopathologic information for all cases is presented.

METHODS AND RESULTS:

Forty-seven candidate cases were screened for presence of HPV. Six cases were identified and genotyped as HPV types 33, 35 and 56. All six cases had areas of dysplastic mucosal lining and showed remarkable heterogeneous morphologies. MYB, MYBL1, and NFIB genes were intact and, interestingly, staining for MYB protein was largely negative in contrast to what was found in ACC. One patient experienced a local recurrence 11 years after initial treatment and the remaining five patients were alive without evidence of disease.

CONCLUSION:

We report six new cases of HPV-related ACC-like carcinoma and found that, although in a small material, the prognosis for these patients seems more favourable than for ACC. For the distinction between ACC and HPV-related ACC-like carcinoma, p16, MYB immunohistochemistry, or investigation of MYB, MYBL1, and NFIB gene status are valuable. This article is protected by copyright. All rights reserved.

Abstract

BACKGROUND:

The incidence of oropharyngeal squamous cell carcinoma (OPSCC) is increasing among older adults. It is unknown whether these trends can be explained by human papillomavirus (HPV) and whether HPV-related tumors remain associated with an improved prognosis among older patients.

METHODS:

In a retrospective study of OPSCCs diagnosed from 1995 to 2013 at 2 National Comprehensive Cancer Network-designated cancer centers, p16 immunohistochemistry and in situ hybridization (ISH) for HPV-16, high-risk DNA, and/or E6/E7 RNA were performed. The median age at diagnosis was compared by p16 and ISH tumor status. Trends in age were analyzed with nonparametric trends. Survival was analyzed with the Kaplan-Meier method and Cox proportional hazards models.

RESULTS:

Among 239 patients, 144 (60%) were p16-positive. During 1998-2013, the median age increased among p16-positive patients (Ptrend = .01) but not among p16-negative patients (Ptrend = .71). The median age of p16-positive patients increased from 53 years (interquartile range [IQR] in 1995-2000, 45-65 years) to 58 years (IQR for 2001-2013, 53-64 years). Among patients ≥ 65 years old, the proportion of OPSCCs that were p16-positive increased from 41% during 1995-2000 to 75% during 2007-2013 (Ptrend = .04). Among all age groups, including older patients, a p16-positive tumor status conferred improved overall survival in comparison with a p16-negative status.

CONCLUSIONS:

The median age at diagnosis for HPV-related OPSCC is increasing as the proportion of OPSCCs caused by HPV rises among older adults. The favorable survival conferred by an HPV-positive tumor status persists in older adults. Cancer 2018. © 2018 American Cancer Society.