Probes for HPV

Abstract

Purpose

High-risk human papillomavirus (HR-HPV) infections was the causal factor in the development of cervical cancer, but the significance of HPV viral load in the prediction of the response to current therapeutic approaches had not reached consensus. The present study was performed to assess the high risk HPV viral load of cervical cancer patients who underwent radiotherapy alone or in combination with chemotherapy or hyperthermotherapy or both in correlation to long-term survival.

Methods

116 cervical cancer patients were recruited and assigned into four groups of different therapeutic modalities. The prevalent high risk types of HPV 16, 18, 58 were detected by type specific in situ hybridization (ISH), and HPV mRNA was detected by RNA scope assay using RNA scope 2.0 FFPE Reagent Kit. Semi-quantification of the HR-HPV viral load was measured based on the intensity of ISH signal captured from the tumor nests in the grey scale.

Results

The HR-HPV viral load had a significant negative correlation with survival (r_s = −0.368,P = 0.001). The 15-year survival rate of low viral load group was 68.18%, moderate viral load group was 52.17%, and high viral load group was 34.69% (P = 0.001). HPV mRNA expression was strongly consistent with HPV viral load. The 15-year survival rates of different therapeutic groups were 39.29%, 58.62%, 50.00%, 55.17%, respectively (P = 0.545). Combinatorial treatment modalities improved the actual survival, which demonstrated no significant difference among 5,10 and 15 years comparison. Cox regression analysis showed that the relative risk of death was obviously higher in the HPV 18 single positive group and high HPV viral load group.

Conclusions

The semi-quantitive viral load assessment in situ is a feasible approach in clinical practice. The more the HPV viral load was, the worse the survival of patients would be. The combinational treatments were in favor of the disease-stabilization.

Abstract

OBJECTIVES/HYPOTHESIS:

Human papillomavirus (HPV)-positive and HPV-negative oropharyngeal squamous cell carcinoma (OPSCC) are distinct disease entities. Prognostic factors specific to each entity have not been adequately explored. Goals for this study were: 1) to determine whether HPV-positive and HPV-negative OPSCCs have distinct prognostic factors, and 2) to explore the prognostic significance of sex and race in OPSCC after HPV stratification STUDY DESIGN: Retrospective case series.

METHODS:

A retrospective review of 239 incident OPSCC patients from 1995 to 2012, treated at Johns Hopkins and University of California-San Francisco was conducted. Women and nonwhite races were oversampled. All analyses were stratified by tumor HPV in situ hybridization status. The effects of sex and race on survival were considered in Kaplan-Meier and unadjusted and adjusted Cox regression models.

RESULTS:

One hundred thirty-four (56.1%) OPSCC patients were HPV positive. On univariate analysis, women had better overall survival than men among HPV-positive (hazard ratio [HR]: 0.47, 95% confidence interval [CI]: 0.20-1.07; P = .06) but not HPV-negative (HR: 0.73, 95% CI: 0.43-1.24; P = .24) OPSCCs. On multivariate analysis, women with HPV-positive OPSCCs remained at lower risk of death (adjusted hazard ratio [aHR]: 0.34, 95% CI: 0.12-0.96; P = .04). Survival did not vary significantly by race among HPV-positive patients. Among HPV-negative patients, Hispanic patients had significantly better survival in unadjusted (HR: 0.27, 95% CI: 0.08-0.91; P = .04) but not adjusted (aHR: 0.93, 95% CI: 0.11-7.36; P = .94) analysis.

CONCLUSIONS:

Women with HPV-positive OPSCC may have improved overall survival compared to men. Sex does not play a prognostic role in HPV-negative OPSCC. There are no differences in prognosis by race among HPV-positive or HPV-negative patients.

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.