Prognostic significance of cell cycle-associated proteins p16, pRB, cyclin D1 and p53 in resected oropharyngeal carcinoma

Abstract

Background: Human papillomavirus (HPV)-related oropharyngeal squamous cell carcinoma (OPSCC) has an improved outcome and may allow for treatment de-escalation. High-risk HPV (HR-HPV) infection is associated with deregulated expression of the cell cycle-associated proteins p16^INK4, pRB, cyclin D1 and p53. The objective of this study was to assess cell cycle proteins as potential surrogate markers for HR-HPV DNA testing to identify OPSCC with favorable prognosis after resection.

Methods: Tissue microarray cores of 313 surgically treated OPSCC were stained for p16^INK4a, pRB, cyclin D1 and p53 using immunohistochemistry. Protein expression was scored as high or low based on the proportion of positive carcinoma cells. Tumor samples were analysed for HR-HPV DNA with polymerase chain reaction-based testing. Associations between cell cycle protein expression and HR-HPV DNA status were evaluated by calculating sensitivity, specificity, predictive values, and diagnostic odds ratios (DOR). Kaplan-Meier and Cox regression analysis were applied to evaluate associations between cell cycle protein expression and patient outcome.

Results: High expression of p16^INK4a, cyclin D1, pRB and p53 in tumor cells were observed in 51.8%, 51.4%, 41.9% and 33.5% of OPSCC, respectively. HR-HPV DNA positive were 158/313 (50.5%) tumor samples (HPV16: 147, HPV18: 1, HPV33: 5, HPV35: 2, HPV56: 2, and HPV59: 1). P16^INK4a showed a higher DOR to predict HR-HPV DNA positivity than pRB, cyclin D1 and p53. Both the p16^INK4a/pRB and the p16^INK4a/pRB/cyclin D1/p53 signatures had lower DOR than p16^INK4a alone. Improved 5-year overall and disease-specific survival were associated with HR-HPV DNA positivity, high p16^INK4a, low pRB, low cyclin D1, and low p53 expression. Associations with improved outcome were also observed for the marker combinations high p16^INK4a/positive HR-HPV DNA, high p16^INK4a/low pRB and high p16^INK4a/low pRB/low cyclin D1/low p53. In a multivariate analysis adjusted for age, smoking history, pT and pN category, high p16^INK4a expression showed the lowest hazard ratio for death.

Conclusions: High p16^INK4a expression is a reliable marker for survival prognostication in surgically treated OPSCC patients. Protein signatures including the pRB, cyclin D1 and p53 proteins do not further increase the prognostic performance of p16^INK4a as a single marker.

Keywords: Cyclin D1; Human papillomavirus; Oropharyngeal squamous cell carcinoma; Prognosis; Retinoblastoma protein; p16; p53.

Fig. 1

Expression of cell cycle-associated proteins in oropharyngeal squamous cell carcinoma. Histological findings (a, b) and expression of the p16^INK4a (c, d), pRB (e, f), cyclin D1 (g, h) and p53 (i, j) proteins in representative HR-HPV positive (b, d, f, h, j) and HR-HPV negative OPSCC (a, c, e, g, i). Original magnification, 400×

Fig. 2

Overall survival after resection in patients with oropharyngeal squamous cell carcinoma. Kaplan-Meier analyses revealed associations between improved survival and HR-HPV DNA positivity (a), high p16^INK4a (b), low pRB (c), high p16^INK4a/low pRB signature (d), low cyclin D1 (e), and low p53 (f)