Human Papillomavirus 16 Infection and TP53 Mutation: Two Distinct Pathogeneses for Oropharyngeal Squamous Cell Carcinoma in an Eastern Chinese Population

Abstract

Objectives: To investigate the clinicopathological characteristics, human papillomavirus (HPV) infection, p53 expression, and TP53 mutations in oropharyngeal squamous cell carcinoma (OPSCC) and determine their utility as prognostic predictors in a primarily eastern Chinese population.

Methods: The HPV infection status was tested via p16INK4A immunohistochemistry and validated using PCR, reverse blot hybridization and in situ hybridization (ISH) in 188 OPSCC samples. p53 expression levels and TP53 gene mutations were assessed through immunohistochemistry and sequencing, respectively. Clinicopathological characteristics and follow-up information were collected. Overall survival was estimated using the Log-rank test.

Results: Overall, 22 of the 188 OPSCC samples were associated with HPV infection. HPV16 was identified in all 22 samples, whereas no samples were positive for HPV18. All 22 HPV-associated OPSCC samples were p53 negative and lacked TP53 mutations. HPV16 positivity, female patients, non-smokers, and patients with histological grade I and stage N0 diseases showed better overall survival (p = 0.009, 0.003, 0.048, 0.009, and 0.004, respectively). No significant differences in overall survival between smoking and non-smoking patients were observed in the HPV-associated OPSCC group. Patients without mutations in TP53 exons 5-8 had better prognoses (p = 0.031) among the 43 sequenced specimens. Multivariate analysis indicated that HPV16 infection status (p = 0.011), histological grade (p = 0.017), and N stage (p = 0.019) were independent prognostic factors for patients with OPSCC.

Conclusions: Distinct from the situation in Europe and America, for the patients with OPSCC in this study, HPV16 infection was relatively low, although it was still the most important independent prognostic predictor for the disease. In addition to the high smoking and drinking rate in this population, HPV16 infection and TP53 dysfunction appear to be two distinct pathogens for OPSCC patients in the eastern Chinese population.

Fig 1. The Incidence of OPSCC from 2008 to 2013.

(A) The distribution of OPSCC patients treated at Shanghai 9th People’s Hospital in Mainland China. (B) The age distribution of the 188 primary OPSCC patients. (C) The number of cases of primary OSCC patients each year from 2008 to 2013. (D) The proportional trend of OPSCC numbers relative to OSCC each year from 2008 to 2013.

Fig 2. Histologic Features of HPV-Associated OPSCC and HPV Status.

(A, B) HE staining of HPV-associated OPSCC with poorly differentiated tumor epithelial nests and a large number of lymphocytic infiltrations (200×). (C, D) Strong diffuse brown signals showing p16INK4A expression in the tumor cell plasma and nuclei by immunohistochemistry in ≥70% of malignant cells. (E) Native PAGE results for HPV16/18 E6: N, negative; P, positive; B, Blank; P16, HPV16 positive control; P18, HPV18 positive control. (F) 1000× magnification (hybridization dots within tumor cell nuclei, inset), integrated form. (G) 400× magnification (diffuse hybridization signals within tumor cell nuclei, inset), episomal form. (H) The proportion of HPV16-associated OPSCC/OPSCC cases from 2008 to 2013.

Fig 3. p53 IHC and TP53 exons 5–8 Sequencing.

(A, B) Accumulation of abnormal p53 protein in tumor cell nuclei. (C, D) An atlas of direct sequencing of TP53 exons 5–8. Upper photographs show different classifications of mutants, and lower photographs are normal controls; TP53 exons 5–8 point mutation are marked with red arrow in picture C; Heterozygous TP53 exons 5–8 frame-shift mutations are shown in picture D.

Fig 4. Kaplan–Meier Overall Survival and Survival Curves.

(A) HPV status, (B) clinical stage, (C) nodal stage, (D) smoking status, (E) histological grade, and (F) gender were compared using categorical data analysis, and overall-survival was estimated using the Kaplan-Meier method among all 188 OPSCC cases. (G) Smoking status (among 22 HPV-associated OPSCC patients) and (H) TP53 exons 5–8 mutation status (among 43 sequencing specimen) were compared by categorical data analysis, and overall-survival was estimated using the Kaplan-Meier method.