Prognostic significance of human papillomavirus 16 viral load level in patients with oropharyngeal cancer

Abstract

Human papillomavirus (HPV) infection in patients with oropharyngeal squamous cell carcinoma (OPSCC) is a major determinant for better prognosis. However, there remain HPV-positive patients who have poor outcomes. The stratification strategy for detecting high-risk patients among those with HPV-positive OPSCC has not been well delineated, especially for Asian patients. We undertook a retrospective cohort study on the survival rate of 89 Japanese patients diagnosed with primary OPSCC. The tumors were concurrently analyzed for the presence of HPV E6 DNA/mRNA, viral DNA load, p16 expression, viral physical status, and viral variant lineage. Human papillomavirus 16 viral DNA was found in 45 (51%) OPSCCs. Human papillomavirus 16 DNA-positive OPSCCs with higher viral load (classified as HPV16 DNA-medium/high OPSCCs) showed significantly favorable overall survival and progression-free survival compared with HPV16 DNA-positive OPSCCs with lower viral load (<10 copies/cell; HPV16 DNA-low OPSCCs) and HPV16 DNA-negative OPSCCs. E6 mRNA expression was observed in all HPV16 DNA-medium/high OPSCCs but not in HPV16 DNA-low OPSCCs. Notably, p16-positive and HPV16 DNA-negative/low OPSCCs showed significantly worse survival than p16-positive and HPV16 DNA-medium/high OPSCCs and resembled HPV-unrelated OPSCCs with regard to survival and risk factor profile. Although not significant, a trend toward shorter survival was observed for HPV16-integrated OPSCCs. Phylogenetic analysis revealed two major types of HPV16 variants termed Asian (A4) and European (A1/A2/A3) variants, but no difference in survival between these variants was observed. Altogether, these findings suggest that HPV viral load is a potentially informative factor for more accurate risk stratification of patients with OPSCC.

Keywords: Japanese cohort; human papillomavirus; oropharyngeal cancer; prognosis; viral load.

FIGURE 1

Assessment of viral DNA load and mRNA expression of human papillomavirus 16 (HPV16) in oropharyngeal squamous cell carcinoma. A, Distribution of HPV16 viral load. Tumors were categorized by viral load level as follows: L, HPV DNA_low; M, HPV DNA_medium; and H, HPV DNA_high. Horizontal lines in box plots indicate median values, and the bars extend to the maximum and minimum values. P values were obtained using the Mann‐Whitney U test. B, Kaplan‐Meier curves showing overall survival (OS) and progression‐free survival (PFS) of tumors categorized by HPV16 DNA status. C, Kaplan‐Meier curves of tumors after being further categorized into four subgroups by viral load. D, Kaplan‐Meier curves of tumors after being recategorized by combining the HPV DNA_high group with the HPV DNA_medium group (HPV DNA_high/medium). Note that the OS was not significantly different when only HPV16 DNA status was assessed. Grouping by viral load revealed a significant difference in both OS and PFS. P values were calculated using the log‐rank test. E, Relative HPV16 E6 mRNA expression level stratified by viral load. P value was obtained using the Mann‐Whitney U test

FIGURE 2

Kaplan‐Meier curves showing overall survival (OS) and progression‐free survival (PFS) of patients with oropharyngeal squamous cell carcinoma. A, Tumors were stratified by p16 expression and human papillomavirus 16 (HPV16) DNA status. B, Tumors were further categorized by p16 expression and HPV DNA viral load. C, Tumors were recategorized into three groups by combining the HPV DNA_low group with the HPV DNA⁻ group. P values were calculated using the log‐rank test

FIGURE 3

Assessment of the viral physical status of human papillomavirus 16 (HPV16) in oropharyngeal squamous cell carcinoma (OPSCC). A, Kaplan‐Meier curves showing overall survival (OS) and progression‐free survival (PFS) of patients with OPSCC tumors by viral physical status. P values were calculated using the log‐rank test. B, Box plot showing HPV16 DNA loads stratified by viral physical status. P values were calculated using the Mann‐Whitney U test. C, Proportion of viral physical status stratified by viral load. H, HPV DNA_high; L, HPV DNA_low; M, HPV DNA_medium

FIGURE 4

Phylogenetic analysis of human papillomavirus 16 (HPV16). A phylogenetic tree was generated using the maximum likelihood method. It was constructed on the basis of concatenated 790‐bp fragments of the E6 and long control region. It included 24 sequences that were successfully recovered from tumor specimens of our patients with oropharyngeal squamous cell carcinoma and 50 GenBank‐retrieved sequences from cervical specimens of various geographic origins. Sequences analyzed in this study are colored in red and underlined. HPV16 viral load levels in our specimens are shown in parentheses: H, high; L, low; M, medium. Scale bars represent the numbers of substitutions per site. AA, Asian‐American; Af, African; NA, North American