Molecular and Immunohistochemical Cognizance of HPV16 in Oral Leukoplakia, Oral Squamous Cell Carcinoma and Oropharyngeal Squamous Cell Carcinoma

Abstract

Prior studies have established the carcinogenic role of HPV16 and also demonstrated its unique biological behavior in cervical and oropharyngeal squamous cell carcinoma (OPSCC) but its role in oral leukoplakia (OL) and oral squamous cell carcinoma (OSCC) is not well explored. Therefore, in the present study, we assessed HPV16 prevalence using PCR and Anti-HPV16 antibodies for the first time and correlated its biological behavior using p16^INK4a and Ki67 proliferation index (PI) in OL, OSCC, and OPSCC. This study included 63 subjects comprising of 25 OL, 26 OSCC, and 12 OPSCC cases. Exfoliated cells were collected and processed for PCR followed by immunohistochemistry with primary antibodies p16^INK4a, Anti-HPV16, and Ki67. The expressions were evaluated and statistical analysis included Chi-square and Spearman's test. Cumulatively 37% (OL-7%, OSCC-14% & OPSCC-16%) of cases showed positive PCR expression. PCR positivity was observed to be significantly higher (p 0.00) in OPSCC (9/12) than OSCC (9/26) and OL (5/25) cases. Overall immunohistochemical expression of p16^INK4a, Anti-HPV16, and Ki67 were significantly (p 0.02) higher in HPV16 (PCR) positive cases. HPV16 + OSCC cases showed higher grades of p16^INK4a and Ki67 expression. We have demonstrated a prevalence of HPV16 in OL, OSCC, and OPSCC through PCR, which may be concluded as a gold standard for the detection of HPV16 DNA.

Keywords: Anti-HPV16; Human papillomavirus 16; Ki67; Leukoplakia; Oral squamous cell carcinoma; PCR; p16INK4a.

Fig. 1

Photograph depicting PCR reaction results (Lane No. 1, 2, 3 & 7-negative, Lane No. 4, 5 & 6—positive)

Fig. 2

Photomicrograph depicting positive nuclear cytoplasmic immunohistochemical expression of p16^INK4a a in positive control (tonsil); b Grade 1 expression; c Grade 2 expression; d Grade 3 expression (200×)

Fig. 3

Photomicrograph showing positive cytoplasmic expression of Anti-HPV16 a in positive control (cervical carcinoma); b 50% positive expression (200×)

Fig. 4

Photomicrograph depicting positive immunohistochemical expression of Ki67 a calculation of percentage expression of Ki67 (positive cells—red, negative cells—blue); b Low PI; c Moderate PI; d High PI (×200)