No evidence of HPV DNA in esophageal squamous cell carcinoma in a population of Southern Brazil

Abstract

Aim: To investigate the association between human papillomavirus (HPV) and esophageal squamous cell carcinoma (ESCC) in southern Brazil.

Methods: We studied 189 esophageal samples from 125 patients from three different groups: (1) 102 biopsies from 51 patients with ESCC, with one sample from the tumor and another from normal esophageal mucosa distant from the tumor; (2) 50 esophageal biopsies from 37 patients with a previous diagnosis of head and neck squamous cell carcinoma (HNSCC); and (3) 37 biopsies from esophageal mucosa with normal appearance from 37 dyspeptic patients, not exposed to smoking or alcohol consumption. Nested-polymerase chain reaction (PCR) with the MY09/11 and GP5/6 L1 primers was used to detect HPV L1 in samples fixed in formalin and stored in paraffin blocks. All PCR reactions were performed with a positive control (cervicovaginal samples), with a negative control (Human Genomic DNA) and with a blank reaction containing all reagents except DNA. We took extreme care to prevent DNA contamination in sample collection, processing, and testing.

Results: The histological biopsies confirmed the diagnosis of ESCC in 52 samples (51 from ESCC group and 1 from the HNSCC group) and classified as well differentiated (12/52, 23.1%), moderately differentiated (27/52, 51.9%) or poorly differentiated (7/52, 13.5%). One hundred twenty-eight esophageal biopsies were considered normal (51 from the ESCC group, 42 from the HNSCC group and 35 from dyspeptic patients). Nine had esophagitis (7 from the HNSCC and 2 from dyspeptic patients). Of a total of 189 samples, only 6 samples had insufficient material for PCR analysis: 1 from mucosa distant from the tumor in a patient with ESCC, 3 from patients with HNSCC and 2 from patients without cancer. In 183 samples (96.8%) GAPDH, G3PDH and/or β-globin were amplified, thus indicating the adequacy of the DNA in those samples. HPV DNA was negative in all the 183 samples tested: 52 with ESCC, 9 with esophagitis and 122 with normal esophageal mucosa.

Conclusion: There was no evidence of HPV infection in different ESCC from southern Brazil.

Keywords: Esophageal cancer; Esophageal squamous cell carcinoma; Head and neck cancer; Human papillomavirus; Nested-polymerase chain reaction; Polymerase chain reactions.

Figure 1

Analysis of DNA from esophageal tumor tissue for human papillomavirus using nested- polymerase chain reaction for human papillomavirus L1 gene. The MY09/11 primer pair was used in the first step (A) and the GP5/GP6 primer pair was used in the second step (B). 1: DNA size marker (100 bp DNA ladder); 2: Patient tumor DNA: [human papillomavirus (HPV)]-negative; 3: HPV DNA-negative control (Human Genomic DNA); 4: HPV DNA-positive control (cervicovaginal sample); 5: Negative control containing all polymerase chain reaction reagents except DNA.