S. Noori, A. Monabati, A.A. Ghaderi Figure 2: Photograph (negative version) of ethidium-bromide stained agarose gel On UV transilluminator showing results of PCR assay using general human papilloma virus Primer set with internal control. Lane 1: DNA size marker (pUC 19 DNA/M spI); Lane 2: Positive control; Lane 3 to9: Samples negative for HPV DNA but showing amplified Beta-globin gene as internal control in the reaction. Thus, the possibility of inhibitory effect of competition between two primer sets (general HPV primer set and beta globin gene specific primer set) on each other as a possible cause of negative results in previous PCR assay was ruled out. In addi- tion, heteroduplex PCR assay using both HPV-16 and HPV-18 specific primer sets was performed for half of tumor cases, and no evidence of infection by these high risk types of HPV was found. Discussion Esophageal squamous cell carcinoma is one of the most common causes of cancer death worldwide. In western countries, where the risk of ESCC is generally low, consumption of tobacco and alcohol could explain great majority of the cases of ESCC. However, in regions such as northern Iran, which has a high rate of ESCC, only a small proportion of ESCC cases could be attributed to smoking or alcohol consumption. So, other risk factors must be responsible for the high incidence of ESCC in these areas.19 According to the results of previous stud- ies in Iran, the main suspected risk factors include low intake of fruits and vegetables, drinking hot tea, consumption of opium products and tobacco, Helicobacter pylori infection of the stomach, drink- ing contaminated water from cisterns, and genetic susceptibility.19 The main suspected mutagens are polycyclic aromatic hydrocarbons and N-nitroso compounds.20 Microbial agents, especially HPV may also be one of the factors that could explain part of such a high incidence rate in northern Iran as was shown by Farhadi and colleagues.19 They showed a prevalence of 36.8% for HPV infection in ESCC cases. The prevalence of HPV16 was significantly higher in ESCC cases (13.2%) than that in con- trols (0%) (P=0.05), but there was no statistically significant difference in the prevalence of HPV18 130 between cases and controls. They concluded that only HPV16, but not HPV18, may be considered as a risk factor for ESCC in northern Iran. While northern Iran is considered a high incidence area, remaining parts of Iran including southern Iran appear to have much lower annual incidence of ESCCs. Although, epidemiologic study and cancer registry of ESCC cases show that esophageal cancer is among the ten most prevalent cancers in Fars province, its annual incidence rate is 1.3 to 2.8 per 100,000. However, in northern Iran, annual incidence of ESCC is much higher (108 per 100,000) in Mazandaran and even higher in Torkamansahra (108.8 per 100,000 in men and 174 per 100,000 in women). Shiraz Medical Centers in the past 2-3 decades have been the major referral centers for referral of patients from large areas of south and south west of Iran. Therefore, registered cases of ESCC may be used to estimate the incidence of ESCC in such areas. Therefore, Fars province and neighboring provinces in south of Iran can be considered as low incidence areas. Human papilloma virus DNA has been found frequently in ESCCs from high incidence areas.13,14,15,21 As mentioned earlier, de Villier and colleagues demonstrated presence of HPV DNA in 17.1% of the ESCC cases.13 Li and colleagues evaluated specimens of balloon cytol- ogy examination from volunteers in two regions with significantly different incidence of esophageal carcinoma. PCR results showed that prevalence of HPV-16 E6 gene in a high incidence area was 1.9 fold higher than that of low incidence area (72% and 37%, respectively, P<0.01). Similar results were obtained with HPV-16 E7 gene using ISH. They suggested that HPV-16 plays a causative role in the pathogenesis of esophageal cancer, especially in the high incidence area of China.14 Si and colleagues evaluated some HPV-16 positive cases of ESCC to determine physical Iran J Med Sci June 2012; Vol 37 No 2