status of HPV-16 in these cases. They showed a predominance of integrated form of viral genes in HPV-16 positive ESCCs from china implying that the HPV infection may play a role in the patho- genesis of ESCC.15 In contrast, HPV infection was infrequent in cancer-bearing patients of north America,4 and even undetectable in many other low incidence regions.17,18 Our study shows that there was no associa- tion between HPV infection and development of esophageal squamous cell carcinoma. Thus, the absence of HPV DNA in this region is similar to that in most of other low incidence areas, and indicates that HPV infection is not likely to be involved in esophageal tumorigenesis in this region. However, there are some other factors such as dietary and environmental factors as well as an ill-defined contribution from genetic predisposition that may be involved in pathogenesis of esophageal carci- nomas.2,3 Esophageal cancers in south of Iran may be attributed to dietary deficiencies of vitamins and essential metals, presence of carcinogens such as fungus-contaminated and nitrosamine-containing food stuffs, drink of hot tea, chronic esophagitis, and tobacco usage. Although alcoholic drinks and their carcinogenic components such as poly cyclic hydrocarbons, fusel oil and nitrosamine along with other mutagenic compounds and associated nutritional deficiencies are considered to play a significant role in some low incidence areas such as Europe and United States, they may not a risk factor in southern parts of Iran.2 The possibility of any technical error in reach- ing negative results in PCR assay is not appre- ciable. Good quantity and quality of extracted DNA confirmed by spectrophotometery and agarose gel electrophoresis, strong positive reaction in posi- tive controls as well as successful amplification of beta globin gene, as internal control in all cases, clearly rule out the possibility of any technical error. Negative results in PCR assay with and without internal control in tumoral and non-tumoral cases also rule out the possibility of competitive inhibition of general HPV primer set by beta globin gene specific primer set. Formalin, which is the most usual fixative, has profound effects on nucleic acids. However, its effects depend on concen- tration, pH, temperature, duration of fixation as well as type of tissue storage.22 Obviously, DNA extracted from tissue left in formalin solution for a long period is more likely to be damaged by forma- lin compared to that extracted from tissue embed- ded in paraffin blocks after initial fixation only for one to two days.23 However, several studies have shown that formalin fixed tissues, even those left in formalin for a long duration, are quantitatively and qualitatively good enough to yield DNA to succeed in PCR assays.24 However, few studies Iran J Med Sci June 2012; Vol 37 No 2 Papilloma virus in esophageal squamous cell carcinoma have reported yield of high molecular weight DNA from formalin fixed tissues.13 Thus, the effect of formalin on DNA as a proba- ble cause of negative results in PCR assay is ruled out. On the other hand, several studies mostly from low incidence areas used extracted DNA from non fixed, frozen tissues as well as cell lines of ESCC (not exposed to formalin) and similarly showed negative results in PCR or southern blot hybridization techniques.18, 25 Moreover, using the same materials, primers and instruments, a PCR assay of cases with uterine cervical carcinoma in this center successfully revealed the presence of common and type specific HPV DNA sequences in many of the cases. So, negative findings in our study can’t be attributable to a sensitivity problem in the PCR assay. Conclusion The findings of the present study indicate that there is no association between HPV infection and devel- opment of esophageal squamous cell carcinoma in the cases evaluated. Acknowledgement The authors would like to thank Mr. Abdul-Moham- mad Pezeshki for his great cooperation and tech- nical support. This study was supported by a grant (number; 81-1528) from Vice Chancellor for Research, Shiraz University of Medical Sciences. Confilct of interest: None declared. References 1 Crawford JM. The gastrointestinal tract. In: Cotran RS, Kumar V, Collins T. Robbins pathologic basis of disease. 6th ed. Philadel- phia: W.B. Saunders; 1999. p. 776-87. 2 Bagheri Lankarani K, Mowla A, Asadian F, Tabei SZ, Panjeshahin MR. Changing epide- miology of esophageal cancer in Fars prov- ince, Iran. Iran J Med Sci. 2002;27:4-10. 3 Riddell RH. Early detection of neoplasia of the esophagus and gastroesophageal junc- tion. Am J Gastroenterol. 1996;91:853-63. PubMed PMID: 8633572. 4 Turner JR, Shen LH, Crum CP, Dean PJ, Odze RD. Low prevalence of human pap- illomavirus infection in esophageal squa- mous cell carcinomas from North America: analysis by a highly sensitive and specific polymerase chain reaction-based approach. Hum Pathol. 1997;28:174-8. doi: 10.1016/ S0046-8177(97)90102-7. 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