HUMAN PAPILLOMAVIRUS 16 E6 POLYMORPHISMS IN CERVICAL LESIONS FROM DIFFERENT EUROPEAN POPULATIONS AND THEIR CORRELATION WITH HUMAN LEUKOCYTE ANTIGEN CLASS II HAPLOTYPES Ingeborg ZEHBE 1 , Ruth TACHEZY 2 , Joannis MYTILINEOS 3 , Gianfranco VOGLINO 4 , Iva MIKYˇ SKOVA 2 , Hajo DELIUS 1 , Antonella MARONGIU 4 , Lutz GISSMANN 1 , Erik WILANDER 5 and Massimo TOMMASINO 1 * 1 Angewandte Tumorvirologie, Deutsches Krebsforschungszentrum, Heidelberg, Germany 2 Department of Experimental Virology, Institute of Hematology and Blood Transfusion, Prague, Czech Republic 3 Department of Transplantation Immunology, University of Heidelberg, Heidelberg, Germany 4 Servizio di Anatomia Patologica, Ospedale Sant’Anna, Torino, Italy 5 Department of Genetics and Pathology, University of Uppsala, Uppsala, Sweden Infection with high-risk human papillomavirus (HPV) is necessary for the development of a cervical lesion, but only a fraction of precursor lesions progress to cancer. Additional factors, other than HPV type per se, are likely to increase the probability for progression. Intratype genome variations have been reported to be associated with viral persistence and the development of a major cervical disease. We have recently shown that the prevalence of specific HPV16-E6 variants in invasive cervical cancer (ICC) varies between Italian and Swedish women. To extend our initial study we have analyzed E6 variants in cervical lesions from Czech women, ranging from low-grade cervical intraepithelial neo- plasia (LCIN) to ICC and scaled up the sample size of our initial study of Swedish and Italian women. In addition, we have correlated the cases of cancers with human leukocyte antigen (HLA) class II haplotypes. In line with our earlier observation, the distribution of specific HPV16-E6 genotypes in CIN and ICC varied in the 3 cohorts. For instance, the HPV16-E6 L83V variant, which has been found to be posi- tively associated with ICC in Swedish women (p  0.002), was more prevalent in LCIN than in ICC in Italian and Czech women (p  0.01 and  0.03, respectively). These data indi- cate that host genetic factors, such as HLA polymorphism, may determine the potential oncogenicity of the HPV16-E6 L83V variant. Indeed, the DR04-DQ03 haplotype, which is approximately 3-fold more abundant in the normal Swedish population than in those in Italy and the Czech Republic, was found to be positively associated with HPV16-E6 L83V in the 3 cohorts investigated (p  0.01). This observation may ex- plain why L83V is a risk factor more in Sweden than in the other 2 countries. © 2001 Wiley-Liss, Inc. Key words: HPV16; E6 and E7 genotypes; polymorphism; cervical lesion; human leukocyte antigen Certain human papillomavirus (HPV) genotypes are etiologic agents for cervical cancer development. 1 HPV16 is the most fre- quently detected genotype in cervical carcinoma and its precur- sors. 2–5 Biochemical data have demonstrated that the products of 2 early genes, E6 and E7, are the major oncoproteins of HPV16. 6 HPV16 E6 and E7 associate with and inactivate the biologic function of several cellular proteins, including the regulators of cell cycle checkpoints, p53 and pRb. 7 Infection with high-risk HPV is the principal risk factor for the development of an invasive lesion. However, most HPV infections regress spontaneously and, for the cases that do progress to cancer, a long period of latency is normally required. It is clear that additional risk factors play a role in disease progression. Recently, independent studies have provided evidence that specific intratype HPV genome variations may influence the persistence of the infection and the progression of a precursor lesion to cancer. 8 –10 Several North American and South American studies have shown that nonprototype-like HPV16 variants, which harbor several nu- cleotide changes within the long control region and the E6 gene, confer a higher risk for the development of anogenital high-grade lesions than the prototype-like variants, which contain no or only few nucleotide changes within the same regions. 11–15 In a fol- low-up study, an HPV16 E6 variant, E-G350, harboring an amino acid change at position 83 substituting a leucine for a valine (L83V), was found to be associated with progression from cervical intraepithelial neoplasia-I (CIN-I) to CIN-III. 8 We have recently shown that the same E6 variation (L83V) is more prevalent in cases of invasive cervical carcinoma (ICC) from Swedish women than the prototype. 10,16 Our initial findings in an Italian cohort have indicated that in this group the HPV16-E6 L83V variant is mainly associated with precursor lesions. 9 Thus, the potential oncogenicity of L83V appears to be dependent on genetic differ- ences between populations. However, a full confirmation of this hypothesis requires an analysis of HPV16-E6 polymorphisms in other cohorts. To assess the role of viral genome variations in cervical disease, we analyzed the sequences of the E6 gene in HPV16-positive cervical biopsies from 169 Swedish, 91 Italian and 107 Czech women. The samples range from low-grade lesion to cancer. In addition, we analyzed human leukocyte antigen (HLA) class II haplotypes in the cases of cancers. We have conducted a cross- sectional investigation because prospective studies of the progres- sion from high-grade lesion to cancer cannot be conducted due to ethical reasons. Here, we have shown that HPV16-E6 genotypes have a different distribution in various grades of cervical lesions in the same cohort and that the prevalence of specific HPV16-E6 polymorphisms varies among European countries. In addition, a positive association between the HLA class II DR04-DQ03 hap- lotype and the L83V variant was found in the cohorts investigated. MATERIAL AND METHODS Clinical specimens We have undertaken 3 cross-sectional studies and included exclusively HPV16-positive biopsies, which were available at the Department of Genetics and Pathology, University Hospital Upp- sala, Sweden (n = 169); at the Servizio di Anatomia Patologica, Ospedale Sant’Anna in Turin, Italy (n = 91); and at the Depart- ment of Experimental Virology, Institute of Hematology and Grant sponsor: Wilhelm Sander-Stiftung, Munich, Germany; Grant number: 98.065.1; Grant sponsor: Granting Agency, Ministry of Health, Prague, Czech Republic; Grant number: 5959-3; Grant sponsor: NATO Collaborative Linkage; Grant number: LST.CLG 975794. *Correspondence to: Angewandte Tumorvirologie, Deutsches Krebsfor- schungszentrum, Im Neuenheimer Feld 242, 69120 Heidelberg, Germany. Fax: +49-6221-424932. E-mail: m.tommasino@dkfz-heidelberg.de Received 7 March 2001; Revised 21 May, 27 June 2001; Accepted 29 June 2001 Int. J. Cancer: 94, 711–716 (2001) © 2001 Wiley-Liss, Inc. Publication of the International Union Against Cancer