Persistent HPV infection plays a major role in cervical cancer. This study was undertaken to identify HPV types in a cohort of Indian women with locally advanced cervical cancer as well as to determine the physical state and/or site of viral integration in the host genome. Pretreatment biopsies (n = 270) from patients were screened for HPV infection by a high throughput HPV genotyping assay based on luminex xMAP technology as well as MY09/11 PCR and SPF1/2 PCR. Overall HPV positivity was observed to be 95%, with HPV16 being most common (63%) followed by infection with HPV18. Integration status of the virus was identified using Amplification of Papillomavirus Oncogene Transcripts (APOT) assay in a subset of samples positive for HPV16 and/or HPV18 (n = 86) and with an adequate follow-up. The data was correlated with clinical outcome of the patients. Integration of the viral genome was observed in 79% of the cases and a preference for integration into the chromosomal loci 1p, 3q, 6q, 11q, 13q and 20q was seen. Clinical data revealed that the physical state of the virus (integrated or episomal) could be an important prognostic marker for cervical cancer.
References
[1]
Munoz N, Bosch FX, de Sanjose S, Herrero R, Castellsague X, et al. (2003) Epidemiologic classification of human papillomavirus types associated with cervical cancer. N Engl J Med 348: 518–527.
[2]
Schmitt M, Bravo IG, Snijders PJ, Gissmann L, Pawlita M, et al. (2006) Bead-based multiplex genotyping of human papillomaviruses. J Clin Microbiol 44: 504–512.
[3]
Wentzensen N, Vinokurova S, von Knebel Doeberitz M (2004) Systematic review of genomic integration sites of human papillomavirus genomes in epithelial dysplasia and invasive cancer of the female lower genital tract. Cancer Res 64: 3878–3884.
[4]
Bechtold V, Beard P, Raj K (2003) Human papillomavirus type 16 E2 protein has no effect on transcription from episomal viral DNA. J Virol 77: 2021–2028.
[5]
Romanczuk H, Howley PM (1992) Disruption of either the E1 or the E2 regulatory gene of human papillomavirus type 16 increases viral immortalization capacity. Proc Natl Acad Sci U S A 89: 3159–3163.
[6]
Klaes R, Woerner SM, Ridder R, Wentzensen N, Duerst M, et al. (1999) Detection of high-risk cervical intraepithelial neoplasia and cervical cancer by amplification of transcripts derived from integrated papillomavirus oncogenes. Cancer Res 59: 6132–6136.
[7]
Couturier J, Sastre-Garau X, Schneider-Maunoury S, Labib A, Orth G (1991) Integration of papillomavirus DNA near myc genes in genital carcinomas and its consequences for proto-oncogene expression. J Virol 65: 4534–4538.
[8]
Choo KB, Pan CC, Han SH (1987) Integration of human papillomavirus type 16 into cellular DNA of cervical carcinoma: preferential deletion of the E2 gene and invariable retention of the long control region and the E6/E7 open reading frames. Virology 161: 259–261.
[9]
Di Luca D, Pilotti S, Stefanon B, Rotola A, Monini P, et al. (1986) Human papillomavirus type 16 DNA in genital tumours: a pathological and molecular analysis. J Gen Virol 67 ( Pt 3): 583–589.
[10]
Jeon S, Allen-Hoffmann BL, Lambert PF (1995) Integration of human papillomavirus type 16 into the human genome correlates with a selective growth advantage of cells. J Virol 69: 2989–2997.
[11]
Jeon S, Lambert PF (1995) Integration of human papillomavirus type 16 DNA into the human genome leads to increased stability of E6 and E7 mRNAs: implications for cervical carcinogenesis. Proc Natl Acad Sci U S A 92: 1654–1658.
[12]
Yu T, Ferber MJ, Cheung TH, Chung TK, Wong YF, et al. (2005) The role of viral integration in the development of cervical cancer. Cancer Genet Cytogenet 158: 27–34.
[13]
Ziegert C, Wentzensen N, Vinokurova S, Kisseljov F, Einenkel J, et al. (2003) A comprehensive analysis of HPV integration loci in anogenital lesions combining transcript and genome-based amplification techniques. Oncogene 22: 3977–3984.
[14]
Koopman LA, Szuhai K, van Eendenburg JD, Bezrookove V, Kenter GG, et al. (1999) Recurrent integration of human papillomaviruses 16, 45, and 67 near translocation breakpoints in new cervical cancer cell lines. Cancer Res 59: 5615–5624.
[15]
Thorland EC, Myers SL, Persing DH, Sarkar G, McGovern RM, et al. (2000) Human papillomavirus type 16 integrations in cervical tumors frequently occur in common fragile sites. Cancer Res 60: 5916–5921.
[16]
Kleter B, van Doorn LJ, ter Schegget J, Schrauwen L, van Krimpen K, et al. (1998) Novel short-fragment PCR assay for highly sensitive broad-spectrum detection of anogenital human papillomaviruses. Am J Pathol 153: 1731–1739.
[17]
Frohman MA, Dush MK, Martin GR (1988) Rapid production of full-length cDNAs from rare transcripts: amplification using a single gene-specific oligonucleotide primer. Proc Natl Acad Sci U S A 85: 8998–9002.
[18]
Basu P, Chandna P, Bamezai RN, Siddiqi M, Saranath D, et al. (2011) MassARRAY spectrometry is more sensitive than PreTect HPV-Proofer and consensus PCR for type-specific detection of high-risk oncogenic human papillomavirus genotypes in cervical cancer. J Clin Microbiol 49: 3537–3544.
[19]
Basu P, Roychowdhury S, Bafna UD, Chaudhury S, Kothari S, et al. (2009) Human papillomavirus genotype distribution in cervical cancer in India: results from a multi-center study. Asian Pac J Cancer Prev 10: 27–34.
[20]
Bhatla N, Lal N, Bao YP, Ng T, Qiao YL (2008) A meta-analysis of human papillomavirus type-distribution in women from South Asia: implications for vaccination. Vaccine 26: 2811–2817.
[21]
Chatterjee R, Mandal B, Bandyopadhyay S (2003) Detection of HPV DNA in cervical carcinomas by PCR and hybrid capture assay. Indian J Pathol Microbiol 46: 596–599.
[22]
Franceschi S, Rajkumar T, Vaccarella S, Gajalakshmi V, Sharmila A, et al. (2003) Human papillomavirus and risk factors for cervical cancer in Chennai, India: a case-control study. Int J Cancer 107: 127–133.
[23]
Peedicayil A, Abraham P, Sathish N, John S, Shah K, et al. (2006) Human papillomavirus genotypes associated with cervical neoplasia in India. Int J Gynecol Cancer 16: 1591–1595.
[24]
Pillai RM, Babu JM, Jissa VT, Lakshmi S, Chiplunkar SV, et al. (2010) Region-wise distribution of high-risk human papillomavirus types in squamous cell carcinomas of the cervix in India. Int J Gynecol Cancer 20: 1046–1051.
[25]
Saranath D, Khan Z, Tandle AT, Dedhia P, Sharma B, et al. (2002) HPV16/18 prevalence in cervical lesions/cancers and p53 genotypes in cervical cancer patients from India. Gynecol Oncol 86: 157–162.
[26]
Travasso CM, Anand M, Samarth M, Deshpande A, Kumar-Sinha C (2008) Human papillomavirus genotyping by multiplex pyrosequencing in cervical cancer patients from India. J Biosci 33: 73–80.
[27]
Singh A, Datta P, Jain SK, Bhatla N, Dutta Gupta S, et al. (2009) Human papilloma virus genotyping, variants and viral load in tumors, squamous intraepithelial lesions, and controls in a north Indian population subset. Int J Gynecol Cancer 19: 1642–1648.
[28]
Deodhar K, Gheit T, Vaccarella S, Romao CC, Tenet V, et al. (2012) Prevalence of human papillomavirus types in cervical lesions from women in rural Western India. J Med Virol 84: 1054–1060.
[29]
Grace Nirmala J, Narendhirakannan (2012) RT Detection and genotyping of high-risk HPV and evaluation of anti-oxidant status in cervical carcinoma patients in Tamil Nadu State, India – a case control study. Asian Pac J Cancer Prev 12: 2689–2695.
[30]
Sowjanya AP, Jain M, Poli UR, Padma S, Das M, et al. (2005) Prevalence and distribution of high-risk human papilloma virus (HPV) types in invasive squamous cell carcinoma of the cervix and in normal women in Andhra Pradesh, India. BMC Infect Dis 5: 116.
[31]
Xue Y, Bellanger S, Zhang W, Lim D, Low J, et al. (2010) HPV16 E2 is an immediate early marker of viral infection, preceding E7 expression in precursor structures of cervical carcinoma. Cancer Res 70: 5316–5325.
[32]
Luft F, Klaes R, Nees M, Durst M, Heilmann V, et al. (2001) Detection of integrated papillomavirus sequences by ligation-mediated PCR (DIPS-PCR) and molecular characterization in cervical cancer cells. Int J Cancer 92: 9–17.
[33]
Vinokurova S, Wentzensen N, Kraus I, Klaes R, Driesch C, et al. (2008) Type-dependent integration frequency of human papillomavirus genomes in cervical lesions. Cancer Res 68: 307–313.
[34]
Corden SA, Sant-Cassia LJ, Easton AJ, Morris AG (1999) The integration of HPV-18 DNA in cervical carcinoma. Mol Pathol 52: 275–282.
[35]
Melsheimer P, Vinokurova S, Wentzensen N, Bastert G, von Knebel Doeberitz M (2004) DNA aneuploidy and integration of human papillomavirus type 16 e6/e7 oncogenes in intraepithelial neoplasia and invasive squamous cell carcinoma of the cervix uteri. Clin Cancer Res 10: 3059–3063.
[36]
Gray E, Pett MR, Ward D, Winder DM, Stanley MA, et al. (2010) In vitro progression of human papillomavirus 16 episome-associated cervical neoplasia displays fundamental similarities to integrant-associated carcinogenesis. Cancer Res 70: 4081–4091.
[37]
Arias-Pulido H, Peyton CL, Joste NE, Vargas H, Wheeler CM (2006) Human papillomavirus type 16 integration in cervical carcinoma in situ and in invasive cervical cancer. J Clin Microbiol 44: 1755–1762.
[38]
Pett MR, Herdman MT, Palmer RD, Yeo GS, Shivji MK, et al. (2006) Selection of cervical keratinocytes containing integrated HPV16 associates with episome loss and an endogenous antiviral response. Proc Natl Acad Sci U S A 103: 3822–3827.
[39]
Bellanger S, Tan CL, Nei W, He PP, Thierry F (2009) The human papillomavirus type 18 E2 protein is a cell cycle-dependent target of the SCFSkp2 ubiquitin ligase. J Virol 84: 437–444.
[40]
Hamid NA, Brown C, Gaston K (2009) The regulation of cell proliferation by the papillomavirus early proteins. Cell Mol Life Sci 66: 1700–1717.
[41]
Kalantari M, Karlsen F, Kristensen G, Holm R, Hagmar B, et al. (1998) Disruption of the E1 and E2 reading frames of HPV 16 in cervical carcinoma is associated with poor prognosis. Int J Gynecol Pathol 17: 146–153.
[42]
Vernon SD, Unger ER, Miller DL, Lee DR, Reeves WC (1997) Association of human papillomavirus type 16 integration in the E2 gene with poor disease-free survival from cervical cancer. Int J Cancer 74: 50–56.
[43]
Holm R, Kraus I, Skomedal H, Langerod A, Kristensen GB, et al. (2008) Human papillomavirus DNA and e6/e7 mRNA status in relation to survival of patients treated for cervical squamous cell carcinoma. Open Virol J 2: 74–81.
[44]
Nambaru L, Meenakumari B, Swaminathan R, Rajkumar T (2009) Prognostic significance of HPV physical status and integration sites in cervical cancer. Asian Pac J Cancer Prev 10: 355–360.
[45]
Kraus I, Driesch C, Vinokurova S, Hovig E, Schneider A, et al. (2008) The majority of viral-cellular fusion transcripts in cervical carcinomas cotranscribe cellular sequences of known or predicted genes. Cancer Res 68: 2514–2522.
[46]
Peter M, Stransky N, Couturier J, Hupe P, Barillot E, et al. (2010) Frequent genomic structural alterations at HPV insertion sites in cervical carcinoma. J Pathol 221: 320–330.
[47]
Rao PH, Arias-Pulido H, Lu XY, Harris CP, Vargas H, et al. (2004) Chromosomal amplifications, 3q gain and deletions of 2q33–q37 are the frequent genetic changes in cervical carcinoma. BMC Cancer 4: 5.
[48]
Scotto L, Narayan G, Nandula SV, Arias-Pulido H, Subramaniyam S, et al. (2008) Identification of copy number gain and overexpressed genes on chromosome arm 20q by an integrative genomic approach in cervical cancer: potential role in progression. Genes Chromosomes Cancer 47: 755–765.
[49]
Wilting SM, de Wilde J, Meijer CJ, Berkhof J, Yi Y, et al. (2008) Integrated genomic and transcriptional profiling identifies chromosomal loci with altered gene expression in cervical cancer. Genes Chromosomes Cancer 47: 890–905.
[50]
Wentzensen N, Ridder R, Klaes R, Vinokurova S, Schaefer U, et al. (2002) Characterization of viral-cellular fusion transcripts in a large series of HPV16 and 18 positive anogenital lesions. Oncogene 21: 419–426.
[51]
Matovina M, Sabol I, Grubisic G, Gasperov NM, Grce M (2009) Identification of human papillomavirus type 16 integration sites in high-grade precancerous cervical lesions. Gynecol Oncol 113: 120–127.
[52]
Dall KL, Scarpini CG, Roberts I, Winder DM, Stanley MA, et al. (2008) Characterization of naturally occurring HPV16 integration sites isolated from cervical keratinocytes under noncompetitive conditions. Cancer Res 68: 8249–8259.
