Impact of sampling origin on molecular detection of high-risk human papillomavirus and oncogene expression

Purpose of investigation: The recognition of high-risk human papillomavirus (FIR-HPV) as an etiological agent of cervical cancer has increased the importance of testing for HPV, and this might contribute to better risk stratification. Methods: Eighty-eight randomly selected cervical cancer specimens including biopsies and their respective smears were used in this study. Control scrapings were obtained from ten healthy women. The presence of HPV 16 and HPV 18 was investigated using the technique of polymerase chain reaction (PCR) with the specific primers for the L1 region, while mRNA expression of HPV16 E6-E7 was evaluated by a reverse transcription PCR method (RT-PCR). Results: The positivity for the viral genotype was influenced by the quantity of amplified DNA used. In tumor biopsies the higher positivity for HPV16 (54.5%) and HPV18 (15.9%) was obtained using 687.4 ng of DNA. At smears level solely 31.8% of HPV16 was detected using an average DNA quantity of about 157.2 ng. The revelation of HPV types depends on clinicopathologic data; HPV16 was detected more in advanced stages of squamous carcinoma (SC) samples (20% Stage I, 62% Stage II and 80% Stage III), while HPV18 and double infection were found exclusively at advanced stages of SC and in adenocarcinoma (AC), respectively (60%, 40% Stage III SC and 80%, 20% Stage II A and C). The prevalence of HPV16 E6-E7 transcripts was evaluated at tumor biopsy with frequencies of 50%. Conclusion: Our data provide prospective evidence that HPV16/18L1 revelation at biopsy toward pathological types is efficien: and correlates well with oncogenic transcript findings. Subtle changes in viral oncogene dynamics highlight the presence of other regulating proteins serving as additional biomarkers.

Biopsies; Cervical cancer; DNA load; E6-E7 oncogenes; Human papillomavirus; L1 gene; Smears

[1] Parkin D.M., Bray F., Ferlay J., Pisani P.: “Global cancer statistics, 2002”. CA Cancer J. Clin., 2005, 55, 74.

[2] Maalej M., Mrad K., Kochbati L., Guigua A., Ben Abdallah M., Ben Ayed F., Ben Romdhane K.: “Cervical cancer in Tunisia: an epidemiological, clinical and pathological study”. Eur. J. Obstet. Gynecol. Reprod. Biol., 2004, 113, 226.

[3] Kim S.W., Yang J.S.: “Human Papillomavirus type 16 E5 protein as a therapeutic target”. Yonsei Med. J., 2006, 47, 1.

[4] Lagunas-Martinez A., Madrid-Marina V., Gariglio P.: “Modulation of apoptosis by early human papillomavirus proteins in cervical cancer”. Biochim. Biophys. Acta, 2010, 1805, 6.

[5] Sánchez-Anguiano L.F., Alvarado-Esquivel C., Reyes-Romero M.A., Carrera-Rodriguez M.: “Human papillomavirus infections in women seeking cervical Papanicolaou cytology of Durango, Mexico: prevalence and genotypes”. BMC Infect. Dis., 2006, 6, 27.

[6] McMurray H.R., Nguyen D., Westbrook T.F., McAnce D.J.: “Biology of human papillomaviruses”. Int. J. Exp. Pathol., 2001, 82, 15.

[7] Yoshida T., Sano T., Oyama T., Kanuma T., Fukuda T.: “Prevalence, viral load, and physical status of HPV 16 and 18 in cervical adenosquamous carcinoma”. Virchows Arch., 2009, 455, 253.

[8] Im S.S., Wilczynski S.P., Burger R.A., Monk B.J.: “Early stage cervical cancers containing human papillomavirus type 18 DNA have more nodal metastasis and deeper stromal invasion”. Clin. Cancer Res., 2003, 9, 4145.

[9] Abudukadeer A., Ding Y., Niyazi M., Ababaikeli A., Abudula A.: Distribution of HPV genotypes in uterine cervical lesions among the Uighur women in Xinjiang province of China. Eur J. Gynaecol. Oncol., 2010, 31, 315.

[10] Bory JP., Cucherousset J., Lorenzato M., Gabriel R., Quereux C., Birembaut P., Clavel C.: “Recurrent human papillomavirus infection detected with the hybrid capture II assay selects women with normal cervical smears at risk for developing high grade cervical lesions: a longitudinal study of 3,091women”. Int. J. Cancer, 2002, 102, 519.

[11] Lie A.K., Risberg B., Borge B., Sandstad B., Delabie J., Remala R. et al.: “DNA-versus RNA-based methods for human papillomavirus detection in cervical neoplasia”. Gynecol. Oncol., 2005, 97, 908.

[12] Albrecht V., Chevallier A., Magnone V., Barbry P., Vandenbos F., Bongain A. et al.: “Easy and fast detection and genotyping of highrisk human papillomavirus by dedicated DNA microarrays”. J. Virol. Methods., 2006, 137, 236.

[13] zur Hausen H.: “Papillomaviruses and cancer: from basic studies to clinical application”. Nat. Rev. Cancer, 2002, 2, 342.

[14] Ma B., Xu Y., Hung C.F., Wu T.C.: “HPV and therapeutic vaccines: where are we in 2010?”. Curr. Cancer Ther. Rev., 2010, 6, 81.

[15] Yang Y.Y., Koh L.W., Tsai J.H., Tsai C.H., Wong E.F., Lin S.J., Yang C.C.: “Correlation of viral factors with cervical cancer in Taiwan”. J. Microbiol. Immunol. Infect., 2004, 37, 282.

[16] Sima N., Wang S., Wang W., Kong D., Xu Q., Tian X. et al.: “Antisense targeting human papillomavirus type 16 E6 and E7 genes contributes to apoptosis and senescence in SiHa cervical carcinoma cells”. Gynecol. Oncol., 2007, 106, 299.

[17] Franco E.L.: “Cancer causes revisited: human papillomavirus and cervical neoplasia”. J. Natl. Cancer Inst., 1995, 87, 779.

[18] Muñoz N., Mendez F., Posso H., Molano M., van den Brule A.J., Ronderos M. et al., Instituto National de Cancerologia HPV Study Group: “Incidence, duration, and determinants of cervical human papillomavirus infection in a cohort of colombian women with normal cytological results”. J. Infect. Dis., 2004, 190, 2077.

[19] Walboomers J.M., Jacobs M.V., Manos M.M., Bosch F.X., Kummer J.A., Shah K.V. et al.: “Human papillomavirus is a necessary cause of invasive cervical cancer worldwide”. J. Pathol., 1999, 189, 12.

[20] Castle P.E., Lorincz A.T., Mielzynska-Lohnas I., Scott D.R., Glass A.G., Sherman M.E. et al.: “Results of human papillomavirus DNA testing with the hybrid capture 2 assay are reproducible”. J. Clin. Microbiol., 2002, 40, 1088.

[21] Bosch F.X., Lorinecz A., Muñoz N., Meijer CJ., Shah KV.: “The causal relation between human papillomavirus and cervical cancer”. J. Clin. Pathol., 2002, 55, 244.

[22] Clifford G.M., Smith J.S., Plummer M., Munoz N., Franceschis S.: “Human papillomavirus types in invasive cervical cancer worldwide: a meta-analysis”. Br. J. Cancer, 2003, 88, 63.

[23] Thomas J.O., Herrero R., Omigbodun A.A., Ojemakinde K., Ajayi I.O., Fawole A. et al.: “Prevalence of papillomavirus in women in Ibadan, Nigeria: a population-based study”. Br. J. Cancer, 2004, 90, 638.

[24] Dictor M., Warenholt J.: “Single-tube multiplex PCR using typespecific E6/E7 primers and capillary electrophoresis genotypes 21 human papillomaviruses in neoplasia”. Infect. Agent. Cancer, 2011, 6, 1.

[25] Pittayakhajonwut D., Angeletti P.C.: “Viral trans-factor independent replication of human papillomavirus genomes”. Virol. J., 2010, 7, 123.

[26] Herrero R., Hildesheim A., Bratti C., Sherman M.E., Hutchinson M., Morales J. et al.: “Population-based study of human papillomavirus infection and cervical neoplasia in rural Costa Rica”. J. Natl. Cancer Inst., 2000, 92, 464.

[27] Huang L.W., Chao S.L., Chen P.H., Chou H.P.: “Multiple HPV genotypes in cervical carcinomas: improved DNA detection and typing in archival tissues”. J. Clin. Virol., 2004, 29, 271.

[28] Chaturvedi A.K., Katki H.A., Hildesheim A., Rodríguez A.C., Quint W., Schiffman M. et al.: “Human Papillomavirus Infection with Multiple Types: Pattern of Coinfection and Risk of Cervical Disease”. J. Infect. Dis., 2011, 203, 910.

[29] Nobbenhuis M.A.E., Walboomers J.M.M., Helmerhorst T.J.M., Rozendaal L., Remmink A.J., Risse E.K. et al.: “Relation of human papillomavirus status to cervical lesions and consequences for cervical-cancer screening: a prospective study”. Lancet, 1999, 354, 20.

[30] Twu N.F., Yen M.S., Lau H.Y., Chen Y.J., Yu B.K., Lin C.Y.: Typespecific human papillomavirus DNA testing with the genotyping array: a comparison of cervical and vaginal sampling. Eur. J. Obstet. Gynecol. Reprod. Biol., 2011, 156, 96.

[31] Prétet J.L., Dalstein V., Monnier-Benoit S., Delpeut S., Mougin C.: “High risk HPV load estimated by Hybrid capture II correlates with HPV16 load measured by real-time PCR in cervical smears of HPV16-infected women”. J. Clin. Virol., 2004, 31, 140.

[32] Burk R.D., Terai M., Gravitt P.E., Brinton L.A., Kurman R.J., Barnes W.A. et al.: “Distribution of human papillomavirus type 16 and 18 variants in squamous cell carcinomas and adenocarcinomas of the cervix”. Cancer Res., 2003, 63, 7215.

[33] Arias-Pulido H., Peyton C.L., Torrez-Martinez N., Anderson D.N., Wheeler C.M.: “Human papillomavirus type 18 variant lineages in United States populations characterized by sequence analysis of LCR-E6, E2, and L1 regions”. Virology, 2005, 338, 22.

[34] Bray F., Carstensen B., Moller H., Zappa M., Zakelj M.P., Lawrence G. et al.: “Incidence trends of adenocarcinoma of the cervix in 13 European countries”. Cancer Epidemiol. Biomarkers Prev., 2005, 14, 2191.

[35] Murillo R., Molano M., Martínez G., Mejía J.C., Gamboa O.: “HPV prevalence in Colombian women with cervical cancer: implications for vaccination in a developing country”. Infect. Dis. Obstet. Gynecol., 2009, 2009, 653598.

[36] Bulk S., Berkhof J., Bulkmans N.W., Zielinski G.D., Rozendaal L., van Kemenade F.J. et al.: “Preferential risk of HPV16 for squamous cell carcinoma and of HPV18 for adenocarcinoma of the cervix compared to women to normal cytology in the Netherlands”. Br. J. Cancer, 2006, 94, 171.

[37] Kudelova M., Krivos V., Raslova H., Valovicova M., Belvoncikova P., Matis J.: “Occurrence of Human papillomavirus 16 and 18 in smears from the two cervix regions of oncogynecological patients in Slovakia”. Acta Virol., 2008, 52, 17.

[38] Kuramoto H., Sugimoto N., Iida M.: “Screening for cancer of the cervix with simultaneous pap smear and colposcopy”. The efficacy of pap smear and colposcopy. Eur. J. Gynaecol. Oncol., 2011, 32, 73.

[39] Schlecht N.F., Kulaga S., Robitaille J., Ferreira S., Santos M., Miyamura R.A. et al.: “Persistent human papillomavirus infection as a predictor of cervical intraepithelial neoplasia”. JAMA, 2001, 286, 3106.

[40] Song S., Liem A., Miller J.A., Lambert P.F.: “Human papillomavirus types 16 E6 and E7 contribute differently to carcinogenesis”. Virology, 2000, 267, 141.

[41] Cuschieri K.S., Whitley M.J., Cubie H.A.: “Human papillomavirus type specific DNA and RNA persistence-implications for cervical disease progression and monitoring”. J. Med. Virol., 2004, 73, 65.