Human papillomavirus genotypes in cervical cancers in Mozambique

Pontus Naucler1, Flora Mabota da Costa2, Otto Ljungberg3, Antonio Bugalho2 and Joakim Dillner1

1 Department of Medical Microbiology, University of Lund, S-20502 Malmö, Sweden
2 Department of Gynaecology and Obstetrics, Central Hospital of Maputo, Mozambique
3 Department of Pathology, Malmö University Hospital, Sweden

Correspondence
Joakim Dillner
joakim.dillner{at}mikrobiol.mas.lu.se


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The distribution of human papillomavirus (HPV) types in cervical cancers is essential for design and evaluation of HPV type-specific vaccines. To follow up on a previous report that HPV types 35 and 58 were the dominant HPV types in cervical neoplasia in Mozambique, the HPV types in a consecutive case series of 74 invasive cervical cancers in Mozambique were determined. The most common worldwide major oncogenic HPV types 16 and 18 were present in 69 % of cervical cancers, suggesting that a vaccine targeting HPV-16 and -18 would have a substantial impact on cervical cancer also in Mozambique.


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A previous study on human papillomavirus (HPV) genotypes in Mozambique, reported that the HPV type distribution was unusual. The most common worldwide oncogenic HPV type 16 was infrequent and HPV type 35 was dominant (Castellsague et al., 2001). The authors drew attention to the fact that type-specific HPV vaccines are currently being produced (Koutsky et al., 2002) and proposed that the type composition of such vaccines should be modified for different populations to reflect the HPV type distribution in the population. To investigate further this important issue, we analysed the HPV types present in a comprehensive case series of invasive cervical cancers from Mozambique.

From June 2002 to April 2003, all women with suspected cervical cancer at the Department of Gynaecology at the Central Hospital of Maputo, the capital of Mozambique, were asked to participate. Following written consent, two biopsies were taken from each tumour, one was formalin-fixed and used for histological diagnosis, the other biopsy was frozen for HPV DNA detection. Human immunodeficiency virus (HIV) testing was performed on serum samples and a questionnaire recorded socio-demographic factors. Ethical approval was obtained from the Commission of Investigation and Ethics at the Central Hospital in Maputo and from Lund University Research Ethics Committee in Sweden.

Biopsies and serum samples were successfully obtained from 88 of 94 patients and transported to Sweden for HPV analysis. Histopathological diagnosis was evaluated in Mozambique and in Sweden. Fourteen patients were excluded because invasive cervical cancer could not be confirmed and two samples were negative in the specimen adequacy test ({beta}-globin PCR). One patient had adenosquamous carcinoma, the others squamous carcinoma. The mean age was 46 years. HPV DNA was detected using general primer PCR (GP5+/GP6+ primers) and HPV DNA typing was performed by reverse dot-blot hybridization (RDBH) assay with full-genomic clones (Forslund et al., 2002) of HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68. GP5+/6+ PCR-negative samples were analysed further using type-specific PCR for HPV-16 and -18 and with another HPV general primer system (MY09/11), followed by HPV typing by sequencing. The last few still HPV-negative samples were also amplified with multiply primed rolling-circle amplification (RCA) (Rector et al., 2004) before GP5+/6+ PCR and RDBH. A repeat analysis of 25 % of the samples found identical results. HIV testing was performed using Axsym (Abbot), followed by confirmation using RIBA HIV-1/HIV-2 SIA (Chiron).

The HPV prevalence was 66 of 72 (92 %) using GP5+/GP6+ primers. This increased to 68 of 72 (94 %) after testing with type-specific PCR and MY09/11 PCRs and reached 70 of 72 (97 %) with prior amplification with RCA before the PCR. It appears that the combination of RCA and PCR is a promising new strategy to minimize false-negative results in diagnostic virology.

The most common HPV types were 16 and 18, being present in 69 % of tumours (Table 1). Although coexistence with other HPV types was common (13, 7, 3 and 1 women were infected with 2, 3, 4 and 5 HPV types, respectively), a substantial proportion of cases (36 %) had a single HPV infection with either HPV-16 or -18.


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Table 1. HPV genotype distribution among 72 invasive cervical cancer patients in Mozambique

 
Suggested reasons for common multiple HPV infections include impaired immune tolerance and concomitant CIN (cervical intraepithelial neoplasia) lesions within the specimen (Davies et al., 2001). Thirteen of 72 (18 %) patients were HIV-positive, but multiple HPV infections were not more common among HIV-positive women (age-adjusted odds ratio, 1·0; 95 % CI, 0·25–4·0). Almost all patients had advanced stage tumours that may have resulted in immunosuppression.

Our findings that HPV-16 and -18 are the most frequent HPV infections associated with cervical cancer in Mozambique is in concordance with the overall world distribution of HPV types in cervical cancer (Clifford et al., 2003), but is not in line with a study by Castellsague et al. (2001) who investigated 239 healthy women and 23 women with cervical dysplasia in Mozambique, and found HPV-35 and 58 to be the most common HPV types. We investigated the HPV type distribution in invasive cervical cancer. Different HPV types are associated with different cancer risks (Clifford et al., 2003), and circulation of HPV types among women without cancer may therefore not accurately reflect which HPV types cause invasive cervical cancer in a population. Furthermore, in many countries it is difficult to obtain representative population-based samples from healthy women or women with asymptomatic lesions. Recruitment of symptomatic women with cancer at the major site offering health care in the country may therefore have resulted in a more representative sample of oncogenic HPV types. However, it should be noted that HPV-35 was not uncommon in our series, being found in 19 % of patients.

Continued investigation and monitoring of which HPV types that actually cause cervical cancer in different countries is undoubtedly an important endeavour. Our study suggests that HPV vaccines targeting HPV-16 and -18, the two worldwide most frequent oncogenic HPV types, would have a substantial impact on cervical cancer also in Mozambique.


   ACKNOWLEDGEMENTS
 
Financial support was provided by the Swedish Cancer Society. Conflict of interest: Professor Dillner has acted as consultant for Merck, a company pursuing research on HPV vaccines.


   REFERENCES
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ABSTRACT
MAIN TEXT
REFERENCES
 
Castellsague, X., Menendez, C., Loscertales, M. P. & 9 other authors (2001). Human papillomavirus genotypes in rural Mozambique. Lancet 358, 1429–1430.[CrossRef][Medline]

Clifford, G. M., Smith, J. S., Aguado, T. & Franceschi, S. (2003). Comparison of HPV type distribution in high-grade cervical lesions and cervical cancer: a meta-analysis. Br J Cancer 89, 101–105.[CrossRef][Medline]

Davies, P., Kornegay, J. & Iftner, T. (2001). Current methods of testing for human papillomavirus. Best Pract Res Clin Obstet Gynaecol 15, 677–700.[CrossRef][Medline]

Forslund, O., Antonsson, A., Edlund, K. & 9 other authors (2002). Population-based type-specific prevalence of high-risk human papillomavirus infection in middle-aged Swedish women. J Med Virol 66, 535–541.[CrossRef][Medline]

Koutsky, L. A., Ault, K. A., Wheeler, C. M., Brown, D. R., Barr, E., Alvarez, F. B., Chiacchierini, L. M. & Jansen, K. U. (2002). A controlled trial of a human papillomavirus type 16 vaccine. N Engl J Med 347, 1645–1651.[Abstract/Free Full Text]

Rector, A., Tachezy, R. & Van Ranst, M. (2004). A sequence-independent strategy for detection and cloning of circular DNA virus genomes by using multiply primed rolling-circle amplification. J Virol 78, 4993–4998.[Abstract/Free Full Text]

Received 30 January 2004; accepted 13 May 2004.