No abstract

Human papillomaviruses (HPVs) are aetiological agents for cervical cancer. More than 70 different HPV types that infect genital mucosa have been found. In order to develop a sensitive and specific detection and typing assay, a PCR/direct sequencing approach was used. Two pairs of consensus primers were used for amplification of HPV DNA and the PCR products obtained were analysed by automated sequencing. Sequences were compared with those in GenBank by using the BLAST program. In this study, 2916 cytological samples were screened for HPV, as well as for triage. Nine hundred and forty-eight (32 . 5 %) samples were positive for HPV, of which 134 harboured more than one HPV type. Of the 948 PCR-positive samples, 648 were typed. Thirty-nine different HPV types were identified by sequencing. The two most frequently found HPV types, 16 and 31, together accounted for 36 . 3 % of the sequences (26 . 2 and 10 . 1 %, respectively). This group was followed by HPV types 6 (5 . 7 %), 18 (5 . 3 %), 58 (4 . 5 %), 61 (4 . 5 %), 53 (4 . 4 %), 42 (4 . 3 %) and 51 (4 . 0 %). All other types were detected at frequencies ,4 % and eight types were detected only once. PCR/ direct sequencing is a reliable method for routine detection of HPV in cytological samples. The data presented here suggest a complex distribution of HPV types in the population tested. The results accentuate the importance of PCR-based techniques in HPV diagnosis, as hybridization-based methods can only detect a limited number of infections. This method can also be applied easily to the analysis of tissue samples and it therefore also allows type-specific follow-up of women who have been treated for cervical intraepithelial neoplasia.

Our results indicate that non-classic cytomorphological signs can improve the sensitivity of cytology for detecting HPV. Minimally abnormal Pap smears lacking mild nuclear changes (16%) in the present study--do not require further molecular HPV testing.

Based on our results, we urgently recommend for any histological report on excision of anal lesions to include a statement whether histological markers of HPV infection were detected. In individual cases, validation via HPV PCR must be considered.

BACKGROUND.The Bethesda System of cervical cytologic findings introduced the term ASCUS (atypical squamous cells of undetermined significance) to cover the broad zone separating normal cytomorphology from definitive squamous intraepithelial lesions (SILs ). The management of patients with ASCUS is particularly problematic as approximately 10% of ASCUS patients develop SIL and 1 per 1000 develop cervical carcinoma. METHODS.Our aim was to demonstrate the combined use of polymerase chain reaction for human papillomavirus (HPV) typing and laser scanning cytometry for DNA content measurements in the subcategorization of ASCUS cases according to the risk for progression toward cancer. Liquid-based monolayer preparation (ThinPrep, Cytyc, Boston, MA) of the cytologic material was used for cytomorphologic analysis. DNA content measurements using laser scanning cytometry and direct sequencing of HPV using the consensus primers GP5ϩ/GP6ϩ and MY09/ MY11 were performed from the same material. RESULTS.Twelve of the 44 cases (27.2%) with ASCUS carried a high-risk HPV genome whereas only 3 of the 195 normal control cases (1.5%) showed positivity for a high-risk HPV genome. Six of 12 (50%) of the high-risk HPV-positive ASCUS cases presented isolated cells with a DNA content above 5c, whereas cells with a DNA content above 9c were found in 3 of 12 cases (25%) and were exclusively found in combination with high-risk HPV infection. In these three cases, the histologic follow-up resulted in cervical intraepithelial neoplasia (CIN) I (one case) and CIN III (two cases). None of the other ASCUS or normal cases displayed DNA aneuploidy above 9c. They returned to normal cytology (within normal limits/ benign cellular changes) in the follow-up. CONCLUSIONS.Human papillomavirus typing and DNA content measurements may delineate a distinct group of ASCUS. Our preliminary data suggest that ASCUS cases with high-risk HPV positivity and with rare cells with abnormally high DNA content represent similar biologic features as high-grade SIL and are at elevated