An exploration of molecular markers in prognosis of cervical intraepithelial neoplasia
Cervical intraepithelial neoplasia (CIN) lesions of the same morphological grade show variable clinical behaviour; some progress and others regress. Currently, there are no biochemical or molecular markers which can distinguish CIN lesions with different prognosis. We have optimised several molecular methods on archival cervical smears and screened a number of molecular markers that may aid prognosis of CIN. By systematically validating different protocols, we have established that crude DNA preparations from a small number of microdissected cells from cervical smears are adequate for various PCR-based investigations. Furthermore, the crude DNA preparations could be further purified and used for PCR-based clonality analysis of the X-linked genes. Using PCR-based clonality analysis of the androgen receptor gene, we have shown that CIN3 and the majority of CIN2 lesions are monoclonal, whereas CINl lesions are polyclonal. Importantly, patients with monoclonal CIN2 show disease persistence or progression, while polyclonal CIN2 regress after treatment and remain negative during follow-up. To further identify molecular markers that are technically easy to apply and potentially suitable for prognosis assessment of all CIN grades, we have investigated the prognostic value of gene deletions. In a pilot study, we screened 12 microsatellite markers, which showed high frequencies of loss of heterozygosity (LOH) in cervical carcinomas, and identified four, including D3S1300 , D3S1260, D11S35 and D11S528, that were significantly associated with CIN persistence or progression. The 4 markers were further investigated in a larger cohort. Combined analysis of LOH at these 4 loci permitted the identification of 22-47% of CIN lesions of various histological grades, that were associated with disease persistence or progression with 100% specificity. LOH at these loci was significantly associated with HPV16 infection. Bioinformatics analysis identified several candidate genes including the fragile histidine triad and progesterone receptor gene that may be the target of deletions.