p16^INK4A, CDC6, and MCM5: predictive biomarkers in cervical preinvasive neoplasia and cervical cancer

Abstract

Aim: To analyse and compare expression patterns of three potential biomarkers-p16 INK4A , CDC6, and MCM5-and evaluate their use as predictive biomarkers in squamous and glandular cervical preinvasive neoplasia. Methods: Immunocytochemical analysis of p16 INK4A, MCM5, and CDC6 expression was performed on 20 normal, 38 cervical intraepithelial neoplasia 1 (CIN1), 33 CIN2, 46 CIN3, 10 squamous cell carcinoma, 19 cervical glandular intraepithelial neoplasia (cGIN), and 10 adenocarcinoma samples. Staining intensity was assessed using a 0-3 scoring system. p16INK4A , MCM5, and CDC6 expression was also examined in ThinPrep slides exhibiting mild, moderate, and severe dyskaryosis. Human papillomavirus (HPV) was detected using a modified SYBR green assay. Fluorogenic polymerase chain reaction (PCR) and solution phase PCR were used for specific HPV typing. Results: All three markers showed a linear correlation between expression and grade of dysplasia. p16INK4A and MCM5 protein expression was upregulated in all grades of squamous and glandular dysplasia. CDC6 protein was preferentially expressed in high grade lesions and in invasive squamous cell carcinoma. Conclusion: p16INK4A expression was closely associated with high risk HPV infection-all grades of squamous and glandular cervical lesions were immunohistochemically positive. MCM5 staining intensity was independent of high risk HPV infection, highlighting its potential as a biomarker in both HPV dependent and independent cervical dysplasia. CDC6 may be a biomarker of high grade and invasive lesions of the cervix, with limited use in low grade dysplasia. p16INK4A was the most reliable marker of cervical dysplasia. Combinations of dysplastic biomarkers may be useful in difficult diagnostic cases.

Quantitation of CDC6 and MCM5 mRNA in cervical intraepithelial neoplasia and invasive squamous cell carcinoma of the cervix

“…CDC6 appears to be preferentially unregulated in highgrade preinvasive neoplasia (CIN3) and in invasive cervical carcinoma and in adenocarcinoma. 19,20 In this study, simple linear regression analysis revealed a linear increase (estimated slope ¼ 12.1, R 2 ¼ 0.085, P-value o0.045) in the level of CDC6 mRNA expression in the normal cervix, CIN3 and invasive carcinoma. These results at the mRNA level correlate strongly with previous findings for CDC6 phenotypic protein expression using immunohistochemical analysis, which demonstrated a linear relationship between increasing CDC6 protein expression and increasing severity of cervical dysplasia.…”

“…Indeed, MCM5 mRNA expression closely correlates with findings at the phenotypic protein expression level, which demonstrated an increase in MCM5 protein expression with increasing severity of dysplasia, using immunohistochemical techniques. 19 These findings suggest that increased MCM5 protein expression in cervical dysplasia is a consequence of transcriptional upregulation. One possible suggestion for upregulation of MCM5 mRNA in cervical dysplasia put forward by Ishimi et al 14 could be that high-risk human papillomavirus oncoprotein E7 binding to the retinoblastoma protein releases inhibition on E2F allowing transcriptional upregu- CDC6 and MCM5 mRNA expression in CIN N Murphy et al lation of MCM5.…”

“…Increased MCM5 protein expression is detectable in all grades of glandular and squamous cervical dysplasia and this increase is proportional to the severity of dysplasia. 14,[19][20][21][22] This study has for the first time examined MCM5 mRNA expression in normal and dysplastic squamous cells of the cervix using quantitative TaqMan RT-PCR. In this study, a linear increase in MCM5 mRNA expression was observed in the normal cervix, CIN3 and invasive carcinoma (estimated slope ¼ 6.8, R 2 ¼ 0.3, P-value o0.000).…”

“…18 Increased expression of CDC6 and MCM5 proteins has been demonstrated using immunohistochemical detection methods in preinvasive and invasive cells of the cervix. 14,[19][20][21][22] This aberrant overexpression of MCM5 and CDC6 has been suggested as a potential molecular marker of cervical squamous and glandular neoplasia both in biopsy sections and in cytology smears. [19][20][21][22] The mRNA expression profile of CDC6 and MCM5 and its significance in normal, dysplastic and malignant cervical cells remains to be elucidated.…”

“…14,[19][20][21][22] This aberrant overexpression of MCM5 and CDC6 has been suggested as a potential molecular marker of cervical squamous and glandular neoplasia both in biopsy sections and in cytology smears. [19][20][21][22] The mRNA expression profile of CDC6 and MCM5 and its significance in normal, dysplastic and malignant cervical cells remains to be elucidated. The aim of this study was to examine and compare the mRNA expression profiles of MCM5 and CDC6 in the normal cervix, preinvasive lesions and invasive carcinomas of the cervix using quantitative real-time PCR assays.…”