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.

The presence of ECS appears to be associated with survival in OPSCC and CUP according to p16 status. Our findings raise questions regarding the benefits of postoperative chemoradiation therapy in p16-positive patients with ECS.

A wide array of immunohistochemical markers have been evaluated with respect to their specificity in staining dysplastic cervical cells in cervical biopsies and cervical cytological smears. However, there is still a significant demand for better biomarkers to identify neoplastic cervical glandular and squamous epithelial cells precisely. The CDKN2A gene, located on chromosome 9p21, encodes the tumour suppressor protein, p16INK4A, which decelerates the cell cycle by inactivating CDK4 and CDK6. The aim of this study was to compare and contrast the expression pattern of p16INK4A in benign and neoplastic glandular lesions and tubo-endometrioid metaplasia. All cases in each category displayed some p16INK4A expression. Adenocarcinoma and in situ cases showed a combination of intense nuclear and cytoplasmic staining. It was observed that all cases of tubo-endometrioid metaplasia showed occasional nuclear positivity and definite cytoplasmic staining. These findings may have important implications for the potential utility of p16INK4A as a biomarker for glandular dysplastic lesions. While p16INK4A has been demonstrated to be an excellent marker of cervical dysplasia in squamous neoplastic lesions of the cervix, it has potential pitfalls in cervical glandular lesions that may limit the utility of this biomarker in resolving the nature of suspicious glandular lesions, particularly in cytopathology.

Patients with p16-positive oropharyngeal SCC and positive margins after excision maintain a low risk of recurrence despite most receiving RT alone as adjuvant treatment. These findings raise questions regarding the additional benefit of postoperative CRT in this group.

CDC6 and MCM5 play essential roles in eukaryotic DNA replication. Several studies have highlighted the potential of these proteins as molecular markers of dysplastic and malignant cells in histopathological diagnosis. The mode of expression of CDC6 and MCM5 mRNA and their significance in normal, dysplastic and malignant cervical cells remains to be elucidated. Using a quantitative real-time RT PCR assay, we compared CDC6 and MCM5 mRNA expression in normal cervical epithelium, cervical intraepithelial neoplasia and invasive squamous cell carcinoma of the cervix. Our study cohort comprised 20 normal cervical biopsies, 20 CIN3 and eight invasive squamous cell carcinomas. All samples were formalin fixed and paraffin embedded. Total RNA was extracted and analysed for expression of GAPDH, CDC6 and MCM5 using real-time quantitative TaqMan RT-PCR. A linear increase in MCM5 and CDC6 mRNA expression is observed in normal cervix, CIN3 and invasive cervical carcinoma. The overall difference in MCM5 mRNA expression in the normal cervix, CIN3 and invasive cohort groups is highly statistically significant (P ¼ 0.001). An increase in CDC6 mRNA expression in CIN3 and invasive cervical squamous cell carcinoma was observed; however, the overall difference between cohort groups was not found to be statistically significant (P ¼ 0.104). Increased transcription of MCM5 and CDC6 occurs as a consequence of cervical neoplastic progression. This pattern of increased mRNA expression in CIN3 and invasive cervical carcinoma directly correlates with findings at the phenotypic protein expression level. This study further confirms the importance of MCM5 and CDC6 in malignant transformation and in the pathogenesis of cervical dysplasia. In all eukaryotes, a conserved mechanism of DNA replication exists, which ensures that DNA replication occurs only once in a single cell cycle. 1 This mechanism is often termed the 'licensing' of DNA replication. 2 DNA replication requires the regulated assembly of pre-replicative complexes (pre-RC) onto DNA during the G1 phase of the cell cycle. Pre-RCs render the chromatin competent or 'licensed' to replicate. Among the proteins known to assemble to form the pre-RC are cell division cycle protein 6 (CDC6) and mini chromosome maintenance (MCM) proteins. 3,4 Biological analysis of CDC6 suggests that it functions as a clamp loader in which ATP binding and hydrolysis induce conformational changes, which result in the recruitment or loading of MCMs onto DNA. 3 Once the MCMs are recruited to the pre-RC, CDC6 protein is then phosphorylated by Cyclin A/CDK2 in S phase of the cell cycle. This results in the translocation of CDC6 from its chromatin sites to the cytoplasm where it is degraded by the anaphase promoting complex (APC)/cyclosome. 5-8 Relocalisation of CDC6 to the cytoplasm prevents reinitiation of replication. In addition to conferring replication competence, studies also suggest that the level/ modification of CDC6 in G2 phase functions as a checkpoint control, which ensures that the S phase nucleus has com...