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. p16^INK4A, 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. p16^INK4A 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.

It is well established that high risk human papillomavirus plays a pivotal role in the development and progression of cervical cancer.³ In fact, HPV infection has been detected in almost all preneoplastic and neoplastic lesions of the cervix.^4,5 HPV contributes to neoplastic progression predominantly through the action of two viral oncoproteins—E6 and E7⁶—and is manifested by changes in the expression of host cell cycle regulatory proteins. Such differentially expressed host proteins and nucleic acids may have a role as “biomarkers” of dysplastic cells. To date, a wide array of molecular markers has been evaluated. Three markers that have shown the greatest potential are the cyclin dependant kinase inhibitor p16^INK4,7–9 and the DNA replication licensing proteins CDC6 (cell division cycle protein 6) and MCM5 (mini chromosome maintenance 5).^10–13

The CDKN2A gene product, p16^INK4A, is a tumour suppressor protein that inhibits cyclin dependant kinases 4 and 6, which phosphorylate the retinoblastoma (Rb) protein. A reciprocal association between p16^INK4A and Rb expression has been found, suggesting the presence of a negative feedback loop that allows Rb to limit levels of p16^INK4A.^14–16 p16^INK4A overexpression has been demonstrated in cervical cancers, and is thought to be the result of inactivation of Rb by the HPV E7 protein.¹⁷ p16^INK4A may also be directly induced by the transcription factor E2F, which is released from the Rb protein after the binding of HPV E7.¹⁸ The expression pattern of p16^INK4A in dysplastic squamous and glandular cervical cells in tissue sections and in cervical smears has been investigated extensively.^{4,7–9,19–23} It is now widely accepted that p16^INK4A is a sensitive and specific marker of squamous and glandular dysplastic cells of the cervix, and is a valuable adjunctive test in cervical cancer lesion diagnosis and cervical screening.⁸

In all eukaryotes, a conserved mechanism of DNA replication exists, which ensures that DNA replication occurs only once in a single cell cycle.²⁴ This mechanism is often termed the “licensing” of DNA replication.²⁵ DNA replication requires the regulated assembly of prereplicative complexes on to DNA during the G1 phase of the cell cycle. Prereplicative complexes render the chromatin competent or “licensed” to replicate. Among the proteins known to assemble to form the prereplicative complexes are CDC6 and the mini chromosome maintenance (MCM) proteins.^26,27 Deregulation of DNA replication results in genomic instability and contributes to the malignant transformation of cells.^25,28 Not surprisingly then, increased expression of MCM5 and CDC6 is seen in dysplastic cells. In normal cells, MCM5 and CDC6 are present only during the cell cycle and are lost from the cell during quiescence and differentiation. As a consequence, MCM5 and CDC6 are specific biomarkers of proliferating cells. Several studies have highlighted their potential use in the identification and/or diagnosis of a variety of dysplastic and neoplastic conditions.^13,29–35 In 1998, Williams et al first identified CDC6 and MCM5 as potential molecular biomarkers of precursor malignant cells of the cervix.

The aim of our study was to compare and contrast the expression pattern of p16^INK4A, MCM5, and CDC6 and to assess their potential as markers of dysplastic squamous and glandular cells of the cervix. Immunohistochemical analysis of MCM5, CDC6, and p16^INK4A was carried out using mouse monoclonal antibodies on formalin fixed and paraffin wax embedded samples of normal cervix, cervical intraepithelial neoplasia 1 (CIN1), CIN2, CIN3, cervical glandular intraepithelial neoplasia (cGIN), invasive cervical squamous carcinoma, and adenocarcinoma. The expression of p16^INK4A, MCM5, and CDC6 was also analysed on a series of ThinPrep smears exhibiting mild, moderate, and severe dyskaryosis. HPV detection was carried out by means of a modified SYBR green assay system, which uses a “degenerate” primer set that detects sequences within the L1 reading frame of at least nine HPV types. Fluorogenic polymerase chain reaction (PCR) and solution phase PCR were used for type specific HPV typing.

Of the three markers assessed, p16^INK4A showed the greatest diagnostic utility. Our findings clearly support numerous other reports confirming the hypothesis that p16^INK4A is overexpressed in squamous and glandular dysplastic cells of the cervix and is a useful adjunctive test in lesion diagnosis and cervical screening.^4,8,9,21,36 We found a strong correlation between HPV positivity and p16^INK4A positivity, although it should be noted that p16^INK4A expression was also seen in a limited number of HPV negative cases. These lesions typically showed weaker p16^INK4A expression. Milde-Langosch et al also reported p16^INK4A overexpression in 41% of HPV negative adenocarcinomas.³⁷ These findings, coupled with the observed upregulated expression of p16^INK4A in the HPV negative cell line C33A, could indicate that a non-HPV E7 mediated mechanism of p16^INK4A upregulation may also exist.³⁷ Loss of transcriptional repression in the presence of inactivating mutations in the Rb gene is the most well defined non-HPV related mechanism of p16^INK4A upregulation. Indeed, p16^INK4A expression may in some cases be independent of pRb. Henshall et al suggested that continued proliferation, despite the growth inhibitory effects of increased p16^INK4A expression, may be possible when cells also overexpress cyclin E or when p27^Kip is unavailable.³⁸

Of importance was the identification of an isolated HPV negative CIN3 lesion that was negative for p16^INK4A protein expression but positive for both MCM5 and CDC6. One possible explanation for the absence of p16^INK4A expression in this lesion could be methylation of the p16^INK4A promoter, resulting in silencing of the p16^INK4A gene.^39–45 Promoter hypermethylation of p16^INK4A may be an alternative pathway resulting in disruption of the Rb–p16^INK4A regulatory pathway in HPV independent cervical dyskaryosis. This case demonstrates that, although rare, p16^INK4A negative cervical dyskaryosis does occur. This may have important implications for the use of p16^INKA staining as a “stand alone test”, and supports the use of combinations of markers of cervical dyskaryosis.

p16^INK4A is strongly expressed in cervical glandular dysplastic cells. However, in addition to recognising cGIN, to be an effective biomarker, it is also essential that p16^INK4A should distinguish these lesions from benign glandular mimic lesions, such as tubo-endometrioid metaplasia. The reported presence of p16^INK4A expression in tubo-endometrioid metaplasia lesions may limit the use of p16^INK4A as a stand alone biomarker for the diagnosis of cervical glandular dysplasia.⁴⁶ As was suggested by Cameron et al, p16^INK4A should be used in combination with other biomarkers.⁴⁷

MCM5 showed a strong correlation between its expression and the grade of dysplasia. We also saw very occasional intermittent staining of stromal cells and normal endocervical glands. Superficial differentiating cells were negative in all cases examined. Strong immunopositivity was seen in all invasive squamous carcinoma and in 88 of 94 CIN cases examined. In the glandular lesions examined, 14 of 17 cGIN cases were positive for MCM5 expression. All adenocarcinoma cases examined were positive for MCM5 protein expression. MCM5 also stains exfoliated dysplastic cells within ThinPrep slides and may have potential as a marker of exfoliated cells exhibiting moderate and severe dyskaryosis. Sporadic staining of morphologically normal metaplastic cells was identified. However, these cells can be easily identified by morphological criteria using the Papanicolaou stain. A large scale study of MCM5 staining in ThinPrep smears is currently under way.

Throughout our study, a striking increase in MCM5 protein expression was seen in cells showing histological HPV features. This may be caused by the release of Rb inhibition on E2F via binding of HPV E7 oncoproteins. The transcription factor E2F facilitates increased transcription of MCM5 by binding to MCM5 promoter sites.⁴⁸ A high rate of high risk HPV infection was present among all grades of dysplasia in our study. Because the Coombe Women’s Hospital is located within a high risk area and is a tertiary referral centre for cervical disease this is not unexpected. Interestingly, however, there did not appear to be a significant relation between the grade of MCM5 staining intensity and HPV status among diagnostic categories. As was suggested by Davidson et al, this lack of correlation between HPV positivity and staining intensity indicates that MCM5 protein expression is a measure of proliferation and is independent of the presence of HPV.²⁹ Indeed, MCM5 upregulation is described in a variety of non HPV related neoplasms, indicating that although in the cervix MCM5 upregulation may be a consequence of HPV infection, it is not solely dependent on it. This highlights the potential of MCM as a biomarker in both HPV dependent and HPV independent cervical dysplasia. This may be particularly relevant in the case of glandular cervical lesions, the pathogenesis of which appears to be less associated with HPV infection.³⁷

CDC6 showed a strong correlation between its expression and grade of dysplasia present. In C33A, HeLa, and CaSki cervical carcinoma cell lines, CDC6 showed intense nuclear and cytoplasmic staining, which is in keeping with previous studies.¹¹ Using immunofluoresent staining, Fujita et al demonstrated nuclear and cytoplasmic staining of CDC6 in asynchronous HeLa cervical carcinoma cells. Fujita et al reported that approximately 60–70% of cells showed dominant nuclear staining, with some cytoplasmic positivity, whereas remaining interphase cells showed strong cytoplasmic staining.¹¹ This pattern of staining closely correlates with our own findings for CDC6 expression in HeLa, CaSki, and C33A cells. In our study, a dominant nuclear staining pattern was seen in all dysplastic lesions, with some high grade lesions and squamous cell carcinomas showing cytoplasmic staining. After phosphorylation by cyclin A–cyclin dependent kinase 2, the CDC6 protein is translocated from its chromatin sites to the cytoplasm during the replication phase (S phase) of the cell cycle.^49,50 CDC6 is then degraded by ubiquitin dependant proteolysis by the anaphase promoting complex/cyclosome.^51,52 Relocalisation of CDC6 to the cytoplasm prevents re-initiation of replication and is necessary for coupling S phase with the following mitosis.^53,54 The presence of CDC6 cytoplasmic staining in several high grade lesions and in squamous cell carcinomas is probably the result of the accumulation of CDC6 protein in the cytoplasm after repeated and prolonged S phases in dysplastic cells.

Although CDC6 showed a significant linear association between staining and dysplastic grade, the numbers of CIN1 and to a lesser extent CIN2/CIN3 lesions positive for CDC6 expression were significantly lower than previously described by Williams et al, who used an alternative CDC6 polyclonal antibody.¹³ However, our results do correlate well with a more recent study published by Bonds et al, which used an alternative mouse monoclonal anti-CDC6 antibody (Neomarkers, Freemont, California, USA).¹⁰ Similar to us, they found decreased CDC6 positivity in lower grade lesions. In addition, although the proportion of cells positive for CDC6 expression increased with increasing grade of glandular and squamous dysplasia, the proportion of positive dysplastic cells was lower when compared with p16^INK4A and MCM5. However, all squamous cell carcinomas examined showed strong nuclear and some cytoplasmic staining. CDC6 expression was present in 11 of 14 cGIN and seven of 10 adenocarcinoma cases. This correlates closely with the study of Bonds et al, in which 11 of 14 adenocarcinoma in situ cases and eight of 10 adenocarcinomas were positive for CDC6 expression.¹⁰ The low or absent expression of CDC6 in low grade lesions may be a function of its role as a G2/M phase checkpoint regulator. Degradation of CDC6 is necessary for entry into M phase of the cell cycle.⁵⁵ This may explain why CDC6 is either undetectable or expressed at relatively low levels in early neoplastic cells. It is possible that as a consequence of neoplastic progression, higher grade neoplastic cells may find a mechanism to evade CDC6 G2/M phase regulation, and thus continue proliferating in the presence of increased amounts of CDC6 protein. CDC6 immunostaining in ThinPrep smears showed that CDC6 expression is preferentially upregulated in higher grade exfoliated dysplastic cells. These results are in keeping with findings at the histological level.

We found a correlation between CDC6 positivity and high risk HPV positivity. However, as for MCM5, no correlation was seen between high risk HPV positivity and the grade of staining intensity. Similar to Bonds et al, we found that CDC6 was preferentially expressed in areas showing histological HPV changes.¹⁰ This may suggest activation of genomic DNA replication processes by HPV associated oncoproteins. Inactivation of Rb by HPV E7 releases the inhibition of E2F and may result in transcriptional upregulation of CDC6. A recent publication by Vaziri et al reported that overexpression of CDC6 in combination with Cdt1 promotes re-replication in human cancer cells with inactive p53 but not in cells with functional p53.⁵⁶ Re-replication results in genomic instability and DNA damage, which causes DNA damage checkpoint pathways to activate the tumour suppressor protein p53. p53 prevents re-replication by inducing G1 phase cell cycle arrest or alternatively by the induction of apoptosis. High risk HPV E6 oncoprotein targets p53 for proteolytic degradation. Thus, HPV infected cells have developed a strategy that permits continued and prolonged re-replication, despite the presence of DNA damage and overexpression of CDC6 protein. Interestingly, the expression pattern of CDC6 closely mirrors that of the high risk HPV E6 oncoprotein, which is typically strongly expressed in high grade lesions and invasive carcinomas. These results indicate that although CDC6 undoubtedly plays a crucial role in the malignant transformation of cells, its potential role as a biomarker of cervical glandular and squamous dysplastic lesions is limited with respect to lower grade lesions. CDC6 may function as a biomarker of high grade and invasive cervical lesions.