Improved cervical smear assessment using antibodies against proteins that regulate DNA replication

Abstract

Carcinoma of the cervix is one of the most common malignancies. Papanicolaou (Pap) smear tests have reduced mortality by up to 70%. Nevertheless their interpretation is notoriously difficult with high false-negative rates and frequently fatal consequences. We have addressed this problem by using affinity-purified antibodies against human proteins that regulate DNA replication, namely Cdc6 and Mcm5. These antibodies were applied to sections and smears of normal and diseased uterine cervix by using immunoperoxidase or immunof luorescence to detect abnormal precursor malignant cells. Antibodies against Cdc6 and Mcm5 stain abnormal cells in cervical smears and sections with remarkably high specificity and sensitivity. Proliferation markers Ki-67 and proliferating cell nuclear antigen are much less effective. The majority of abnormal precursor malignant cells are stained in both low-grade and high-grade squamous intraepithelial lesions. Immunostaining of cervical smears can be combined with the conventional Pap stain so that all the morphological information from the conventional method is conserved. Thus antibodies against proteins that regulate DNA replication can reduce the high false-negative rate of the Pap smear test and may facilitate mass automated screening.

The cell cycle and cancer

“…Identification of MCM proteins in pathological specimens using immunodetection methods has been shown to be an accurate and simple method for determining the growth fraction in dynamic tumour cell populations [39,[68][69][70] ( Figure 1B). Moreover, Mcm2-7 expression levels are powerful prognostic indicators in diverse tumour types, including cancers of the lung, breast, kidney, bladder, prostate and ovary [71][72][73][74][75][76][77].…”

“…These early, non-invasive lesions are characterized by the emergence of cytologically abnormal cells, abrogation of the normal differentiation programme with loss of epithelial polarity, and an increase in the size of the proliferative compartment. Intriguingly, the switch to the dysplastic state is associated with a failure to downregulate the licensing system, resulting in high MCM protein expression in all epithelial layers, including the surface cells [3,39,68,69]. This expression pattern indicates that the majority of cells in premalignant lesions are locked into the cell division cycle.…”

“…The detection of exfoliated MCM positive cells in body fluids (eg in urine, prostatic secretions, stool samples or gastro-oesophageal aspirates) or active sampling by swabbing or brushing (eg cervical smears or ERCP brushings for pancreaticobiliary tract sampling) provides a sensitive and specific method for the detection of premalignant and malignant lesions in a range of organ systems [39,[91][92][93][94][95][96][97][98]. For example, the immunostaining of cervical Pap smears for Mcm2-7 has potential to increase both the sensitivity and specificity of this error-prone test [39,[99][100][101]. Clinical trials are ongoing, combining MCM biomarkers with liquid-based cervical cytology and automated microscopy platforms (eg BD ProEx C/FocalPoint GS Imaging System).…”

“…Using this approach to study breast cancer, a complex and highly heterogeneous tumour type with respect to cancer genetics and clinicopathological parameters, has revealed three discrete cell cycle phenotypes [110,111] (Figure 3B). These include: (a) an out-ofcycle state composed predominantly of MCM-negative cells; (b) a G 1 -delayed/arrested state composed of cells The receiver operating characteristics curves show the high sensitivity and specificity of MCM-based tests for detection of oesophageal, bladder and prostate cancer [95,96,98], while the detection of (pre)malignant cells in cervical smears is illustrative of the potential for cytology-based testing [39,93].…”

“…An alternative approach is to focus on the cell cycle machinery, which acts as an integration point for information transduced through upstream signalling networks [3,39,40]. Notably, many of the current most effective neoadjuvant and adjuvant therapeutic interventions in the clinic are cell cycle directed agents (Table 1).…”