Cervical cancer is one of the leading causes of cancer morbidity and mortality in women, with more than 98% related to a human papilloma virus (HPV) infection origin. HPV is known to infect basal keratinocytes found within the cervical transformation zone.
The stratified squamous epithelium of the cervix consists of multiple layers of cells. In a typical cervical sampling, it cannot be ruled out that only the most superficial layers are scraped away for analysis.
Promising new HPV tests based on the detection of viral oncoproteins of HPV high risk types are in the pipeline, but their diagnostic capabilities may be limited without a way to assess specimen validity. Hence, there is a dire need to reduce false negative results of these tests due to unreliable sampling.
Here we describe a new Sandwich ELISA that captures specific intermediate filament proteins (cytokeratins 5, 8 and 18) from potentially target cells located within or originating from the cervical transformation zone as a means of normalizing cervical specimens.
Cervical samples were analyzed for presence of HPV and carefully characterized via microscopy for content of cellular material and cell morphology. All samples were tested in both a newly developed keratin 5/8/18 ELISA and a pan-keratin control ELISA.
Cervical samples collected from patients participating in studies of the PIPAVIR program were used to test the feasibility of the 5/8/18 ELISA in a screening population and in patients referred to colposcopy showing various stages of cervical intraepithelial neoplasia (CIN).
Suitable for measurement of Keratin in both ELISA systems are liquid-based cytological samples (ThinPrep) as well as frozen samples.
The ELISA was successfully validated with cell lysates of different cell lines of cervical origin.
The proof of concept was shown by measurement of well characterized clinical samples: with samples containing cells of the cervical transformation zone which were likely to be found and with samples showing only intermediate and/or superficial cells for demonstrating the likely absence of keratins 8, 18, and 5 in differentiated squamous cells.
In HPV positive samples of all stages of CIN, Keratin 5, 8, 18 could be detected with a similar signal distribution when compared to normal HPV positive samples.
Our results demonstrate the presence and detectability by ELISA of keratins 5, 8, and 18 in parabasal, squamous metaplastic, and endocervical cells, while simultaneously suggesting their absence in differentiated squamous cells. We also validate with ELISA the expression of these keratins in individuals with HPV-induced dysplasia.
Smedts et al, Am J Pathol. 1992 Aug;141(2):497-511.