P19-01A NEW GENERATION OF VALIDITY TESTING FOR ONCOPROTEIN-BASED CERVICAL CANCER SCREENING

19. New technologies
I. Koch 1, C. Reichhuber 1, S. Mc Namara 1, A.M. Kaufmann 2, E. Boschetti 2, I. Drechsler 2, I. Hagemann 3, K. Chatzistamatiou 4, T. Agorastos 4, E. Soutschek 1, O. Böcher 1.
1Mikrogen GmbH (Germany), 2Charité-Universitaetsmedizin Berlin CBF, Clinic for Gynaecology (Germany), 3abts+partner (Germany), 4Aristotle University of Thessaloniki, Depts of Obstetrics and Gynecology Hippokrateio Hospital (Greece)

Background / Objectives

HPV is known to infect basal keratinocytes found within the cervical transformation zone. Promising new HPV tests are based on the detection of viral oncoproteins of hrHPV types, but their diagnostic capabilities may be limited without a way to assess specimen validity. Hence, there is a need to reduce false negative results of these tests due to unreliable sampling. Here we describe a new assay that captures cytokeratins 5, 8 and 18 from potential target cells as a means of normalizing cervical specimens.


Methods

A keratin 5/8/18 sandwich ELISA – recomWell Keratin 5/8/18 - was developed for detection of cells located within or originating from the cervical transformation zone. Content of different cell types was validated microscopically. Suitable for measurement of Keratin are liquid-based cytological samples in PreserveCyte.


Results

The Keratin ELISA was successfully validated with cell lysates of HPV positive and negative cell lines of cervical origin. The proof of concept was shown by measurement of well characterized clinical samples. In 335 HPV positive samples of all stages of CIN, Keratin 5/8/18 could be detected with a similar signal distribution when compared to 1484 normal samples (OD 0.96 +/-0.17). 94.5% of all samples and 96.1% of normal samples showed signals for Keratin 5/8/18 above cut off. On the contrary, 90.4% of samples with CIN2+ and 89.1% with CIN3+ were positive for Keratin 5/8/18.


Conclusion

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 validated the expression of these Keratins in individuals with HPV-induced dysplasia and found differences in the proportion of valid samples between healthy woman and those which developed CIN2+. Furthermore, recommendations for interpretation of the results of the combined test systems (validity testing by recomWell Keratin ELISA and HPV E7 oncoprotein testing by recomWell HPV 16/18/45) were set.

The recomWell Keratin 5/8/18 allows validity testing of cervical samples by detection of potential HPV target cells and could therefore be a means to decrease the rate of false negative HPV results due to unreliable sampling.


References