Diagnostic and prognostic potential of new markers for cervical cancer

Abstract

The fight against cervical cancer is a major priority in public health in the world. Alternative methods to the traditional Pap smear are needed. Cervical cancer is closely related to human papillomavirus (HPV). HPV testing for primary screening is being introduced in several countries. However, tests are expensive and need to be processed in specialized laboratories, limiting their implementation in developing countries. Therefore, it is vital to develop low-cost exams with simple technology that can benefit a large number of patients. The gastrin releasing peptide receptor (GRPR) has shown to be overexpressed in the presence of neoplastic lesions of the cervix, being found in both precursor lesions and invasive carcinomas. Human Kallikrein 7 (HK7) is also a potential marker for cervical lesions, and is particularly related with tumor metastasis and invasion. Superoxide dismutase-2 (SOD2) is implicated in carcinogenesis of various cancers and showed increased expression according to the severity of cervical disease. The relationship between HPV and the above markers has not yet been evaluated. We estimate they can be surrogate markers for the presence of HPV by different mechanisms. There are no rapid tests for HPV detection so far. The objective of this study is to evaluate the relationship of GRPR, HK7 and SOD2 with HPV, to develop a rapid test that can aid in the initial screening of cervical lesions. This analysis will be performed through studies using various techniques. Immunohistochemistry: cervical lesions samples previously tested for HPV will be incubated with anti-GRPR, anti-HK7 and anti-SOD2 antibodies. As a result, it is hoped to correlate the expression of the markers with HPV and to correlate to the severity of the lesions. Cell cultures: SiHa (HPV 16), HeLa (HPV 18), CaSki (HPV 16 and 18), C33 (negative HPV) and human keratinocytes (QPH) cell lines will be tested for the presence of GRPR, HK7 and SOD2 by real-time PCR. As a result, we expect to establish a pattern of markers expression in these cultures, to be used as a control for the phase two tests. Organotypic cultures will be established with fibroblast cell line. The dermal equivalent will be added to normal QPH or to QPH transduced with retroviral vectors containing the mutant E6, E7 and HPV16. After this, histological sections will be obtained and to be submitted to IHC using the antibodies of the study. As a result, we hope to understand the relation of each marker in distinct models of cervical carcinogenesis. Real-time PCR: this technique will be used to establish and quantify the presence of the markers in cell lines. The qPCR will be critical in the next phase of the project in order to determine the positivity cut-off point of the markers in patient samples. If this project leads to positive results, the company Ziel Biosciences aims to develop in a second phase a multiplex rapid test, innovating the cervical cancer screening market. (AU)