Abstract A118: Optimization of end point PCR for the determination of ESR1, PGR, and ERBB2 expression level in resource-limited settings: Ethiopian context
Authors: Woldesonbet Zelalem Desalegn , Vetter Martina , Nigussie Meron Yohannes , Zeleke Tamrat Abebe , Bekuretsion Yonas , Arayselassie Mahlet , Assefa Mathewos , Bekele Abebe , Anberber1 Endale , Wickenhauser Claudia , Kantelhardt Eva J , Bukur Jürgen , Barbara Seliger 
Published: August 2020

Abstract
Background: Breast cancer is becoming a major public health problem in developing countries. Accumulated evidence noted that the majority of breast cancer patients are in a younger age group. Moreover, most breast cancer patients appear in health institutions at late stages of the disease, which compromises the clinical outcome and disease management. With regard breast cancer, it solely depends on pathologic and clinical diagnoses. The characterization of hormone receptors and HER-2 status using immunohistochemistry (IHC) has been introduced in Ethiopia; however, there is critical problem with the provision of requirements for the test. As a result, the development and establishment of a rapid, reproducible, and alternative diagnostic method in resource limited setting is essential. The current research work aims to optimize endpoint PCR as a tool for determination of ESR1, PGR, and ERBB2 expression level using breast cancer specimen in Ethiopian patients. Furthermore, the project work will be extended to implement the method into clinical practice in the Ethiopian settings.
Methods: The experiments were started with known ESR1, PGR, and ERBB2 RNA expression in different breast cancer cell lines such as MCF-7, MDA-MB-231, BT-20, and SKBR-3 .cDNA from the breast cancer cell lines and FFPE derived-RNAof BC lesions was synthesized using BiozymcDNA synthesis kit. Variable concentrations (25ng, 50ng, and 100ng) of breast cancer cell lines and FFPE-derived total RNA were used for ESR1, PGR, and ERBB2 mRNA transcript detection using the corresponding primer pairs of each gene. GAPDH served as a reference gene. The following PCR program was applied: denaturation 950c (1min), 620c annealing temperature (20sec), 720c synthesis/extension temperature (20sec) for 35 cycles. The experiments were performed by including non-template controls in each PCR run. The PCR products were subjected to gel electrophoresis using SERVA agarose standard electro endosmosis (EEO)followed by documentation with the Image QuantTM LAS4000.
Results: Multiple PCR experiments were carried out with different counts of PCR cycles, with and without extension, with various RNA concentrations and using breast cancer cell lines and FFPE-derived RNA. With the PCR conditions, testing of ESR1, PGR, and ERBB2 expression was possible. In particular, the MCF-7, MDA-MB-231 cell lines PCR run produced concordant data with IHC. However, some BC cell lines (BT-20 and SKBR-3) known as negative for the respective hormone receptor showed discordant results.
Conclusion: In summary, the determination of the expression of ESR1, PGR and ERBB2 level in BC was successful. However, it requires further tests using different PCR conditions and a large set of known human breast tissues. From the experiment, we have learnt that endpoint PCR based ESR1, PGR, and ERBB2 detection can be used as an alternative method for better BC receptor diagnosis, treatment plan, and management in resource-limited countries.
Citation Format: Zelalem Desalegn Woldesonbet, Martina Vetter, Meron Yohannes Nigussie, Tamrat Abebe Zeleke, Yonas Bekuretsion, Mahlet Arayselassie, Mathewos Assefa, Abebe Bekele, Endale Anberber1, Claudia Wickenhauser, Eva J Kantelhardt, Jürgen Bukur, Seliger Barbara. Optimization of end point PCR for the determination of ESR1, PGR, and ERBB2 expression level in resource-limited settings: Ethiopian context [abstract]. In: Proceedings of the Eleventh AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2018 Nov 2-5; New Orleans, LA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl):Abstract nr A118.