Advanced breast cancer diagnosis: Multiplex RT-qPCR for precise typing and angiogenesis profiling

Abstract

Breast cancer (BC) remains the foremost cause of cancer-related mortality, with an estimated 2.3 million new cases anticipated globally. The timely diagnosis of BC is pivotal for effective treatment. Currently, BC diagnosis predominantly relies on Immunohistochemistry (IHC), a method known for its sluggishness, expense, and dependence on proficient pathologists for confident cancer typing. In this study, we introduce a novel approach to enhance the accuracy, speed, and cost-effectiveness of BC diagnosis. We employ multiplex Reverse Transcription quantitative Polymerase Chain Reaction (RT-qPCR) with touch-down methods, which consistently yield significantly lower Cycle Threshold (CT) values. The study evaluates gene expression profiles of HER2, PGR, ESR, and Ki67 genes across 61 samples representing four BC subtypes, using RPL13A as the endogenous control gene. The results demonstrate that our method offers remarkable precision, nearly equivalent to IHC, in detecting gene expressions vital for BC diagnosis and subtyping. Moreover, we explore the gene expression of Hif1A, ANG, and VEGFR genes involved in angiogenesis, shedding light on the metastatic potential of the tested BC tumours. Notably, numerous samples exhibit elevated levels of Hif1A and VEGFR, indicating their potential as valuable biomarkers for assessing metastatic status. Collectively, our RT-qPCR methodology emerges as a powerful diagnostic tool for swiftly identifying BC subtypes and can be complemented with other essential tumorigenic biomarker assessments, such as angiogenesis, to further refine cancer characterisation and inform personalised therapeutic strategies for BC patients. This innovation holds the promise of revolutionising BC diagnosis and treatment, offering expedited and reliable insights for improved patient care.

Keywords

• Breast cancer types
• Touch-down PCR
• Multiplex PCR
• Angiogenesis