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INTERNATIONAL CONGRESS OF

PharmacoGenetics

• TaqMan Probe Real-Time PCR Assay for Quantifying BCL-2 Expression in Breast Cancer Tissues: Advancing Personalized Therapy

• Jamileh Saberzadeh,^1,* Rita Arabsolghar,² Mohammadreza haji jafari,³
  1. Division of Medical Biotechnology, Department of Laboratory Sciences, School of Paramedical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran
  2. Division of Medical Biotechnology, Department of Laboratory Sciences, School of Paramedical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran
  3. Division of Medical Biotechnology, Department of Laboratory Sciences, School of Paramedical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran
  
  
  
• Introduction: Breast cancer (BC) represents approximately 25% of all malignancies in females, making it the most prevalent type of cancer worldwide. It is responsible for about 15% of all cancer-related deaths among women. The hallmarks of cancer, including angiogenesis, uncontrolled cell proliferation, and evasion of apoptosis, are present in all tumor cells, regardless of the underlying cause or type. Central to the regulation of apoptosis are the BCL-2 protein family members, where even minor alterations in the balance of these proteins can lead to either the inhibition or promotion of cell death. Approximately 75% of early-stage breast cancer cases display elevated BCL-2 levels, with 85% of these tumors exhibiting estrogen receptor (ER) positivity and 50% expressing HER-2. The variability in BCL-2 expression across different breast cancer subtypes underscores its potential as a biomarker for prognosis and treatment decisions. High BCL-2 expression is often associated with poor clinical outcomes in breast cancer patient, serving as a prognostic biomarker that indicating increased risk of tumor progression and lower survival rates. High levels of BCL-2 are also associated with resistance to traditional therapies, including chemotherapy. Consequently, BCL-2 inhibitors, such as BH3 mimetics, can be utilized to enhance the sensitivity of tumors to chemotherapy, thereby potentially improving treatment efficacy and overcoming resistance. Moreover, a reduction in BCL-2 levels during treatment may indicate effective therapeutic response, whereas stable or rising levels could suggest the presence of resistance. Currently, assessing BCL-2 expression levels can assist physicians in selecting optimal treatment strategies and pharmacological interventions such as BCL-2 protein inhibitors. BCL-2 protein inhibitors represent a novel therapeutic approach tailored to tumor characteristics, designed to inhibit the anti-apoptotic functions of BCL-2 and counteract tumor cell resistance to apoptosis. BCL-2 inhibitors represent a promising avenue for enhancing therapeutic responses in breast cancer patients, especially those whose cancer cells exhibit resistance to standard therapies due to elevated BCL-2 levels. Consequently, understanding BCL-2 expression levels in various breast cancer subtypes can facilitate the development of personalized therapeutic strategies by identifying patients who may benefit from therapies targeting apoptotic pathways. In this study, we developed a robust method for quantifying BCL-2 gene expression levels in formalin-fixed paraffin-embedded (FFPE) breast cancer tissues.
• Methods: This study utilized one hundred untreated early-stage invasive breast cancer samples representing various subtypes, alongside twelve marginal non-tumor breast tissue samples which were all pathologically diagnosed and preserved in formalin-fixed paraffin-embedded (FFPE) format. Specific primers and a TaqMan probe for the BCL-2 target gene, as well as for beta-actin as the internal control gene, were designed using Allel ID software, and their efficiencies were determined using standard curves. Following RNA extraction from the FFPE samples, a one-step TaqMan probe RT-qPCR assay was conducted. The relative fold change (RFC) of BCL-2 expression in each sample was calculated using the Livak formula. An RFC greater than 2 was classified as high expression, while an RFC of 2 or lower indicated normal expression. The differences in mean BCL-2 RFC between tumor and normal samples were assessed using the Mann-Whitney test.
• Results: The developed TaqMan probe Real-Time PCR assay demonstrated acceptable efficiency for assessing BCL-2 gene expression levels. The results revealed that high expression of BCL-2 was observed in 49 out of 100 (49%) tumor samples and in 3 out of 12 (25%) normal samples. While BCL-2 expression was found to be higher in tumor tissues compared to normal tissues, the difference between the two groups was not statistically significant (p > 0.05).
• Conclusion: As the expression levels of BCL-2 in breast cancer have several potential implications for prognosis, treatment resistance, and therapeutic strategies, highlighting the need for a quantitative method to assess its expression in breast cancer tissues. Our developed assay is capable of accurately measuring BCL-2 gene expression levels in both tumor and non-tumor samples. Consequently, this method has the potential to be implemented in clinical laboratories for the evaluation of breast cancer patients.
• Keywords: Breast cancer BCL-2 TaqMan Probe Real-Time PCR BCL-2 protein inhibitor