Designing an immunofluorescence-based biomarker assay for detecting Rb and Phospho-Rb expression in HR+/HER2-breast cancer

Abstract Introduction: Cyclin-dependent kinases 4/6 inhibitors (CDK4/6i) have emerged as a major advance in treating hormone receptor-positive/human epidermal growth factor receptor-2-negative breast cancer (HR+/HER2- BC), which accounts for over 60% of all breast tumors. However, response rate to CDK4/6i combined with endocrine therapy (ET) is only 50% or less, with de novo resistance seen in 15-30% in patients with HR+/HER2- metastatic BC. Therefore, predictive biomarkers that can help select patients who are likely to derive clinical benefit from adding CDK4/6i to ET are urgently needed to spare non-responders from toxicities and cost. The long-term objective of our study is to design and develop a predictive biomarker assay that can be used on circulating tumor cells for evaluating tumor response to CDK4/6i [palbociclib, ribociclib, and abemaciclib]. In this research, we aimed to select internal controls that can be used for the immunofluorescence (IF) assay to evaluate the expression of Rb and phospho-Rb as potential predictive biomarkers for CDK4/6i therapy. Methods: MCF7 and T47D parental (P) cells were treated with various concentrations of abemaciclib and palbociclib to determine their effects on cell growth by automated cell counting using Ensight® Multimode Plate Reader and on Rb and phospho-Rb expression by western blotting (WB) and IF using Leica SP8 STED confocal microscope. We also utilized derivatives of MCF7 and T47D cells resistant to estrogen deprivation (EDR) and/or Palbociclib (PalboR). Results: Abemaciclib and palbociclib treatment resulted in a concentration-dependent decrease in cell growth of MCF7 P (IC50: 37.4 and 254.7 nM, respectively) and T47D P (IC50: 2.7 and 59.1 nM, respectively) cells. Abemaciclib also reduced the expression phospho/total-Rb in a concentration-dependent manner by both WB and IF. The IC50 for abemaciclib was 19 nM in MCF7 P and 24 nM in T47D P cells when detected by WB. Similarly, there was a concentration-dependent decrease in the phospho/total-Rb with abemaciclib treatment as detected by IF. Expression of both total Rb and phospho-Rb was undetectable in the P/PalboR T47D cells compared to T47D P cells by both WB and IF. Therefore, this cell line can be used as a control for lack of Rb expression. EDR/PalboR derivative of MCF7 had ~50% reduction in Rb and ~70% reduction in phospho-Rb by IF, supporting its use as a control for reduced Rb expression. Conclusion: In summary, we have identified cell line models that can be used as internal controls for detecting the expression of total and phospho-Rb as predictive biomarkers of CDK4/6i treatment response in HR+/HER2- MBC patients. Ongoing performance validation studies are underway, including automated quantitation using digital whole slide fluorescent imaging to optimize the throughput and dynamic range of the assay. Citation Format: Mantasha Tabassum, Alexis Ruiz, Noor M. Abdulkareem, Mothaffar F. Rimawi, George E. Miles, Meghana V. Trivedi. Designing an immunofluorescence-based biomarker assay for detecting Rb and Phospho-Rb expression in HR+/HER2-breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2136.