Abstract 6707: Optimization of miRNA isolation from plasma biopsies of proton therapy prostate cancer patients
Authors: Velasquez Johnny I. , Bass Andrew , Rajaei Moein , Bryant Curtic , Mendenhall Nancy , Ricks-Santi Luisel 
Published: April 2023

Abstract
Introduction: Radiotherapy (RT) is a treatment option for men with Prostate Cancer (PCa). While minimizing damage to proximal healthy tissue, RT can still result in severe toxicities. Studies have shown that exposure to ionizing radiation leads to changes of miRNA expression at the cellular level, making cells more radioresistant or radiosensitive which could result in toxicity, recurrence, and metastatic spread. Importantly, RT induced miRNA changes have been found to be associated with RT response in clinical contexts and may serve as diagnostic tools to assess PCa risk stratification that inform precision medicine approaches. However, miRNAs have not been assessed in patients treated with proton therapy (PT), a type of particle-based RT which purports improved dose distribution and reduced toxicity. This is attributed to the proton’s unique ability to deposit most of its energy at a narrow bragg peak with minimal energy deposition beyond the peak, into surrounding organs. Because miRNAs are ubiquitous in the human body, they are stable in many environments including the bloodstream and are becoming attractive biomarkers of treatment response, including RT. Still, blood-derived biomarkers for diagnosis, prognosis, and therapy response is an emerging field and there remains a lack of standardization and normalization of sample preparation and protocol for isolating human derived miRNA from plasma. The objective is to optimize RNA isolation procedures so that it is achievable to accurately identify miRNA biomarkers in PCa patients undergoing PT.
Methods: The results of 2 RNA isolation kits were compared. To assess the effectiveness of each optimization strategy performed, a number of metrics were used to quantitate and determine the quality of the RNA: fluorometry (Qubit 2.0 and 4.0), spectrophotometry (nano-drop), and qRT-PCR amplification (Taqman).
Results: Fluorometry methods were not sensitive enough for RNA quantification for either kit. Of the two RNA isolation kits, Kit A outperformed Kit B in every metric output when 100 uL of plasma was initially used. Furthermore, amelioration of metric output was observed when the original starting plasma amount of 100 uL was increased to 200 uL in Kit A. Nanodrop quantities ranged from 8.85 - 32.65 ng/uL and -72.82 - 4.98 ng/uL using Kit A and Kit B, respectively. The quality (260/280 ratios) ranged from -7.65 - 3.17 and -41.65 - 6.13 for Kit A and Kit B, respectively. Taqman amplification was achieved for both kits.
Conclusions: Although total RNA isolation was effectively performed, there is a need for properly optimizing miRNA detection in these blood-based biopsies. Enhancing miRNA detection to determine treatment outcomes may result in miRNA’s becoming a powerful predictive biomarker. The goal is to utilize miRNAs as a diagnostic tool for real time monitoring of individualized therapy response, detecting developing metastases, and predicting relapse.
Citation Format: Johnny I. Velasquez, Andrew Bass, Moein Rajaei, Curtic Bryant, Nancy Mendenhall, Luisel Ricks-Santi. Optimization of miRNA isolation from plasma biopsies of proton therapy prostate cancer patients [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 6707.