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Measuring disease biomarkers in biofluids to detect and monitor pathophysiologic events in tissues has enormous appeal. While detecting specific DNA sequences in blood plasma has been successful in informing about the presence of cancers, viruses, and a range of pathogens, this approach does not provide gene expression information, which could reveal more detailed information about disease activity. Measuring cell-free RNA in plasma would provide such information and has the potential to be more sensitive, given that one DNA molecule in a cell gives rise often to thousands or more RNA molecules. However, with the exception of microRNAs, broad sequencing of cell-free messenger RNA (mRNA) profiles in plasma has been generally challenging. We discovered that a major barrier to cell-free RNA sequencing from plasma is that the large majority of mRNAs are both fragmented and have modified phosphorylation states at their 5’ and 3’ ends that make them invisible to standard small RNA-seq methods. We present a modified RNA-seq methodology, called phospho-RNA-seq, which revealed thousands of mRNA in blood plasma. The key to this approach is (i) the incorporation of T4-polynucleotide kinase treatment to change the phosphorylation states at the RNA ends, and (ii) a stringent bioinformatics analysis pipeline to reduce false positive alignments. Phospho-RNA-seq identified cohorts of gene transcripts in plasma, including ones expressed in a tissue-specific manner. As proof-of-concept validation of the approach for biomarker identification, we used phospho-RNA-seq to longitudinally profile plasma specimens collected from patients undergoing hematopoietic stem cell transplantation. We detected bone marrow-enriched and liver-enriched transcript sets in plasma, which tracked with bone marrow recovery and hepatic injury, respectively. By providing expanded access to the transcriptome in plasma and potentially other biofluids, phospho-RNA-seq enables the discovery of new cell-free RNA biomarker signatures for a wide variety of liquid biopsy applications. Citation Format: Maria D. Giraldez, Ryan M. Spengler, Alton Etheridge, Annika J. Goicochea, Missy Tuck, Sung W. Choi, David J. Galas, Muneesh Tewari. Phospho-RNA-seq: A liquid biopsy approach for cell-free mRNA/lncRNA profiling [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr IA23.

Introduction and Objective: Our previous studies demonstrated that some circulation-bound tumor-derived factors are taken up and concentrated by circulating platelets, and are released upon platelet activation. MicroRNAs (miRNAs) are small, non-coding nucleic acid molecules involved in regulation of gene expression that have potential as biomarkers in prostate cancer. Prior descriptions of miRNA presence in platelets of healthy individuals indicates that uptake of tumor-derived miRNAs may be reflected by changes in platelet miRNA signature. To date, in the prostate cancer setting, the role of platelets as a source of miRNA-based biomarkers has not been investigated. The aim of this study was to ascertain the presence of prostate cancer-associated miRNAs in platelets from patients with aggressive disease and explore the potential use of platelets as a source of miRNA-based biomarkers. Methods Patients with aggressive prostate cancer (n = 12) were identified at a large, tertiary care medical center, informed of the study and consented to provide whole blood samples. Platelets were isolated from venous blood using citrate containing tubes and purified by serial centrifugations and washings. Platelet purification was confirmed at >99.9% by flow cytometry and platelet specific antibodies. Total RNA was extracted from platelets of men with aggressive prostate cancer (n = 2), low risk disease (n = 1) and age matched (50+) controls (n = 2). By performing a Real Time-quantitative PCR (RT-qPCR) profiling using the Human Panel I (Exiqon) (which encompasses 373 microRNAs representing the best-established and well-annotated human miRNAs) we identified several miRNAs as potential candidates of the aggressive disease. TaqMan miRNA Reverse Transcription kit and miRNA-specific stem loop primers were then used to validate the presence of miRNA candidates identified by the array in platelets from patients with aggressive disease (n = 10). All RT-qPCR reactions were performed in triplicates using TaqMan 2x Universal PCR Master Mix. Results Eleven of 12 patients in our initial study had castrate-resistant prostate cancer (the remaining one was characterized as having low risk prostate cancer). Patient ages ranged from 57 to 82. Upregulated expression of the following prostate cancer-specific miRNA molecules in platelets were identified: mir-141 (n = 7/12 patients), mir-134 (6/12), mir-181c (7/12), mir-193 (3/12),and mir-132 (7/12). Conclusions Our findings indicate that platelets may potentially represent a novel source of biomarkers in prostate cancer. Citation Format: Brady Miller, Mackenzie Adams, Harene Venghatakrishnan, Maria Giraldez, Muneesh Tewari, Ganesh Palapattu, Alexander Zaslavsky. Platelets possess prostate cancer specific miRNAs: Implications for a novel source of biomarkers. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3966. doi:10.1158/1538-7445.AM2015-3966