Evaluation of full spectrum cell-free RNA in peripheral blood as potential biomarker for acute myeloid leukemia disease profiling

Abstract

ntroduction: Acute myeloid leukemia is one of the most aggressive haematological malignancies with high relapse/ refractory rate. To date, studies are lacking on extracellular RNA profiling in AML as cell free RNA is relatively unstable but could contain potential biomarkers. In this study, we investigated the potential of total cell-free RNA from small volumes of blood plasma as a potential biomarker for AML disease profiling. Methods: Peripheral blood was obtained after informed consent from de novo AML patients. Plasma was separated and cfRNA isolated using four different platforms. cfRNA quality was checked by bioanalyzer. Low input total RNA library was generated using cfRNA (QIAseq FastSelect RNA Library kit). In parallel, RNA isolated from paired PBMCs was subjected to library preparation using the same protocol. Sequencing was performed on Novaseq 6000 and data analysis was done using R software. Results: The yield of cfRNA from peripheral blood ranged between 64 to 380ng/ml plasma. With an average RIN value of 2.2, the cfRNA showed low molecular weight RNA profile. Next generation sequencing analysis of paired cfRNA and cell-associated peripheral blood RNA revealed >2000 protein coding transcripts with >10 TPM value and a high concordance (0.84-0.94) between the paired samples was observed (Figure below). Amongst the transcripts with highest TPM values were ferroptosis related (FTL, FTH1) and extracellular S100 proteins (S100A8 and S100A9), that regulate immune homeostasis, inflammation and serve as danger signals. In addition, long noncoding RNA (MALAT1, RMRP_1, GAS5) and small nucleolar RNA known to play a crucial role in the development of cancer were detected. Conclusion: Our findings provided the basis for the utilization of cfRNA to enhance our understanding of the AML biology as well as identify potential biomarkers relevant to the diagnosis and prognosis of AML patients.