Blood Cell Origin of Circulating MicroRNAs: A Cautionary Note for Cancer Biomarker Studies

Abstract

Circulating, cell-free microRNAs (miRNAs) hold great promise as a new class of cancer biomarkers due to their surprisingly high stability in plasma, association with disease states, and ease of sensitive measurement. Yet little is known about the origin of circulating miRNAs in either healthy or sick people, or what factors influence levels of circulating miRNA biomarkers. Of 79 solid tumor circulating miRNA biomarkers reported in the literature, we found that fifty-eight percent (47/79) are highly expressed in one or more blood cell type. Plasma levels of miRNA biomarkers expressed by myeloid (e.g., miR-223, miR-197, miR-574-3p, let-7a) and lymphoid (e.g., miR-150) blood cells tightly correlated with corresponding white blood cell counts. Plasma miRNA biomarkers expressed by red blood cells (e.g., miR-486-5p, miR-451, miR-92a, miR-16) could not be correlated to red cell counts due to limited variation in hematocrit in the cohort studied, but were significantly increased in hemolyzed specimens (20-30 fold plasma increase; p<0.0000001). Finally, in a patient undergoing autologous hematopoietic cell transplantation, plasma levels of myeloid- and lymphoid-expressed miRNAs (miR-223 and miR-150, respectively) tracked closely with changes in corresponding blood counts. We present evidence that blood cells are a major contributor to circulating miRNA, and that perturbations in blood cell counts and hemolysis can alter plasma miRNA biomarker levels by up to 50-fold. Given that a majority of reported circulating miRNA cancer biomarkers are highly expressed in blood cells, we suggest caution in interpretation of such results as they may reflect a blood cell-based phenomenon rather than a cancer-specific origin.

Circulating microRNAs as diagnostic biomarkers for pancreatic cancer

“…Small nuclear U6 is less stable in fluids than in tissues, [7] whilst miR-16 is a red blood cell expressed miRNA, and variations in blood cell count and/or any hemolysis could have important implications. [19] Interestingly, highly expressed circulating miR-16 [14] and peripheral blood mononuclear cell miR-16 [15] levels have themselves been shown to discriminate PDAC from HC, and those with benign disease. Therefore, the combined use of snU6 and miR-16 might not be appropriate as endogenous control, whereas a miRNA with the least change in expression across the various samples in the array could have been chosen as a normalizer.…”

“…Importantly, the majority of circulating miRNAs might originate from blood rather than cancer cells. [19] Furthermore, co-expression of miRNAs in different diseases might reflect a general inflammatory response. Hence, bloodbased miRNA expression could be a non-specific indicator of a tumor, rather than tissue-origin-specific.…”

The Potential Role of miR-451 in Cancer Diagnosis, Prognosis, and Therapy

“…And, most of the studies so far have evaluated the expression of miRNAs using formalin-fixed paraffin-embedded (FFPE) samples. Pritchard and colleagues found that 58% of the solid tumor-associated circulating miRNA biomarkers were highly expressed in blood cells, and levels of these biomarkers were correlated with blood counts (68). Therefore, they hypothesized that the levels of circulating miRNAs might reflect blood counts, rather than correlate with a cancer-specific origin.…”

MicroRNA levels quantified in whole blood varies from PBMCs

“…Erythrocyte haemolysis has been reported to alter miRNA measurements in whole blood, plasma, serum and tissues ( McDonald et al , 2011; Pritchard et al , 2012). The total RNA extracted using the three different methods was examined for degree of haemolysis by quantifying miR-451-5p and miR-23a-3p ( Dataset b).…”