MicroRNA molecular profiling from matched tumor and bio-fluids in bladder cancer

Abstract

Background: MicroRNAs have been identified as potential cancer biomarkers due to their presence and stability in many body fluids including urine and plasma, but the relationship of the pattern of expression of these messengers across various biological media has not been addressed and could provide important information in order to translate these biomarkers for epidemiologic or clinical use.

Methods: We analyzed microRNA of matched FFPE-tumor tissue, plasma, urine exosomes (n = 16) and WBCs (n = 11) from patients with bladder cancer, using Nanostring miRNA assays and droplet digital PCR for validation. Pearson correlations were used to compare expression between media.

Results: Numerous microRNAs were detected and overlapping from specific bio-specimen sources. MiR-4454 and miR-21 overexpression was found in three sources: tumor, WBCs and urine. Additionally, miR-15b-5p, miR-126-3p, miR-93-5p, and miR-150-5p were common to tumor/WBCs, while miR-720/3007a, miR-205, miR-200c-3p and miR-29b-3p common to tumor/urine. Significant associations were noted between the log-adjusted average miRNA counts in tumor vs. WBCs (r = 0.418 p < 0.001), and tumor vs. urine (r = 0.38 p < 0.001). No association was seen tumor vs. plasma exosome miRs (r = 0.07 p = 0.06).

Conclusions: MicroRNA profiling from matched samples in patients shows a significant number of microRNAs up regulated in bladder tumors are identifiable in urine exosomes and WBCs of the same patient, but not in blood plasma. This study demonstrated varying relationships between miRNA detected in biological media from the same patient, and serves to inform the potential of urine-based microRNAs as biomarkers for bladder cancer and potentially other malignancies.

Fig. 1

Association of FFPE-derived miRNA with bio-specimen-derived miRNA. MicroRNA abundance measured by NanoString miR assays in FFPE tumor tissue was compared to microRNA abundance in each additional bio-specimen. a urine exosomes (n = 16)(y-axis) and FFPE (n = 16) (x-axis). b plasma (n = 16) (y-axis) and FFPE (n = 16) (x-axis). c enriched-buffy coat (n = 11) (y-axis) and FFPE (n = 16) (x-axis)

Fig. 2

MicroRNAs common among bio-specimen sources in NMIBC. Overlapping distribution of microRNAs between the bio-specimens profiled by NanoString nCounter microRNA assay. Nine miRNAs are common between tumor and enriched-WBCs, seven miRNAs are common between tumor and urine exosomes, and two miRNAs are common between the three bio-specimens miR-4454 and miR-21

Fig. 3

NanoString Counts Comparing FFPE-tumor with Urine Exosomes and Correlation of NanoString Counts to ddPCR Counts. NanoString nCounter miRNA assays were used to profile microRNAs in tumor and cell-free urine and subsequently compared in three of the highest abundance miRNAs (miR-4454, miR-720, and miR-21) (a). ddPCR/ Taqman assays were used for independent validation of specific microRNAs. Correlations were high between NanoString and ddPCR for miR-4454 (b), miR-720 (c) and miR-21 (d)

Fig. 4

Correlation of miR-451a versus miR-4454 & miR-720/3007a in FFPE, urine exosomes and enriched-buffy coat. NanoString nCounter microRNA assay was used to determine the abundance of miR-451a, miR-4454 and miR-720/3007a across patient samples in FFPE-derived RNA, urine exome miRs and enriched-WBCs RNA. Moderate correlations were seen in FFPE: miR-4454 vs miR-451a (a) (r = 0.32 p = 0.19), and miR-720 vs miR-451a (d) (r = 0.35 p = 0.16). Weak to no correlation seen in urine exosomes: miR-4454 vs miR-451a (b) (r = 0.01 p = 0.96) and miR-720 vs miR-451a (e) (r = 0.13 p = 0.59). Moderate negative correlation was seen in enriched-buffy coat : : miR-4454 vs miR-451a (c) (r = −0.21 p = 0.51) and miR-720 vs miR-451a (f) (r = − 0.23 p = 0.47)