Abstract 3669

Background

MicroRNAs (miRNAs) are short, non-coding RNA molecules that average 18–24 nucleotides long and act as important post-transcriptional regulators. It has been demonstrated that miRNAs stably circulate in the blood and can be detected in plasma and serum. Numerous disease states, including both solid tumor and hematologic malignancies, have been shown to release miRNAs into the circulation. Furthermore, these tumor cells possess their own characteristic expression patterns of miRNAs. This presents an opportunity for specific miRNAs to serve as biomarkers for different diseases. Previous studies have shown microRNA-155 (miR-155) to be elevated in low grade lymphomas such as Chronic Lymphocytic Leukemia (CLL) and Waldenstrom Macroglobulinemia (WM). Currently, an established biomarker for diagnostic or prognostic applications does not exist for these diseases. In our study, we investigate miR-155 as a potential biomarker for CLL and WM in plasma.

Methods

Both plasma and serum samples were collected from CLL patients, WM patients, and healthy individuals. In order to quantify miR-155 levels, 100 μl of each sample went through a round of RNA isolation, reverse transcription (RT) reaction, and real-time quantitative PCR (qPCR) by using miRNeasy mini kit (Qiagen), Taqman microRNA reverse transcription kit (Applied Biosystem) and RT2 Fast SYBR® Green/ROX qPCR Master Mixes (SABiosciences). Based on the RT-qPCR results, samples were classified as having either detectable miR-155 (Ct value less than 40) or undetectable miR-155 (Ct value more than 40). The ratio of detectable to undetectable samples was used to compare groups of samples: i) CLL plasma, ii) CLL serum, iii) WM plasma, iv) WM serum v) Normal plasma, and vi) Normal serum. To adjust for variations in efficiency of RNA extraction among samples, C. elegans synthetic microRNA-39 was added specifically after denaturation of the sample and used as a spiked-in control. By using the CD63 antigen as a marker, exosomes in the plasma and serum were also detected through flow cytometry.

Results

A total of 20 CLL plasma samples showed a 100% positive ratio of detectable miR-155 while 20 CLL serum samples had only a 50% positive ratio. In addition, a total of 96 WM plasma samples had a positive ratio of 74% while 30 WM serum samples showed a much lower positive ratio of 3%. Of these 96 samples, 13 were obtained from upfront WM patients while 83 were from relapsed/refractory WM patients. The 10 plasma and 10 serum samples from healthy individuals were all negative for miR-155. Lastly, preliminary data indicated that a higher level of exosomes may exist in plasma compared to serum.

Summary

This finding suggests that miR-155 in peripheral blood plasma could potentially be used as a clinical biomarker for WM and/or CLL. The considerable difference in detectable miR-155 between patient plasma and serum was unexpected. Reasons for this disparity are still under investigation. Our preliminary data indicated that this may be due to a difference in exosomes, which are known to carry miRNAs, between plasma and serum. Ultimately, further investigation is warranted within a larger cohort to confirm the status of miR-155 as a reliable plasma biomarker for these low grade B cell malignancies.

Disclosures:

Roccaro:Roche. Ghobrial:Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Research Funding; Noxxon: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Author notes

*

Asterisk with author names denotes non-ASH members.

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