Plasma Microrna Predicts B-Cell Lymphoma up to 12 Months before Diagnosis – Data from the Danish Blood Donor Study
Authors: Jørgensen Steffen , Tholstrup Dorte , Hansen Jakob Werner , Keld Homburg Mikkelsen , Hjalgrim Henrik , Brown Peter de Nully , Erikstrup Christian , Petersen Mikkel Steen , Nielsen Kaspar Rene , Sørensen Erik , Troelsen Jesper , Ullum Henrik , Grønbæk Kirsten , Pedersen Ole Birger 
Published: October 2019

Abstract
Introduction
MicroRNAs are small regulatory RNAs which have been shown to be deregulated in a wide variety of human cancers, including different types of B-cell lymphoma. Circulating microRNAs can be isolated from plasma and are promising biomarkers for early detection of B-cell lymphomas.
The Danish Blood Donor Study (DBDS) was initiated in 2010 and has currently enrolled more than 90,000 blood donors. Plasma repositories are kept from every blood donation allowing the study of biomarkers up to 5 years ahead of disease development.
The aim of the present study was to determine whether it is possible to detect alterations in expression profile of specific microRNAs one year before diagnosis of B-cell lymphoma using plasma repositories from healthy blood donors.
Methods
The study was done on plasma samples taken at the time of diagnosis from 17 diffuse large B-cell lymphoma (DLBCL) patients and on plasma repository samples from 13 DBDS blood donors drawn up to more than a year before they were diagnosed with B-cell lymphoma. DBDS cases with B-cell lymphoma were identified among the first 40,000 participating donors using public registries and the diagnoses were confirmed by medical records (Table 1). The expression levels of 184 different circulating microRNAs were determined in plasma from the 17 DLBCL patients and 14 age-matched healthy controls in order to design a B-cell lymphoma specific microRNA panel. This custom microRNA panel was subsequently tested on plasma repository samples from the 13 cases of B-cell lymphoma and 14 age-matched healthy controls. All plasma samples from DBDS cases and controls were isolated, frozen, stored, and trawled the same way.
Total RNA, including microRNAs, was extracted from plasma using MiRNeasy Serum/Plasma kit (Qiagen) and the expression levels were determined by Real Time RT-PCR using Serum/Plasma Focus microRNA PCR Panel (Exiqon) and Custom Pick-&-Mix microRNA PCR Panels (Exiqon). The relative expression levels of microRNA in plasma were analyzed using the ΔCt method. For normalization mir-23a and Let-7d-3p were used in addition to UniSP3 inter plate calibrator.
Results
32 microRNAs were more than 3 fold up- or down-regulated in plasma when comparing patients with DLBCL to healthy blood donors. The following microRNA were most significantly down-regulated (mir-17-92 cluster, mir-10b, mir-190a, mir-346, mir-363, mir-205) or up-regulated (mir-326, mir-328, mir-33a) (Table 2). Applying the same microRNA profile in a customized panel to plasma samples from donors that were diagnosed with B-cell lymphoma revealed that the B-cell lymphoma specific pattern could be identified in all donor samples at the time of their last donation. Thus, B-cell lymphoma specific microRNAs were detected in plasma more than a year prior to diagnosis.
Conclusion
This first study of circulating microRNA in plasma taken from healthy individuals more than a year ahead of their diagnosis of B-cell lymphoma indicates that it is feasible to develop early and non-invasive tools for diagnosis of this disease.
Table 1 Blood donors that developed B-cell lymphoma. Diagnosis Number of blood donors Interval between last donation and diagnosis (range in days) Follicular lymphoma 4 39 – 384 days DLBCL 8 56 – 698 days Hodgkin lymphoma 1 138 days
Table 2 Up- and down-regulated microRNAs in plasma from DLBCL patients compared to healthy controls. MicroRNA Fold change P-Value hsa-miR-326 51,67547 1,00E-08 hsa-miR-92a-3p -5,03677 1,00E-08 hsa-miR-363-3p -7,80529 5,30E-08 hsa-miR-10b-5p -12,89477 7,90E-08 hsa-miR-19b-3p -5,14066 1,59E-07 hsa-miR-328 5,90068 3,73E-07 hsa-miR-205-5p -39,20498 4,12E-07 hsa-miR-19a-3p -4,97523 1,10E-06 hsa-miR-33a-5p 10,51611 3,04E-06 hsa-miR-190a -8,69103 2,69E-05 hsa-miR-20a-3p -10,94205 4,15E-05 Positive values indicate up-regulated and negative values down-regulated microRNAs.

Disclosures
No relevant conflicts of interest to declare.