Platelet RNA Splicing Profiles Can Distinguish IgM MGUS Patients from Healthy Individuals
Authors: Van Eijs Mick J.M. , Notermans Nicolette C. , Würdinger Tom , Minnema Monique C. 
Published: November 2019

Introduction
Within the general population over 50 years, 3% is affected with a monoclonal gammopathy of undetermined significance (MGUS). Although MGUS generally demands no treatment, interest in treating premalignant stages to prevent progression to overt malignancies has grown. Better insight in processes underlying progression of MGUS is therefore warranted. Platelet RNA profiles have been shown to reflect information on the topical tumor environment in various cancer types, including multiple myeloma (MM). Our aim was to investigate whether IgM MGUS patients can be distinguished from healthy controls (HCs) using platelet RNA splicing profiles, and to relate IgM MGUS splicing profiles to genes associated with Waldenström's Macroglobulinemia (WM).
Methods
Twenty-four IgM MGUS patients with presence of a paraprotein confirmed through immunofixation consented to participation. The thromboSeq protocol for platelet RNA sequencing (RNA-Seq) based classification (Best et al. Nat Protoc 2019) was used to acquire platelet RNA profiles from all MGUS subjects. RNA was extracted from platelets isolated from whole blood. After reverse transcription and SMARTer amplification, 100 bp single-read sequencing was performed on the Illumina Hiseq 2500 platform. Raw sequencing data were processed with Trimmomatic, aligned to a reference genome with STAR and mapped with HTSeq. Mapping data of 29 age and blood storage time matched HCs were also included in the analysis. Differential expression of spliced RNAs expressed as log2 fold change (logFC) was assessed with a likelihood-ratio ANOVA and visualized in a heatmap constructed with differentially spliced RNAs that passed the particle swarm optimized (PSO) false discovery rate (FDR) threshold. RNA profiles were further explored and related to WM and MM by DAVID gene ontology enrichment analyses.
Results
Mean age in the MGUS and HC groups was 67.2 (SD 7.7) and 66.9 (SD 6.7) years. Thirteen of 24 MGUS patients suffered from IgM mediated anti-myelin associated glycoprotein (MAG) neuropathy; the other 11 were asymptomatic. Of 3,426 high-abundant spliced RNAs detected, 1,371 demonstrated significant differential expression (FDR <0.05) and allowed perfect distinction of MGUS from HCs after PSO (Fig. 1; P <0.0001). The two distinct MGUS phenotypes (neuropathy vs. asymptomatic) could not be distinguished. Top 50 overexpressed RNA isoforms were mainly involved in apoptotic processes, transcription regulation, cellular defense response and immune response regulation. WM associated genes (logFC in parentheses) BCL7C (1.35), CASP8 (1.75), CD79A (2.65), CD81 (1.58), DUSP1 (2.32), HLA-DRB1 (1.83), JAK3 (0.65), NFKBIA (3.59), NFKB2 (1.63), REL (1.35) and TNFRSF14 (0.75), as well as MM associated JUND (4.61) and CCNL1 (3.11), were overexpressed in MGUS. TNFAIP3, negatively regulating the NF-κB pathway, was highly overexpressed (logFC 4.07). JAK1 and JAK2 mRNAs were mildly though significantly depleted in the MGUS group. We also found overexpression of CCNL2 (1.81), MS4A1 (2.17) and WNK1 (0.79), which have been shown to be enriched in WM relative to IgM MGUS. Top 50 depleted RNAs were mainly involved in protein phosphorylation, intracellular signal transduction and response to tumor necrosis factor. CASP3 (-1.43), CASP4 (-0.62) and TNFSF4 (-0.99) mRNAs, of which overexpression is associated with WM, were depleted in MGUS.
Conclusions
For the first time, we have demonstrated widespread differential expression of spliced RNAs in platelets of MGUS patients as compared to healthy controls. We found evidence of overexpression of various genes that have been associated with WM. The differences were mainly in pathways related to apoptosis, signaling and immune response regulation. This is in line with findings that balanced elevation of free light chains, possibly due to a chronic inflammatory state, increases risk of developing a monoclonal gammopathy (Kumar et al. Blood Cancer J 2019). Overexpression of NFKBIA, NFKB2 andTNFAIP3 in MGUS possiblysuggests activation of the NF-κB pathway, a key step in MYD88 mediated progression to WM. JAK subtype expression levels may reflect that the JAK/STAT pathway, also important for progression to WM, has not (yet) been aberrantly activated in our MGUS subjects. Future research could be focused on studying the development of MGUS and MGUS progression to WM and MM using differentially expressed splice junctions in platelets.

Disclosures
Minnema: Amgen: Honoraria; Celgene Corporation: Honoraria, Research Funding; Gilead: Honoraria; Jansen Cilag: Honoraria; Servier: Honoraria.