Circulating exosomal long noncoding RNA PRINS—First findings in monoclonal gammopathies

Abstract Multiple myeloma is the second most common hematological malignancy characterized by focal lesions of malignant plasma cells in the bone marrow. These lesions contain subclones that directly influence survival of patients. Bone marrow biopsies are single‐site biopsies and thus cannot contain all information about the tumor. In contrast, liquid biopsies analyze circulating cells and molecules that are secreted from all sites of the tumor. Long noncoding RNA molecules are one class of these molecules. We performed a two‐phase biomarker study investigating lncRNA expression profiles in exosomes of peripheral blood serum of newly diagnosed multiple myeloma (MM) patients, monoclonal gammopathy of undetermined significance (MGUS) patients in comparison with healthy donors (HD). Surprisingly, this analysis revealed dysregulation of only one exosomal lncRNA PRINS in MM vs HD. Overall, MM and MGUS patients were distinguished from HD with sensitivity of 84.9% and specificity of 83.3%. Our study suggests a possible diagnostic role for exosomal lncRNA PRINS in monoclonal gammopathies patients.


| INTRODUCTION
Monoclonal gammopathies (MG), including multiple myeloma (MM) and monoclonal gammopathy of undetermined significance (MGUS), are diseases characterized by malignant proliferation of clonal plasma cells in the bone marrow (BM). 1 Multiple myeloma is a heterogeneous disease with focal lesions in the BM but also elsewhere in the body; these lesions contain subclones that directly influence survival of patients as well as response to treatment. 1 Therefore, analysis of biopsy specimen obtained from a single site in the BM does not contain information about all pathological clones. 2 Liquid biopsies (biopsies of peripheral blood) represent a real promise for such diseases since circulating molecules detectable in peripheral blood (PB) mirror the complex heterogeneity of MG and can serve as potential diagnostic, prognostic, and predictive markers. We and others showed that these  [4][5][6] and long noncoding RNA (lncRNA). 7 LncRNA expression is tissue specific and implicated in diverse biological functions. 8 LncRNA are involved not only in tumorigenesis but also in tumor progression and metastases. 9 These molecules also circulate in body fluids. 2,7 This two-phased biomarker study focused on circulating lncRNA as potential diagnostic markers of MG. target lncRNA were determined as 2 −ΔCt . Average values of three most stable reference genes-B2M, RPLP0, and RN7SK-were used for normalization.

| Statistical evaluation
Expression data from lncRNA profiling were statistically evaluated in the environment of statistical language R by use of Bioconductor

| Correlation of PRINS expression with biochemical parameters
In  (Table S1).
We did not observe dysregulation of PRINS expression levels between patients at different DS and ISS stages.
No cytogenetics data were available for MGUS patients.  phase, only two lncRNA, UCA1 and PRINS, were detected-and only PRINS remained statistically significant.

| Analysis of overall survival
PRINS (psoriasis susceptibility-related RNA gene induced by stress) is an lncRNA that has been described in stress-induced psoriasis; its higher expression may increase susceptibility to this disease. [12][13][14] The gene is located on chromosome 10 (10p12.1); the transcript is about 3.6-kb long. Increased expression of this lncRNA occurs with respect to proliferation and differentiation of keratinocytes and stress factors (UVB, viral infection, translation inhibition). In cells exposed to stress, PRINS has a protective role. Expression of this lncRNA was demonstrated in adrenocortical carcinoma. 15 However, expression of PRINS has not been described in hematological malignancies.
In In our previous study, 10  In the second study, the authors analyzed expression of only one circulating lncRNA-PCAT-1. They showed higher expression of circulating PCAT-1 in MM patients than in HD by qPCR. PCAT-1 was able to distinguish these two groups with 71.7% sensitivity and 93.8% specificity; its levels correlated with serum β 2 -microglobulin levels. 16 On contrary, our study used a more comprehensive approach of analysis of 84 lncRNA on a commercial platform and found deregulated expression of PRINS in exosomes of MM and MGUS patients.
The last study published recently showed elevated expression of lncRNA H19 in a cohort of MM patients by qRT-PCR. H19 was shown to correlate with MM staging. 17 However, this lncRNA was not expressed in our cohort.

| CONCLUSION
Our study indicates a potential of lncRNA as a possible minimally invasive marker of MM and MGUS. However, in order to use lncRNA molecules in the so-called liquid biopsies, further studies are needed.

ACKNOWLEDGMENTS
The authors would like to thank all the patients and their caregivers for participating in this study. We would like to thank our laboratory