Measurement of Serum microRNAs in US Veterans with Monoclonal Gammopathy of Undetermined Significance
Authors: Wang Weixin , Shim Youn K , Michalek Joel E , Barber Emily , Saleh Layla M , Choi Byeong Yeob , Wang Chen-Pin , Ketchum Norma , Costello Rene , Marti Gerald E , Vogt Robert , Landgren Ola , Calvo Katherine R. 
Published: October 2019

Abstract
Background
Monoclonal gammopathy of undetermined significance (MGUS) is an asymptomatic condition arising from clonal plasma cells and has the risk of transformation to multiple myeloma (MM). Dysregulated expression of microRNAs (miRs) has been well demonstrated in MM, but miRs are not as well characterized in MGUS. We previously found an increased risk for MGUS in Vietnam veterans exposed to Agent Orange and its contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Here we report the effect of age on serum levels of 13 miRs in MGUS cases and controls from that study cohort.
Methods
The study base population comprised 958 US veterans who participated in the 2002 follow-up of the Air Force Health Study (AFHS). A total of 49 MGUS cases were identified by using their serum stored at the AFHS 2002 follow-up (Landgren O et al. JAMA Oncology, 2015). The current study included 47 MGUS cases and 85 controls. The controls were selected from the veterans who did not have MGUS, did not have a history of tumors, and whose TCDD level was below the third quartile value among the study base.
Quantitative real-time PCR (qPCR) was used to determine the serum levels of 13 miRs (let-7a, let-7i, miR-16, miR-20a, miR-21, miR-34a, miR-106b, miR-146a, miR-181a, miR-192, miR-205, miR-335 and miR-361) that were previously shown to be dysregulated in MM or other cancers . The cycle threshold (Ct) values obtained from qPCR were normalized by using spiked-in cel-miR-39 as a control.
Univariate rank regression was used to examine the relationships between relative expression of each miR and age, and each miR and MGUS status (MGUS vs control); each miR-MGUS relation was also examined with adjustment for age. All miR values were analyzed in log2 units. Continuously distributed age was summarized by the median and interquartile range (IQR): age variation with MGUS status was assessed with Kruskal-Wallis test. All statistical testing was two-sided with a significance level of p<0.1. Corrections for multiple comparisons were not made. We used R for rank regression analysis and SAS Version 9.4 for Windows for all other analyses.
Results
Overall MGUS cases were older than controls (median, controls 66 years [IQR 61, 69] versus MGUS 67 years [IQR 64, 71], p=0.03). Rank regression analysis showed that age was significantly and negatively correlated with the serum levels of 9 (let-7a, let-7i, miR-146a, miR-361, miR-106b, miR-16, miR-20a, miR-21, and miR-335) of the 13 miRs examined in controls. Among MGUS cases, age was significantly negatively correlated with only 5 (let-7a, miR-146a, miR-106b, miR-16, and miR-20a) of the 13 miRs and positively correlated with miR-181a and miR-205 (Table 1).
In the univariate analysis of the miR-MGUS relationship, the serum levels of 4 miRs (let-7a, miR-106b, miR-16, and miR-21) were significantly associated with MGUS. However, none of these miRs remained significant after adjusting for age. In both adjusted and unadjusted analyses, the miR levels were lower in MGUS cases than in controls with the exceptions of miR-205 (unadjusted) and miR-335 (adjusted).
Conclusions and Discussions
In this study population, serum levels of the majority of miRs tested were negatively correlated with age in controls. Most of the same miRs were similarly decreased in MGUS with age, with the exception of miR-181a and miR-205 which were positively correlated with age. miR-181a is an important regulator of apoptosis, that is altered in many cancers, and was previously shown to be elevated in plasma cells of MM and MGUS. miR-205 inhibits tumor suppressors PTEN and SMAD4, and has been shown to be increased in several cancers. Of note, based on univariate analysis, several miRs were significantly differentially expressed in MGUS, but failed to remain significant after adjusting for age effect. This finding underscores the importance of assessing the need for age adjustment when analyzing serum miR data. We are currently exploring this data set to determine whether TCDD levels had any independent effect on miR levels in this population.
Note: The first two authors contributed equally.

Disclosures
Landgren: Pfizer: Consultancy; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Consultancy, Research Funding; Amgen: Consultancy, Research Funding; Karyopharm: Consultancy; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Merck: Membership on an entity's Board of Directors or advisory committees.