Comparison of serum and plasma <scp>microRNA</scp> expression levels in patients with germ cell tumours

The serum tumour markers (STMs) β-HCG, alphafetoprotein (AFP) and lactate dehydrogenase (LDH) are used for the diagnosis, treatment and surveillance of patients with germ cell tumours (GCTs). However, many patients with normal STMs harbour viable disease. Although circulating microRNAs (miRNAs) are a potentially more sensitive and specific marker [1], there is a lack of consensus regarding the ideal method of collecting and measuring miRNA levels, and particularly on whether a patient's peripheral blood serum or plasma should be utilised. While the majority of studies have used serum samples, some groups favour plasma. The heterogeneity in measurement techniques has limited the comparability of results across studies and is a barrier to the widespread clinical adoption of miRNA assays for the management of GCTs. To assess the comparability of miRNA results taken from serum and plasma, we prospectively collected paired samples from a convenience sample of patients just prior to starting chemotherapy for active GCT or while on surveillance with no evidence of disease. We measured miR-371a-3p and miR-372-3p, with comparative threshold cycle (CT) and relative values calculated as previously described [2]. Informed consent for blood collection was obtained under an approved institutional review board protocol. Thirty patients with paired samples from our institution were included in this analysis. Samples were taken prior to treatment on Cycle 1, Day 1 of chemotherapy from 15 patients with active disease determined via either elevated traditional STMs or radiographic evidence of metastases. Of the 15 patients with active disease, six had non-seminomatous GCT (NSGCT) and nine had pure seminoma. Of the 15 patients without active disease, all samples were taken while on surveillance with no evidence of recurrent disease. Ten patients in the surveillance cohort had clinical stage (CS) I seminoma, four patients had CS I NSGCT and one patient had CS IIA NSGCT with no evidence of disease after three cycles of bleomycin, etoposide, and cisplatin (BEP) and post-chemotherapy retroperitoneal lymph node dissection. For the patients with CS I who were on surveillance, the median time from orchiectomy was 8 months and all patients without disease remained free from relapse following their miRNA draw, with a median post-draw follow-up time of 5 months (Table S1). The CT values measured in serum and plasma from simultaneously collected, paired samples showed excellent concordance for miR-371a-3p (concordance correlation coefficient [CCC] 0.95, 95% CI 0.88, 0.98) and miR-372-3p (CCC 0.90, 95% CI 0.78, 0.96). Similarly, relative values showed high concordance for both miR-371a-3p (CCC 0.93, 95% CI 0.83, 0.97) and miR-372-3p (CCC 0.90, 95% CI 0.77, 0.95 [Fig. 1]). Using our previously reported predefined assay positivity cutoffs [2], of the 30 paired samples, 25 paired plasma and serum samples produced the same result (positive or negative; 83%, 95% CI 70, 97 [Table S2]). Of the five discordant combined miRNA assay results, all five had discordant miRNA-372-3p levels and one of these also had discordant miRNA-371-3p levels. In each of these cases, the discordant miRNA values were within or very close to the prespecified indeterminant range (Fig. 1). Had we compared serum and plasma results of only miRNA-371a-3p, the concordance would have been 97% (95% CI 90, 100 [Table S3]). The calculation of sensitivity, specificity and receiver-operating characteristics for the detection of viable GCT was limited by the small sample size, but results were consistent across serum and plasma samples (Table S4) and in line with our prior studies, which utilised serum samples [2, 3]. For miRNA assays to disseminate into widespread clinical use, the biomarker will need to be accurate, reliable, and generalisable. There will also need to be a reasonable consensus on marker interpretation, especially if specimens are processed in different manners. In this study, we aimed to better understand potential variation in results from plasma and serum assays as a step towards improving comparability of results across studies. We demonstrate good qualitative concordance (83%) between plasma and serum combined miR-371a-3p and miR-372-3p samples in terms of disease status, which increases to 97% when considering only miRNA-371a-3p results. Prior studies have demonstrated that some miRNAs, including miR-371a-3p, may have higher raw expression levels when measured in plasma, perhaps due to dilution or interaction with plasma clotting factors [4]. Additionally, miRNA levels may have higher variability in plasma depending on the concentration of platelets [5]. In their study of serum and plasma miR-371-373 expression levels, Mørup et al. [5] found levels of the control miR-20a-5p were more variable in plasma than in serum. In matched serum and plasma from male donors without GCT, Lobo et al. [6] found a significant difference in the CT values of the endogenous reference miRNA, miR-30b-5b, with plasma samples showing lower values. Although our assay did not assess raw expression levels, we found that CT and relative values were comparable between plasma and serum samples. The relative value calculation involves normalisation against endogenous and exogenous controls and healthy donors that could compensate for intra- and inter-kinetic RT-PCR variations (sample to sample and run to run variants) and allow meaningful biological comparison between serum and plasma assays [7]. In addition, our study also demonstrates that the relative values of both miR-371a-3p and miR-372-3p measured slightly higher in the serum assay compared with the plasma assay. This finding could be attributable to under-quantification as a result of a higher relative proportion of fluid volume and clotting factors present in plasma samples leading to dilution and sequestration of miRNAs, respectively. Therefore, serum samples may be preferred for miRNA quantification. This study is limited by a modest sample size and heterogeneous disease state among patients in the cohort, but this is balanced by a mixture of patients with and without metastatic disease. Importantly, the study did not focus on patients in whom borderline elevations of miRNA are likely to be more common, such as those with small testicular masses and no other signs of disease or those with early signs of recurrence during follow-up. Further validation will be needed in larger cohorts, with special attention to clinical scenarios (localised vs advanced disease, peri-operative vs pre-/post-chemotherapy) where plasma and serum result interpretation may differ. Importantly, our results may allow increased collaboration across institutions with different sample types (serum vs plasma), thus enabling the multi-institutional prospective trials that will be needed to validate miRNA assays for clinical decision making. R.S.M., J.S., S.A.F. and D.R.F. were supported by a Cancer Center Program Grant (P30 CA008748) and a 2023 MSK Department of Surgery Faculty Research Award. S.A.F. was supported by the American Cancer Society Research Scholars Grant (ACS RSG-22-105-01-CSCT). The funding sources had no role in the study or preparation of the manuscript. S.A.F. has stock interests in UroGen Pharma, Allogene Therapeutics, Kronos Bio, Vida Ventures, IconOVir Bio, Doximity, 76Bio and ByHeart, has consulted for Merck, Immunai, BioNtech and Generate Biomedicines, and has research funding from Genentech/Roche (Inst), AstraZeneca (Inst), Decibel Therapeutics (Inst), ALX Oncology (Inst) and Merck (Inst). D.R.F. consulted for BioNTech, Telix Pharmaceuticals, Xencor and Renibus Therapeutics, has research funding from Decibel Therapeutics (Inst), Telix Pharmaceuticals, Bristol Myers Squibb/Roche and Exelixis, and received royalties from UpToDate. A.K. has consulted for ByHeart. Table S1. Patient and disease characteristics for cohort of patients on surveillance (n = 15) and those with disease prior to treatment (n = 15). Table S2. Agreement of serum and plasma combined miR-371a-3p and miR-372-3p assay results. Table S3. Agreement of serum and plasma miRNA-371a-3p and miRNA-372-3p results, individually. Table S4. Serum and plasma combined miRNA 371a-3p and 372-3p result for detection of viable GCT. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.