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Journal of Biotechnology
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Journal of BIOTECHNOLOGY
Comparison of plasma and urinary microRNA-483-5p for the diagnosis of adrenocortical malignancy
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Abel Decmanna, Irina Bancosb, Aakanksha Khannab, Melinda A. Thomasb, Péter Turaia, Pál Pergeª, József Zsolt Pintérª, Miklós Tóthª, Attila Patócs“, Peter Igaza,d,*
a 2nd Department of Internal Medicine, Faculty of Medicine, Semmelweis University, Szentkirályi str. 46., 1088 Budapest, Hungary
b Division of Endocrinology, Diabetes, Metabolism and Nutrition, Department of Internal Medicine, Mayo Clinic, 200 First Street SW, Rochester, MN 55905 USA
” Hereditary Endocrine Tumors Research Group, Hungarian Academy of Sciences and Semmelweis University, Szentkirályi str. 46., 1088 Budapest, Hungary d MTA-SE Molecular Medicine Research Group, Hungarian Academy of Sciences and Semmelweis University, Szentkirályi str. 46., 1088 Budapest, Hungary
ARTICLE INFO
Keywords:
microRNA
miR-483-5p Adrenocortical cancer
Plasma Urinary
ABSTRACT
Introduction: Minimally invasive circulating microRNAs might be used for the preoperative differentiation of adrenocortical carcinoma (ACC) and adrenocortical adenoma (ACA). So far, the best blood-borne microRNA biomarker of ACC is circulating hsa-miR-483-5p. The expression of urinary hsa-miR-483-5p as a non-invasive marker of malignancy and its correlation with plasma hsa-miR-483-5p, has not been investigated, yet. Aim: Our aim was to investigate the expression of urinary hsa-miR-483-5p and its correlation with its plasma counterpart.
Methods: Plasma and urinary samples from 23 ACC and 23 ACA patients were analysed using real-time RT-qPCR. To evaluate the diagnostic applicability of hsa-miR-483-5p, ROC-analysis was performed.
Results: Significant overexpression of hsa-miR-483-5p was observed in carcinoma patients’ plasma samples compared to adenoma patients’ (p < 0.0001, sensitivity: 87%, specificity: 78.3%). In urinary samples, however, no significant difference could be detected between ACC and ACA patients.
Conclusions: Plasma hsa-miR-483-5p has been confirmed as significantly overexpressed in adrenocortical cancer patients and thus might be exploited as a minimally invasive preoperative marker of malignancy. The applic- ability of urinary hsa-miR-483-5p for the diagnosis of adrenocortical malignancy could not be confirmed.
1. Introduction
Tumors of the adrenal cortex are relatively common in humans. The most common adrenocortical tumor is adrenocortical adenoma (ACA), representing 60-70% of all adrenal incidentaloma (Fassnacht et al., 2016; Terzolo et al., 2014), while adrenocortical carcinoma (ACC) is a very rare disease with an annual incidence of 0.7-2 cases per million population (Fassnacht et al., 2018). ACC has a poor prognosis, as the 5- year survival rate in stage IV tumors is 0-28% (Fassnacht et al., 2018; Libé et al., 2015).
The differential diagnosis of ACC and ACA is often difficult. Imaging features characteristic for benign and malignant adrenocortical tumors are mostly exploited in the preoperative diagnosis, but there is no re- liable blood-borne marker of malignancy to date. Urinary steroid me- tabolomics is a promising novel approach, but it is not widely available (Bancos and Arlt, 2017). Adrenal biopsy is rarely performed due to difficulties of histological diagnosis and fear for tumor spread
(Fassnacht et al., 2016).
MicroRNAs (miRNA, miR) are short, 19-25 nucleotide long single- stranded non-coding RNA molecules in their mature form. They are involved in the regulation of gene expression mostly at the post-tran- scriptional level (Gebert and MacRae, 2019). miRNAs are expressed in a tissue-specific manner and secreted in body fluids (Malumbres, 2013). In the blood, miRNAs circulate in extracellular vesicles (EV) (e.g. mi- crovesicles, apoptotic bodies, exosomes) and bound to macromolecular complexes, like the Argonaute 2 protein or lipoproteins (Arroyo et al., 2011; Vickers et al., 2011). miRNAs can be used as biomarkers in various diseases, including neoplasms.
Recent studies, including ours, have found significant differences in circulating miRNA expression profiles between ACC and ACA (Chabre et al., 2013; Igaz et al., 2015; Patel et al., 2013; Zheng et al., 2016). Among several miRNAs, hsa-miR-483-5p was identified as the best marker of ACC both in unfractionated plasma and in extracellular ve- sicle preparations (Decmann et al., 2018; Perge et al., 2017).
* Corresponding author at: 2nd Department of Internal Medicine, Faculty of Medicine, Semmelweis University, Szentkirályi str. 46., H-1088 Budapest, Hungary. E-mail address: igaz.peter@med.semmelweis-univ.hu (P. Igaz).
Blood-borne circulating miRNAs could serve as minimally invasive biomarkers of diseases, and, accordingly, urinary miRNAs might serve as non-invasive biomarkers. Several studies have shown that urinary miRNAs might be potential biomarkers in urinary tract diseases (Kutwin et al., 2018). Moreover, some studies have reported alterations in urinary miRNA expression in diseases of other organs, such as, myocardial infarction (Cheng et al., 2012; Gidlöf et al., 2011; Zhou et al., 2013), diabetes mellitus (Bacon et al., 2015; Osipova et al., 2014), and tumors (gastric (Kao et al., 2017), hepatocellular (Abdalla and Haj-Ahmad, 2012), breast (Erbes et al., 2015) and ovarian cancer (Záveský et al., 2015; Zhou et al., 2015)). In a few studies, some cor- relation between urinary and plasma miRNA expression has been ob- served, as well (Erbes et al., 2015; Kao et al., 2017).
Our aim was to study the expression of urinary hsa-miR-483-5p in ACC and ACA, to correlate it with its plasma counterpart, and to ex- amine its potential utility in adrenocortical cancer diagnosis as a non- invasive marker.
2. Materials and methods
2.1. Tissue collection and ethics approval
Altogether 23 ACC and 23 ACA patients were involved in the study. All ACC samples were histologically proven. The diagnosis of ACA left non-operated, was based mostly on imaging and follow-up. Hormonally inactive and cortisol-secreting tumors were studied. From all patients, EDTA-anticoagulated plasma and random urine sample were taken (for ACC patients before therapy) (Table 1). Preoperative biochemical testing was performed for all patients involving basal cortisol, ACTH (adrenocorticotropin), dehydroepiandrosterone sulphate (DHEAS), ur- inary free cortisol and low-dose dexamethasone test (cut-off: 1.8 µg/dl). Urinary creatinine levels were measured using Beckman Coulter AU5800 (Beckmann Coulter Inc, 92821, Brea, CA, USA). The study was approved by the Ethical Committee of the Hungarian Health Council. All experiments were performed in accordance with relevant guidelines and regulations, and informed consent was obtained from the involved patients.
2.2. Sample processing and RNA isolation
Total RNA was isolated from all plasma and urine samples by miRNeasy Serum/Plasma Kit (Qiagen GmbH, 40724 Hilden, Germany). As a spike-in control for purification efficiency, 5 ul of 5 nM syn-cel-miR- 39 miScript miRNA mimic (Qiagen GmbH) was added before the ad- dition of Acid-Phenol : Chloroform. Until further processing, total RNA was stored at -80 ℃.
2.3. Analysis of miR-483-5p expression by real-time RT-qPCR
TaqMan microRNA Reverse Transcription Kit (Thermo Fisher Scientific, 02451 Waltham, MA, USA) and individual TaqMan MicroRNA Assay (CN: 4427975, Thermo Fisher Scientific) were used to reverse-transcribe RNA. The expression of hsa-miR-483-5p (ID: 002338) was measured and as an internal control, cel-miR-39 (ID: 000200) was used. Quantitative real-time PCR was performed by TaqMan Fast Universal PCR Master Mix (2x) (CN: 4352042, Thermo Fisher Scientific) on a Quantstudio 7 Flex Real-Time PCR System (Thermo Fisher Scientific) according to the protocol of manufacturer for TaqMan MicroRNA Assay with minor modifications. Negative control reactions contained no cDNA templates and the samples were always run in tri- plicate. For data evaluation, dCT method (ACT value equals target miRNA’s CT minus internal control miRNA’s CT) was used with Microsoft Excel 2016.
2.4. Statistical analysis
To adjust for urine density, relative levels of miRNA expression (2-dCT) were normalised to urinary creatinine. Real-time qPCR data was analysed by GraphPad Prism 7.00 (GraphPad Software, Inc., La Jolla, CA, USA). For differentiating between ACC and ACA groups, Student’s t-test or Mann-Whitney test was used depending on the results of Shapiro-Wilk normality test. Spearman correlation test was used for correlating plasma and urinary miR-483-5p. ROC-analysis was also performed on hsa-miR-483-5p. P-values less than 0.05 were considered to be significant.
3. Results
3.1. Analysis of miR-483-5p expression by RT-qPCR
Plasma and urine samples from 23 ACC and 23 ACA patients were included in the study. Significant overexpression of hsa-miR-483-5p was observed in carcinoma patients’ plasma samples compared to adenoma patients’ (p < 0.0001) (Fig. 1a).
In urinary samples, however, no significant difference could be detected between ACC and ACA patients (Fig. 1b).
No correlation between plasma and urinary miR-483-5p could be established. There has been no correlation between miR-483-5p and cortisol parameters (basal, cortisol after dexamethasone in plasma and urinary free cortisol), either. By subdividing tumors into hormonally inactive and cortisol-secreting subgroups, we have not found differ- ences in miR-483-5p expression either in plasma or in urine.
3.2. Diagnostic performance of miR-483-5p
To examine the diagnostic applicability of plasma miR-483-5p as a minimally invasive biomarker, ROC analysis was performed. When differentiating between ACC and ACA samples, the area under curve (AUC) was 0.88 (Fig. 1c). By selecting 12.05 as cut-off point, sensitivity was 87%, while specificity was 78.3%.
4. Discussion
ACC is a rare but serious disease that may represent a significant differential diagnostic dilemma. The preoperative differentiation of large benign tumors from ACC, as well as an early diagnosis of adre- nocortical cancer in a patient with a smaller indeterminate mass is ra- ther difficult. Former and recent studies have identified blood-borne hsa-miR-483-5p as the so far best potential minimally invasive bio- marker of adrenocortical carcinoma. In this study we have measured the expression of urinary hsa-miR-483-5p in ACA and ACC patients, and investigated its correlation to its plasma counterpart. We have not studied normal individuals as our primary aim was to study the utility of this miRNA in adrenocortical tumor differential diagnosis. The ex- pression of serum miR-483-5p in normal subjects was reported in one study and it turned out to be very low (Chabre et al., 2013). To the best of our knowledge, the expression of urinary hsa-miR-483-5p has not yet been investigated.
As expected, we observed a significant up-regulation of plasma hsa- miR-483-5p in ACC compared to ACA (Decmann et al., 2018; Patel et al., 2013). On the other hand, the expression of miR-483-5p in ur- inary samples showed no difference between ACC and ACA samples. In the literature, with the exception of diseases of the urinary tract, the concordance between plasma/serum and urinary miRNAs is rarely ob- servable. Overexpressed miR-21-5p is found in gastric cancer in urinary samples (Kao et al., 2017), but the up-regulation of its blood-borne counterpart is only found in 9 from 40 articles concerning this disease. A group of investigators found significantly elevated urinary miR-155 in breast cancer patients (Erbes et al., 2015), while others reported on both significant serum miR-155 overexpression (Mar-Aguilar et al.,
| Sample | Tumor type | Sample type | Sex | Age at sample taking | Hormonal activity | Tumor size (mm) | Ki-67 (%) | ENSAT stage |
|---|---|---|---|---|---|---|---|---|
| 1 | ACC | Plasma/urine | Female | 22 | Non-functioning | 39 | 10 | 1 |
| 2 | ACC | Plasma/urine | Male | 59 | Cortisol | 128 | 60 | 4 |
| 3 | ACC | Plasma/urine | Female | 63 | Non-functioning | 30 | 50 | 4 |
| 4 | ACC | Plasma/urine | Female | 46 | Non-functioning | 52 | 5 | 1 |
| 5 | ACC | Plasma/urine | Female | 65 | Non-functioning | 79 | 15 | 2 |
| 6 | ACC | Plasma/urine | Female | 68 | Cortisol | 173 | n.d. | 4 |
| 7 | ACC | Plasma/urine | Male | 52 | Non-functioning | 180 | 30 | 4 |
| 8 | ACC | Plasma/urine | Female | 30 | Non-functioning | 75 | 5 | 2 |
| 9 | ACC | Plasma/urine | Male | 44 | Non-functioning | 64 | 70 | 4 |
| 10 | ACC | Plasma/urine | Female | 71 | Non-functioning | 150 | 70 | 4 |
| 11 | ACC | Plasma/urine | Female | 63 | Non-functioning | 14 | 15 | 4 |
| 12 | ACC | Plasma/urine | Female | 39 | Non-functioning | 43 | 15 | 1 |
| 13 | ACC | Plasma/urine | Female | 54 | Non-functioning | 15 | 50 | 4 |
| 14 | ACC | Plasma/urine | Male | 60 | Cortisol | 71 | n.d. | 4 |
| 15 | ACC | Plasma/urine | Female | 61 | Non-functioning | 105 | 40 | 4 |
| 16 | ACC | Plasma/urine | Male | 58 | Cortisol | 110 | 30 | 4 |
| 17 | ACC | Plasma/urine | Female | 34 | Non-functioning | 100 | n.d. | 4 |
| 18 | ACC | Plasma/urine | Female | 39 | Non-functioning | 90 | 10 | 4 |
| 19 | ACC | Plasma/urine | Male | 26 | Cortisol | 115 | 10-20 | 3 |
| 20 | ACC | Plasma/urine | Female | 75 | Cortisol | 75 | 40-50 | 4 |
| 21 | ACC | Plasma/urine | Male | 56 | Non-functioning | 65 | 20 | 2 |
| 22 | ACC | Plasma/urine | Female | 58 | Non-functioning | 180 | n.d. | 4 |
| 23 | ACC | Plasma/urine | Female | 64 | Cortisol | 80 | 20-30 | 4 |
| 24 | ACA | Plasma/urine | Male | 73 | Non-functioning | 19 | ||
| 25 | ACA | Plasma/urine | Female | 38 | Non-functioning | 45 | ||
| 26 | ACA | Plasma/urine | Female | 59 | Non-functioning | 32 | ||
| 27 | ACA | Plasma/urine | Male | 71 | Non-functioning | 45 | ||
| 28 | ACA | Plasma/urine | Female | 67 | Non-functioning | 38 | ||
| 29 | ACA | Plasma/urine | Female | 55 | Non-functioning | 26 | ||
| 30 | ACA | Plasma/urine | Male | 31 | Non-functioning | 34 | ||
| 31 | ACA | Plasma/urine | Female | 51 | Non-functioning | 4 | ||
| 32 | ACA | Plasma/urine | Female | 29 | Non-functioning | 20 | ||
| 33 | ACA | Plasma/urine | Female | 59 | Cortisol | 37 | ||
| 34 | ACA | Plasma/urine | Female | 45 | Cortisol | 19 | ||
| 35 | ACA | Plasma/urine | Female | 43 | Cortisol | 22 | ||
| 36 | ACA | Plasma/urine | Female | 68 | Cortisol | 12 | ||
| 37 | ACA | Plasma/urine | Female | 72 | Cortisol | 50 | ||
| 38 | ACA | Plasma/urine | Male | 68 | Cortisol | 62 | ||
| 39 | ACA | Plasma/urine | Female | 55 | Cortisol | 32 | ||
| 40 | ACA | Plasma/urine | Female | 26 | Cortisol | 42 | ||
| 41 | ACA | Plasma/urine | Female | 44 | Cortisol | 36 | ||
| 42 | ACA | Plasma/urine | Female | 60 | Cortisol | 46 | ||
| 43 | ACA | Plasma/urine | Female | 28 | Cortisol | 16 | ||
| 44 | ACA | Plasma/urine | Male | 50 | Cortisol | 30 | ||
| 45 | ACA | Plasma/urine | Female | 48 | Cortisol | 40 | ||
| 46 | ACA | Plasma/urine | Female | 51 | Cortisol | 29 |
n.d .: no data, ENSAT: European Network for the Study of Adrenal Tumors.
2013) and underexpression (Eichelser et al., 2013). In other tumors, such as hepatocellular carcinoma, no concordance between urinary and plasma/serum miRNAs has been revealed so far (Abdalla and Haj- Ahmad, 2012). The regulation of miRNA release in body fluids is very poorly elucidated (Turchinovich et al., 2012), and the mechanism of miRNA passage from the blood to the urine is unknown.
For normalizing real-time PCR data, we have used an exogenous spike-in control, as in our previous studies this turned out to be reliable. (Decmann et al., 2018; Perge et al., 2018, 2017). Since there are no reliable biological controls for urinary miRNA, we considered cel-miR- 39 as a good option.
The diagnostic accuracy of miR-483-5p for the differentiation of ACC and ACA from unfractionated plasma was similar to our previous study on unfractionated plasma samples (sensitivity and specificity 88.9 and 75.0%), whereas in the present study 87% and 78.3% has been ob- served, respectively (Szabó et al., 2013). Higher specificity (94.4%) value was achieved in our former study on miRNA isolated from plasma extracellular vesicles (Perge et al., 2017). Moreover, no correlation between urinary and plasma cortisol parameters and miR-483-5p ex- pression was established, similar to our previous study on extracellular vesicles (Perge et al., 2018). We have not studied extracellular-vesicle
associated miRNA in the present study as extracellular vesicles in the urine mostly come from the adjacent urinary tract cells and tissues (Pisitkun et al., 2004).
In conclusion, this is the first study to report the performance of a urinary miRNA (miR-483-5p) in adrenocortical tumors. Unfortunately, we were not able to demonstrate a diagnostic value of this non-invasive marker in differentiating ACC from benign tumors.
Declaration of interest
None.
Author contributions
P.I. designed the research. A.D., P.P. and P.T. performed the re- search. I.B. A.K., J.Z.P. and M.A.T provided patient samples. A.P and M.T. assisted writing. A.D and P.I. wrote the manuscript. All authors approved the manuscript.
a
hsa-miR-483-5p relative to cel-miR-39
-8.
-10-
-dCT value
[-(Ctnsa-miR-483-5p-Ctcel-miR-39)]
-12.
-14.
-16
ACC
ACA
b
hsa-miR-483-5p relative to cel-miR-39
5.
4
-dCT value
[-(Ctnsa-miR-483-5p-Ctcel-miR-39)]
3
2
1
0
ACC
ACA
c
100-
Sensitivity%
50
AUC: 0.876
0
0
50
100
100% - Specificity%
Acknowledgements
The study has been supported by a grant from the Hungarian National Research, Development and Innovation Office (NKFIH K115398) to Dr. Peter Igaz. The presented research activities were also financed by the Higher Education Institutional Excellence Programme of the Ministry of Human Capacities in Hungary, within the framework of the molecular biology thematic programme of the Semmelweis University. The study was performed as a collaborative study in the framework of the European Network for the Study of Adrenal Tumors (ENS@T). Aakanksha Khanna MD is currently a first year resident of internal medicine at the Univ. of Buffalo. The authors thank Dr. Balázs Szalay MD PhD and Prof. Barna Vásárhelyi MD PhD DSc (Institute of Laboratory Medicine, Semmelweis University) for their help in per- forming the measurements for urinary creatinine.
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