LiKidMiRs: A ddPCR-Based Panel of 4 Circulating miRNAs for Detection of Renal Cell Carcinoma

Simple Summary Early detection of renal cell carcinoma (RCC) significantly increases the likelihood of curative treatment, avoiding the need of adjuvant therapies, associated side effects and comorbidities. Thus, we aimed to discover circulating microRNAs that might aid in early, minimally invasive, RCC detection/diagnosis. Abstract Background: Decreased renal cell cancer-related mortality is an important societal goal, embodied by efforts to develop effective biomarkers enabling early detection and increasing the likelihood of curative treatment. Herein, we sought to develop a new biomarker for early and minimally invasive detection of renal cell carcinoma (RCC) based on a microRNA panel assessed by ddPCR. Methods: Plasma samples from patients with RCC (n = 124) or oncocytomas (n = 15), and 64 healthy donors, were selected. Hsa-miR-21-5p, hsa-miR-126-3p, hsa-miR-155-5p and hsa-miR-200b-3p levels were evaluated using a ddPCR protocol. Results: RCC patients disclosed significantly higher circulating levels of hsa-miR-155-5p compared to healthy donors, whereas the opposite was observed for hsa-miR-21-5p levels. Furthermore, hsa-miR-21-5p and hsa-miR-155-5p panels detected RCC with high sensitivity (82.66%) and accuracy (71.89%). The hsa-miR-126-3p/hsa-miR-200b-3p panel identified the most common RCC subtype (clear cell, ccRCC) with 74.78% sensitivity. Conclusion: Variable combinations of plasma miR levels assessed by ddPCR enable accurate detection of RCC in general, and of ccRCC. These findings, if confirmed in larger studies, provide evidence for a novel ancillary tool which might aid in early detection of RCC.


Introduction
Renal cancer remains one of the leading urologic cancers worldwide, being listed as one of the twenty most common and deadly cancers, especially among men (1.5:1) [1,2].
Renal cell tumors (RCTs) correspond to a set of benign and malignant neoplasms, with extensive diversity at epigenetic, molecular, and clinical levels [3,4]. Among them, about 10% correspond to benign tumors, with oncocytomas constituting the most common benign tumor [1,4]. Concerning malignant RCTs, clear-cell renal cell carcinoma (ccRCC) is the most common subtype (65-75% of all RCCs) [5], followed by papillary renal cell carcinomas (pRCC,~16%) and chromophobe renal cell carcinomas (chRCC,~7%) [5]. RCCs derive from nephron epithelial cells [1,6,7] and are characterized by their heterogeneity, both morphological and molecular. Whereas localized RCC is mostly cured by surgery, locally advanced or systemic disease constitute major therapeutic challenges, entailing the need for development not only of biomarkers for early detection, but also novel therapies [8].
In recent years, several studies have been published concerning the use of circulating microRNAs (miRNAs) for early and minimally invasive detection of RCC [1,9]. MiRNAs are small non-coding RNAs involved in cell differentiation, growth, apoptosis, and proliferation, and have been implicated in suppressing gene expression after translation [10,11]. MicroRNA dysregulation has been extensively described in various cancers, including RCC [4,[10][11][12][13]. Frequently, miRNA levels differ between cancerous and normal tissues, representing an opportunity for biomarker development, both in tissue samples and in liquid biopsies [10,11]. Nonetheless, the biomarker performance of most candidate miRNAs remains suboptimal, and concerns remain as to the most adequate methods for assessment and normalization [14,15]. Indeed, all published studies on the assessment of miRNAs in the liquid biopsies of RCC patients have used qRT-PCR [1,9], a technique which provides relative quantification, thus requiring normalization of the results. Although miR-16 should be the preferential normalizer due to its stability in RCC [15][16][17][18], many of the published studies used RNU44, U6, or other similar RNA species instead, which are unstable in liquid biopsies, eventually leading to biased results [14,[19][20][21][22][23][24][25][26][27]. This problem might be solved using a different technology, droplet digital PCR (ddPCR), as it obviates the need for normalization and preamplification. DdPCR is a recent technology that appears to improve miRNA detection, as it is based on sample partitioning before the PCR reaction and on the Poisson distribution, allowing for absolute quantification, in a time-cost effective and reliable manner [28,29]. Furthermore, the time point of data acquisition increases the precision and robustness of the method [28,29].

Samples
A total of five plasma samples were included in the technical optimization phase of the study, in which the ddPCR methodology was tested: one oncocytoma, one stage I pRCC, one stage I ccRCC, one stage I chRCC, and one healthy adult blood donor.
After optimizing the ddPCR pipeline, a cohort of 203 plasma samples was assessed, comprising 139 samples collected from RCT patients at the time of diagnosis and 64 healthy blood donors. Regarding RCT patients, 87 corresponded to ccRCC, 22 to chRCC and 15 to pRCC, whereas oncocytoma was diagnosed in the remaining 15. All patients were treated at IPO Porto by the same multidisciplinary team between 2015 and 2021. After peripheral blood collection into EDTA-containing tubes, plasma was separated by centrifugation at Cancers 2022, 14, 858 3 of 15 2500 g for 30 min at 4 • C, and subsequently stored at −80 • C in the institutional biobank until further use. All blood samples were processed within 4 h from the time of collection. Relevant clinical and pathological data were analyzed from clinical charts and grouped in an anonymized database specifically constructed for the analysis.

RNA Extraction and cDNA Synthesis
Total RNA was extracted from 100 µL plasma using a MagMAX mirVana Total RNA Isolation kit (Thermo Fisher, Waltham, MA, USA, A27828), according to the manufacturer's protocol. As a technical control, a non-human synthetic spike-in, ath-miR-159a (0.2 µL per sample of a stock solution at 0.2 nM), was added to the lysis buffer in all samples. The final 50 µL of RNA was collected to a 1.5 mL RNase-free tube. All steps were performed at room temperature, and extracted RNA was stored at −80 • C until further use.
The limit of the blank (LOB) and the limit of detection (LOD) were calculated for each target miRNA according to Armsbruster et al. [2]. Additionally, the limit of quantification (LOQ) for the five miRNAs was assessed by performing a 2-fold dilution series of an RCT sample.

Quality Control Steps
All plasma samples were inspected for hemolysis as previously reported by others [31,32]. Hence, from 238 initial samples, 35 samples that presented absorbance higher than 0.25 at 414 nm were excluded. Appropriate engineering and manual controls were used to prevent contaminations-including a master mix made using a clean hood, clean gloves, PCR reagents and consumables-and reactions were performed in separate dedicated labs. RNA previously extracted from RCC cell lines (HKC8 was obtained from Expasy and Caki-1, 769-P, Caki-2, ACHN, A-498, HEK-293, 786-O were from ATCC), and a pool of them was used as positive control for the four candidate miRNAs. A no-template control (NTC) and no-enzyme control (NEC) were included in all cDNA synthesis and ddPCR stages as negative controls. For ddPCR pipeline optimization, further negative controls ("no-cDNA control" and "no-Supermix control") were included, as recommended [33]. All samples were run in a single reaction for each target.

Statistical Analysis
Non-parametric tests were performed to compare levels of each miRNA among histologic subtypes and to evaluate associations with clinicopathological features. A Spearman test was used for correlation analyses between two variables. A Mann-Whitney U test was used for comparisons between two groups, whereas a Kruskal-Wallis test was used for multiple groups, followed by a Mann-Whitney U test with Bonferroni's correction for pairwise comparisons. A result was considered statistically significant when the p-value < 0.05.
For each miRNA, samples were categorized as positive or negative based on the cut-off values established using Youden's J index [34,35] (value combining the highest sensitivity and specificity), through Receiver-Operating Characteristic (ROC) curve analysis. Validity estimates (sensitivity, specificity, and accuracy) were determined to assess the detection biomarker performance. To improve the detection performance of the selected miRNAs, panels were constructed considering a positive result whenever at least one target miRNA was plotted as positive in an individual analysis.
A two-tailed p-value calculation and ROC curve analyses (without resampling analysis) were performed using SPSS 27.0 software for Windows (IBM-SPSS Inc., Chicago, IL, USA). All graphics were assembled using GraphPad Prism 8.0 software for Windows (GraphPad Software Inc., LA Jolla, CA, USA). To increase the statistical power through a resampling analysis, multiple ROC curves were constructed to calculate validity estimates for the best miRNA panels, as previously described [36,37]. In brief, samples were randomly divided into training (70%) and validation (30%) sets. Subsequently, the cut-off value was estimated in the training set considering the highest sensitivity and specificity and using this calculated cut-off, validity estimates were calculated in the validation set. The procedure was repeated 1000 times and the mean of the parameters (sensitivity and specificity) were calculated. These calculations were performed using R v3.4.4.

Patients' Cohort Characterization
The relevant clinical-pathological features of optimization and validation cohorts are depicted in Table 1.

Distribution of Circulating miRNA Levels and Biomarkers Performance for Detection of Malignancy
Initially, target miRNA levels were compared between oncocytoma (a benign tumor) and healthy donor samples, and no significant differences between these groups were found for any of the tested hsa-miRNAs, except for hsa-miR-155-5p (p-value = 0.037).
Due to the clinical relevance of discriminating malignant disease (RCC) from healthy individuals, this comparison was subsequently performed. Interestingly, circulating levels of hsa-miR-21-5p and hsa-miR-155-5p significantly differed between these two groups (p-value < 0.001 and p-value = 0.013, respectively) ( Figure 1). Circulating levels of hsa-miR-21-5p disclosed the highest accuracy for identifying malignant tumors, although hsa-miR-155-5p depicted the best specificity (90.63%). Remarkably, a panel comprising hsa-miR-21-5p/hsa-miR-155-5p detected about 83% of the three major RCC subtypes, with 71.89% accuracy (Table 2). Importantly, the same two hsa-miRNAs could discriminate RCTs from healthy individuals (Figures S1 and S2 and Table S1).   When the analysis was restricted to early-stage disease (patients with an organ-confined tumor) and healthy donor samples, hsa-miR-21-5p and hsa-miR-155-5p, but not the other miRNAs, retained statistical difference (p-value < 0.01 for both miRNAs) between  When the analysis was restricted to early-stage disease (patients with an organconfined tumor) and healthy donor samples, hsa-miR-21-5p and hsa-miR-155-5p, but not the other miRNAs, retained statistical difference (p-value < 0.01 for both miRNAs) between these two groups (Figure 2A,B). Hence, these two miRNAs were able to detect small RCC (tumors limited to the kidney, without regional lymph node metastasis) with 89.04% sensitivity and high negative predictive value (NPV) (77.68%) ( Table 3). Remarkably, the AUC for both miRNAs was higher than 65.00% ( Figure 2C,D).

Discussion
RCC remains a leading cause of cancer-related death worldwide. Alongside prostate and bladder cancers, RCC is one of the most common urological malignancies [38]. Early detection of RCC (ideally at stage I or II) significantly increases the likelihood of a cure through surgical treatment, with a 5-year survival rate of 98%, averting the need for sub-

Discussion
RCC remains a leading cause of cancer-related death worldwide. Alongside prostate and bladder cancers, RCC is one of the most common urological malignancies [38]. Early detection of RCC (ideally at stage I or II) significantly increases the likelihood of a cure through surgical treatment, with a 5-year survival rate of 98%, averting the need for subsequent therapies, which are not curative and often carry significant adverse side effects [15]. Nonetheless, 20-30% of patients display metastatic disease at diagnosis [38,39], and even following curative-intent nephrectomy, the standard of care for localized RCC, metastases develop in up to 20-40% of patients [39]. Notably, the response to medical treatment (mainly targeted therapy or immunotherapy) is rather limited, with a 5-year survival rate lower than 10%. Among RCCs, ccRCC, pRCC, and chRCC represent more than 90% of cases, emphasizing the importance of accurately detecting these tumor subtypes and discriminating them from benign conditions [39,40].
Circulating miRNAs are emergent cancer biomarkers which might be assessed using minimally invasive strategies, eventually constituting promising RCC biomarkers. Nevertheless, only a few studies have addressed this issue, mostly using conventional qPCR techniques [14,15,[17][18][19][20][21][22][23][24][25]41,42]. Owing to the diversity of the results of those studies and the need to overcome the limitations of normalization, we assessed the clinical potential of a circulating miRNA-based panel for RCC detection using ddPCR.
Accurate identification of patients harboring RCC and discrimination from healthy individuals, as well as from carriers of benign renal lesions (including tumors), is pivotal to reliably establishing therapeutic vs. monitoring strategies. Thus, after a first analysis between oncocytomas and healthy donors, we compared healthy donors with RCC patients. Remarkably, two (hsa-miR-21-5p and hsa-miR-155-5p) out of the four candidate miRNAs disclosed statistically significant differences in plasma levels. Although hsa-miR-21-5p has been described to act as oncomiR, we observed lower circulating levels in RCC patients [20,[43][44][45]. This might be due to the distinct miRNAs levels in the different clinical samples. Indeed, higher miRNA levels may be found in tissues compared to body fluid samples [46]. Importantly, increased hsa-miR-21-5p levels were also found in serum samples of RCC patients, further supporting that circulating miRNA levels in serum and plasma may be different [20]. Moreover, differences were also reported for hsa-miR-21-5p levels in serum and plasma among patients with Non-ST-elevation myocardial infarction, a non-cancer-related pathology [47]. Herein, higher hsa-miR-21-5p levels were found in serum when compared with respective control samples, whereas lower levels were observed in plasma samples from the same patients [47]. Of note, plasma has been reported to be the sample of election for translational studies [47][48][49], as red blood cell lysis during the coagulation process increases discharging of RNA and platelets to the serum, increasing the non-tumor derived circulating miRNAs present in each sample [48]. Importantly, hsa-miR-21-5p is expressed in platelets [47,50] and, thus, an increase of platelets in serum might explain the higher levels found for this miRNA. Furthermore, in breast cancer, lower hsa-miR-30b-5p levels were found in tissue compared with plasma, unveiling the disparities between these two sample sources [51]. Moreover, inadequate normalization and biased results may occur if the normalizer used is not the most suitable. Indeed, U6 is more prone to degradation by serum RNases [1]. Interestingly, in a previous study we found that hsa-miR-21-5p miRNA was significantly downregulated in tissue samples from RCT patients, discriminating RCT patients from healthy donors [13].
Concerning hsa-miR-155-5p, upregulation of this circulating hsa-miR was found in RCC patients, and a panel comprising hsa-miR-155-5p and hsa-miR-21-5p could identify 82.66% of RCC patients with 71.89% accuracy. Interestingly, hsa-miR-155-5p was shown to be upregulated in tissue [13,52] and ccRCC serum samples [18], and is also associated with cancer development [52]. Moreover, an hsa-miR-21-5p/hsa-miR-155-5p panel depicted high sensitivity (89.04%) for identifying organ-confined carcinomas, which might allow for reducing false-negative results and increase the likelihood of curative-intent treatment. To the best of our knowledge, this is the first study that evaluated the biomarker performance of plasma circulating hsa-miRs to detect early-stage RCC. Previously, Wang and colleagues described a 5-miRNA panel (miR-193a-3p, miR-362, miR-572, miR-378, and miR-28-5p) that was able to identify early-stage RCC, albeit in serum samples [22]. Furthermore, our panel achieved a higher NPV than that reported by Wang et al. [22].
We further evaluated whether circulating hsa-miRNAs might also convey relevant information to discriminate ccRCC from the remainder RCC subtypes. Indeed, all four miRNAs were able to differentiate this major RCC subtype from the others. The panel constituted by hsa-miR-126-3p and hsa-miR-200b-3p disclosed the best performance, with 74.78% sensitivity and 52.95% specificity. Since ccRCC is an aggressive RCC subtype, early detection is of major importance, and its accurate identification might improve patient outcomes [20,53]. Although stratification by stage was not performed due to a limited number of cases with advanced stages, for early stages, hsa-miR-126-3p and hsa-miR-200b-3p levels also differed significantly between ccRCC and the remainder RCC subtypes.
Considering that various studies have reported other strategies for RCC identification (including imaging and epigenetic biomarkers), our results seem to offer the best sensitivity for RCC detection [9,54]. Indeed, the methodology we developed uses a lower initial sample volume [15,17,20,22,25,41,42], which is more cost-effective, and the procedure to obtain the sample is better tolerated by patients. Molecular imaging such as 18 F-fluorodeoxt-glucose (FDG) positron emission tomography/computed tomography (PET/CT) was reported to detect localized RCC, but it discloses lower sensitivity (only 22%) [54,55]. Despite the superior specificity (85.9%) of 124 I-cG250 PET for RCC detection, when compared to our hsa-miR-21-5p/hsa-miR-155-5p panel (51.13%), this monoclonal antibody has a half-life of several days, constituting a significant disadvantage in relation to the protocol reported by us [56]. Moreover, diffusion magnetic resonance imaging was reported to characterize malignant lesions with similar sensitivity (86%) to our panel but with higher specificity (78%) [57]. Nevertheless, it should be noted that despite the better performance, these imaging biomarkers are more costly and less well-tolerated by the patient compared to liquid biopsies [54].
As previously reported, most circulating miRNA studies are based on blood-based liquid biopsies [1]. When compared with our protocol, only a few studies included more than 100 RCC patients, which might, at the least partially, explain the differences in results [9]. Additionally, the discrepant results might also be explained, as described above, by the biased normalization (e.g., spike-in as normalizer miRNA, U6, RNU48) [14,19,20,23,24]. Nevertheless, the sensitivity reported for the most widely studied serum miRNAs (miR-210, miR-1233, and miR-378) was generally lower than our plasma panel [14,17,25]. Indeed, using this less time-consuming and more cost-effective approach, we were able to detect RCC using a minimally invasive technique, with a lower initial quantity of plasma than serum-based studies (although detecting other miRNAs), and obtained similar or even better results, obviating the need for normalization and the associated bias (due to ddPCR absolute quantification) [15,17,20,22,25,41,42]. Hence, our results from multiple ROC curve analysis demonstrate a potential clinical application of this technology to identify RCC, and is the first study to quantify circulating miRNAs in these patients using ddPCR ( Figure 6).
These results require validation in more extensive prospective studies. Overall, and notwithstanding our promising results for RCC detection, it should be acknowledged that the lack of long-term follow-up constitutes a significant limitation. Further studies using liquid biopsies should also be considered to further subtype RCC, namely, to distinguish oncocytomas from chRCCs, which will lead to a prioritization of treatments for patients with malignant tumors.

Conclusions
Our findings support the research question that a minimally invasive test can be developed to detect RCC, improving patient survival through increased diagnosis at earlier stages. This might help to reduce the morbidity and mortality associated with advanced disease, as well as the lack of curative treatment at those stages. Furthermore, and to the best of our knowledge, this work is the first to report a novel tool to quantify circulating miRNAs in plasma using ddPCR in RCC patients.

Supplementary Materials:
The following are available online at www.mdpi.com/xxx/s1, Table S1: Performance of miRNAs as biomarkers for detection of Renal Cell Tumors., Figure   Data Availability Statement: All data generated or analyzed during this study are included in this article.

Conflicts of Interest:
The authors declare that they have no competing interests.

Conclusions
Our findings support the research question that a minimally invasive test can be developed to detect RCC, improving patient survival through increased diagnosis at earlier stages. This might help to reduce the morbidity and mortality associated with advanced disease, as well as the lack of curative treatment at those stages. Furthermore, and to the best of our knowledge, this work is the first to report a novel tool to quantify circulating miRNAs in plasma using ddPCR in RCC patients.

Supplementary Materials:
The following are available online at https://www.mdpi.com/article/ 10.3390/cancers14040858/s1, Table S1: Performance of miRNAs as biomarkers for detection of Renal Cell Tumors., Figure   Informed Consent Statement: Written informed consent was obtained from all subjects involved in the study.
Data Availability Statement: All data generated or analyzed during this study are included in this article.