MicroRNA-30a-5p^me: a novel diagnostic and prognostic biomarker for clear cell renal cell carcinoma in tissue and urine samples

Renal cell carcinoma (RCC) is the third most prevalent urologic malignancy [1], twice more common in men than in women [2] disclosing rising incidence (2–4% per year) worldwide. RCC accounts for 2–3% of all malignant tumors in adults, displaying the highest mortality rate among urinary tract cancers [1, 3]. Notwithstanding, renal cell tumors are morphologically and genetically heterogeneous [4]. The three main subtypes of RCC - clear cell RCC (ccRCC), papillary RCC and chromophobe RCC [1] - have distinct clinical behaviors, which should be considered for adequate patient management [4].

CcRCC is simultaneously the most common and one of the most aggressive RCC subtypes, being prone to local invasion, metastization and death [2, 5]. It comprises 70–75% of all RCC cases and is characterized by several distinct genetic and epigenetic alterations [6]. About 25% of ccRCC patients present distant metastases at time of diagnosis, and in 20–50%, metastatic disease develops few years after diagnosis and surgical treatment of the primary tumor [3, 5, 7, 8]. Furthermore, ccRCC is extremely resistant to radiation and to conventional chemotherapy [3]. Therefore, biomarkers allowing for earlier diagnosis and accurate prognostication are required, improving current treatment and follow-up strategies [9]. Indeed, metastatic dissemination is the most important prognostic factor in ccRCC [10], highlighting the importance of accurately identifying patients at high risk of disease progression. Moreover, the identification of molecular biomarkers that might indicate risk of disease progression (recurrence, metastization) at the time of diagnosis might improve clinical management [3, 7] effectively contributing to implementation of Precision Medicine [10].

MicroRNAs (miRs) are small non-coding RNAs, 18–25 nucleotides long, that repress specific genes’ expression by targeting its 3′ untranslated region [11‐13]. MiRs’ deregulation has been shown to participate in tumorigenesis, affecting differentiation, invasion, migration and apoptosis [10, 14] and has been implicated in urological tumors [15]. Several studies have associated microRNAs (miRs) deregulation with ccRCC clinicopathological features, suggesting a role in tumor initiation and progression [5, 7, 16, 17]. MiR-30a-5p, an intergenic miR (chromosome 6, 71,403,551–71,403,621 [− strand]), was suggested to play a role in cellular differentiation and development [18], but its precise role remains largely unknown [19, 20]. In ccRCC, an onco-suppressor function was proposed for miR-30a-5p, since its downregulation was associated with metastasis development [5, 9]. Moreover, miR-30a-5p was found to inhibit autophagy, by targeting BECN1, the gene encoding for beclin-1, a key protein for autophagosome formation [3]. In addition, miR-30a-5p was shown to decrease tumor microvessel density, by targeting endothelial DLL4, which is enrolled in tumor angiogenesis [5]. However, the mechanism underlying miR-30a-5p downregulation in ccRCC remains elusive [21]. Similarly to protein coding genes, miRs’ downregulation might be associated with aberrant promoter methylation, a common feature of urological tumors [22‐25]. Thus, we sought to investigate for the first time, whether miR-30a-5p expression is regulated by promoter hypermethylation in ccRCC and evaluate its value as diagnostic and prognostic biomarker, both in tissue and urine samples.

Over the last decade, the frequency of incidentally detected RCC has significantly increased, mostly due to the widespread use of imaging techniques. CcRCC, the most prevalent RCC, carries worse prognosis than other common RCC subtypes, as approximately 20–40% of cases develop distant metastases [7], which are the main cause of RCC-related mortality, the highest among urologic cancers [26, 27]. Thus, biomarkers capable of accurately identifying ccRCC cases prone to metastasize, mostly among early stage tumors at diagnosis, would be a major clinical breakthrough. MiR-30a-5p expression downregulation has been reported in ccRCC, associated with metastatic disease and adverse prognosis. This being said, we aimed to determine whether (a) miR-30a-5p expression silencing was due to aberrant promoter methylation and (b) miR-30a-5p^me levels might not only accurately detect ccRCC in tissue and urine samples, but also identify patients at increased risk to develop metastatic disease independently of standard clinicopathological parameters.

Firstly, TCGA dataset was surveyed and two CpG loci at miR-30a promoter were identified as putative regulators of its expression in ccRCC. Further, miR-30a-5p^me inversely correlated with miR-30a-5p expression levels in ccRCC. Moreover, these results were mostly corroborated in IPO Porto ccRCC cohort #1, also confirming previous reports [28, 29], and providing compelling evidence that miR-30a-5p downregulation in ccRCC might be caused by aberrant promoter methylation. Thus, our results add miR-30a-5p to the growing list of epigenetically-deregulated microRNAs in urologic malignancies [22‐25], reinforcing the contribution of epigenetic alterations to renal carcinogenesis.

Notwithstanding the mechanism underlying miR-30a-5p downregulation in ccRCC, our study firstly demonstrated that miR-30a-5p^me levels might be a specific biomarker for this cancer type. Indeed, since high methylation levels are cancer-specific, they may be used as a tool for ccRCC identification, both in tissue (e.g., as an ancillary tool for histopathological or cytopathological workup of renal mass) and urine samples, providing, in the latter case, a non-invasive tool for early disease detection in high-risk populations [30, 31] (e.g., patients with end-stage chronic renal disease undergoing haemodialysis). Although other hypermethylated miRs (miR-9, miR-124-3) have been proposed as molecular biomarkers for ccRCC [32, 33], their performance in urine samples has not been assessed, yet. Thus, to the best of our knowledge, this is the first miR methylation-based urine biomarker to be proposed for ccRCC. Importantly, it should be emphasized that miR^me assessment has several advantages, including higher stability, reduced amount of clinical material requirements and methodological celerity compared to RNA expression assays. Thus, methylation analysis is more robust, enabling the development of tests for use in clinical practice [34, 35].

The rising incidence of incidentally detected RCC presents a significant clinical challenge owing to the risk of overtreatment. Thus, perfecting prognostic models through the inclusion of molecular biomarkers might contribute to reduce that risk. Remarkably, we demonstrated that higher miR-30a-5p^me levels assessed in tissue samples independently predicted shorter time to relapse, showing promise as biomarker for risk-stratification among ccRCC and more accurate identification of the high-risk patient subset which may require alternative therapeutic interventions.

Although miR-30a-5p^me biomarker performance in Cohort #3 was not impressive, it should be highlighted that miR-30a-5p^me levels were able to identify six out of each ten ccRCC in urine samples and, notably, correctly classified seven out of each ten suspects. This simple and cost-effective method is likely to increase patient compliance while reducing the risk of mistreatment. Moreover, since metastatic patients disclosed significantly higher miR-30a-5p^me levels than non-metastatic ccRCC, this non-invasive test might also provide relevant information concerning patient monitoring after curative-intent surgery.

The main limitation of our study is the relatively small number of urine samples tested. Moreover, accuracy might be improved by adding additional markers to the panel, as we previously demonstrated for other urologic cancers [22‐25]. Nonetheless, the novelty of using a miRNA methylation marker with diagnostic and prognostic value, amenable for non-invasive detection, constitutes, in our view, a relevant contribution to the field and, hopefully, will stimulate the design of validation studies in larger and independent series.

We demonstrated that miR-30a-5p downregulation, probably due to aberrant promoter methylation, is common in ccRCC. Importantly, miR-30a-5p^me levels might aid for diagnostic and prognostic purposes, helping to identify patients at higher risk for disease progression and metastization, although additional markers should be included to improve its overall performance as diagnostic/prognostic biomarker.