Promoter hypermethylation of HS3ST2, SEPTIN9 and SLIT2 combined with FGFR3 mutations as a sensitive/specific urinary assay for diagnosis and surveillance in patients with low or high-risk non-muscle-invasive bladder cancer

Bladder cancer (BCa) ranks among the five most common malignancies worldwide [1]. In a majority of the cases (more than 80 %), Bca is non-muscle-invasive bladder cancer (NMIBC) with low-stage (CIS, pTa, pT1) and low or high-grade [2]. The pTa tumors are associated with a high rate of recurrence (50–75 %) but a low probability (5 %) of progression to lamina propria-invasive (pT1) after resection, whereas carcinoma in-situ (CIS) may be the most common precursor of invasive bladder cancer because it shows a strong tendency to progress (40–50 %) towards muscle-invasive bladder cancer (MIBC) [3]. Cystoscopy and urine cytology are the standard exams for diagnosis and surveillance of NMIBC. The monitoring of NMIBC patients consists of cystoscopic evaluations performed periodically, making it the most expensive of all cancers [4]. The sensitivity of cystoscopy is, however, limited to the tumors that can be identified visually, and the sensitivity of cytology is relatively low in low-stage/low-grade tumors [5]. Therefore, other methods (e.g. NMP22, BTA test, ImmunoCyt and Urovysion) have been developed to reduce the need for cystoscopy with a considerable benefit to both patients and healthcare systems. These noninvasive molecular tests are not recommended for use in diagnosis and monitoring of Bca because of their low diagnostic accuracy [6]. Genetic and epigenetic factors are known to contribute to the occurrence of BCa [7]. FGFR3 mutations were observed in over 50 % of patients with NMIBC [8] and proposed as a urine prognostic marker for the early diagnosis and detection of recurrences in low-grade tumors [9, 10]. DNA hypermethylation of the CpG islands located in the promoter regions of tumor-suppressor genes has been associated with tumor development in many human cancers [11]. Studies have suggested that measurement of the methylation level in urine sample can aid to early diagnosis of BCa [12‐14]. Several recent works showed that the detection of FGFR3 mutations in combination with methylation analysis could be a promising method for the sensitive detection of primary and recurrent NMIBC [15, 16]. The purpose of this present study is to investigate whether combining methylation measurement of a novel set of DNA methylation markers (HS3ST2, SEPTIN9 and SLIT2) to the detection of FGFR3 mutations can lead to the development a sensitive/specific urine test for the initial diagnosis and the surveillance of low, intermediate, and high-risk NMIBC.

In this present study, we present a set of markers for a new noninvasive urine testing affording, to our knowledge, the best accuracy for initial diagnosis, surveillance, and risk stratification of NMIBC patients. With a high sensitivity and high NPV, our set of markers is useful for avoiding biopsies and decreasing the frequency of cystoscopic surveillance, thereby allowing for both patient quality of life improvement and cost reduction. Andersson and co-workers showed that the exfoliation of tumor cells into the urine depends on tumor characteristics such as size, stage, grade, and that the filtration of urine samples, as compared to the centrifugation step, increased the diagnostic accuracy of BCa [21]. This observation could be due to the removal of contaminant leucocytes that are smaller size than bladder cells, and this size difference could be exploited to enrich samples for tumor cells. In order to optimize the accuracy of our combined test, we chosen to perform the same procedure of urine samples filtration.

FGFR3 is a transmembrane tyrosine kinase receptor that binds fibroblast growth factors. Rieger-Christ [22] and Oers [23] showed an overall frequency of FGFR3 mutations in urine samples for pTa low-grade NMIBC, ranging 43 % to 62 % and confirmed with our two studies (53.0 % in the primary tumor and 46.8 % in detection of early recurrence). As it has been demonstrated by Zuiverloon [24], sensitivity for FGFR3 assay is correlated with the number of shed low-grade tumor cells. He also showed that low-grade tumors are less likely to shed many cells into the urine as their high-grade counterparts because the high-grade tumors have weaker intercellular attachments with in consequence the difficulty to detect the FGFR3 mutations. As has been shown by the author, a 24-h collection urines would optimize performance in pTa low-grade tumors and thereby increase the sensitivity of FGFR3 assay. Consequently, we are likely to have underestimated the FGFR3 assay sensitivity, performed on a simple urine sample (100 ml). It has been shown that high-risk tumors, generally, have generally more hypermethylated genes than low-risk groups [25]. Consistent with all these observations, we showed the existence of a strong complementarity between detection of FGFR3 mutations and methylation analysis of HS3ST2, SEPTIN9 and SLIT2 genes and that their complementarity has afforded the best diagnostic accuracy for low-risk NMIBC. HS3ST2, a heparin sulfate sulfotransferase, is expressed predominantly in brain and may play a role in the nervous system [26]. Silencing of the HS3ST2 gene by promoter hypermethylation has been observed in a variety of cancers [27‐29], such as BCa [12]. The SEPTIN9 gene encodes a member of the conserved septin family of GTP-binding protein that function in key processes including vesicle trafficking, apoptosis, cytoskeletal remodelling and cell division [30]. SEPTIN9 plays a role in multiple cancers including ovarian, prostate and breast cancer as either an oncogene or a tumor suppressor gene [31]. Hypermethylation of SEPTIN9 has been observed also in colorectal cancer (CRC) and a commercially available assay for that marker has been developed by the Epigenomics company, affording a sensitivity of 70 % for a specificity of 81 % [32]. As it has been observed for HS3ST2 methylated gene, we showed its informativeness in NMIBC. The SLIT gene family is a recently characterized family of secreted repellents in axon guidance and neuronal migration during the development of the central nervous system [33]. Several studies have showed that SLIT2, a tumor suppressor gene, is epigenetically silenced by hypermethylation of the promoter region in many tumors [34‐37], and recently in bladder urothelial carcinoma [12]. While the case for informativeness of the methylation and mutation status of well-chosen biomarkers is clearly shown by the literature and our results, we should also mention the considerable potential that the next-generation sequencing (NGS) is showing as compared to previous techniques. This has been highlighted by Ward and colleagues for the detection of low frequency FGFR3 and TERT mutations in the urine of BCa patients from a few nanograms of DNA [38].

We showed that our noninvasive urinary test, combining the use of genetic and epigenetic alterations, is at the same time highly sensitive and highly specific in diagnosis, surveillance and can improve risk stratification of NMIBC patients. In the surveillance of NMIBC two directions of use are possible: (1) with low-risk tumors one could reduce the frequency of follow-up cystoscopies, providing a major benefit on the patient’s life quality as well as a positive effect on the medical costs, and (2) with intermediate or high-risk tumors, it could provide earlier detection of tumor recurrence, resulting in improved patient survival.