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

Abstract

BackgroundNon-muscle-invasive bladder cancer (NMIBC) is a high incidence form of bladder cancer (BCa), where genetic and epigenetic alterations occur frequently. We assessed the performance of associating a FGFR3 mutation assay and a DNA methylation analysis to improve bladder cancer detection and to predict disease recurrence of NMIBC patients.MethodsWe used allele specific PCR to determine the FGFR3 mutation status for R248C, S249C, G372C, and Y375C. We preselected 18 candidate genes reported in the literature as being hypermethylated in cancer and measured their methylation levels by quantitative multiplex-methylation specific PCR. We selected HS3ST2, SLIT2 and SEPTIN9 as the most discriminative between control and NMIBC patients and we assayed these markers on urine DNA from a diagnostic study consisting of 167 NMIBC and 105 controls and a follow-up study consisting of 158 NMIBC at diagnosis time’s and 425 at follow-up time. ROC analysis was performed to evaluate the diagnostic accuracy of each assay alone and in combination.ResultsFor Diagnosis: Using a logistic regression analysis with a model consisting of the 3 markers’ methylation values, FGFR3 status, age and known smoker status at the diagnosis time we obtained sensitivity/specificity of 97.6 %/84.8 % and an optimism-corrected AUC of 0.96. With an estimated BCa prevalence of 12.1 % in a hematuria cohort, this corresponds to a negative predictive value (NPV) of 99.6 %. For Follow-up: Using a logistic regression with FGFR3 mutation and the CMI at two time points (beginning of the follow-up and current time point), we got sensitivity/specificity/NPV of 90.3 %/65.1 %/97.0 % and a corrected AUC of 0.84. We also tested a thresholding algorithm with FGFR3 mutation and the two time points as described above, obtaining sensitivity/specificity/NPV values of, respectively, 94.5 %/75.9 %/98.5 % and an AUC of 0.82.ConclusionsWe showed that combined analysis of FGFR3 mutation and DNA methylation markers on urine can be a useful strategy in diagnosis, surveillance and for risk stratification of patients with NMIBC. These results provide the basis for a highly accurate noninvasive test for population screening and allowing to decrease the frequency of cystoscopy, an important feature for both patient quality of life improvement and care cost reduction.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-016-2748-5) contains supplementary material, which is available to authorized users.

A Multiplex Test Assessing MiR663ame and VIMme in Urine Accurately Discriminates Bladder Cancer from Inflammatory Conditions

“…Although some of those previous studies report an apparently superior performance to the panel reported herein, it should be recalled that in most cases the patients' series were smaller, only healthy donors were included as controls or these were comprised of a mixed group of healthy donors and patients with diverse urological diseases, and/or did not use a multiplex approach, which might impact in sample availability, testing time length and cost [22][23][24][25][26][27][28]. Roperch et al proposed a three gene multiplex methylation panel (HS3ST2, SEPTIN9 and SLIT2) combined with FGFR3 mutations assessment, age and smoking-status at time of diagnosis in a multivariate model, for diagnosis of NMIBC in urine samples, disclosing 97.6% sensitivity and 84.8% specificity, in a smaller control cohort [29]. Nonetheless, this strategy might be more difficult to implement in clinical practice, since it requires both mutation and methylation analyses, in which the multiplex is performed in two distinct gene duplex reactions.…”

A novel ultra-sensitive method for the detection of FGFR3 mutations in urine of bladder cancer patients – Design of the Urodiag® PCR Kit for surveillance of patients with non-muscle-invasive bladder cancer (NMIBC)

“…Consistently with all these observations, we have identified and clinically validated a combination of genetic and epigenetic urinary markers (mutations of FGFR3 added to hypermethylation of HS3ST2 (heparin sulfate sulfotransferase), SEPTIN9 and SLIT2) which, due to their strong complementarity, afforded the best diagnostic accuracy for the surveillance in NMIBC patients presenting low-, intermediate-and highrisk of recurrence. In comparison with the tests above mentioned, our combined test gives the best clinical performances: sensitivity/specificity/NPV respectively equal to or greater than 95%/76%/99% [18]. During the present study, we designed the Urodiag ® Kit so that it contains all the necessary components to use our test in clinical routine.…”

“…Prior works have shown that the detection of FGFR3 mutations added with methylation analysis could be a promising method for the diagnosis and surveillance of NMIBC patients [17]. In a previous work, we have shown that an integrated genetic/epigenetic analysis could lead to the development of high accurate urine-based test for the surveillance of patients with NMIBC [18]. Here, we developed and clinically validated the MASO-PCR assay, generating cost-effective, 4 simple, fast and clinically applicable assay for the detection of FGFR3 mutations in urine.…”

A novel ultra-sensitive method for the detection of FGFR3 mutations in urine of bladder cancer patients – Design of the Urodiag® PCR Kit for surveillance of patients with non-muscle-invasive bladder cancer (NMIBC)

“…We used the QM-MSPCR, a highly sensitive and specific PCR developed previously by our team [18]. The QM-MSP technology is presented in Figure 2b.…”

“…Prior works have shown that the detection of FGFR3 mutations added with methylation analysis could be a promising method for the diagnosis and surveillance of NMIBC patients [17]. In a previous work, we have shown that an integrated genetic/epigenetic analysis could lead to the development of highly accurate urine-based test for the surveillance of patients with NMIBC [18]. Here, we developed and clinically validated the MASO-PCR assay, generating a cost-effective, simple, fast and clinically applicable assay for the detection of FGFR3 mutations in urine.…”