Multiplex PCR and Next Generation Sequencing for the Non-Invasive Detection of Bladder Cancer

Abstract

BackgroundHighly sensitive and specific urine-based tests to detect either primary or recurrent bladder cancer have proved elusive to date. Our ever increasing knowledge of the genomic aberrations in bladder cancer should enable the development of such tests based on urinary DNA.MethodsDNA was extracted from urine cell pellets and PCR used to amplify the regions of the TERT promoter and coding regions of FGFR3, PIK3CA, TP53, HRAS, KDM6A and RXRA which are frequently mutated in bladder cancer. The PCR products were barcoded, pooled and paired-end 2 x 250 bp sequencing performed on an Illumina MiSeq. Urinary DNA was analysed from 20 non-cancer controls, 120 primary bladder cancer patients (41 pTa, 40 pT1, 39 pT2+) and 91 bladder cancer patients post-TURBT (89 cancer-free).ResultsDespite the small quantities of DNA extracted from some urine cell pellets, 96% of the samples yielded mean read depths >500. Analysing only previously reported point mutations, TERT mutations were found in 55% of patients with bladder cancer (independent of stage), FGFR3 mutations in 30% of patients with bladder cancer, PIK3CA in 14% and TP53 mutations in 12% of patients with bladder cancer. Overall, these previously reported bladder cancer mutations were detected in 86 out of 122 bladder cancer patients (70% sensitivity) and in only 3 out of 109 patients with no detectable bladder cancer (97% specificity).ConclusionThis simple, cost-effective approach could be used for the non-invasive surveillance of patients with non-muscle-invasive bladder cancers harbouring these mutations. The method has a low DNA input requirement and can detect low levels of mutant DNA in a large excess of normal DNA. These genes represent a minimal biomarker panel to which extra markers could be added to develop a highly sensitive diagnostic test for bladder cancer.

Detection of multiple mutations in urinary exfoliated cells from male bladder cancer patients at diagnosis and during follow-up

“…The distribution of mutations among genes is similar to other previous studies on tumor [22, 26–29] and voided urine [22, 30, 31], although TERT mutations are lower [22, 32]. Two very recent studies have reported the same TERT mutation frequencies in urine of BC patients as us by different methodologies [26, 33]. In the small group of MIBC patients, sensitivity of the TERT assay was 80% while mutations in the other genes were less represented, in line with the distribution found in other studies [22].…”

Targeted deep sequencing of urothelial bladder cancers and associated urinary DNA: a 23‐gene panel with utility for non‐invasive diagnosis and risk stratification

“…Amplicon sequencing reads were aligned to the human genome (Hg19) using bowtie , and reference and non‐reference read depths extracted using bamreadcount . Only Q >30 base‐calls were considered, and variant detection was based on the non‐reference reads >2.5% of the total read depth and a minimum of 10 non‐reference reads, as described previously . All mutations included in the 23‐gene panel had to meet the criteria of ≥10% MAF in ≥1 tumour and <2.5% in germline DNA.…”

Recent progress with next‐generation biomarkers in muscle‐invasive bladder cancer

“…Most studies have focused on ctDNA in plasma, although ctDNA in urine is of particular interest in BCa. One of the main challenges of this technology is to distinguish tumor‐specific DNA from normal DNA, which is typically present in great excess …”

“…A second study developed a PCR/NGS method to analyze urinary ctDNA and a number of frequently mutated genes in BCa ( FGFR3 , TERT , PIK3CA , TP53 , HRAS , RXRA and KDM6A ) . The authors detected very low levels of urinary ctDNA and mutations that were frequently found in coding regions of FGFR3 , with the highest frequency in NMIBC, as well as mutations in PIK3CA and TERT across all BCa stages.…”