John M. Millholland

A noninvasive multi-analyte diagnostic assay: Combining protein and DNA markers to stratify bladder cancer patients

Purpose The authors recently reported the development of a noninvasive diagnostic assay using urinary matrix metalloproteinases (MMPs) as monitors of disease-free status and bladder cancer in high-risk populations. Using an approach called clinical intervention determining diagnostic (CIDD), they identified with high confidence those patients who could be excluded from additional intervention. To maximize performance, MMPs were combined with DNA-based markers and CIDD was applied to a population of patients undergoing monitoring for recurrence. Patients and methods Urine samples were obtained from 323 patients, 48 of whom had a recurrence and 275 of whom did not have cancer upon cytoscopic evaluation. Twist1 and Nid2 methylation status was determined using methylation-specific polymerase chain reaction, FGFR3 mutational status by quantitative PCR, and MMP levels by enzyme-linked immunosorbent assay. Results Using a combination of these DNA and protein markers, the authors identified with high confidence (97% negative predicted value) those patients who do not have cancer. Cutoffs were adjusted such that at 92% sensitivity, 51% of disease-free patients might be triaged from receiving further tests. Conclusion The multi-analyte diagnostic readout assay described here is the first to combine protein and DNA biomarkers into one assay for optimal clinical performance. Using this approach, the detection of FGFR3 mutations and Twist1 and Nid2 methylation in the urine of patients undergoing bladder cancer recurrence screening increase the sensitivity and negative predictive value at an established MMP protein cutoff. This noninvasive urinary diagnostic assay could lead to the more efficient triage of patients undergoing recurrence monitoring.

Detection of low frequency FGFR3 mutations in the urine of bladder cancer patients using next-generation deep sequencing

Biological fluid-based noninvasive biomarker assays for monitoring and diagnosing disease are clinically powerful. A major technical hurdle for developing these assays is the requirement of high analytical sensitivity so that biomarkers present at very low levels can be consistently detected. In the case of biological fluid-based cancer diagnostic assays, sensitivities similar to those of tissue-based assays are difficult to achieve with DNA markers due to the high abundance of normal DNA background present in the sample. Here we describe a new urine-based assay that uses ultradeep sequencing technology to detect single mutant molecules of fibroblast growth factor receptor 3 (FGFR3) DNA that are indicative of bladder cancer. Detection of FGFR3 mutations in urine would provide clinicians with a noninvasive means of diagnosing early-stage bladder cancer. The single-molecule assay detects FGFR3 mutant DNA when present at as low as 0.02% of total urine DNA and results in 91% concordance with the frequency that FGFR3 mutations are detected in bladder cancer tumors, significantly improving diagnostic performance. To our knowledge, this is the first practical application of next-generation sequencing technology for noninvasive cancer diagnostics.

Combining next-gen sequencing of TP53 and FGFR3 for detecting bladder cancer-related mutations.

304 Background: We have recently described an ultra-deep amplicon sequencing method for FGFR3 in urine that closely replicates the sensitivity found in tissue. While FGFR3 mutations are present in a large fraction of non-invasive tumors, these mutations are rarely detected in invasive bladder tumors. To complement our existing FGFR3 deep sequencing assay, we have developed a sequencing assay for TP53. Mutations in TP53 are commonly found in advanced bladder cancer, and show little overlap with FGFR3 mutations. This proof of concept study demonstrates detection of FGFR3 and TP53 in bladder cancer tissue. METHODS The FGFR3 sequencing assay was performed as described previously, permitting the detection of 9 mutations associated with bladder cancer. Similarly, amplicons were designed against TP53 exons 5-8 using, permitting the detection of 133 unique TP53 mutations previously detected in bladder cancer. Primary amplification was performed on DNA isolated from 3 10µ tissue sections. The resulting PCR products were used as template for emulsion PCR and sequenced using the Ion Torrent PGM. Samples were analyzed for total DNA reads and number of mutant sequencing reads to determine percent mutation. RESULTS Previously we analyzed 151 non-invasive bladder tumor samples for the presence of FGFR3 mutations. Of these samples, 93 out of 151 (61.5%) were positive for FGFR3 mutations. An additional set of 10 high stage bladder tumor samples were analyzed for mutations in TP53. Of these samples, 5 out of 10 (50.0%) were positive for TP53 mutations, while a separate sample was found to be positive for FGFR3 only. Importantly, a TP53 mutation was detected in a Tis carcinoma in situ sample. These studies will be expanded to a larger set of bladder cancer tissues and urine samples to more accurately assess clinical performance of this FGFR3/TP53 deep sequencing assay. CONCLUSIONS We have developed a multiplexed FGFR3 and TP53 sequencing assay that can detect mutations in a broad range of bladder cancer stages. The complementarity of mutations found in these two genes may allow for the detection of a larger fraction of patients with undiagnosed bladder cancer.

Abstract 52: Next-Gen deep amplicon sequencing of TP53 complements FGFR3 for the detection of bladder cancer-related mutations.

Background: We have recently described an ultra-deep amplicon sequencing method for detection of FGFR3 mutations in urine that closely matches the frequency of mutations found in bladder cancer tissue. While FGFR3 mutations are present in a large fraction of non-invasive tumors, these mutations are rarely detected in invasive bladder tumors. To complement our existing FGFR3 deep sequencing assay, we have developed a sequencing assay for TP53. Mutations in TP53 are commonly found in advanced bladder cancer, and show little overlap with FGFR3 mutations. Here, we demonstrate the detection of FGFR3 and TP53 mutations in bladder cancer tissue using a Next-Gen sequencing platform. Methods: The FGFR3 sequencing assay was performed as described previously, permitting the detection of 9 mutations associated with bladder cancer. Similarly, amplicons were designed against TP53 exons 5-8, permitting the detection of 133 TP53 mutations previously reported in bladder cancer. Primary amplification was performed on DNA isolated from FFPE or fresh frozen tissue. The resulting PCR products were used as template for emulsion PCR and sequenced using the Ion Torrent PGM. Samples were analyzed for total DNA reads and number of mutant sequencing reads to determine percent mutation. Results: A set of 57 bladder cancer tissue samples, comprised of 38 non-muscle invasive (NMIBC) and 19 muscle invasive (MIBC) tumors, was analyzed for mutations in both FGFR3 and TP53. Of these samples, 18 out of 38 (47.4%) NMIBC tumors were positive for FGFR3 mutations, and 2 of 38 (5.3%) were positive for TP53 mutations. 9 out of 19 (47.4%) MIBC tumors were positive for TP53 mutations, with an additional 2/19 (10.9%) positive for FGFR3 mutations. Across all tumor stages tested, 35.1% (20/57) of samples were positive for FGFR3 mutations, and 19.3% (11/57) were positive for TP53 mutations. No overlap was seen between samples positive for FGFR3 or TP53 mutations. In addition, we have now shown that these assays can detect both FGFR3 and TP53 mutations in the urine of bladder cancer patients. These studies will be expanded to a large set of urine samples to more accurately assess clinical performance of this FGFR3/TP53 deep sequencing assay in bodily fluids. Conclusions: We have developed FGFR3 and TP53 sequencing assays that can detect mutations in a broad range of bladder cancer stages. The complementarity of mutations found in these two genes may allow for the non-invasive detection of bladder cancer in a larger fraction of undiagnosed patients. Citation Format: John M. Millholland, Maria C. Campo, Lydia D. Anderson, Cecilia A. Fernandez, Anthony P. Shuber. Next-Gen deep amplicon sequencing of TP53 complements FGFR3 for the detection of bladder cancer-related mutations. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 52. doi:10.1158/1538-7445.AM2013-52

Detection of low-frequency FGFR3 mutations in the urine of patients with bladder cancer using next-generation deep sequencing.

59 Background: FGFR3 mutations have been identified in ~60-70% of low-stage, non-invasive tumors. Our group and others have developed assays to detect FGFR3 mutations in the urine of bladder cancer patients. However, urine-based assays have been limited by the technical ability to detect rare events in a dilute medium where there is a high background of normal DNA. In these assays, FGFR3 mutations are generally found in ~30% of the urine samples, which is < 50% concordance with the expected detection in tissue. We have now developed an ultra-deep amplicon sequencing technique that increases FGFR3 mutation detection in urine to ~67%, close to the expected detection frequency if every mutation found in tissue could be detected in urine. METHODS Amplicons were designed against FGFR3 exons 7, 10, and 15 using PCR primers containing the adapter sequences for unidirectional sequencing. Primary amplification was performed from DNA isolated from 4 ml of urine. The resulting PCR products were used as template for emulsion PCR and these were then sequenced using the Roche 454 GS Junior. Samples were analyzed for total DNA reads per sample and number of mutant sequencing reads to determine percent mutation. RESULTS Urine samples from 43 patients with bladder cancer were analyzed by both our previously described qPCR method and the new ultra-deep sequencing approach. Using ultra-deep amplicon sequencing, 24 out of 43 (55.8%) were positive for FGFR3 mutations, while only 5 out of 43 (11.6%) were positive for mutations by qPCR. The urine samples from the 15 newly identified mutations using deep sequencing contained FGFR3 mutations as low as 0.05% mutant DNA. The sensitivity achieved using deep sequencing was 91% concordant with the FGFR3 mutations observed in tissue. CONCLUSIONS We have developed a highly sensitive non-invasive urine based assay that can detect FGFR3 mutant DNA when present at < 1% of the sample and is > 90% concordance with the mutations found in tumor tissues. To our knowledge, this is the first practical application of next generation sequencing technology for diagnostic use.

Use of next-generation deep sequencing to improve FGFR3 mutation detection in the urine of patients with bladder cancer.

289 Background: FGFR3 mutations have been identified in ∼60-70% of low-stage, non-invasive tumors. Our group and others have developed assays to detect FGFR3 mutations in the urine of bladder cancer patients. However, urine-based assays have been limited by the technical ability to detect rare events in a dilute medium where there is a high background of normal DNA. In these assays, FGFR3 mutations are generally found in ∼30% of the urine samples, which is < 50% concordance with the expected detection in tissue. We have now developed an ultra-deep amplicon sequencing technique that increases FGFR3 mutation detection in urine to ∼67%, near the expected detection if every mutation found in tissue could be detected in urine. METHODS Amplicons were designed against FGFR3 exons 7, 10, and 15 using PCR primers containing the adapter sequences for unidirectional sequencing. Taqman probes were used to determine if sufficient DNA was present in each sample. Primary amplification was performed from DNA isolated from 4 ml of urine. The resulting PCR products were used as template for emulsion PCR and these were then sequenced using the Roche 454 GS Junior. Samples were analyzed for total DNA reads per sample and number of mutant sequencing reads to determine percent mutation. RESULTS Urine samples from 29 patients with stage Ta bladder cancer were analyzed by both our previously described qPCR method and the new ultra-deep sequencing approach. Of the 29 samples, 2 did not have sufficient DNA for analysis by sequencing. Using ultra-deep amplicon sequencing, 18 out of 27 (66.7%) were positive for FGFR3 mutations, while only 3 out of 27 (11.1%) were positive for mutations by qPCR. The urine samples from the 15 newly identified mutations using deep sequencing contained FGFR3 mutations as low as 0.05%. The sensitivity achieved using deep sequencing approximates the FGFR3 mutations observed in tissue. CONCLUSIONS We have developed a highly sensitive non-invasive urine based assay that can detect FGFR3 mutant DNA when present at < 1% of the sample and suggests > 90% concordance with the expected mutations in Ta tumor tissues. To our knowledge, this is the first practical application of next generation sequencing technology for diagnostic use.

Abstract 2093: Next-gen deep sequencing improves FGFR3 mutation detection in the urine of bladder cancer patients

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL FGFR3 mutations have been identified in ∼60-70% of low-stage, non-invasive tumors. Our group and others have developed assays to detect FGFR3 mutations in the urine of bladder cancer patients. However, urine-based assays have been limited by the technical ability to detect rare events in a dilute medium where there is a high background of normal DNA. In these assays, FGFR3 mutations are generally found in ∼30% of the urine samples, which is 90% concordance with tumor tissues. To our knowledge, this is the first practical application of next generation sequencing technology for diagnostic use. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2093. doi:1538-7445.AM2012-2093

Abstract 3870: Improved PCR amplification of DNA biomarkers with the use of chimeric oligonucleotides

Methylation of the Twist1 gene has been linked to various cancers, including bladder cancer. We are developing a non-invasive diagnostic assay utilizing urinary protein and DNA biomarkers, including Twist1, as triage monitors in high-risk bladder cancer populations. Since final clinical assay performance is dependent on the optimal technical performance of each individual marker, we incorporated the use of chimeric PCR primers to establish PCR conditions for all DNA markers that would greatly simplify the process of optimizing PCR performance. Amplification of difficult DNA templates can involve significant optimization of PCR conditions, and is often complicated by mispriming and other non-specific amplification events. The chimeric primers we designed have a variable 3’ target-specific sequence, and a common 22 nucleotide 5’ sequence that is unrelated to any human DNA. The common 5’ sequence generates similar melting temperatures for all primers (Tm 68-72oC) that permits uniform PCR conditions regardless of the characteristics of the template specific sequence. In addition, the chimeric primers permit a higher annealing temperature, which minimizes non-specific PCR amplification. In the study described here, conventional methylation specific primers were designed against a highly methylated region of the Twist1 promoter with and without the 5’ chimeric DNA sequence, and PCR conditions were optimized for each primer pair. Performance was assessed using DNA isolated from 50 bladder cancer tissues. After bisulfite conversion, PCR amplifications were carried out using the Bio-Rad C1000 platform and subsequently analyzed by agarose gel electrophoresis. Using traditional primers, Twist1 sensitivity was comparable to previous reports (68%, Renard et al, 2010), whereas using the chimeric primers, sensitivity increased to 90%. Incorporation of chimeric primers greatly simplifies PCR optimization and permits the use of higher annealing temperatures, resulting in higher specificity. We have applied this method to the detection of Twist1 methylation in bladder cancer tissues and demonstrate significant improvement in performance, when compared to traditional, non-chimeric oligonucleotide primers. In addition, this primer modification can be applied to multiple assay formats and DNA targets to improve sensitivity for use in non-invasive diagnostic assays, where mutant DNA is expected to be limiting. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3870. doi:10.1158/1538-7445.AM2011-3870

Abstract LB-313: Development of a non-invasive, multi-analyte assay for the triage of patients presenting with hematuria

Introduction The presence of blood in the urine (hematuria) is one of the hallmark symptoms of bladder cancer. Thus, the current best practice policy suggests that patients with hematuria undergo cystoscopic examination. However, hematuria can be present in healthy individuals as well as in individuals with various non-life threatening conditions such that a majority of patients with blood in the urine (∼95%) who are screened by cystoscopy do not have bladder cancer. In addition, recent studies have found that a majority of patients with hematuria are not referred on to a specialist for further invasive tests, despite the guidelines. This suggests that the prevalence of cancer in this population may be lower than 5%, making screening by cystoscopy inefficient and, in a large number of cases, unnecessary. We are developing a non-invasive diagnostic assay utilizing urinary protein and DNA biomarkers to triage those patients who do not have cancer from those who should go on to be existing invasive screening modalities. Methods Urine samples were obtained from 48 cancer patients and 256 patients who were evaluated for hematuria but who did not have cancer upon cystoscopic evaluation (Hem+/Cysto−). Twist1 and Nid2 methylation status was assessed using methylation-specific PCR primers and FGFR3 mutational status was determined by quantitative PCR. Matrix Metalloproteinases (MMPs) levels were determined by ELISA. Results Using a novel combination of these DNA and protein markers we identified with high confidence (99% negative predictive value) those patients who do not have cancer and who could be excluded from further testing. Cutoffs could be adjusted such that at 94% sensitivity, 65% of patients who do not have cancer could be excluded. Conclusions The Multi-Analyte Diagnostic Readout (MADR) assay described here combines the best performance characteristics of protein and DNA biomarkers into one assay for optimal clinical performance. Using this approach, the detection of FGFR3 mutations and Twist1 and Nid2 methylation in the urine of hematuria patients undergoing screening for bladder cancer effectively increases the sensitivity and negative predictive value at an established MMP protein cutoff. This non-invasive urinary diagnostic assay could lead to the more efficient triage of hematuria patients by identifying those patients who do not have cancer and could be excluded from receiving invasive procedures. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-313. doi:10.1158/1538-7445.AM2011-LB-313

Abstract 4161: Development of a real-time multiplex PCR assay for the detection of FGFR3 mutations in urine

271 Background: We have recently reported the development of a Multi-Analyte Diagnostic Readout (MADR) non-invasive assay using urinary matrix metalloproteinases (MMPs) and FGFR3 as triage monitors in high-risk bladder cancer populations. This concept combines the marker performance characteristics of protein and DNA biomarkers into one assay for optimal performance. Eight common FGFR3 mutations in 3 exons have been associated with bladder cancer. Analysis of mutational status for each single mutation required 8 amplification steps, which were costly and time consuming. We have now developed a real-time multiplexed FGFR3 assay, generating a cost-effective, clinically applicable assay for the detection of FGFR3 mutations in urine. METHODS Our approach involves a two-step PCR amplification process. The initial round generates exon specific PCR products, which are then used as template for real-time PCR mutation detection utilizing locked nucleic acid (LNA) oligonucleotides. The LNA suppress wild-type DNA amplification. To convert our existing FGFR3 assay to a multiplex format, primary amplifications of exons 7, 10, and 15 were combined into a single real-time PCR assay for exon specific amplification and DNA quantitation. The LNA-mediated mutation detection was then converted from 4 reactions to 2 duplex amplifications. All multiplex assays were carried out on the Roche LC 480 real-time PCR platform. RESULTS To validate the new multiplex format, FGFR3 multiplex analysis was performed on DNA isolated from 50 Ta stage bladder tumors. FGFR3 mutations were detected in 90% (48/50) of the tumors. To directly compare performance with single mutation analysis, 40 urine samples previously analyzed using the singleplex format were again tested using the multiplex FGFR3 assay. 100% concordance was seen between the two assay formats. CONCLUSIONS By multiplexing the FGFR3 mutation analysis we reduced the number of amplification steps, improving assay turnaround time and throughput, without compromising assay performance. The FGFR3 multiplex analysis provides a robust, cost-effective DNA assay that in combination with MMP protein analysis delivers a clinically applicable assay with optimal performance. [Table: see text].