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

Every week, the Editor-in-Chief selects an Article of the Week from the current issue of BJUI. The abstract is reproduced below and you can click on the button to read the full article, which is freely available to all readers for at least 30 days from the time of this post.

In addition to the article itself, there is an editorial written by a prominent member of the urological community and a video prepared by the authors. These are intended to provoke comment and discussion and we invite you to use the comment tools at the bottom of each post to join the conversation. 

If you only have time to read one article this week, it should be this one.

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

Douglas G. Ward*, Naheema S. Gordon*, Rebecca H. Boucher*, Sarah J. Pirrie*, Laura Baxter, Sascha Ott, Lee Silcock, Celina M. Whalley*, Joanne D. Stockton*, Andrew D. Beggs*, Mike Griffiths§, Ben Abbotts*, Hanieh Ijakipour*, Fathimath N.Latheef*, Robert A. Robinson*, Andrew J. White*, Nicholas D. James*, Maurice P.Zeegers, K. K. Cheng** and Richard T. Bryan*

 

*Institute of Cancer and Genomic Sciences, University of Birmingham, Birmingham, Department of Computer Science, University of Warwick, Coventry, Nonacus Limited, Birmingham Research Park, §West Midlands Regional Genetics Laboratory, Birmingham Women’s and Children’s NHS Foundation Trust, Birmingham, UK, NUTRIM School for Nutrition and Translational Research in Metabolism and CAPHRI Care and Public Health Research Institute, Maastricht University, Maastricht, The Netherlands and **Institute of Applied Health Research, University of Birmingham, Birmingham, UK

Abstract

Objectives

To develop a focused panel of somatic mutations (SMs) present in the majority of urothelial bladder cancers (UBCs), to investigate the diagnostic and prognostic utility of this panel, and to compare the identification of SMs in urinary cell‐pellet (cp) DNA and cell‐free (cf) DNA as part of the development of a non‐invasive clinical assay.

Patients and Methods

A panel of SMs was validated by targeted deep‐sequencing of tumour DNA from 956 patients with UBC. In addition, amplicon and capture‐based targeted sequencing measured mutant allele frequencies (MAFs) of SMs in 314 urine cpDNAs and 153 urine cfDNAs. The association of SMs with grade, stage and clinical outcomes was investigated by univariate and multivariate Cox models. Concordance between SMs detected in tumour tissue and cpDNA and cfDNA was assessed.

 

Results

The panel comprised SMs in 23 genes: TERT (promoter), FGFR3, PIK3CA, TP53, ERCC2, RHOB, ERBB2, HRAS, RXRA, ELF3, CDKN1A, KRAS, KDM6A, AKT1, FBXW7, ERBB3, SF3B1, CTNNB1, BRAF, C3orf70, CREBBP, CDKN2A and NRAS; 93.5–98.3% of UBCs of all grades and stages harboured ≥1 SM (mean: 2.5 SMs/tumour). RAS mutations were associated with better overall survival (P = 0.04). Mutations in RXRA, RHOB and TERT (promoter) were associated with shorter time to recurrence (P < 0.05). MAFs in urinary cfDNA and cpDNA were highly correlated; using a capture‐based approach, >94% of tumour SMs were detected in both cpDNA and cfDNA.

Conclusions

SMs are reliably detected in urinary cpDNA and cfDNA. The technical capability to identify very low MAFs is essential to reliably detect UBC, regardless of the use of cpDNA or cfDNA. This 23‐gene panel shows promise for the non‐invasive diagnosis and risk stratification of UBC.

 

Editorial: Non‐invasive diagnosis and monitoring of urothelial bladder cancer: are we there yet?

In this issue of BJUI, Ward et al. [1] describe the development of DNA‐based urinary biomarkers for urothelial carcinoma (UC). The genomics of UC have been well characterized through interrogation of tumour issues in institutional series (e.g. the Memorial Sloan Kettering Cancer Center [MSKCC] experience), multi‐institutional collaborations (e.g. The Cancer Genome Atlas [TCGA]) and commercial platforms (e.g. the Foundation Medicine experience) [2]. Until recently, these have been largely academic pursuits, with possible impact on prognostication but limited clinical applicability and utility for therapy selection and monitoring of response; however, with the US Food and Drug Administration approval of erdafitinib several weeks ago, patients with advanced UC will routinely receive genomic assessment for FGFR2/3 mutation or fusion, the targets for this therapy [3]. In due time, it is anticipated that multiple other putative targets with associated therapies (e.g. ERBB2, CDKN2A), as well as potential predictive biomarkers, may also warrant testing.

The evolving landscape in advanced UC makes a non‐invasive biomarker particularly attractive. The authors of the present commentary have previously reported results from a series of 369 patients with advanced UC, demonstrating that genomic alterations in ctDNA could be identified in 91% of patients using a commercially available 73-gene panel [4]. More recently, Christensen et al. [5] assessed a cohort of 68 patients receiving neoadjuvant chemotherapy for muscle‐invasive disease, demonstrating 100% sensitivity and 98% specificity for the detection of relapsed disease with a patient‐specific ctDNA assessment (sequenced to a median target coverage of 105 000×) after cystectomy. Impressively, the data also showed that the dynamics of ctDNA appeared to be more useful than pathological downstaging in predicting relapse.

In contrast to these studies, Ward et al. have developed a 23‐gene panel based on frequently expressed genes in a cohort of 916 UC tissue specimens, largely derived from patients with non‐muscle‐invasive disease. Ultimately, with a cohort of 314 patients with DNA derived from a urinary cell pellet, sequencing identified 645 (71.4%) of 903 mutations detected in tumour. Using urinary supernatant, 353 (80.7%) of 437 mutations were detected. These relatively high sensitivities, if they can be interpreted as such, are promising but do not rise to the level of replacing existing strategies for UC detection, staging and monitoring. Notably, another study demonstrated that urinary ctDNA can be detected with high sensitivity and specificity in patients with localized early‐stage bladder cancer and for after‐treatment surveillance, providing the foundation for further studies evaluating the role of ctDNA in non‐invasive detection, genotyping and monitoring [6].

Beyond its use as a diagnostic tool, it is hoped that urinary ctDNA may also find applications in the selection of therapeutics. To this end, Ward et al. identified FGFR3, PIK3CA, ERCC2 and ERBB2 mutations in 45%, 32%, 14% and 7% of patients, respectively. The frequency of FGFR3 alteration decreased with increasing stage and grade, ranging from 72% in pTaG1 disease to just 13% in ≥pT2 disease, consistent with other reports [7]. These results may guide forthcoming studies evaluating FGFR inhibitors in non‐muscle‐invasive, muscle‐invasive and metastatic disease, where studies are ongoing. In reviewing the potential link between genomic alterations and clinical outcomes, perhaps the most curious finding is that between RAS mutations and improved overall survival (P = 0.04), the only such association found in multivariate analysis. These results stand in sharp contrast to reports in lung cancer, colorectal cancer and multiple other tumour types [8]. A closer look at the deleterious nature and functional impact of NRAS and KRAS mutations seen in this series is certainly warranted, along with further external validation in a more homogenous and larger patient population. There is also the potential application of monitoring treatment response by assessing eradication of urinary ctDNA, a hypothesis that is being evaluated in ongoing studies [9].

How will the results of this and other emerging urinary biomarker studies eventually make their way to the clinic? The answer is simple: incorporation of these biomarkers in prospective therapeutic trials. As the bladder cancer investigative community formulates novel trials for non‐muscle‐invasive and muscle‐invasive disease using targeted therapies, an excellent opportunity exists to correlate urinary, blood and tissue‐based biomarkers and to assess their relative predictive capabilities and clinical utility. Furthermore, with clinical surrogate endpoints likely to drive regulatory approval (e.g. landmark complete response rates for non‐muscle‐invasive disease, or pT0N0 rate for muscle‐invasive disease), a validated urinary biomarker could ultimately offer an alternative biological surrogate endpoint [10]. In an era of genomic revolution, prospective validation can help establish the potential clinical utility of promising biomarkers and help realize the dream of ‘precision oncology’.

by Rohit K. Jain, Petros Grivas and Sumanta K. Pal

References

  1. Ward DGGordon NSBoucher RH et al. Targeted deep sequencing of urothelial bladder cancers and associated urinary DNA: a 23‐gene panel with utility for non‐invasive diagnosis and risk stratification. BJU Int 2019
  2. Schiff JPBarata PCYu EYGrivas PPrecision therapy in advanced urothelial cancer. Expert Rev Precis Med Drug Dev 2019481– 93
  3. FDA grants accelerated approval to erdafitinib for metastatic urothelial carcinoma [press release] 2019.
  4. Agarwal NPal SKHahn AW et al. Characterization of metastatic urothelial carcinoma via comprehensive genomic profiling of circulating tumor DNA. Cancer 20181242115– 24
  5. Christensen EBirkenkamp‐Demtroder KSethi H et al. Early detection of metastatic relapse and monitoring of therapeutic efficacy by ultra‐deep sequencing of plasma cell‐free DNA in patients with urothelial bladder carcinoma. J Clin Oncol 2019371547– 57
  6. Dudley JCSchroers‐Martin JLazzareschi DV et al. Detection and surveillance of bladder cancer using urine tumor DNA. Cancer Discov 20199500– 9
  7. Tomlinson DCBaldo OHarnden PKnowles MAFGFR3 protein expression and its relationship to mutation status and prognostic variables in bladder cancer. J Pathol 200721391– 8
  8. Zhuang RLi SLi Q et al. The prognostic value of KRAS mutation by cell‐free DNA in cancer patients: a systematic review and meta‐analysis. PLoS One 201712e0182562
  9. Abbosh PHPlimack ERMolecular and clinical insights into the role and significance of mutated DNA repair genes in bladder cancer. Bladder Cancer 201849– 18
  10. Jarow JPLerner SPKluetz PG et al. Clinical trial design for the development of new therapies for nonmuscle‐invasive bladder cancer: report of a Food and Drug Administration and American Urological Association public workshop. Urology 201483262– 4

 

 

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