Development and validation of a next-generation sequencing panel for the non-invasive detection of urothelial bladder cancer utilising common somatic mutations in urine DNA
BAUS ePoster online library. Ward D. 11/10/20; 304219; P12-1
Dr. Douglas Ward
Dr. Douglas Ward
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Development and validation of a next-generation sequencing panel for the non-invasive detection of urothelial bladder cancer utilising common somatic mutations in urine DNA

Ward D1, Gordon N1, Baxter L2, Wang J3, Ott S2, Zeegers M4, Cheng K1, James N1, Bryan R1
1University of Birmingham, United Kingdom, 2University of Warwick, Coventry, United Kingdom, 3University Hospitals Birmingham NHS Foundation Trust, United Kingdom, 4Maastricht University, The Netherlands

Background:
Next generation sequencing (NGS) of urine DNA may enable reliable non-invasive detection of urothelial bladder cancer (UBC).

Objectives:
1) To develop a panel of somatic mutations for detection of UBC, 2) to optimise methodology to generate a non-invasive detection test, and 3) to prospectively assess the panel in haematuria and surveillance clinic patients.

Methods:
A panel of mutations was developed and tested with DNA from 956 UBCs using multiplex-PCR NGS. The utility of the panel for non-invasive UBC detection was refined using a 'training set' of urine pellet DNAs from 403 incident UBC patients and 102 UBC-free control subjects. The panel was then tested using two 'test sets' of urine pellet DNAs from >400 haematuria clinic patients and >400 surveillance clinic patients. We also evaluated the advantages of capture-based library preparation incorporating unique molecular identifiers (UMIs).

Results:
We have developed a panel of 451 mutations in 23 genes that are suitable for UBC detection. In the training set we obtain 85% sensitivity (95% CI 81-88%) at 90% specificity (83-95%) based on multiplex-PCR NGS data. Test set performance based on multiplex-PCR NGS data will be reported. Inclusion of non-coding mutation hotspots in 5 additional genes improves sensitivity and the evaluation of UMIs shows successful noise suppression and improved mutation detection leading to higher sensitivity and specificity.

Conclusions:
Multiplex-PCR NGS analysis of urine pellet DNA achieves UBC detection at sensitivity and specificity that approaches clinical utility; improved library preparation incorporating UMIs improves performance further.
Development and validation of a next-generation sequencing panel for the non-invasive detection of urothelial bladder cancer utilising common somatic mutations in urine DNA

Ward D1, Gordon N1, Baxter L2, Wang J3, Ott S2, Zeegers M4, Cheng K1, James N1, Bryan R1
1University of Birmingham, United Kingdom, 2University of Warwick, Coventry, United Kingdom, 3University Hospitals Birmingham NHS Foundation Trust, United Kingdom, 4Maastricht University, The Netherlands

Background:
Next generation sequencing (NGS) of urine DNA may enable reliable non-invasive detection of urothelial bladder cancer (UBC).

Objectives:
1) To develop a panel of somatic mutations for detection of UBC, 2) to optimise methodology to generate a non-invasive detection test, and 3) to prospectively assess the panel in haematuria and surveillance clinic patients.

Methods:
A panel of mutations was developed and tested with DNA from 956 UBCs using multiplex-PCR NGS. The utility of the panel for non-invasive UBC detection was refined using a 'training set' of urine pellet DNAs from 403 incident UBC patients and 102 UBC-free control subjects. The panel was then tested using two 'test sets' of urine pellet DNAs from >400 haematuria clinic patients and >400 surveillance clinic patients. We also evaluated the advantages of capture-based library preparation incorporating unique molecular identifiers (UMIs).

Results:
We have developed a panel of 451 mutations in 23 genes that are suitable for UBC detection. In the training set we obtain 85% sensitivity (95% CI 81-88%) at 90% specificity (83-95%) based on multiplex-PCR NGS data. Test set performance based on multiplex-PCR NGS data will be reported. Inclusion of non-coding mutation hotspots in 5 additional genes improves sensitivity and the evaluation of UMIs shows successful noise suppression and improved mutation detection leading to higher sensitivity and specificity.

Conclusions:
Multiplex-PCR NGS analysis of urine pellet DNA achieves UBC detection at sensitivity and specificity that approaches clinical utility; improved library preparation incorporating UMIs improves performance further.
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