Monitoring of urothelial cancer disease status after treatment by digital droplet PCR liquid biopsy assays

Urol Oncol. 2020 Sep;38(9):737.e1-737.e10. doi: 10.1016/j.urolonc.2020.05.012. Epub 2020 Jun 10.

Abstract

Objectives: Real-time monitoring of disease status would be beneficial for timely decision making in the treatment of urothelial cancer (UC), and may accelerate the evaluation of clinical trials. Use of cell free tumor DNA (cftDNA) as a biomarker in liquid biopsy is minimally invasive and its successful use has been reported in various cancer types, including UC. The objective of this study was to evaluate the use of digital droplet PCR (ddPCR)-based assays to monitor UC after treatment.

Method and materials: Blood, urine and matching formalin fixed, paraffin embedded diagnostic specimens were collected from 20 patients diagnosed with stage T1 (n = 2) and T2/T3 (n = 18) disease. SNaPshot assays, Sanger sequencing and whole exome sequencing were used to identify tumor-specific mutations, and somatic mutation status was confirmed using patient-matched DNAs extracted from buffy coats and peripheral blood mononucleocytes. The ddPCR assays of the tumor-specific mutations were used to detect the fractional abundance of cftDNA in plasma and urine.

Results: SNaPshot and Sanger sequencing identified point mutations in 70% of the patients that were assayable by ddPCR. Cases of remission and relapse monitored by assays for PIK3CA E542K and TP53 Y163C mutations in plasma and urine concurred with clinical observations up to 48 months from the start of chemotherapy. A new ddPCR assay for the telomerase reverse transcriptase (TERT) promoter (-124) mutation was developed. The TERT assay was able to detect mutations in cases below the limit of detection by SNaPshot. Whole exome sequencing identified a novel mutation, CNTNAP4 G727*. A ddPCR assay designed to detect this mutation was able to distinguish mutant from wild-type alleles.

Conclusions: The study demonstrated that ddPCR assays could be used to detect cftDNA in liquid biopsy monitoring of the post-therapy disease status in patients with UC. Overall, 70% of the patients in our study harbored mutations that were assayable by ddPCR.

Keywords: Cell free DNA; Circulating tumor DNA; Digital droplet PCR; Molecular diagnosis; Urothelial carcinoma; Whole exome sequencing.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adult
  • Aged
  • Aged, 80 and over
  • Biomarkers, Tumor
  • Carcinoma, Transitional Cell / chemistry
  • Carcinoma, Transitional Cell / genetics*
  • Carcinoma, Transitional Cell / metabolism
  • Carcinoma, Transitional Cell / pathology*
  • Circulating Tumor DNA / analysis*
  • Circulating Tumor DNA / blood
  • Circulating Tumor DNA / urine
  • Feasibility Studies
  • Female
  • Humans
  • Liquid Biopsy
  • Male
  • Middle Aged
  • Mutation
  • Polymerase Chain Reaction / methods*
  • Urinary Bladder Neoplasms / chemistry
  • Urinary Bladder Neoplasms / genetics*
  • Urinary Bladder Neoplasms / metabolism
  • Urinary Bladder Neoplasms / pathology*

Substances

  • Biomarkers, Tumor
  • Circulating Tumor DNA