CLINICAL EVIDENCE

Methodology for Single Copy Detection and Quantitative Monitoring of Clinically Actionable Circulating Tumor DNA Mutations in Urine from Cancer Patients

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Methodology for Single Copy Detection and Quantitative Monitoring of Clinically Actionable Circulating Tumor DNA Mutations in Urine from Cancer Patients

Introduction

  • Non-invasive detection and monitoring of ctDNA mutations for personalized treatment of cancer patients can be realized by combining practical advantages of urine as a ctDNA sample source with high throughput of next-generation sequencing (NGS).
  • We developed a platform that combines an extraction process capable of isolating transrenal ctDNA from the entire void volume of a urine sample with an ultra- sensitive NGS-integrated mutation enrichment method with single copy detection sensitivity.
  • Assays have been developed and validated to interrogate clinically actionable mutations/deletions in the KRAS G12/13, BRAF V600E and EGFR (Exons 19, 20, 21) oncogenes in both urine and plasma ctDNA samples.

Urinary ctDNA Extraction and Comparison to Plasma

Urinary ctDNA extraction

  • Urinary ctDNA extraction method that utilizes anion-exchange-based DNA isolation methodology for selective enrichment of highly fragmented urinary ctDNA was developed. An upstream urine concentration step enables isolation of DNA from an entire void of urine (~100mL).

Highlighted Tables and Graphs

Analytical Performance Characterization

Urinary ctDNA Extraction and Comparison to Plasma

Monitoring Urine ctDNA EGFR for Response to Therapy

Conclusions

  • Quantitative ctDNA assays using enrichment PCR followed by NGS were developed to detect/monitor KRAS and EGFR mutational load in urine and plasma. These ultrasensitive assays have a LLOD of 1 (KRAS, EGFR L858R, EGFR ex19del) or 2 (EGFR T790M) copies in a background of ≈ 20,000 wild-type genome equivalents (0.0055% – 0.011% sensitivity). Accurate quantitation and linearity are achieved across verified reportable range.
  • In KRAS tissue positive patients, the median amount of total ctDNA as well as mutant ctDNA was 10 times higher in a sample of urine versus sample of blood plasma.
  • Clinical utility of the Trovagene platform is supported by ongoing clinical studies that demonstrate correlation of urinary and plasma ctDNA levels with tumor burden, response to therapy, disease progression, and monitoring of minimal residual disease.
  • As a patient-friendly specimen, urine enables frequent monitoring of ctDNA, and this accessibility can be applied to investigating mechanisms of action of targeted therapeutics and, ultimately, cancer management.
Trovera

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