# Pre-Amplification of Cell-Free DNA: Balancing Amplification Errors with Enhanced Sensitivity

## TL;DR

This paper explores a method to improve detection of cancer DNA in blood by amplifying it, while managing errors introduced during the process.

## Contribution

The study introduces optimized pre-amplification parameters for cell-free DNA using TOP-PCR to enhance ctDNA detection sensitivity.

## Key findings

- TOP-PCR pre-amplification preserved DNA size profiles with a consistent 22 bp increase.
- Optimized pre-amplification improved ctDNA detection sensitivity and sample availability for multiple tumor mutations.
- PCR errors in pre-amplified samples highlight the need for negative controls and strict mutation thresholds.

## Abstract

Circulating tumour DNA (ctDNA) is a promising biomarker for personalised oncology. However, its clinical utility is limited by detection sensitivity, particularly in early-stage disease. T-Oligo Primed Polymerase Chain Reaction (TOP-PCR) is a commercial amplification approach utilising an efficient “half-adapter” ligation design and a single-primer-based PCR strategy. This study evaluated the clinical value and application of cell-free DNA (cfDNA) pre-amplification. cfDNA amplification with TOP-PCR preserved DNA size profiles and resulted in a 22 bp size increase due to the half-adaptor ligation. Gene target amplification rates varied, showing lower efficiency for the GC-rich TERT promoter amplicon and higher efficiency for the BRAF and TP53 amplicons. Optimised pre-amplification (20 ng cfDNA input and 5–7 cycles of PCR) enhanced ctDNA detection sensitivity and expanded sample availability for the detection of multiple tumour-informed mutations. Importantly, PCR errors emerged in pre-amplified cfDNA samples, underscoring the necessity for negative controls and the establishment of stringent mutation positivity thresholds.