# Establishment of Reference Measurement Procedure for TP53 R175H/R248W Detection and a Novel Preparation Method for ctDNA Reference Material

## TL;DR

This study creates reliable methods to detect TP53 mutations in ctDNA and prepares reference materials to improve cancer biomarker testing accuracy.

## Contribution

The paper introduces a new dPCR method for TP53 variant detection and a novel ctDNA reference material preparation protocol.

## Key findings

- dPCR assays showed high repeatability (RSD 0.16%-7.65%) and excellent linearity (R² 1.0000-0.9981) for TP53 variants.
- ctDNA reference materials had dominant peaks at 128 bp (R175H) and 143 bp (R248W), mimicking clinical ctDNA properties.
- The dPCR assays successfully detected TP53 variants in prepared reference materials.

## Abstract

Background/Aims: Circulating tumor DNA (ctDNA) is becoming a valuable cancer biomarker for clinical decision-making. Nevertheless, the lack of quality control materials to assess the reliability of test results remains a challenge. This study aimed to establish digital PCR (dPCR) assays for detecting TP53 variants (R175H and R248W) and develop a preparation method for ctDNA reference materials to improve detection reliability. Methods: Two dPCR assays targeting TP53-R175H and TP53-R248W variants were developed and validated for repeatability, sensitivity, and linearity. Additionally, a ctDNA reference material preparation protocol was developed by digesting nucleosomes from cultured cancer cell lines with micrococcal nuclease, followed by magnetic beads purification. The size distribution and quality of the generated ctDNA fragments was analyzed, and the developed dPCR assays were applied to detect the variants in the ctDNA samples. Results: The dPCR assays demonstrated high repeatability (RSD of 0.16% to 7.65%) and excellent linearity (R2 values of 1.0000 and 0.9981) across variant allele frequencies of 50%–0.1%. The limits of detection (LOD) and quantification (LOQ) were 0.143% (R175H) and 0.092% (R248W). The ctDNA reference materials exhibited single dominant peaks at 128 bp (R175H) and 143 bp (R248W). The dPCR assays successfully detected variants in these reference materials, confirming their applicability for ctDNA samples. onclusion: Firstly, accurate measurement procedures for TP53-R175H and TP53-R248W variants based on dPCR were established in this study. Furthermore, a protocol for preparing ctDNA reference material was established here. By digesting nucleosomal DNA derived from cancer cell lines with micrococcal nuclease, this method can closely mimic the properties of clinical ctDNA. The dPCR method and ctDNA reference material preparation approach established here could be used in ctDNA detection and for improving its reliability.