Proficiency testing of PIK3CA mutations in HR+/HER2-breast cancer on liquid biopsy and tissue
Claudia Vollbrecht1, Inga Hoffmann2, Annika Lehmann2
1Charité-Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt-Universität zu Berlin and Berlin Institute of Health, Institute of Pathology, Berlin, Germany. claudia.vollbrecht@charite.de.
Abstract:
Precision oncology based on specific molecular alterations requires precise and reliable detection of therapeutic targets in order to initiate the optimal treatment. In many European countries-including Germany-assays employed for this purpose are highly diverse and not prescribed by authorities, making inter-laboratory comparison difficult. To ensure reproducible molecular diagnostic results across many laboratories and different assays, ring trials are essential and a well-established tool. Here, we describe the design and results of the ring trial for the detection of therapeutically relevant PIK3CA hotspot mutations in HR+/HER2-breast cancer tissue and liquid biopsy (LB). For PIK3CA mutation detection in tissue samples, 43 of the 54 participants (80%) provided results compliant with the reference values. Participants using NGS-based assays showed higher success rate (82%) than those employing Sanger sequencing (57%). LB testing was performed with two reference materials differing in the length of the mutated DNA fragments. Most participants used NGS-based or commercial real-time PCR assays (70%). The 167 bp fragments led to a successful PIK3CA mutation detection by only 31% of participants whereas longer fragments of 490 bp were detectable even by non-optimal assays (83%). In conclusion, the first ring trial for PIK3CA mutation detection in Germany showed that PIK3CA mutation analysis is broadly established for tissue samples and that NGS-based tests seem to be more suitable than Sanger sequencing. PIK3CA mutation detection in LB should be carried out with assays specifically designed for this purpose in order to avoid false-negative results.
Insights
This study evaluated PIK3CA mutation detection in breast cancer, finding NGS assays superior for tissue and liquid biopsies. Ring trials are crucial for ensuring accurate, reproducible molecular diagnostics across laboratories.
Area of Science:
- Oncology
- Molecular Diagnostics
- Genetics
Background:
- Precision oncology relies on accurate detection of molecular targets for optimal breast cancer treatment.
- Diverse diagnostic assays and lack of regulatory oversight in Germany hinder inter-laboratory comparisons.
- Ring trials are essential for ensuring reproducible molecular diagnostic results across laboratories and assays.
Purpose of the Study:
- To assess the accuracy and reliability of PIK3CA hotspot mutation detection in HR+/HER2- breast cancer tissue and liquid biopsies.
- To compare the performance of different molecular detection methods, including Next-Generation Sequencing (NGS) and Sanger sequencing.
- To evaluate the impact of DNA fragment length on PIK3CA mutation detection in liquid biopsies.
Main Methods:
- A ring trial was conducted involving 54 laboratories in Germany for PIK3CA mutation detection.
- Participants analyzed both tissue samples and liquid biopsy materials with varying mutated DNA fragment lengths.
- Assay types included Next-Generation Sequencing (NGS), Sanger sequencing, and real-time PCR.
Main Results:
- 80% of participants accurately detected PIK3CA mutations in tissue samples.
- NGS-based assays achieved a higher success rate (82%) compared to Sanger sequencing (57%) for tissue analysis.
- Liquid biopsy testing showed significantly lower success rates with shorter DNA fragments (31% for 167 bp) versus longer fragments (83% for 490 bp).
Conclusions:
- PIK3CA mutation analysis is well-established for breast cancer tissue samples, with NGS assays demonstrating superior performance.
- Liquid biopsy analysis for PIK3CA mutations requires specialized assays to avoid false-negative results, particularly concerning DNA fragment length.
- The ring trial highlighted the importance of standardized methods and appropriate assay selection for reliable molecular diagnostics in precision oncology.
