Background: Multigene next-generation sequencing (NGS) has transformed molecular diagnostics in oncology over the past decade, increasingly replaced single-gene approaches such as Sanger sequencing. Although NGS is now integral to precision oncology, differences in laboratory infrastructure, workflow standardization, and quality assurance persist. This study evaluated regional laboratory performance in pan-cancer NGS testing, examined practical barriers to implementation, and identified strategies to improve diagnostic accuracy.

Methods: A multicenter RING Trial including external quality assessment (EQA) was conducted across five laboratories in Argentina, Brazil, Chile, Colombia, and Peru. Each laboratory analyzed 30 artificial FFPE reference samples containing predefined oncogenic variants using routine local workflows. Performance was centrally assessed by concordance between expected and reported variants. Findings were further contextualized through an expert discussion and a follow-up structured survey examining workflows, technical limitations, and bioinformatics practices.

Results: All five laboratories completed testing of the full sample set, yielding 150 evaluable laboratory-sample observations. The overall mean genotyping score was 1.46 out of 2.00 (range: 0.20–2.00). Performance was highest for less complex samples and declined for specimens with multiple concurrent alterations, splice-related events, or fusion-rich profiles. Thirty-five critical genotyping errors were recorded overall, an error rate of 23.3%. Expert discussion and survey findings indicated pre-analytical variability, early-stage wet-lab implementation, differences in bioinformatics filtering, and inconsistent reporting practices were major contributors to performance variability.

Conclusions: This regional EQA ring trial demonstrated that important implementation gaps in multigene NGS persist across Latin America, particularly for detecting molecularly complex alterations. Findings underscore EQA's value not only as a benchmarking tool but as a practical implementation support mechanism for laboratories introducing comprehensive genomic profiling. Strengthening pre-analytical standardization, bioinformatics governance, interpretive review, and regionally adapted quality frameworks may improve diagnostic accuracy and support more equitable access to high-quality precision oncology across Latin America.

Next-generation sequencing, Latin America, Precision oncology