Analytical Performance of Next-Generation Sequencing and RT-PCR on Formalin-Fixed Paraffin-Embedded Tumor Tissues for Testing in HR+/HER2- Breast Cancer.

Somatic mutations in are present in ~40% breast cancers (BC); their detection in hormone receptor (HR)+/HER2- tumors allows for selecting patients with advanced disease eligible for targeting with alpelisib. The choice of what type of testing approach to adopt and which tissue sample to analyze is a new task in breast pathology. In this methodological study, we sought to assess the performance of next-generation sequencing (NGS) and RT-PCR for testing on archival formalin-fixed paraffin-embedded (FFPE) primary tumors and corresponding metastases. Sixteen HR+/HER2- BC with known -mutated status (ex. 7, 9, and 20) on metastatic samples by means of amplicon-based targeted NGS were selected, and = 13 of these samples were re-tested with a commercially available CE-IVD RT-PCR assay. All available primary tumors ( = 8) were tested with both methods. NGS detected mutations in all samples, while RT-PCR in = 2 sample-pairs and overall, in = 5/8 (62.5%) primary tumors and 7/13 (53.8%) metastases (κ = 0.09; 95% CI, -0.69-0.87). Slight agreement (κ = 0; 95% CI, -0.59-0.59) was observed between NGS and RT-PCR, with the former being generally more sensitive in cases with low DNA quality and quantity. Post hoc visual inspection of the RT-PCR data increased the concordance to 76.9%. Targeted NGS offers reliable and robust testing on both tumor and metastasis FFPE samples; the accuracy of RT-PCR depends on the DNA quantity and quality. In HR+/HER2- BC, both the selection of the testing strategy of FFPE tissues and which sample to analyze should consider several technical parameters and should be tailored for each case.