Cytology smears can be Effectively Used on Two Different Fully Automated Real-Time PCR Platforms that have been Validated for Use Only with FFPE Tissue

Summary: Introduction: NSCLC requires accurate biomarker information for optimal treatment selection. Current guidelines mandate testing at least for EGFR, BRAF V600, KRAS, MET, HER2 mutations, ALK, ROS1, NTRK, RET translocations and PD-L1 expression. This testing can be performed on any cytological specimens. Although NGS is the standard procedure, Automated Real-Time PCR Systems are available to analyze limited gene panels with faster response times. In this study, we perform molecular testing on cytological smears, after DNA and RNA extraction, with both IDYLLA TM System (ID) and AmoyDx® Pan Lung Cancer Panel (PLC). Comparison between both was performed and against NGS when available. Materials and Methods: Thirty-two NSCLC patients diagnosed on cytological smears were analyzed. Each smear was classified as having high, medium, or low cellularity before nucleic acid extraction. An in-house protocol was applied for extraction from 34 Diff-Quik and 60 Papanicolau-stained smears. Biomarker analysis was performed using ID and PLC. NGS was available on 6 patients. Results: The average concentration of DNA and RNA was 90,21ng/µL and 122,9ng/µL, respectively, in the high cellularity category smears; 41,18ng/µL and 38,16ng/µL in the medium; and 22,98ng/µL and 62,42ng/µL in the low cellular cases, respectively. Both ID and PLC ID detected the same 6 EGFR and 12 KRAS mutations, along with fusions involving MET in 1 case, RET in 1 case, and ALK in 3 cases, respectively. In addition, ID detected two KRAS mutations not studied by PLC. A comparison between both systems showed 100% concordance, as seen in cases where NGS data was available. Conclusions: Cytological stained smears (DQ and PAP) are feasible for performing Fully Automated Real-Time PCR Systems such as ID or PLC with an adequate protocol to obtain high-yield DNA and RNA. Both systems showed 100% concordance, as did NGS when available. While these systems do not replace NGS, they offer faster response times, enabling early targeted treatment.