CHALLENGES FOR THE IDENTIFICATION ON EGFR EXON 20 INSERTION MUTATIONS IN LUNG CANCER: COMPARATIVE ANALYSIS NGS VERSUS QPCR DETECTION METHODS IN A LARGE BRAZILIAN COHORT

Introduction: EGFR exon 20 insertions (ins20) are found in about 2% of non-squamous non-small cell lung cancer (nsNSCLC) patients, representing close to 10% of all EGFR activating mutations. Although there is some heterogeneity, ins20 mutations are not sensitive to 1st to 3rd generation tyrosine kinase inhibitors (TKI). Novel targeted therapy approaches have significantly changed clinical outcomes in this subgroup of patients. However, for a long time, the identification of EGFR ins20 was a challenge. Aim: To assess the prevalence of EGFR ins20 in Brazilian patients with nsNSCLC using gold-standard large NGS panel (GS180 Archer DNA, OCPM lab) and discordances (not detected or “false negatives”) with orthogonal EGFR qPCR assay (Cobas®). Methods: nsNSCLC were prospectively recruited in the OCPM Lung Mapping platform from March 2020 to August 2022. The archived formalin-fixed paraffin-embedded (FFPE) tumor material from these cases underwent DNA extraction for EGFR ins20 evaluation by the GS180 and Cobas® in parallel. Results: Among the 400 cases of nsNSCLC evaluated for the EGFR gene, fifteen cases presented ins20 mutations (3.7%) by GS180. The p.Ser768_Asp770dup mutation was the most frequent in 4/15 cases, followed by p.Asn771_His773dup (2/15) and p.His773_Val774dup (2/15). All 7 remaining were unique. Out of these 15 cases, in 3 Cobas® failed and in 12 cases it was available for comparison: 2 p.Ser768_Asp770dup cases were detected by qPCR and additional 10 variants were negative for EGFR ins20 mut (not detected because variants were not covered by qPCR assay). Overall sensitivity of qPCR for EGFR ins20 was 13% and discordance rate with NGS was 83%. In eight cases (8/15 [53%]) the EGFR ins20 mutations were single drivers and in other cases we found coexisting alterations in TP53, ATM, PALP2 or RAD54L genes. Conclusion: The prevalence of EGFR ins20 mutations in Brazilian patients with nsNSCLC is slightly higher that data published elsewhere. Unlike other EGFR mutations, ins20 alterations present unique diagnostic challenges due to variant complexity that mandate high-quality NGS and optimized bioinformatics pipelines for accurate detection. A negative EGFR qPCR Cobas® must be followed by large NGS panel to complete the diagnostic journey.