EGFR Mutation Analysis by PCR

Clinical Significance
Recurrent somatic mutations in the tyrosine kinase domain ﴾TKD﴿ of EGFR are observed in approximately 10‐20% of lung adenocarcinoma, and at higher frequencies in never‐smoker, female, andAsian populations[PMID: 20388509; PMID: 25079552; PMID: 24071849; PMID: 22588877]. The most common mutations occur near the ATP‐binding pocket of the TKD and include short in‐frame deletions in exon 19 ﴾EGFR exon 19 deletion﴿ and the L858R amino acid substitution in exon 21[PMID: 20887192]. These mutations constitutively activate EGFR resulting in downstream signaling, and represent 80% of the EGFR mutations observed in lung cancer. A second group of less prevalent activating mutations include E709K, G719X, S768I, L861Q, and short in‐frame insertion mutations in exon 20[PMID: 23371856; PMID: 26206867; PMID: 24353160; PMID: 25668120]. EGFR activating mutations in lung cancer tend to be mutually exclusive to KRAS activating mutations[PMID: 23122493]. In contrast, a different set of recurrent activating EGFR mutations in the extracellular domain include R108K, A289V and G598V and are primarily observed in
glioblastoma[PMID: 24120142; PMID: 20887192]. Amplification of EGFR is observed in several cancer types including 30% of glioblastoma, 12% of esophageal cancer, 10% of head and neck cancer, 5% of bladder cancer, and 5% of lung squamous cell carcinoma[PMID: 24120142; PMID: 25631445;PMID: 25079552;PMID: 24071849;PMID: 22588877]. Deletion of exons 2‐7, encoding the extracellular domain of EGFR ﴾EGFRvIII﴿, results in overexpression of a ligand‐independent constitutively active protein and is observed in approximately 30% of glioblastoma[PMID: 19922469; PMID: 19680293; PMID: 19324552].

Approved first‐generation EGFR tyrosine kinase inhibitors﴾TKIs﴿ include erlotinib[FDA ‐ erlotinib: TARCEVA] ﴾2004﴿ and gefitinib[FDA ‐ gefitinib: IRESSA] ﴾2015﴿, which block the activation of downstream signaling by reversible interaction with the ATP ‐ binding site. Although initially approved for advanced lung cancer, the discovery that drug sensitivity was associated with exon 19 and exon 21 activating mutations allowed first ‐ generation TKIs to become subsequently approved for front ‐ line therapy in lung cancer tumors containing exon 19 or exon 21 activating mutations.Second ‐ generation TKIs afatinib[FDA ‐ afatinib: GILOTRIF]﴾2013﴿ and dacomitinib[FDA ‐ dacomitinib: VIZIMPRO]﴾2018﴿ bind EGFR and other ERBB / HER gene family members irreversibly and were subsequently approved. First ‐ and second ‐ generation TKIs afatinib, dacomitinib, erlotinib, and gefitinib are recommended for the treatment NSCLC harboring EGFR exon 19 insertions, exon 19 deletions, point mutations L861Q, L858R, S768I, and codon 719 mutations, whereas most EGFR exon 20 insertions, except p.A763_Y764insFQEA, confer resistance to the same therapies[NCCN ‐ Non ‐ Small Cell Lung Cancer; PMID: 26096453; PMID: 30854234; PMID: 24891042]. However, in 2021, the irreversible tyrosine kinase inhibitor, mobocertinib[FDA ‐ mobocertinib: EXKIVITY]was FDA approved for the treatment of NSCLC with EGFR exon 20 insertion mutations. Additionally, in 2022, the FDA granted breakthrough therapy designation to the
irreversible EGFR inhibitors, CLN ‐ 081﴾TPC ‐ 064﴿[FDA ‐ TPC ‐ 064: Breakthrough] and sunvozertinib[FDA ‐ sunvozertinib: Breakthrough], for locally advanced or metastatic non ‐ small cell lung cancer harboring EGFR exon 20 insertion mutations.

In lung cancer containing EGFR exon 19 or 21 activating mutations, treatment with TKIs is eventually associated with the emergence of drug resistance[PMID: 30647840].The primary resistance mutation that emerges following treatment with first ‐ generation TKI is T790M, accounting for 50 ‐ 60 % of resistant cases[PMID: 20887192].Third generation TKIs were developed to maintain
sensitivity in the presence of T790M.Osimertinib[FDA ‐ osimertinib: TAGRISSO]﴾2015﴿ is an irreversible inhibitor indicated for metastatic EGFR T790M positive lung cancer and for the first‐line treatment of metastatic NSCLC containing EGFR exon 19 deletions or exon 21 L858R mutations. Like first ‐ generation TKIs, treatment with osimertinib is associated with acquired resistance. In this case, resistance is associated with the C797S mutation and occurs in 22 ‐ 44 % of cases[PMID: 30647840].The T790M and C797S mutations may be each selected following sequential treatment with a first ‐ generation TKI followed by a third ‐ generation TKI or vice versa[PMID: 25964297].T790M and C797S can occur in either cis or trans allelic orientation[PMID: 25964297].If C797S is observed following progression after treatment with a third ‐ generation TKI in the first ‐ line setting, sensitivity may be retained to first ‐ generation TKIs[PMID: 25964297].If C797S co ‐ occurs in trans with T790M following sequential treatment with first ‐ and third ‐ generation TKIs, patients may exhibit sensitivity to combination first ‐ and third ‐ generation TKIs, but resistance to third ‐ generation TKIs alone[PMID: 25964297; PMID: 28662863]. However, C797S occurring in cis conformation with T790M, confers resistance to first ‐ and third ‐ generation TKIs[PMID: 25964297].Fourth ‐ generation TKIs are in development to overcome acquired C797S and T790M resistance mutations after osimertinib treatment. EGFR targeting antibodies including cetuximab﴾2004﴿, panitumumab﴾2006﴿, and necitumumab﴾2016﴿ are under investigation in combination with EGFR ‐ targeting TKIs for efficacy against EGFR mutations. The bispecific antibody, amivantamab[FDA ‐ amivantamab: RYBREVANT], targeting EGFR and MET was approved﴾2021﴿ NSCLC tumors harboring EGFR exon 20 insertion mutations.The Oncoprex immunogene therapy quaratusugene ozeplasmid[FDA ‐ quaratusugene ozeplasmid: Fast Track] in combination with osimertinib received a fast track designation from the FDA ﴾2020﴿ for NSCLC tumors harboring EGFR
mutations that progressed on osimertinib alone. BDTX ‐ 189[FDA ‐ BDTX ‐ 189: Fast Track] was granted a fast track designation﴾2020﴿ for the treatment of solid tumors harboring an EGFR exon 20 insertion mutation.

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