EGFR R521K

Our results suggest that EGFR R521K, unlike reported previously, is unable to cause cetuximab resistance in HNSCC patients; therefore, its screening before therapy selection is not justifiable.

Interestingly, the cetuximab-resistant R521K tumors were successfully treated with c-MET tyrosine kinase inhibitor SU11274. Our results suggest that HNSCC cell line expressing the R521K mutant form of EGFR does not respond well to cetuximab treatment in vitro or in vivo, but hopefully might be targeted by c-MET tyrosine kinase inhibitor treatment.

This selectivity was not reflected in immunophenotype or survival data of HNSCC patients, suggesting a more complex mechanism behind. Interestingly, c-Met inhibitor SU11274 was more effective in cetuximab-resistant, EGFR R521K heterozygous cells and xenografts, raising the possible importance of simultaneous targeting of the two receptors.

Computational analysis of the genetic variants revealed a reduction in the stabilization of the EGFR tethered form for both V550M and the common R521K variant with low energetic state (- ∆∆G). Molecular interactions analysis suggested that these mutations might affect the receptor's function and promote tumorigenesis.

It is feasible to isolate high quality CTCs and cfDNA from the same blood collection tube to perform targeted sequencing; this streamlines specimen processing, decreases overall costs, and minimizes required blood volumes. Importantly, there is overlap in the majority of mutations identified in CTC-DNA and cfDNA, but actionable mutations (e.g., PIK3CA, EGFR) were detected in CTC-DNA only. The clinical and theranostic relevance of these findings is unclear and warrants further investigation.