We established two afatinib-resistant sublines, HCC827/BR1-8 (BR1-8) and HCC827/BR2-3 (BR2-3), after exposing the parental lung cancer cell line HCC827, which harbors a mutation of EGFR exon 19 (E746-A750) that activates tyrosine kinase activity, to step-wise increasing concentrations of afatinib up to 1 μmol/L (see Materials and Methods). Here, EGFR is linked to lung carcinoma.