This classification is intended to support clinical practice as well as research investigation and clinical trials.

EGFR-mutant lung cancers eventually become resistant to treatment with EGFR tyrosine kinase inhibitors (TKIs). The combination of EGFR-TKI afatinib and anti-EGFR antibody cetuximab can overcome acquired resistance in mouse models and human patients. Since afatinib is also a potent HER2 inhibitor, we investigated the role of HER2 in EGFR-mutant tumor cells. We show in vitro and in vivo that afatinib plus cetuximab significantly inhibits HER2 phosphorylation. HER2 overexpression or knockdown confers resistance or sensitivity, respectively, in all studied cell line models. Fluorescent in situ hybridization analysis revealed that HER2 was amplified in 12% of tumors with acquired resistance versus only 1% of untreated lung adenocarcinomas. Notably, HER2 amplification and EGFR T790M were mutually exclusive. Collectively, these results reveal a previously unrecognized mechanism of resistance to EGFR TKIs and provide a rationale to assess the status and possibly target HER2 in EGFR mutant tumors with acquired resistance to EGFR TKIs.

The principles governing evolution of tumors exposed to targeted therapy are poorly understood. Here we modeled the selection and propagation of BRAF amplification (BRAFamp) in patient-derived tumor xenografts (PDX) treated with a direct ERK inhibitor, alone or in combination with other pathway inhibitors. Single cell sequencing and multiplex-fluorescence in situ hybridization mapped the emergence of extra-chromosomal amplification in parallel evolutionary tracts, arising in the same tumor shortly after treatment. The evolutionary selection of BRAFamp is determined by the fitness threshold, the barrier subclonal populations need to overcome to regain fitness in the presence of therapy. This differed for ERK signaling inhibitors, suggesting that sequential monotherapy is ineffective and selects for a progressively higher BRAF copy number. Concurrent targeting of RAF, MEK and ERK, however, imposes a sufficiently high fitness threshold to prevent the propagation of subclones with high-level amplification. Administered on an intermittent schedule, this treatment inhibited tumor growth in 11/11-lung cancer and melanoma PDX without apparent toxicity in mice. Thus, gene amplification can be acquired and expanded through parallel evolution, enabling tumors to adapt while maintaining their intratumoral heterogeneity. Treatments that impose the highest fitness threshold will likely prevent the evolution of resistance-causing alterations and merit testing in patients.