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Large-scale screening of patients with lung cancer for EGFR mutations is feasible and can have a role in decisions about treatment.

Purpose PD-1 inhibitors are established agents in the management of non-small cell lung cancer (NSCLC); however, only a subset of patients derives clinical benefit. To determine the activity of PD-1/PD-L1 inhibitors within clinically-relevant molecular subgroups, we retrospectively evaluated response patterns among EGFR-mutant, ALK-positive, and EGFR wild-type/ALK-negative patients. Experimental Design We identified 58 patients treated with PD-1/PD-L1 inhibitors. Objective response rates (ORRs) were assessed using RECIST v1.1. PD-L1 expression and CD8+ tumor infiltrating lymphocytes (TILs) were evaluated by immunohistochemistry. Results Objective responses were observed in 1/28 (3.6%) EGFR-mutant or ALK-positive patients versus 7/30 (23.3%) EGFR wild-type and ALK-negative/unknown patients (P = 0.053). The ORR among never- or light- (≤10 pack years) smokers was 4.2% versus 20.6% among heavy smokers (P = 0.123). In an independent cohort of advanced, EGFR-mutant (N=68) and ALK-positive (N=27) patients, PD-L1 expression was observed in 24%/16%/11% and 63%/47%/26% of pre-tyrosine kinase inhibitor (TKI) biopsies using cutoffs of ≥1%, ≥5% and ≥50% tumor cell staining, respectively. Among EGFR-mutant patients with paired, pre- and post-TKI resistant biopsies (N=57), PD-L1 expression levels changed after resistance in 16 (28%) patients. Concurrent PD-L1 expression (≥5%) and high levels of CD8+ TILs (grade ≥2) were observed in only 1 pre-treatment (2.1%) and 5 resistant (11.6%) EGFR-mutant specimens, and was not observed in any ALK-positive, pre- or post-TKI specimens. Conclusion NSCLCs harboring EGFR mutations or ALK rearrangements are associated with low ORRs to PD-1/PD-L1 inhibitors. Low rates of concurrent PD-L1 expression and CD8+ TILs within the tumor microenvironment may underlie these clinical observations.

Epidermal growth factor receptor (EGFR) gene mutations (G719X, exon 19 deletions/insertions, L858R and L861Q) predict favorable responses to EGFR tyrosine kinase inhibitors (TKIs) in advanced non-small-cell lung cancer (NSCLC). However, EGFR exon 20 insertion mutations (∼10% of all EGFR mutations) are generally associated with insensitivity to available TKIs (gefitinib, erlotinib and afatinib). The basis of this primary resistance is poorly understood.  We study a broad subset of exon 20 insertion mutations, comparing in vitro TKI sensitivity with responses to gefitinib and erlotinib in NSCLC patients; and find that most are resistant to EGFR TKIs. The crystal structure of a representative TKI-insensitive mutant (D770_N771insNPG) reveals an unaltered ATP-binding pocket and the inserted residues form a wedge at the end of the C-helix that promotes the active kinase conformation. Unlike EGFR-L858R, D770_N771insNPG activates EGFR without increasing its affinity for EGFR TKIs. Unexpectedly, we find that EGFR-A763_Y764insFQEA is highly sensitive to EGFR TKIs in vitro; and patients whose NSCLCs harbor this mutation respond to erlotinib. Analysis of the A763_Y764insFQEA mutant indicates that the inserted residues shift the register of the C-helix in the N-terminal direction, altering the structure in the region that is also affected by the TKI-sensitive EGFR-L858R. Our studies reveal intricate differences between EGFR mutations, their biology and their response to EGFR TKIs.

Tyrosine kinase inhibitors (TKIs) are effective treatments for non-small cell lung cancers (NSCLCs) with epidermal growth factor receptor (EGFR) mutations. However, relapse typically occurs after an average of one year of continuous treatment. A fundamental histological transformation from NSCLC to small cell lung cancer (SCLC) is observed in a subset of the resistant cancers, but the molecular changes associated with this transformation remain unknown. Analysis of tumor samples and cell lines derived from resistant EGFR mutant patients revealed that RB is lost in 100% of these SCLC transformed cases, but rarely in those that remain NSCLC. Further, increased neuroendocrine marker and decreased EGFR expression as well as greater sensitivity to BCL2 family inhibition are observed in resistant SCLC transformed cancers compared to resistant NSCLCs. Together, these findings suggest that this subset of resistant cancers ultimately adopt many of the molecular and phenotypic characteristics of classical SCLC.

Acquired resistance to EGF receptor (EGFR) tyrosine kinase inhibitors (TKIs) is inevitable in metastatic EGFR-mutant lung cancers. Here, we modeled disease progression using EGFR-mutant human tumor cell lines. Although five of six models displayed alterations already found in humans, one harbored an unexpected secondary NRAS Q61K mutation; resistant cells were sensitive to concurrent EGFR and MEK inhibition but to neither alone. Prompted by this finding and because RAS/RAF/MEK mutations are known mediators of acquired resistance in other solid tumors (colon cancers, gastrointestinal stromal tumors, and melanomas) responsive to targeted therapies, we analyzed the frequency of secondary KRAS/NRAS/ BRAF/MEK1 gene mutations in the largest collection to date of lung cancers with acquired resistance to EGFR TKIs. No recurrent NRAS, KRAS, or MEK1 mutations were found in 212, 195, or 146 patient samples, respectively, but 2 of 195 (1%) were found to have mutations in BRAF (G469A and V600E). Ectopic expression of mutant NRAS or BRAF in drug-sensitive EGFR-mutant cells conferred resistance to EGFR TKIs that was overcome by addition of a MEK inhibitor. Collectively, these positive and negative results provide deeper insight into mechanisms of acquired resistance to EGFR TKIs in lung cancer and inform ongoing clinical trials designed to overcome resistance. In the context of emerging knowledge about mechanisms of acquired resistance to targeted therapies in various cancers, our data highlight the notion that, even though solid tumors share common signaling cascades, mediators of acquired resistance must be elucidated for each disease separately in the context of treatment.

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Purpose: Advanced non-small-cell lung cancer (NSCLC) patients harboring epidermal growth factor receptor (EGFR) mutations (deletion in exon 19 or L858R) show an impressive progression-free survival of 14 months when treated with erlotinib. However, the presence of EGFR mutations can only imperfectly predict outcome. We hypothesized that progression-free survival could be influenced both by the pretreatment EGFR T790M mutation and by components of DNA repair pathways.Experimental Design: We assessed the T790M mutation in pretreatment diagnostic specimens from 129 erlotinib-treated advanced NSCLC patients with EGFR mutations. The expression of eight genes and two proteins involved in DNA repair and four receptor tyrosine kinases was also examined.Results: The EGFR T790M mutation was observed in 45 of 129 patients (35%). Progression-free survival was 12 months in patients with and 18 months in patients without the T790M mutation (P ¼ 0.05). Progression-free survival was 27 months in patients with low BRCA1 mRNA levels, 18 months in those with intermediate levels, and 10 months in those with high levels (P ¼ 0.02). In the multivariate analysis, the presence of the T790M mutation (HR, 4.35; P ¼ 0.001), intermediate BRCA1 levels (HR, 8.19; P < 0.0001), and high BRCA1 levels (HR, 8.46; P < 0.0001) emerged as markers of shorter progression-free survival.Conclusions: Low BRCA1 levels neutralized the negative effect of the T790M mutation and were associated with longer progression-free survival to erlotinib. We advocate baseline assessment of the T790M mutation and BRCA1 expression to predict outcome and provide alternative individualized treatment to patients based on T790M mutations and BRCA1 expression.