Monitoring of somatic mutations in circulating cell-free DNA by digital PCR and next-generation sequencing during afatinib treatment in patients with lung adenocarcinoma positive for EGFR activating mutations.

Background: Analysis of circulating cell-free DNA (cfDNA) is under intensive investigation for its potential to identify tumor somatic mutations. We have now explored the usefulness of such liquid biopsy testing with both the digital polymerase chain reaction (dPCR) and next-generation sequencing (NGS) during treatment of patients with the epidermal growth factor receptor (EGFR) inhibitor afatinib. Patients and methods: Eligible patients had advanced lung adenocarcinoma with EGFR activating mutations and were treated with afatinib. Plasma samples were collected before and during (4 and 24 weeks) afatinib treatment as well as at disease progression. Tumor and plasma DNA were analyzed by dPCR and NGS. Results: Thirty-five patients were enrolled. The objective response rate and median progression-free survival (PFS) were 77.1% and 13.8 months, respectively. Tumor and plasma DNA were available for 32 patients. dPCR and NGS detected EGFR activating mutations in 81.3% and 71.9% of baseline cfDNA samples, respectively. In 19 patients treated with afatinib for ≥24 weeks, the number of EGFR mutant alleles detected in cfDNA by dPCR declined rapidly and markedly after treatment onset, becoming undetectable or detectable at only a low copy number (<10 copies per milliliter) at 4 weeks. Median PFS was slightly longer for patients with undetectable EGFR mutant alleles in cfDNA at 4 weeks than for those in whom such alleles were detectable (14.3 versus 10.0 months). A total of 45 somatic mutations was identified in baseline tumor DNA, and 30 (66.7%) of these mutations were identified in cfDNA by NGS. Allele frequency for somatic mutations in cfDNA determined by NGS changed concordantly during afatinib treatment with the number of EGFR mutant alleles determined by dPCR. Conclusions: Monitoring of cfDNA by dPCR is informative for prediction of afatinib efficacy, whereas that by NGS is reliable and has the potential to identify mechanisms of treatment resistance.

Association of PD-L1 overexpression with activating EGFR mutations in surgically resected nonsmall-cell lung cancer.

BACKGROUND: Recent clinical trials have shown that immune-checkpoint blockade yields a clinical response in a subset of individuals with advanced nonsmall-cell lung cancer (NSCLC). We examined whether the expression of programmed death-ligand 1 (PD-L1) is related to clinicopathologic or prognostic factors in patients with surgically resected NSCLC. PATIENTS AND METHODS: The expression of PD-L1 was evaluated by immunohistochemical analysis in 164 specimens of surgically resected NSCLC. Cell surface expression of PD-L1 in NSCLC cell lines was quantified by flow cytometry. RESULTS: Expression of PD-L1 in tumor specimens was significantly higher for women than for men, for never smokers than for smokers, and for patients with adenocarcinoma than for those with squamous cell carcinoma. Multivariate analysis revealed that the presence of epidermal growth factor receptor gene (EGFR) mutations and adenocarcinoma histology were significantly associated with increased PD-L1 expression in a manner independent of other factors. Cell surface expression of PD-L1 was also significantly higher in NSCLC cell lines positive for activating EGFR mutations than in those with wild-type EGFR. The EGFR inhibitor erlotinib downregulated PD-L1 expression in the former cell lines but not in the latter, suggesting that PD-L1 expression is increased by EGFR signaling conferred by activating EGFR mutations. A high level of PD-L1 expression in resected tumor tissue was associated with a significantly shorter overall survival for NSCLC patients. CONCLUSIONS: High expression of PD-L1 was associated with the presence of EGFR mutations in surgically resected NSCLC and was an independent negative prognostic factor for this disease.