Stromal PD-L1-Positive Regulatory T cells and PD-1-Positive CD8-Positive T cells Define the Response of Different Subsets of Non-Small Cell Lung Cancer to PD-1/PD-L1 Blockade Immunotherapy.

INTRODUCTION: Inhibition of programmed cell death-1 (PD-1) and its ligand programmed death ligand 1 (PD-L1) by using an immune checkpoint inhibitor has emerged as a promising immunotherapy for NSCLC. The correlation of PD-L1 expression in tumor cells with treatment outcomes has been reported in many pivotal trials; however, the relationship remains unclear. Here, we demonstrate that those patients with both high density of PD-1-positive CD8 and PD-L1-positive CD4-positive CD25-positive (PD-1hi PD-L1hi) regulatory T cells (Tregs) have a better response to PD1/PD-L1 blockade. METHODS: In our study between April 1, 2014, and May 30, 2017, a total of 73 NSCLC peripheral blood samples and fresh tumor specimens were collected for study. Of these, 42 large (10-mm3) fresh tumor specimens were obtained from surgical procedures and checked for expression of immunology biomarkers, including PD-L1, PD-1, CD8, CD4, and CD25, in tumor cells and tumor-infiltrating lymphocytes (TILs) by flow cytometry, immunohistochemistry, and immunofluorescence (IF). Moreover, 31 small biopsy specimens from patients who received immunotherapy (pembrolizumab or nivolumab) were analyzed by immunohistochemistry and IF. The correlation between flow cytometry and IF detected for TILs' density was evaluated by Spearman's rank correlation test; the primary end point was progression-free survival. For the PD-1/PD-L1 blockade assay, the TILs and peripheral blood mononuclear CD8 T cells were cultured (1×105 per well) with anti-PD-1 (clone MIH4), anti-PD-L1 (clone MIH1). The cytotoxic activity of TILs in killing NSCLC cells after stimulation by anti-PD-1 and anti-PD-L1 was measured by a conventional 51Cr release assay. RESULTS: We first identified a population of high-PD-L1-expressing CD25-positive CD4-positive T cells (PD-L1hi Tregs) in the tumor microenvironment. The frequency of PD-L1hi Tregs was higher in tumor tissue (mean 48.6 ± 14.3% in CD25-positive CD3-positive CD4-positive T cells) than in blood (mean 35.4 ± 10.2% in CD25-positive CD3-positive CD4-positive T cells) and normal tissue (mean 38.6 ± 9.7% in CD25-positive CD3-positive CD4-positive T cells) (p < 0.05), as determined by flow cytometry. The frequency of PD-L1hi Tregs was positively correlated with PD-1-positive CD8 in Tregs. In addition, the TILs from these patients (PD-1hi PD-L1hi) showed PD-1/PD-L1 pathway dependence and could induce a greater killing effect of TILs by PD-1/PD-L1 blockade treatment. The patients with PD-L1-positive NSCLC with PD-1hi PD-L1hi TILs showed a better clinical outcome than those with a low frequency of PD-1hi CD8 or PD-L1hi Tregs (median progression-free survival not reached versus 2 months). CONCLUSIONS: Our findings suggested that the density of PD-L1-positive CD4-positive CD25-positive Tregs in the tumor microenvironment can serve as a diagnostic factor to supplement PD-L1 expression in tumor cells and predict the response to PD-1/PD-L1 blockade immunotherapy in NSCLC.

Acquired MET Y1248H and D1246N Mutations Mediate Resistance to MET Inhibitors in Non-Small Cell Lung Cancer.

Purpose:MET amplification, responsible for 20% of acquired resistance to EGFR tyrosine kinase inhibitor (TKI) in patients with advanced non-small cell lung cancer (NSCLC), presents an attractive target. Numerous studies have conferred susceptibility of MET mutations and focal amplification to targeted MET-TKIs. However, the mechanism underlying MET-TKIs-induced resistance remains elusive.Experimental Design: We conducted a cohort of 12 patients with advanced NSCLC who developed resistance to a combinatorial therapy consisting of gefitinib and a type I MET-TKI. We performed capture-based targeted ultra-deep sequencing on serial tumor biopsies and plasmas ctDNA samples to detect and quantify genetic alterations.Results: We identified 2 newly acquired MET mutations, Y1248H and D1246N, in 2 patients and further confirmed their resistance against type I MET-TKIs in silco, in vitro, and in vivo Interestingly, NIH3T3 cells harboring either mutation exhibited responses to type II MET-TKIs, suggesting sequential use of MET-TKIs may offer a more durable response. In addition, we also discovered that EGFR amplification may act as an alternative MET-TKI resistance mechanism.Conclusions: Our study provides insight into the diversity of mechanisms underlying MET-TKI-induced resistance and highlights the potential of sequential use of MET-TKIs. Clin Cancer Res; 23(16); 4929-37. ©2017 AACR.

The Unique Characteristics of MET Exon 14 Mutation in Chinese Patients with NSCLC.

INTRODUCTION: Predictive biomarkers of mesenchymal-to-epithelial transition factor (MET)-targeted therapy remain elusive. Since the discovery of the MNNG HOS Transforming gene (MET) exon 14 mutation, it has been found to have the best potential to become one precise biomarker for MET-targeted therapy. Here, we present the unique characteristics of MET exon 14 mutations in Chinese patients with NSCLC. METHODS: A total of 1296 patients with NSCLC were screened for MET exon 14 mutations. Next-generation sequencing was performed on the DNA of 968 patients and Sanger sequencing was conducted on complementary DNA of the other 328 patients. Immunohistochemical analysis and fluorescence in situ hybridization were also performed on all specimens. RESULTS: Twelve patients had MET exon 14 mutations. These accounted for only 0.9% of adenocarcinoma. Thus, the mutations were present at less than half the frequency of their occurrence in Western patients (0.9% versus 3% in Chinese and white patients, respectively, χ(2) = 15.1, p < 0.001). Samples from six patients with MET exon 14 mutations were analyzed using immunohistochemical analysis and fluorescence in situ hybridization. We found no significant relationships among the mutation, MET amplification, and MET overexpression. In two patients who received crizotinib, only one patient (who exhibited MET amplification) experienced a partial response; the progression-free survival was 9 months. However, it remains unclear whether the sensitivity of this patient to crizotinib was conferred by the MET exon 14 mutation per se or by MET amplification. In the other patient with concomitant MET exon 14 skipping and KRAS G12D mutation, the disease progressed in only 1 month. CONCLUSIONS: MET exon 14 mutation per se may not be sufficiently robust for use in defining a subset of NSCLCs. Further research on MET exon 14 mutations, MET amplification, and MET overexpression is required. Maybe a panel of biomarkers will be necessary in the future.

The coexistence of MET over-expression and an EGFR T790M mutation is related to acquired resistance to EGFR tyrosine kinase inhibitors in advanced non-small cell lung cancer.

MET overexpression and the EGFR T790M mutation are both associated with acquired resistance (AR) to epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) in advanced non-small cell lung cancer (NSCLC). We characterized the frequency, underlying molecular mechanisms, and subsequent treatment for AR in MET overexpressing NSCLC patients with or without the T790M mutation. The study participants were 207 patients with advanced NSCLC and AR to EGFR-TKIs. The percentages of MET-, T790M- and MET/T790M-positive patients were 20.3% (42/207), 34.8% (72/207) and 6.8% (14/207), respectively. The disease control rate was 100% (5/5) for five patients with MET overexpression who received EGFR-TKIs plus a MET inhibitor. Among the MET/T790M-positive patients, seven received EGFR-TKIs plus a MET inhibitor and four received a T790M inhibitor, but no response was observed. The median post-progression survival (PPS) was 14.1, 24.5, and 10.7 months for MET-overexpressing, T790M-positive and MET/T790M-positive patients, respectively (P=0.044). c-Met, p-Met, ERBB3, and p-ERBB3 were highly expressed in MET-positive and MET/T790M-positive patients, but were poorly expressed in T790M-positive patients. EGFR, p-EGFR, AKT, p-AKT, MAPK, and p-MAPK were highly expressed in all three groups. These results suggest that MET/T790M-positive patients are at higher risk of AR to EGFR-TKIs, and have a worse PPS than patients with only MET overexpression or the T790M mutation alone. Clinical trials are needed to determine the best treatment for patients with both MET overexpression and the EGFR T790M mutation.

Blockade of Hedgehog Signaling Synergistically Increases Sensitivity to Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitors in Non-Small-Cell Lung Cancer Cell Lines.

Aberrant activation of the hedgehog (Hh) signaling pathway has been implicated in the epithelial-to-mesenchymal transition (EMT) and cancer stem-like cell (CSC) maintenance; both processes can result in tumor progression and treatment resistance in several types of human cancer. Hh cooperates with the epidermal growth factor receptor (EGFR) signaling pathway in embryogenesis. We found that the Hh signaling pathway was silenced in EGFR-TKI-sensitive non-small-cell lung cancer (NSCLC) cells, while it was inappropriately activated in EGFR-TKI-resistant NSCLC cells, accompanied by EMT induction and ABCG2 overexpression. Upregulation of Hh signaling through extrinsic SHH exposure downregulated E-cadherin expression and elevated Snail and ABCG2 expression, resulting in gefitinib tolerance (P < 0.001) in EGFR-TKI-sensitive cells. Blockade of the Hh signaling pathway using the SMO antagonist SANT-1 restored E-cadherin expression and downregulate Snail and ABCG2 in EGFR-TKI-resistant cells. A combination of SANT-1 and gefitinib markedly inhibited tumorigenesis and proliferation in EGFR-TKI-resistant cells (P < 0.001). These findings indicate that hyperactivity of Hh signaling resulted in EGFR-TKI resistance, by EMT introduction and ABCG2 upregulation, and blockade of Hh signaling synergistically increased sensitivity to EGFR-TKIs in primary and secondary resistant NSCLC cells. E-cadherin expression may be a potential biomarker of the suitability of the combined application of an Hh inhibitor and EGFR-TKIs in EGFR-TKI-resistant NSCLCs.

Differences in driver genes between smoking-related and non-smoking-related lung cancer in the Chinese population.

Recently, non-smoking-related lung cancer was classified as an independent disease entity because it is different from tobacco-associated lung cancer. Non-smoking-related lung cancer occurs more often in women than men, and the predominant histological type is adenocarcinoma (ADC) rather than squamous cell carcinoma. Most of the driver gene alterations that have been identified in ADC in never-smokers include epidermal growth factor receptor mutations, KRAS mutations, echinoderm microtubule-associated protein like 4/anaplastic lymphoma kinase fusion, and ROS1 fusion, among others. Meanwhile, significant progress has been made in the treatment of ADC. However, in comparison with ADC, no such available molecular targets exist for smoking-associated lung cancer, for which treatment strategies are limited. Next-generation sequencing has been widely applied to the discovery of more genetic profiles of lung cancers. This review summarizes the differences between smoking-related and non-smoking-related lung cancer as follows: different somatic mutation burdens, C:G→A:T transversions, common and novel driver genes, and treatment strategies. Overall, smoking-related lung cancer is more complicated than non-smoking-related lung cancer. Furthermore, we review the prevalence of driver genes in smoking-associated and non-smoking-associated lung cancers in the Chinese population.

Prevalence of driver mutations in non-small-cell lung cancers in the People's Republic of China.

Lung cancer is a leading cause of cancer-related mortality worldwide and in the People's Republic of China. Recently, the pathological proportions of the various forms of lung cancer have changed. A shift to a preponderance of adenocarcinoma at the expense of squamous cell carcinoma is observable. Treatment decisions have historically been based on tumor histology, and evolution of our molecular understanding of cancer has led to development of targeted therapeutic agents. It is essential to further understand mutations that drive cancer development (driver mutations) in relevant genes and their effects on cancer cell proliferation and survival. The epidemiology of lung cancer in the People's Republic of China has been extensively reviewed elsewhere. However, molecular epidemiological data from mainland China are scarce. Consequently, we herein review the prevalence of driver mutations in Chinese patients.