Genomic and immune characteristics of HER2-mutated non-small-cell lung cancer and response to immune checkpoint inhibitor-based therapy.

The efficacy of immunotherapy in advanced HER2-mutated non-small-cell lung cancer (NSCLC) remains incomprehensively studied. A total of 107 NSCLC patients with de novo HER2 mutations were retrospectively studied at Guangdong Lung Cancer Institute [GLCI cohort, exon 20 insertions (ex20ins): 71.0%] to compare clinical/molecular features and immune checkpoint inhibitor (ICI)-based therapy efficacy between patients with ex20ins and non-ex20ins. Two external cohorts (TCGA, n = 21; META-ICI, n = 30) were used for validation. In the GLCI cohort, 68.2% of patients displayed programmed death-ligand 1 (PD-L1) expression < 1%. Compared with ex20ins patients, non-ex20ins patients had more concurrent mutations in the GLCI cohort (P < 0.01) and a higher tumour mutation burden in the TCGA cohort (P = 0.03). Under ICI-based therapy, advanced NSCLC patients with non-ex20ins had potentially superior progression-free survival [median: 13.0 vs. 3.6 months, adjusted hazard ratio (HR): 0.31, 95% confidence interval (CI): 0.11-0.83] and overall survival (median: 27.5 vs. 8.1 months, adjusted HR: 0.39, 95% CI: 0.13-1.18) to ex20ins patients, consistent with findings in the META-ICI cohort. ICI-based therapy may serve as an option for advanced HER2-mutated NSCLC, with potentially better efficacy in non-ex20ins patients. Further investigations are warranted in clinical practice.

Prognostic features and comprehensive genomic analysis of NF1 mutations in EGFR mutant lung cancer patients.

OBJECTIVE: NF1 is a tumor suppressor gene that encodes the neurofibromin protein and negatively regulates Ras signaling. This study was aimed to investigate the molecular, clinical characteristics, and prognostic features of NF1 gene in EGFR mutant lung cancer patients. METHOD: The next-generation sequencing (NGS) was used to analyze the data from lung cancer patients in the Guangdong Lung Cancer Institute (GLCI) from June 2016 to December 2020. RESULTS: Somatic NF1 mutations were present in 4.2% (135/3220) of Chinese lung cancer patients. NF1 mutations where clearly enriched in older (p < 0.001), male (p < 0.001), and smoking (p < 0.001) patients. Patients with NF1 mutations were more likely to have TP53 (p = 0.003), BRAF (p = 0.001) and RASA1 (p = 0.026) mutations and mutually exclusive with EGFR mutations (p = 0.006). TP53 mutation had worsen prognosis in cases of NF1 mutant (p = 0.026) or EGFR/NF1 co-mutant (p = 0.031) lung adenocarcinomas (LUAD) patients. There was no effect on overall survival (OS) in LUAD patients with and without NF1 mutations, even in LUAD driver-gene negative patients. NF1/EGFR co-mutation patients had a longer OS than a single mutation of either the EGFR gene (median OS: 47.7 m vs. 30.2 m, hazard ratio [95% CI], 0.47 [0.30-0.74], p = 0.004) or NF1 gene (47.7 m vs. 19.0 m, 0.44 [0.27-0.73], p = 0.003). Furthermore, NF1 mutations significantly prolonged OS in EGFR mutant/TP53 wild-type LUAD patients (106.5 m vs. 25.5 m, 0.28 [0.13-0.59], p = 0.003) but not in patients with EGFR/TP53 co-mutations (36.8 m vs. 30.2 m, 0.70 [0.39-1.26], p = 0.280). CONCLUSION: Our results indicated NF1 mutations served as a good prognostic factor in EGFR mutant/TP53 wild-type lung cancer patients in this single-center study. TP53 mutation was obviously enriched in NF1 mutant patients and had shorter OS.

A novel miR-200c/c-myc negative regulatory feedback loop is essential to the EMT process, CSC biology and drug sensitivity in nasopharyngeal cancer.

Overexpression of the c-Myc oncogene has been implicated in cancer stem cell - like (CSC) phenotypes and epithelial-to-mesenchymal transition (EMT) in cancer. However, the underlying molecular mechanism by which c-Myc regulates EMT and CSC potential in remains unclear. In the present study, we showed that the expression of c-Myc protein is inversely correlated with microRNA (miR)-200c expression in primary tumor samples from nasopharyngeal cancer (NPC) patients. We further demonstrated that Myc and miR-200c negatively regulate the expression each other in NPC cell lines. c-Myc transcriptionally repressed expression of miR-200c by directly binding to two E-box sites located within a 1 kb segment upstream of TSS of the miR-200c. In addition, miR-200c post-transcriptionally repressed expression of c-Myc by binding to its 3'-untranslated region, suggesting the existence of a negative feedback loop between Myc and miR-200c. Overexpression of c-Myc interfered with this feedback loop and activated the EMT program, induced CSC phenotypes, and enhanced drug sensitivity, whereas miR-200c could counteract these biological effects of c-Myc. Our results provide a novel mechanism governing c-Myc and miR-200c expression and indicate that either targeting c-Myc or restoring miR-200c expression would be a promising approach to overcome oncogenic role of c-Myc in NPC.

Clinical relevance of PD-L1 expression and CD8+ T cells infiltration in patients with EGFR-mutated and ALK-rearranged lung cancer.

OBJECTIVES: EGFR-mutated or ALK-rearranged non-small cell lung cancer (NSCLC) often showed unfavorable clinical benefit to checkpoint inhibitors (CPIs). However, few reports exist with integrated analysis, to interpret the underlying mechanism of poor response to PD-1/PD-L1 inhibitors. We have retrospectively analyzed the tumor microenvironment (TME) based on tumor PD-L1 expression and CD8+ T cells infiltration in patients with EGFR mutations and ALK rearrangements, and the prognostic value of TME subtypes on overall survival (OS). MATERIALS AND METHODS: Tumor samples from 715 patients with lung cancer were retrospectively collected at Guangdong Lung Cancer Institute. Tumoral PD-L1 expression (N = 715) and CD8+ T cells infiltration (N = 658) was determined by immunohistochemistry (IHC), based on which TME was categorized into four different subtypes: PD-L1+/CD8+, PD-L1-/CD8+, PD-L1+/CD8-, PD-L1-/CD8-. Proportion of four TME subtypes was determined, and overall survival with PD-L1 expression and TME was analyzed. RESULTS: In patients with EGFR mutations or ALK rearrangements, proportion of PD-L1+/CD8+ tumors was the lowest (5.0%, 17/342), and that of PD-L1-/CD8- tumors was the highest (63.5%, 217/342). In patients with wild-type EGFR and ALK, 14.2% (45/316) tumors were PD-L1+/CD8+ and 50.3% (159/316) tumors were PD-L1-/CD8- (P < 0.001). Median OS of EGFR-mutated or ALK-rearranged lung cancer was 78.6 months in PD-L1 positive group and 93.4 months in PD-L1 negative group (HR 0.47, 95%CI 0.23-0.76, P = 0.005). PD-L1+/CD8+ group exhibited the shortest OS, with 44.3 months, but is likely to respond to CPIs. The PD-L1-/CD8+ group exhibited the longest OS but is unlikely to respond to CPIs. CONCLUSION: Patients with EGFR mutations or ALK rearrangements exhibited lower PD-L1 and CD8 co-expression level in TME, which could be responsible for poor response to CPIs. PD-L1 and CD8 co-expression in EGFR-mutated or ALK-rearranged lung cancer is a biomarker for poor prognosis with shorter OS.

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.

A comprehensive review of uncommon EGFR mutations in patients with non-small cell lung cancer.

INTRODUCTION: Patients with non-small cell lung cancer (NSCLC) harboring uncommon epidermal growth factor receptor (EGFR) mutations are a heterogeneous group exhibiting differential responses to EGFR inhibitors. This retrospective study reviews the prevalence of uncommon EGFR mutations in a Chinese NSCLC cohort and the clinical characteristics and efficacy of EGFR tyrosine kinase inhibitors (TKIs) associated with these patients. MATERIALS ANDMETHODS: A total of 5363 lung cancer patients were screened and underwent EGFR genotyping at the Guangdong Lung Cancer Institute. Of those with uncommon EGFR mutations, the clinical characteristics and responses to EGFR-TKIs were reviewed retrospectively. RESULTS: Uncommon EGFR mutations were observed in 218 patients, comprising 11.9% of all patients with documented EGFR mutations. More smokers (30.7% vs. 24.3%, P=0.039) and males (54.1% vs. 44.4%, P=0.007) were among the patients with uncommon mutations compared with common mutations. The most frequent uncommon mutations were exon 20 insertions (30.7%, n=67), followed by G719X mutations (21.1%, n=46) and compound L858R mutations (17.0%, n=37). Favorable efficacy was observed in patients harboring compound L858R or G719X mutations, with a median progression-free survival (PFS) of 15.2 (95% CI: 8.7-21.7) or 11.6 (95% CI: 3.6-19.6) months, respectively. The median PFS of those with the T790M mutation or an exon 20 insertion was 1.0 (95% CI: 0.0-2.2) and 3.0 (95% CI: 1.3-4.7) months, respectively. CONCLUSION: This study reviewed the prevalence of uncommon EGFR mutations and their sensitivity to EGFR-TKIs. Favorable responses were observed in patients with G719X and compound L858R mutations, indicating that they may benefit from EGFR-TKIs as a first-line therapy.

Epidermal growth factor receptor is associated with the onset of skeletal related events in non-small cell lung cancer.

BACKGROUND: Bone metastasis and skeletal related events (SREs) are common in non-small cell lung cancer (NSCLC). Patients with mutant epidermal growth factor receptor (EGFR) could benefit from tyrosine kinase inhibitors (TKIs). However, it is unclear whether SRE is influenced by EGFR status. We aimed to evaluate the correlation of EGFR status and TKIs with the incidence of SREs. METHODS: We conducted a retrospective study of stage IV NSCLC patients with bone metastasis. Incidence rate of SREs was collected and was compared using chi-square test. Logistic-regression analysis was used to identify the risk factors predicting the incidence of SREs. RESULTS: 410 eligible patients were enrolled in the study. 49.0% were detected with EGFR mutation. 49.8% of patients received EGFR-TKIs therapy prior to the onset of SREs. 42.7% experienced at least one SRE. Patients who were treated with TKIs held lower incidence of SREs than patients who were not treated with TKIs (23.5% vs 61.7%, p<0.001). Multivariate analysis showed that poor performance status (OR 5.550, 95%CI 2.290-13.450; p<0.001) and mutant EGFR (OR 3.050, 95%CI 1.608-5.787, p=0.001) were independent risk factors predicting the onset of SREs, while the usage of TKIs (OR 0.102, 95%CI 0.054-0.193, p<0.001) was a protective factor of SREs in NSCLC patients with bone metastasis. CONCLUSIONS: This study indicates that the incidence of SREs is common in both patients with and without EGFR mutation. Poor performance ability and mutant EGFR imply higher risks of SREs, while the usage of TKIs may be a protective factor of SREs.

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.

Potential Predictive Value of TP53 and KRAS Mutation Status for Response to PD-1 Blockade Immunotherapy in Lung Adenocarcinoma.

Purpose: Although clinical studies have shown promise for targeting programmed cell death protein-1 (PD-1) and ligand (PD-L1) signaling in non-small cell lung cancer (NSCLC), the factors that predict which subtype patients will be responsive to checkpoint blockade are not fully understood.Experimental Design: We performed an integrated analysis on the multiple-dimensional data types including genomic, transcriptomic, proteomic, and clinical data from cohorts of lung adenocarcinoma public (discovery set) and internal (validation set) database and immunotherapeutic patients. Gene set enrichment analysis (GSEA) was used to determine potentially relevant gene expression signatures between specific subgroups.Results: We observed that TP53 mutation significantly increased expression of immune checkpoints and activated T-effector and interferon-γ signature. More importantly, the TP53/KRAS comutated subgroup manifested exclusive increased expression of PD-L1 and a highest proportion of PD-L1+/CD8A+ Meanwhile, TP53- or KRAS-mutated tumors showed prominently increased mutation burden and specifically enriched in the transversion-high (TH) cohort. Further analysis focused on the potential molecular mechanism revealed that TP53 or KRAS mutation altered a group of genes involved in cell-cycle regulating, DNA replication and damage repair. Finally, immunotherapeutic analysis from public clinical trial and prospective observation in our center were further confirmed that TP53 or KRAS mutation patients, especially those with co-occurring TP53/KRAS mutations, showed remarkable clinical benefit to PD-1 inhibitors.Conclusions: This work provides evidence that TP53 and KRAS mutation in lung adenocarcinoma may be served as a pair of potential predictive factors in guiding anti-PD-1/PD-L1 immunotherapy. Clin Cancer Res; 23(12); 3012-24. ©2016 AACR.

Potential biomarker for checkpoint blockade immunotherapy and treatment strategy.

Programmed cell death protein-1 (PD-1) and ligand (PD-L1) provide an important escape mechanism from immune attack, and blockade therapy of these proteins show promising clinical benefits in many types of cancer. PD-L1 can be induced by interferon-gamma (IFN-γ), hypoxia, or toll-like receptor (TLR)-mediated pathways that confer adaptive immune resistance, or upregulated by oncogenic signals leading to constitutive expression and resulting in intrinsic immune resistance. The PD-1/PD-L1 checkpoint blockade, which targets regulatory pathways in T cells to overcome immune resistance, is correlated to PD-L1 expression pattern and the presence of tumor-infiltrating lymphocytes (TILs). Meanwhile, immunogenic mutation loads show significant response to checkpoint blockade, which is probably due to PD-1/L1 status and TIL content. Finally, the clinical strategies to design effective checkpoint-targeting immunotherapies are based on the classification of inducible/constitutive expression of PD-L1 and the presence of TILs.

Nuclear ß-catenin accumulation is associated with increased expression of Nanog protein and predicts poor prognosis of non-small cell lung cancer.

BACKGROUND: Although the prognostic roles of ß-catenin expression in non-small cell lung cancer (NSCLC) have been reported in several immunohistochemical (IHC) studies, the results were not consistent because some studies lack sufficient number of the positive cases or did not evaluate the subcellular localization features of the protein. METHOD: In this study, we have evaluated the expression levels and subcellular localization of ß-catenin and Nanog proteins IHC staining in tissue specimens from 309 patients with NSCLC, and explored their association with clinicopathological features and patient outcome. RESULTS: We showed that patients with negative expression of membranous beta-catenin had a trend towards shorter survival (p=0.064) than those with positive expression. In contrast to previous studies, we found that increased expression of either cytoplasmic or nuclear ß-catenin was strongly associated with poor prognosis and was an independent prognosticator for overall survival (p <0.01). We further found that NSCLC cells frequently exhibited an abundance of nuclear Nanog protein which was significantly correlated with nuclear ß-catenin expression (p <0.01) and poor prognosis (p <0.01). Interestingly, immunofluorescent staining results revealed that increased expression of Nanog and nuclear translocation of ß-catenin occurred concomitantly in response to epidermal growth factor receptor(EGFR) signaling in A549 and H23 cells. Furthermore, western blot analysis show that nuclear ß-catenin rather than cytoplasmic ß-catenin expression in the A549 and H23 cells can be enhanced by adding EGF, Nanog expression in the A549 and H23 cells with knockdown of ß-catenin can not be obviously enhanced by adding EGF. CONCLUSION: We propose that evaluation of subcellular localization of ß-catenin and Nanog expression is of clinical significance for patients with NSCLC.

MYC regulation of CHK1 and CHK2 promotes radioresistance in a stem cell-like population of nasopharyngeal carcinoma cells.

Radiotherapy is the most successful nonsurgical treatment for nasopharyngeal carcinoma (NPC). Despite this, the prognosis remains poor. Although NPCs initially respond well to a full course of radiation, recurrence is frequent. The cancer stem cell (CSC) hypothesis provides a framework for explaining the discrepancy between the response of NPC to therapy and the poor survival rate. In this study, a stem cell-like subpopulation (PKH26+) was identified in NPC cell lines using a label-retention technique. PKH26+ cells were enriched for clonogenicity, sphere formation, side-population cells, and resistance to radiotherapy. Using genomic approaches, we show that the proto-oncogene c-MYC (MYC) regulates radiotolerance through transcriptional activation of CHK1 (CHEK1) and CHK2 (CHEK2) checkpoint kinases through direct binding to the CHK1 and CHK2 promoters. Overexpression of c-MYC in the PKH26+ subpopulation leads to increased expression of CHK1 and CHK2 and subsequent activation of the DNA-damage-checkpoint response, resulting in radioresistance. Furthermore, loss of CHK1 and CHK2 expression reverses radioresistance in PKH26+ (c-MYC high expression) cells in vitro and in vivo. This study elucidates the role of the c-MYC-CHK1/CHK2 axis in regulating DNA-damage-checkpoint responses and stem cell characteristics in the PKH26+ subpopulation. Furthermore, these data reveal a potential therapeutic application in reversal of radioresistance through inhibition of the c-MYC-CHK1/CHK2 pathway.

[Relation of advanced oxidation protein products with VEGF and TGF-ß1 in colon cancer cells exposed to intermittent hypoxia].

OBJECTIVE: To investigate the association of advanced oxidation protein products (AOPP) with oxidative stress in colon cancer cells exposed to intermittent hypoxia (IH). METHODS: Colon cancer SW480 cells were exposed to IH, continuous hypoxia, or normoxia. Enzyme-linked immunosorbent assay (ELISA) was employed to examine the levels of AOPP and vascular endothelial growth factor (VEGF), xanthine oxidase assay was used to determine malonaldehyde (MDA) and glutathione peroxidase (GSH-PX), and Western blotting and immunofluorescence assay were performed for detection of transforming growth factor-ß(1) (TGF-ß(1)) expression. RESULTS: Compared with the normoxia group, the two hypoxia groups showed significantly increased AOPP and MDA levels (P<0.05) and lowered SOD and GSH-PX levels (P<0.05). The concentration of AOPP was positively correlated to MDA, VEGF, and TGF-ß(1) levels (P<0.05), but inversely to SOD. No significant correlation was found between AOPP and GSH-PX levels. CONCLUSION: Compared with continuous hypoxia, IH results in more obvious protein oxidation in relation to oxidative stress. The increased expression of VEGF and TGF-ß(1) in the context of hypoxia is closely related to AOPP level.