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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.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.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).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.

MET inhibitors have shown promising efficacy for MET-dysregulated non-small cell lung cancer (NSCLC). However, quite a few patients cannot benefit from it due to the lack of powerful biomarkers. This study aims to explore the potential role of plasma proteomics-derived biomarkers for patients treated with MET inhibitors using mass spectrometry. We analyzed the plasma proteomics from patients with MET dysregulation (including MET amplification and MET overexpression) treated with MET inhibitors. Thirty-three MET-dysregulated NSCLC patients with longitudinal 89 plasma samples were included. We classified patients into the PD group and non-PD group based on clinical response. The baseline proteomic profiles of patients in the PD group were distinct from those in the non-PD group. Through protein screening, we found that a four-protein signature (MYH9, GNB1, ALOX12B, HSD17B4) could predict the efficacy of patients treated with MET inhibitors, with an area under the curve (AUC) of 0.93, better than conventional fluorescence in situ hybridization (FISH) or immunohistochemistry (IHC) tests. In addition, combining the four-protein signature with FISH or IHC test could also reach higher predictive performance. Further, the combined signature could predict progression-free survival for MET-dysregulated NSCLC (p < 0.001). We also validated the performance of the four-protein signature in another cohort of plasma using an enzyme-linked immunosorbent assay. In conclusion, the four plasma protein signature (MYH9, GNB1, ALOX12B, and HSD17B4 proteins) might play a substitutable or complementary role to conventional MET FISH or IHC tests. This exploration will help select patients who may benefit from MET inhibitors.

Purpose Rearranged during transfection (RET) fusions are important genetic drivers in non-small cell lung cancer (NSCLC). Selective RET inhibitors are setting a new paradigm in RET-driven NSCLC. However, the real-world treatment patterns, outcomes and toxicity remain largely unknown. Methods Data from RET fusion-positive NSCLC patients treated in our centre were retrospectively analysed. Of them, patients diagnosed before and after August 2018 were included in analysis of treatment patterns; and patients received selective RET inhibitors were eligible for analysis of adverse events (AEs). Results Patients diagnosed before August 2018 (n = 30) predominantly received chemotherapy and immunotherapy (83%) as initial therapy, while patients diagnosed after August 2018 (n = 39) mainly received selective RET inhibitors (38.5% at first-line; 50.0% at second-line). In the total 69 patients, overall survival (OS) was prolonged in patients treated with selective RET inhibitors versus untreated patients (median 34.3 versus 17.5 months; p = 0.002) during a median follow-up of 28.7 months. But there was no difference between patients treated with immunotherapy versus untreated patients. In the 38 patients received selective RET inhibition, median progression-free survival (PFS) was 11.9 months. AEs ≥ grade 3 occurred in 42.1% patients and were not associated with PFS (p = 0.63) or OS (p = 0.60). Haematological toxicity ≥ grade 3 occurred in 31.6% patients and was the leading cause of drug discontinuation. Conclusion Selective RET inhibitors are increasingly being adopted into clinical practice and are associated with improved OS. However, treatment-related ≥ grade 3 AEs, especially haematologic AEs, occur frequently in real-world setting.

20–40% of lung cancer patients develop bone metastasis (BM) with significantly decreased overall survival. Currently, BM is mainly diagnosed by computerized tomography (CT) scan or magnetic resonance imaging (MRI) when symptom develops. Novel biomarkers with higher prediction value of BM are needed. Plasma-derived exosomal microRNAs had been isolated and sequenced of total 30 non-small cell lung cancer (NSCLC) patients including 16 with bone metastasis and 14 without bone metastasis. Hierarchical clustering based on the total miRNA profile can clearly separate cancer patients and healthy individuals (H), but not patients with (BM +) or without (BM−) BM. Weight Co-expression network of miRNAs (WGCNA) analyses identified three consensus clusters (A, B, C) of highly correlated miRNAs, among which cluster B (144 miRNAs) showed significantly differential expression in lung cancer patients, especially in BM + group. Pathway analysis of cluster B miRNAs revealed enrichment in metabolic pathways that may involve in preconditioning of the metastatic niche. Three differentially expressed miRNAs between BM + and BM− patients within cluster B were identified as miR-574-5p, a suppressor of Wnt/β-catenin pathway, was down-regulated, while miR-328-3p and miR-423-3p, two activators of the same pathway, were up-regulated in BM + patients. Cluster A miRNAs (n = 49) also showed trend of upregulation in BM + patients. Interestingly, pathway analysis indicated that 43 of them are associated with chromosome14, which has been suggested to promote epithelial-mesenchymal transition (EMT) and bone metastasis. Electronic supplementary material The online version of this article (10.1007/s10585-020-10062-y) contains supplementary material, which is available to authorized users.

Background The development of “precision medicine” needs a novel genetic screening and diagnostic technique for clinical detection. This study aims to establish a method for highly parallel multiplexed detection of genetic mutations in Chinese lung cancer samples through testing 285 genes by customized next generation sequencing (NGS) on Ion-Proton platform. Methods We reviewed the related literature and collected data of genomic alteration that occurred in lung cancer. We identified 285 target genes closely related to the pathogenesis, drug resistance, and metastasis of lung cancer. Targeted hybridization probes were designed using SureDesign software. The detection method was established by analyzing four cell lines and 13 lung cancer specimens which had been validated through Sanger sequencing. The sensitivity and specificity of the proposed method were preliminarily evaluated by comparisons with the Sanger sequencing and a LungCarta mutation-detection method. Results The proposed method was able to detect mutations of 285 genes in lung cancer cell lines and clinical lung cancer specimens. The reads, mapped reads, on target, mean depth and uniformity were 14.90±4.37 (×106), 98.68%±0.61%, 60.49%±10.72%, 714.42±264.13 and 90.51%±6.91%, respectively. The detected mutation result of cell lines was consistent with the observations on previously reported mutations, and the congruence rate was 100%. The proposed method can detect single nucleotide polymorphism (SNP), InDel, Fusion and copy number variation (CNV). The complete congruence rate of detected result of specimens between the proposed method and Sanger sequencing, LungCarta mutation-detection method, immunohistochemistry (IHC), real-time polymerase chain reaction (RT-PCR) method were all 100% regarding mutations in common genes like EGFR, KRAS, or fusions of ALK, RET, etc. In addition, NFE2L3_p.Ser511_Pro513del, ERBB2_E770delinsEAYVM, MET_S701N, PDGFRA_T674I, TP53_G245V, TP53_V274A, TP53_A276F, TP53_G334L, TP53_R337L and TP53_Y220C mutations were detected only through the proposed method. The proposed method can detect mutations from blood, this detection result was consistent with the cancer tissues of the same clinical lung cancer patient. Conclusions The proposed Ion-Proton technology-based NGS method can detect genetic mutations in Chinese lung cancer patients. Therefore, the proposed method could be used to detect mutations in other cancer tissues and plasma, which needs further validation.

Background Tyrosine kinase domain (TKD) mutation and particularly exon 20 insertion mutations of ERBB2 have been extensively reported in non-small cell lung cancer (NSCLC). Due to the increased accessibility of next-generation sequencing, more ERBB2 mutations within the non-TKD can be detected in clinical practice. Nevertheless, the clinical significance of non-TKD mutations remains unknown. Hence, this study was designed to comprehensively outline the landscape and characteristics of ERBB2 mutations in NSCLC. Methods A total of 1934 patients with NSCLC from cBioPortal were included in the study. An ERBB2 mutation cohort was identified, while subsequent analyses revealed clinical and genomic characteristics. Results The frequency of ERBB2 mutation was 4.5%, and it was determined to be more likely to occur in never-smokers. ERBB2 mutations occurring in the non-TKD accounted for 57.5% of ERBB2 mutations. In the non-TKD, missense mutation was the most recurrent mutation type, and S310F was the most recurrent mutation variant. ERBB2 mutations within non-TKD also had a strong oncogenic ability where up to 37.5% of ERBB2 oncogenic mutations were within non-TKD. The co-mutation of EGFR or KRAS was higher in the non-TKD mutation compared to the TKD mutation. Shorter overall survival was observed in ERBB2-mutant patients compared with ERBB2 wild-type patients. There was no significant difference in overall survival between patients with non-TKD mutations and TKD mutations. Conclusions The present study showed that a considerable portion of non-TKD mutations were oncogenic. ERBB2 mutation was a poor prognostic factor. The non-TKD mutation might also be used as a therapeutic target in ERBB2-directed target therapy. Key points • Significant findings of the study ERBB2 mutations were more abundant within a nontyrosine domain than those within the tyrosine domain. Up to 37.5% of ERBB2 oncogenic mutations were within the nontyrosine domain. ERBB2 mutation was a poor prognostic factor. • What this study adds The frequency of EGFR or KRAS co-mutations were significantly higher in ERBB2 mutations within the nontyrosine kinase domain compared to ERBB2 mutations within the tyrosine kinase domain. Nontyrosine domain mutations confer equal overall survival to tyrosine domain mutations.

Immune checkpoint inhibitors (ICIs), especially anti-programmed death 1 (PD-1)/programmed death ligand 1 (PD-L1) antibodies, have made dramatic progress in the treatment of lung cancer, especially for patients with cancers not driven by oncogenes. However, responses are limited to a subset of patients, and which subset of patients will optimally benefit from ICI remains unknown. With the advantage of being minimally invasive and dynamic, noninvasive biomarkers are promising candidates to predict response, monitor resistance, and track the evolution of lung cancer during ICI treatment. In this review, we focus on the application of circulating tumor DNA (ctDNA) in plasma in immunotherapy. We examine the potential of pre- and on-treatment features of ctDNA as biomarkers, and following multiparameter analysis, we determine the potential clinical value of integrating predictive liquid biomarkers of ICIs to optimize patient management. We further discuss the role of ctDNA in monitoring treatment resistance, as well as challenges in clinical translation.

BackgroundEchinoderm microtubule-associated protein-like 4 (EML4) is the canonical anaplastic lymphoma kinase (ALK) fusion partner in non-small cell lung cancer (NSCLC), and ALK-positive patients showed promising responses to ALK tyrosine kinase inhibitors (TKIs). However, studies that comprehensively investigate ALK TKI treatment in patients with different ALK fusion patterns are still lacking.MethodsNinety-eight ALK-positive patients with advanced NSCLC were retrospectively studied for their response to crizotinib and subsequent treatments. Comprehensive genomic profiling (CGP) was conducted to divide patients into different groups based on their ALK fusion patterns. Non-canonical ALK fusions were validated using RNA-sequencing.Results54.1% of patients had pure canonical EML4-ALK fusions, 19.4% carried only non-canonical ALK fusions, and 26.5% harbored complex ALK fusions with coexisting canonical and non-canonical ALK fusions. The objective response rate and median progression-free survival to crizotinib treatment tended to be better in the complex ALK fusion group. Notably, patients with complex ALK fusions had significantly improved overall survival after crizotinib treatment (p = 0.012), especially when compared with the pure canonical EML4-ALK fusion group (p = 0.010). The complex ALK fusion group also tended to respond better to next-generation ALK TKIs, which were used as later-line therapies. Most identified non-canonical ALK fusions were likely to be expressed in tumors, and some of them formed canonical EML4-ALK transcripts during mRNA maturation.ConclusionOur results suggest NSCLC patients with complex ALK fusions could potentially have better treatment outcomes to ALK TKIs therapy. Also, diagnosis using CGP is of great value to identify novel ALK fusions and predict prognosis.

Highlights • Predictive power of PD response for ICIs was superior than traditional biomarkers; • Predictive efficacy was improved by integrating tumor-immune-related features; • When tumor-specific feature was replaced, the model has pan-cancer applicability. • NRAS and PDPK1 have the potential to induce primary resistance to ICIs., Background Treatment by immune checkpoint blockade (ICB) provides a remarkable survival benefit for multiple cancer types. However, disease aggravation occurs in a proportion of patients after the first couple of treatment cycles. Methods RNA sequencing data was retrospectively collected. 6 tumour-immune related features were extracted and combined to build a lung cancer-specific predictive model to distinguish responses as progression disease (PD) or non-PD. This model was trained by 3 public pan-cancer datasets and a lung cancer cohort from our institute, and generated a lung cancer-specific integrated gene expression score, which we call LITES. It was finally tested in another lung cancer dataset. Results LITES is a promising predictor for checkpoint blockade (area under the curve [AUC]=0.86), superior to traditional biomarkers. It is independent of PD-L1 expression and tumour mutation burden. The sensitivity and specificity of LITES was 85.7% and 70.6%, respectively. Progression free survival (PFS) was longer in high-score group than in low-score group (median PFS: 6.0 vs. 2.4 months, hazard ratio=0.45, P=0.01). The mean AUC of 6 features was 0.70 (range=0.61-0.75), lower than in LITES, indicating that the combination of features had synergistic effects. Among the genes identified in the features, patients with high expression of NRAS and PDPK1 tended to have a PD response (P=0.001 and 0.01, respectively). Our model also functioned well for patients with advanced melanoma and was specific for ICB therapy. Conclusions LITES is a promising biomarker for predicting an impaired response in lung cancer patients and for clarifying the biological mechanism underlying ICB therapy.

Background ALK-tyrosine kinase inhibitors (TKIs) have been proven effective for treating ALK-positive non-small cell lung cancer (NSCLC), although patients present with variable responses and disease progression courses. The detailed underlying molecular mechanisms require further investigation to yield a better prognosis. Methods Targeted next-generation sequencing (NGS) mutation profiling was performed on samples from 42 NSCLC patients confirmed positive for ALK rearrangements by fluorescence in situ hybridization or immunohistochemistry who experienced disease progression after crizotinib treatment. Results ALK rearrangements were not confirmed in six patients (14%) with other potential oncogenic drivers identified by NGS, who therefore did not respond to crizotinib and had significantly shorter overall survival (OS) compared to NGS ALK -positive patients. Fifteen ALK activating mutations were detected in 8 out of 26 post-treatment samples (31%), among which ALK L1196M and G1269A were the most common acquired mutations detected in half of the patients with ALK activating mutations. Dynamic monitoring of the genetic evolution in one patient revealed both spatial and temporal heterogeneity of resistant mechanisms during different ALK-TKI treatment courses. Activation of ALK downstream or bypass pathways was detected in patients without ALK activating mutations, such as genetic alterations in PIK3CA, MET, and KRAS. Interestingly, we identified two patients with acquired mutations in the DNA mismatch repair gene POLE, which resulted in a dramatically increased tumor mutation burden, and might contribute to the poor response to crizotinib. Conclusions Heterogeneous resistant mechanisms have been identified and correlate to diverse responses to crizotinib. Comprehensive and dynamic mutation profiling is required to better predict clinical outcomes.

Purpose: Leptomeningeal metastases are more common in non–small cell lung cancer (NSCLC) with EGFR mutations. The diagnosis is difficult by traditional imaging only, and leads to poor understanding of resistance mechanisms of leptomeningeal metastases. Experimental Design: We compared the CellSearch Assay, the Thinprep cytologic test (TCT), and brain magnetic resonance imaging (MRI) in 21 NSCLC patients with suspected leptomeningeal metastases. Next-generation sequencing that included 416 cancer-associated genes was also performed on cerebrospinal fluid circulating tumor cells (CSFCTC) of 19 patients. Results: Twenty-one patients were diagnosed with leptomeningeal metastases, and CSFCTCs were captured by CellSearch in 20 patients (median, 969 CSFCTCs/7.5 mL; range, 27–14,888). CellSearch had a sensitivity of 95.2% for leptomeningeal metastases diagnosis, which was higher than that of TCT (12/21, 57.1%), MRI (10/21, 47.6%), and MRI plus TCT (19/21, 90.5%), respectively. CTCs were found only in 5 of 14 patients (median, 2 CTCs/7.5 mL; range, 2–4), which was a much lower ratio than CSFCTCs. Genetic profiles of CSFCTCs were highly concordant with molecular mutations identified in the primary tumor (17/19, 89.5%). The resistance gene EGFR T790M was detected in 7 of 9 patients with extracranial lesions, but was detected in only 1 of 14 CSFCTC samples. Other potential resistant mutations, such as MET amplification and ERBB2 mutation, were also identified in CSFCTCs. Conclusions: CSFCTCs captured by CellSearch may be a more sensitive and effective way to diagnose leptomeningeal metastases, and may serve as a liquid biopsy medium for gene profiles in NSCLC patients with leptomeningeal metastases. Clin Cancer Res; 23(18); 5480–8. ©2017 AACR.

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.

Type 1 diabetes (T1D) is an autoimmune disease characterized by a selective destruction of insulin-secreting β-cells. Both T cells and B cells serve a crucial role in pathogenesis and development of T1D. CD20 is a specific membrane antigen of B lymphocytes, while interleukin (IL)-10 is an important cytokine secreted by T helper 2 cells and has a short half-life in vivo. The combined effect of anti-CD20 and IL-10 on immune function of mice with T1D remains unknown. In the present study, 30 non-obese diabetic (NOD) mice were treated with anti-CD20 and adenoviral vector-mediated interleukin-10 (Ad-mIL-10) therapy. Alterations in CD4+, CD8+, CD4+CD25+Foxp3+ T cells, T-box expressed in T-cells (T-bet), GATA-binding protein-3 (GATA-3) interferon-γ (IFN-γ) and IL-4 were detected by flow cytometry, reverse transcription-quantitative polymerase chain reaction in NOD mice spleen tissue. The present results suggested that anti-CD20 and IL-10 treatment in NOD mice can modulate the immune functions by upregulating GATA-3 and IL-4 expression as well as downregulating T-bet and IFN-γ expression, which are involved in the pathogenesis of T1D. The current findings may provide a potential method for T1D treatment and a novel preventive therapy for T1D. Combination of anti-CD20 and Ad-mIL-10 treatment had not only immune regulatory effects but also protective effects on islet β-cells in NOD mice with T1DM at the early stages, by regulating T-bet/GATA-3 expression and Th1/Th2 cell differentiation, which has the potential for diabetes prevention and therapy.

Accumulating evidence indicates that epithelial splicing regulatory protein 1 (ESRP1) can inhibit the epithelial-to-mesenchymal transition (EMT), thus playing a central role in regulating the metastatic progression of tumors. However, it is still not clear whether ESRP1 directly influences the cell cycle, or what the possible underlying molecular mechanisms are. In this study, we showed that ESRP1 protein levels were significantly correlated with the Ki-67 proliferative index (r = −0.521, p &lt, 0.01), and that ESRP1 overexpression can significantly inhibit cervical carcinoma cell proliferation and induced G1-phase arrest by downregulating cyclin A2 expression. Importantly, ESRP1 can bind to GGUGGU sequence in the 3′UTR of the cyclin A2 mRNA, and ESRP1 overexpression significantly decreases the stability of the cyclin A2 mRNA. In addition, our experimental results confirm that ESRP1 overexpression results in enhanced CDC20 expression, which is known to be responsible for cyclin A2 degradation. This study provides the first evidence that ESRP1 overexpression induces G1-phase cell cycle arrest via reducing the stability of the cyclin A2 mRNA, and inhibits cervical carcinoma cell proliferation. The findings suggest that the ESRP1/cyclin A2 regulatory axis may be essential as a regulator of cell proliferation, and may thus represent an attractive target for cervical cancer prevention and treatment.

Background Intratumoral epidermal growth factor receptor (EGFR) mutational heterogeneity is yet controversial in non-small cell lung cancer (NSCLC) patients. Single-cell analysis provides the genetic profile of single cancer cells and an in-depth understanding of the heterogeneity of a tumor. Methods Firstly, single H1975 cells harboring the EGFR L858R mutation were submitted to flow cytometry isolation, nested polymerase chain reaction (nested-PCR) amplification, and direct DNA sequencing to assess the feasibility of single-cell direct DNA sequencing. Then, the single cells of patients with lung adenocarcinoma receiving gefitinib were captured by laser capture microdissection and analyzed by the above methods to identify the intratumoral heterogeneity of the EGFR L858R mutant. Three patients with progression-free survival (PFS) > 14 months were categorized as the long PFS group, and 3 patients with PFS

The co-occurrence of epidermal growth factor receptor (EGFR) mutations and anaplastic lymphoma kinase (ALK) rearrangements constitutes a rare molecular subtype of non-small-cell lung cancer (NSCLC). Herein, we assessed the clinical outcomes and incidence of acquired resistance to tyrosine kinase inhibitors (TKIs) in this subtype. So we enrolled 118 advanced NSCLC treated with TKIs. EGFR mutations and ALK rearrangements were detected by DNA sequencing or Scorpion amplification refractory mutation system and fluorescence in situ hybridization respectively. Immunohistochemistry was used to evaluate the activation of associated proteins. We found that nine in ten patients with EGFR/ALK co-alterations had good response with first-line EGFR TKI, and the objective response rate (ORR) of EGFR TKIs was 80% (8/10) for EGFR/ALK co-altered and 65.5% (55/84) for EGFR-mutant (P = 0.57), with a median progression-free survival (PFS) of 11.2 and 13.2 months, (hazard ratio [HR]=0.95, 95% [CI], 0.49-1.84, P= 0.87). ORR of crizotinib was 40% (2/5) for EGFR/ALK co-altered and 73.9% (17/23) for ALK-rearranged (P= 0.29), with a median PFS of 1.9 and 6.9 months (hazard ratio [HR], 0.40; 95% [CI] 0.15-1.10, P = 0.08). The median overall survival (OS) was 21.3, 23.7, and 18.5 months in EGFR-mutant, ALK-rearranged, and EGFR/ALK co-altered (P= 0.06), and there existed a statistically significant difference in OS between ALK-rearranged and EGFR/ALK co-altered (P=0.03). Taken together, the first-line EGFR-TKI might be the reasonable care for advanced NSCLC harbouring EGFR/ALK co-alterations, whether or nor to use sequential crizotinib should be guided by the status of ALK rearrangement and the relative level of phospho-EGFR and phospho-ALK.

Background Our previous study identified rs9387478 as a new susceptibility locus associated with lung cancer in never-smoking women in Asia; however, the clinical and prognostic significance of this finding is not known. Methods We analyzed the relationship between the rs9387478 single nucleotide polymorphism and i) clinical parameters and ii) overall survival time in 505 female nonsmoking lung cancer patients, using the chi-square test and Kaplan-Meier analysis with the log-rank test, respectively. We further established the epidermal growth factor receptor (EGFR) mutation status and assessed its association with rs9387478 genotypes as well as the efficacy of EGFR tyrosine kinase inhibitors. Results The frequency of the AA genotype was significantly higher in the EGFR-mutation-negative group than in the EGFR-mutation-positive group (32% vs. 16%, χ 2 = 13.025, p = 0.011). Patients with the CC genotype had a better overall survival time than patients with the AA/AC genotype (median survival time: 54.2 vs. 32.9 months, χ 2 = 4.593, p = 0.032). The distribution of rs9387478 genotypes differed according to the clinical disease stage. Conclusions This study indicates that the rs9387478 genotype was associated with overall survival in nonsmoking female patients with lung cancer, although it was not significant after adjusting for multiple testing. The identification of the location of the rs9387478 single nucleotide polymorphism in the genomic interval containing the DCBLD1 and ROS1 genes, together with the finding that the rs9387478 polymorphism correlates with EGFR mutation status, may have important implications for therapeutic approaches targeting EGFR or ROS1 in patients with lung cancer.

BackgroundImmunotherapy has become an important treatment option for patients with advanced non-small cell lung cancer (NSCLC). At present, none of these existing biomarkers can effectively stratify true responders and there is an urgent need for identifying novel biomarkers. Exosomes derived from the serum of patients with cancer have been proven to be reliable markers for cancer diagnosis. Here, we explored the possibility of using plasma-derived exosomal microRNAs as potential biomarkers for optimal selection of patients with advancedEGFR/ALKnegative NSCLC to immunotherapy.MethodsFrom June 2017 to February 2019, 30 patients with advancedEGFR/ALKwild-type (WT) NSCLC who received PD-1/PD-L1 inhibitors were enrolled. The efficacy evaluation was conducted after every three cycles of treatment according to RECIST 1.1. Plasma samples of these patients were collected before the administration of PD-1/PD-L1 inhibitors as baseline, and after every three cycles if the patients achieved partial response (PR) or complete response. Plasma from seven healthy individuals was also collected as normal control. Exosomes were prepared by ultracentrifugation followed by total RNA extraction, and exosome-derived miRNAs were profiled using small RNA next-generation sequencing followed by differential expression analysis.ResultsIn order to identify biomarker for better response, all five patients who achieved PR and four patients with progressive disease (PD) at efficacy evaluation were included for differential expression analysis. Based on unsupervised hierarchical clustering, exosomal miRNA expression profile was significantly altered in patients with NSCLC compared with normal controls with a total of 155 differentially expressed exosomal miRNAs. Interestingly, hsa-miR-320d, hsa-miR-320c, and hsa-miR-320b were identified significantly upregulated in the PD groups compared with the PR group at baseline before the treatment. In addition, we identified that hsa-miR-125b-5p, a T-cell suppressor, showed a trend of increased expression in the PD group at baseline and was significantly downregulated in the post-treatment plasma exosomes compared with pre-treatment samples of the PR patients.ConclusionPatients with NSCLC represent unique plasma exosomal miRNA profiles. Hsa-miR-320d, hsa-miR-320c, and hsa-miR-320b were identified as potential biomarkers for predicting the efficacy of immunotherapy in advanced NSCLCs. When T-cell suppressor hsa-miR-125b-5p was downregulated during the treatment, the patients may obtain increased T-cell function and respond well to immunotherapy.

Background The third-generation EGFR tyrosine kinase inhibitor osimertinib has been approved in many countries to treat advanced NSCLC in patients with the EGFR T790M mutation. As the development of acquired resistance is inevitable, it is urgent that the mechanisms of such resistance be clarified. Methods DNA samples from a cohort of 340 patients with lung adenocarcinoma who were taking osimertinib were subjected to next-generation sequencing and screened in terms of the frequencies of the L792H and G796R mutations. Ba/F3 cells stably expressing the EGFR L858R/T790M mutations (in cis) with either the L792H or G796R mutation were created to investigate the impact of the two novel mutations on EGFR tyrosine kinase inhibitors and other potential drug combinations in vitro. Structural analyses were performed by using Schrodinger/Maestro software (version 11.1.012, Schrodinger LLC, Cambridge, MA). Results L792H and G796R were detected in 1.76% (six of 340) and 0.56% (two of 340) patients with lung adenocarcinoma treated with osimertinib, respectively. The introduction of L792H or G796R mutations against an L858R/T790M background caused dramatic reductions in osimertinib sensitivity. Structural modeling showed that mutations in cis with T790M either forced the ligand (osimertinib) to rotate out (breaking the binding) or pulled the hinge loop (breaking the hinge). Various other drug combinations. including cetuximab with EAI045, failed to inhibit either cis mutant effectively. Conclusions The EGFR L858R/T790M/L792H and L858R/T790M/G796R mutations conferred resistance to osimertinib both in vitro and in silico. For patients in whom the two resistance mutations occur at low frequency, more precise treatment strategies and additional combinational approaches are required.

Background Genome-wide association studies (GWAS) have determined a new single nucleotide polymorphism (SNP) called VTI1A (rs7086803) that induces lung cancer susceptibility in nonsmoking women in Asia. This study aimed to evaluate the association between the VTI1A gene and the susceptibility of Chinese patients to lung cancer; it was also conducted to investigate the relationship between VTI1A SNP and adiponectin receptor 1 expression. Methods A total of 887 subjects were enrolled in this study. VTI1A (rs7086803) genotypes were determined by genotyping. Overall survival (OS) was evaluated using Kaplan-Meier analysis with a log-rank test. Results Multivariate regression analysis results indicated that the AA genotype of VTI1A (rs7086803) polymorphism was associated with an increased risk of developing non-small cell lung carcinoma (NSCLC) compared with the GG genotype (AA vs. GG: odds ratio [OR] = 2.020; 95% confidence interval [95% CI], 1.033-3.949, p = 0.037). The AA genotype of VTI1A (rs7086803) in smokers predicted significantly shorter OS (median survival time [MST]: AA 9.8 months, AG 19.3 months, GG 12.2 months, p = 0.017). Adiponectin receptor 1 expression in tumor tissues with the AA genotype was significantly lower than that for other genotypes (mean rank: AA 18.55, AG 25, GG 45.76, p = 0.001). Conclusions The presence of the allele A of VTI1A (rs7086803) may be the allele contributing to the risk of lung cancer susceptibility in Chinese population. Smoking lung cancer patients with the AA genotype of VTI1A gene (rs7086803) had a poor survival rate. Adiponectin receptor 1 expression may be correlated with the susceptibility of the allele A of VTI1A.

The concomitant presence of echinoderm microtubule-associated protein-like 4 anaplastic lymphoma kinase (EML4-ALK) and epidermal growth factor receptor (EGFR) is extremely rare. Previous studies have shown that EGFR mutation and EML4-ALK fusion are mutually exclusive and that the coexistence of these 2 genes are associated with resistance to EGFR tyrosine kinase inhibitors (TKIs) and ALK inhibitors. We report a case of lung adenocarcinoma harboring a concomitant EGFR mutation and EML4-ALK fusion that benefited for a short period from 3 TKIs. Additional study is warranted to clarify the therapeutic strategies in such patients with a concomitant EGFR mutation and EML4-ALK fusion.

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.

Objectives To investigate the clinical characteristics of anaplastic lymphoma kinase (ALK) rearrangements and sequence complexity of the ALK fusion gene in Chinese lung cancer patients. Methods We prospectively screened ALK rearrangements in 1474 lung cancer specimens, including 1387 cases of non-small cell lung cancer (NSCLC), 54 cases of small cell lung cancer (SCLC), and 33 cases of cancer with lung metastasis from other organs by both standard polymerase chain reaction (PCR) and rapid amplification of cDNA ends (RACE)-coupled PCR. Fifteen cases of ALK-positive RACE-coupled PCR products were transformed into Escherichia coli for molecular cloning and sequenced for complexity analysis. Results The overall frequency of ALK rearrangements was 5.1% (71/1387) in NSCLC. In 71 positive cases, the coexistence of epidermal growth factor receptor (EGFR) and ALK variations was found in 6 cases (8.5%), and the coexistence of different ALK variants was found in 2 cases (2.8%) (1 case with variants 1 and 9; the other case with variants 3 and 2) by PCR analysis. Furthermore, through sequence cloning analysis of 15 cases of non-selective ALK-positive samples, two cases with variants 1 and 3 harbored the coexistence of three subtypes (variant 1 subtypes: E13; A20, E13del63; A20 and E7E12E13; A20 and variant 3 subtypes: E6; A20, E6ins33; A20 and E3E6; A20). Variant 3a and 3b subtypes were always coexistent and had the same proportion of ALK variant 3 rearrangements. ALK rearrangement was associated with young age, female gender, never-smokers, those with adenocarcinoma, advanced stage, and EGFR mutations. No ALK fusion was detected in 54 cases of SCLC or 33 cases of cancer with lung metastasis from other organs. Conclusions The identification of novel ALK variants, the coexistence of EGFR mutations and ALK fusions, the coexistence of ALK variants, and the coexistence of subtypes reveal the diversity and sequence complexity of ALK fusions.

Introduction Subtype classification of lung adenocarcinoma (LUAD) divides different survivals and therapeutic vulnerabilities; however, little is known about the disease's underlying molecular mechanism. This study sought to determine the genetic and immune profiles of histologic subtypes and identify the evidence for adjuvant immunotherapy. Methods We performed an integrated analysis of multidimensional data from a discovery set consisting of cohorts of The Cancer Genome Atlas and the Broad Institute data set from the LUAD public database and a validation set from the Guangdong Lung Cancer Institute. Immunohistochemical staining was carried out to determine the expression of the proteins programmed cell death 1 ligand (PD-L1) and CD8. Results Patients with solid predominant LUAD showed poor disease-free survival and a high frequency of relapse/metastasis compared with those with the nonsolid subtype of LUAD. The solid subtype tended to occur more frequently in those with a history of smoking. Solid predominant LUAD exclusively showed increased expression of PD-L1 and a high proportion of dual positive PD-L1– and tumor-infiltrating lymphocytes. Meanwhile, a notable increase in the tumor mutation burden and higher frequency of GC>TA transversions were specifically identified in tumors of the solid subtype. Furthermore, the solid subtype of tumor displayed an active cytotoxic immune signature and increased incidence of genetic mutations related to immunogenicity. Conclusion Solid predominant LUAD was identified as a subtype with adaptive immune resistance, higher cytotoxic activity, and enhanced immunogenicity. These findings suggest that patients with solid predominant LUAD may represent a potential selective group that will benefit from adjuvant programmed cell death 1 blockade immunotherapy.

Background: Drug resistance to targeted therapies occurs in lung cancer, and resistance mechanisms related to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) are continuously being discovered. We aimed to establish a novel method for highly parallel multiplexed detection of genetic mutations related to EGFR TKI-resistant lung cancer using Agena iPLEX chemistry and matrix-assisted laser desorption ionization time-of-flight analysis on the MassARRAY mass spectrometry platform. Methods: A review of the literature revealed 60 mutation hotspots in seven target genes (EGFR, KRAS, PIK3CA, BRAF, ERBB2, NRAS, and BIM) that are closely related to EGFR TKI resistance to lung cancer. A total of 183 primers comprised 61 paired forward and reverse amplification primers, and 61 matched extension primers were designed using Assay Design Software. The detection method was established by analyzing nine cell lines, and by comparison with LungCarta™ kit in ten lung cancer specimens. EGFR, KRAS, and BIM genes in all cell lines and clinical samples were subjected to Sanger sequencing for confirming reproducibility. Results: Our data showed that designed panel was a high-throughput and robust tool, allowing genotyping for sixty hotspots in the same run. Moreover, it made efficient use of patient diagnostic samples for a more accurate EGFR TKIs resistance analysis. The proposed method could accurately detect mutations in lung cancer cell lines and clinical specimens, consistent with those obtained by the LungCarta™ kit and Sanger sequencing. We also established a method for detection of large-fragment deletions based on single-base extension technology of MassARRAY platform. Conclusions: We established an effective method for high-throughput detection of genetic mutations related to EGFR TKI resistance based on the MassARRAY platform, which could provide more accurate information for overcoming cancers with de novo or acquired resistance to EGFR-targeted therapies. Key words: Drug Resistance; Epidermal Growth Factor Receptor; Lung Cancer; MassARRAY; Targeted Molecular Therapy

The objective of this study was to analyze the predictive roles of VEGF/KDR/Ras/MAPK gene expression in patients with advanced non-small-cell lung cancer (NSCLC) treated with bevacizumab plus chemotherapy. Twenty-five patients participating in an open-label phase IV trial (SAiL, MO19390) with available tumor tissues were analyzed. The mRNA expression levels of VEGF, kinase insert domain receptor (KDR), Ras, and mitogen-activated protein kinase (MAPK) in tumor tissues were detected using real-time quantitative PCR methods. The relationships between gene expression and disease control rate (DCR), progression-free survival (PFS), and overall survival (OS) were assessed. Patients with lower Ras expression had a longer PFS and OS than patients with higher expression (median PFS, 9.9 vs 5.5 months, χ(2)=3.944, P=0.047; OS, 19.3 vs 7.1 months, χ(2)=9.384, P=0.002). The PFS and OS of patients with lower and higher MAPK expression exhibited a marginal and significant difference (median PFS, 9.9 vs 5.5 months, χ(2)=3.464, P=0.063; OS, 19.3 vs 9.7 months, χ(2)=5.298, P=0.021), respectively. Multivariate analyses using Cox's proportional hazards model showed that Ras is an independent predictor of OS (χ(2)=9.384, P=0.002). No differences in DCR were found according to Ras expression level. The results indicate that Ras is an independent predictor of OS. Thus, patients with lower Ras expression are most likely to benefit from bevacizumab plus chemotherapy treatment regimen. Patients with higher levels of Ras should receive other inhibitors that target Ras. The results also suggest that gene therapies that decrease RAS expression combined with bevacizumab may improve lung cancer treatment. Although there is a very important implication to patient selection in the target therapy, the data in this study are very preliminary owing to the too small sample size. Therefore, further research involving large numbers of patients and a prospective assessment of low and high RAS mRNA expressions getting the same treatments need to be done before conclusions can be made.

As molecular targets continue to be identified and more targeted inhibitors are developed for personalized treatment of non-small cell lung cancer (NSCLC), multigene mutation determination will be needed for routine oncology practice and for clinical trials. In this study, we evaluated the sensitivity and specificity of multigene mutation testing by using the Snapshot assay in NSCLC. We retrospectively reviewed a cohort of 110 consecutive NSCLC specimens for which epidermal growth factor receptor (EGFR) mutation testing was performed between November 2011 and December 2011 using Sanger sequencing. Using the Snapshot assay, mutation statuses were detected for EGFR, Kirsten rate sarcoma viral oncogene homolog (KRAS), phosphoinositide-3-kinase catalytic alpha polypeptide (PIK3CA), v-Raf murine sarcoma viral oncogene homolog B1 (BRAF), v-ras neuroblastoma viral oncogene homolog (NRAS), dual specificity mitogen activated protein kinase kinase 1 (MEK1), phosphatase and tensin homolog (PTEN), and human epidermal growth factor receptor 2 (HER2) in patient specimens and cell line DNA. Snapshot data were compared to Sanger sequencing data. Of the 110 samples, 51 (46.4%) harbored at least one mutation. The mutation frequency in adenocarcinoma specimens was 55.6%, and the frequencies of EGFR, KRAS, PIK3CA, PTEN, and MEK1 mutations were 35.5%, 9.1%, 3.6%, 0.9%, and 0.9%, respectively. No mutation was found in the HER2, NRAS, or BRAF genes. Three of the 51 mutant samples harbored double mutations: two PIK3CA mutations coexisted with KRAS or EGFR mutations, and another KRAS mutation coexisted with a PTEN mutation. Among the 110 samples, 47 were surgical specimens, 60 were biopsy specimens, and 3 were cytological specimens; the corresponding mutation frequencies were 51.1%, 41.7%, and 66.7%, respectively (P = 0.532). Compared to Sanger sequencing, Snapshot specificity was 98.4% and sensitivity was 100% (positive predictive value, 97.9%; negative predictive value, 100%). The Snapshot assay is a sensitive and easily customized assay for multigene mutation testing in clinical practice.

e14534 Background: 20-40% of lung cancer patients develop bone metastasis (BM) with significantly decreased overall survival. Currently, BM is mainly diagnosed by CT scan or MRI when symptom develop. Novel biomarkers with higher prediction value of BM are needed. Tumor secreted exosomes that are enriched with microRNAs have been recently implicated in site-specific tumor metastasis for establishing a distant pro-metastatic niche. Methods: Plasma exosomes from 39 stage IV non-small cell lung cancer (NSCLC) patients (n = 23 with BM; n = 16 without BM) and 16 healthy individuals were separated by ultracentrifugation. Total RNAs were extracted followed by small RNA-sequencing. miRNA identification was performed by miRDeep2. Weighted correlation network analysis (WGCNA) was used for identifying co-expression networks of highly correlated miRNA clusters. miRNA enrichment analysis and annotation (MIEAA) was applied for pathway analysis. Differential expression of individual miRNA was analyzed by edgeR. Results: Hierarchical clustering based on the total miRNA profile can clearly separate cancer patients and healthy individuals (HP), but not patients with (BM+) or without (BM-) BM. WGCNA identified three consensus clusters (A, B, C) of highly correlated miRNAs, among which cluster A (144 miRNAs) showed significantly differential expression in lung cancer patients, especially in BM+ group. Pathway analysis of cluster B miRNAs revealed enrichment in glycolysis and gluconeogenesis pathways that may involve in metabolic preconditioning of the metastatic niche. Three differentially expressed miRNAs between BM+ and BM- patients within cluster A were identified as miR-574-5p, a suppressor of Wnt/β-catenin pathway, was down-regulated, while miR-328-3p and miR-423-3p, two activators of the same pathway, were up-regulated in BM+ patients. Cluster B miRNAs (n = 49) also showed trend of upregulation in BM+ patients. Interestingly, pathway analysis indicated that 43 of them are associated with chr14, which has been suggested to promote EMT and bone metastasis. Conclusions: NSCLC patients with BM displayed unique plasma exosomal miRNA profiles, which might involve in promoting BM, and be potential biomarkers for predicting BM in NSCLC.

Available study revealed non-small cell lung cancer (NSCLC) patients faced a risk of disease flare after cessation of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) treatment. There was no data concerning the prognostic value of disease flare. This study aimed to investigate the prevalence of disease flare in a Chinese cohort, and analyzed its prediction to survival. A cohort of 227 NSCLC patients with acquired resistance to EGFR TKI was retrospectively analyzed. Prevalence and clinical features of disease flare after TKI cessation were reviewed. Survival data were analyzed between patients with flare and those without flare. EGFR gene mutations in tumors were detected. Twenty of 227 (8.8 %) patients were determined with disease flare after TKI cessation. The median interval from TKI cessation to disease flare was 7 days (range 3-18). Forty percent of patients complained of deteriorated dyspnea attributable to malignant effusion. Thirty percent of patients had progressive lesions in the brain. After TKI cessation 35 % of flare patients died before challenge of subsequent treatment. No response was observed in 30 % of flare patients undergoing subsequent chemotherapy. When compared with the non-flare group, patients with disease flare demonstrated comparable progression-free survival (10.1 vs. 9.9 months; P = 0.973), shorter post-TKI survival (4.1 vs. 6.1 months; P < 0.001), and a significantly poor overall survival (16.6 vs. 21.6 months; P = 0.002). Disease flare after cessation of EGFR TKI occurred in Chinese NSCLC population and predicted a poor survival.

Background Detecting epidermal growth factor receptor (EGFR) activating mutations in plasma could guide EGFR-tyrosine kinase inhibitor (EGFR-TKI) treatment for advanced non-small cell lung cancer (NSCLC). However, dynamic quantitative changes of plasma EGFR mutations during the whole course of EGFR-TKI treatment and its correlation with clinical outcomes were not determined. The aim of this study was to measure changes of plasma EGFR L858R mutation during EGFR-TKI treatment and to determine its correlation with the response and resistance to EGFR-TKI. Methods This study was a pre-planned exploratory analysis of a randomized phase III trial conducted from 2009 to 2014 comparing erlotinib with gefitinib in advanced NSCLC harboring EGFR mutations in tumor (CTONG0901). Totally, 256 patients were enrolled in CTONG0901 and randomized to receive erlotinib or gefitinib. One hundred and eight patients harbored L858R mutation in their tumors and 80 patients provided serial blood samples as pre-planned scheduled. Serial plasma L858R was detected using quantitative polymerase chain reaction. Dynamic types of plasma L858R were analyzed using Ward’s hierarchical clustering method. Progression-free survival (PFS) and overall survival (OS) were compared between different types. Results As a whole, the quantity of L858R decreased and reached the lowest level at the time of best response to EGFR-TKI. After the analysis of Ward’s hierarchical clustering method, two dynamic types were found. In 61 patients, L858R increased to its highest level when disease progressed (ascend type), while in 19 patients, L858R maintained a stable level when disease progressed (stable type). Median PFS was 11.1 months (95 % CI, 6.6–15.6) and 7.5 months (95 % CI, 1.4–13.6) in patients with ascend and stable types, respectively (P = 0.023). Median OS was 19.7 months (95 % CI, 16.5–22.9) and 16.0 months (95 % CI, 13.4–18.5), respectively (P = 0.050). Conclusions This is the first report finding two different dynamic types of plasma L858R mutation during EGFR-TKI treatment based on a prospective randomized study. Different dynamic types were correlated with benefits from EGFR-TKI. The impact of plasma L858R levels at disease progression on subsequent treatment strategy needs further exploration. Trial registration ClinicalTrials.gov, NCT01024413 Electronic supplementary material The online version of this article (doi:10.1186/s13045-016-0316-8) contains supplementary material, which is available to authorized users.

Background According to the literature and our experience, the most common sites of non-small cell lung cancer (NSCLC) metastases include the brain, bone, liver, adrenal glands, contralateral lung and distant lymph nodes. Metastases to other organs are relatively rare. There have been numerous case reports and a few small case series of uncommon metastases derived from NSCLC. Methods We defined all organs except the common metastatic sites mentioned above as uncommon sites of metastasis. Patients with uncommon metastases among 2,872 consecutive NSCLC patients with stage IV disease at the Guangdong Lung Cancer Institute (GLCI) from 2006 to 2012 were included in this study. The diagnosis of uncommon metastases was based on pathology or imaging studies. Results Uncommon metastases were diagnosed in 193 cases at anatomical sites such as the soft tissue, kidney, pancreas, spleen, peritoneum, intestine, bone marrow, eye, ovary, thyroid, heart, breast, tonsil and nasal cavity. Uncommon metastases were identified as independent poor prognostic factors through a multivariate analysis with a HR (hazard ratio) of 1.29 [95 % confidence interval (CI) 1.09–1.52, P

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.

The prognostic and predictive value of KRAS mutations in patients with lung cancer is controversial. Biases in disease stage, treatment regimen, small-scale patient studies, and biomarker status have led to inconsistent results in previous reports. The KRAS and EGFR genes were examined in 1935 consecutive patients with non-small cell lung cancer. All patients were divided into KRAS mutation (KRAS group), EGFR mutation (EGFR group), and KRAS/EGFR wild type (WT group) groups. Randomly selected cases were paired with patients with the KRAS mutation, the EGFR mutation, and KRAS/EGFR wild type patients according to tumor, node, metastasis stage, time of first visit within 1 year, and pathology. Progression-free survival (PFS) and overall survival were evaluated by Kaplan–Meier and Cox models. The KRAS mutation rate for lung adenocarcinoma was 5.90 %. The overall survival was 14.47, 20.57, and 42.73 months for the KRAS group, WT group, and EGFR group, respectively (P

Background. Non-small cell lung cancer patients with epidermal growth factor receptor (EGFR) mutations have mixed responses to tyrosine kinase inhibitors (TKIs). Intertumor heterogeneity in EGFR mutations is one potential explanation for this phenomenon. Methods. We performed direct sequencing to identify EGFR mutations in 180 pairs of lung adenocarcinoma samples (from 3,071 patients). The high-resolution melting method was used in discordant cases to confirm EGFR mutation status. Matching samples were divided into four groups: primary lesions detected at different times, primary tumors with matched metastatic lymph nodes, multiple pulmonary nodules, and primary tumors with matched distant metastases. Multivariate analyses were performed to evaluate correlations between heterogeneity and patient characteristics. Results. In the study population, the discordance rate was 13.9% (25 of 180). The multiple pulmonary nodules group had the highest discordance rate of 24.4% (10 of 41; odds ratio for heterogeneity in primary lesions detected at different times, 6.37; 95% confidence interval, 1.71–23.72; p = .006). Discordance rates in the metachronous and synchronous settings were 15.7% (22 of 140) and 7.5% (three of 40), respectively. In the 34 patients who developed EGFR TKI resistance, 10 (29.4%) cases exhibited heterogeneity and five (14.7%) patients exhibited a mixed response to the drug. Three (8.8%) of the patients with a mixed response also exhibited discordant EGFR mutations. Conclusions. The overall discordance rate of EGFR mutation heterogeneity in Asian patients with pulmonary adenocarcinoma is relatively low, but the rate in patients with multiple pulmonary nodules is significantly higher. This observation may explain the mixed tumor response to EGFR TKIs.

We sought to find blood-based biomarkers that can be used to predict efficacy in advanced non-small cell lung cancer patients treated with bevacizumab plus chemotherapy. Blood was collected before treatment and after 6 weeks of therapy from patients who were participating in a phase 4 trial. Plasma vascular endothelial growth factor (VEGF) levels were evaluated by ELISA. A total of eight single nucleotide polymorphisms in four candidate genes were analyzed by PCR and sequencing. A total of 45 patients enrolled in a clinical trial at Guangdong General Hospital between August 2007 and March 2008 were used as subjects. The median survival times of OS was 25.6 and 13.4 months in the low and high groups, respectively, when the median posttreatment plasma VEGF level (46.63 pg/ml) was used as the cut-off point (P = 0.0284). Patients carrying the AA genotype at the −6C > A polymorphism in laminin 5 (LN5) were more likely to exhibit reduced hemoglobin compared with patients carrying the CA/CC genotype (OR = 8.364, χ2 = 5.34, P = 0.021). Similar associations were found at the −89A > G and −260C > A polymorphisms in LN5. Patients with the CC genotype at the −6C > A polymorphism in LN5 had an increased risk of neutropenia than those with the CA/AA genotype (OR = 4.444, χ2 = 5.116, P = 0.030). Our results show improved survival in patients with lower posttreatment plasma VEGF levels treated with bevacizumab plus chemotherapy; thus, the posttreatment plasma VEGF level may be a promising biomarker to predict clinical benefit early in the course of therapy. Polymorphisms in LN5 were associated with a reduced level of hemoglobin and neutropenia.

Although the kinase insert domain-containing receptor (KDR) gene play an very important role in the metastasis of cancer and is also as one of the molecular targets used in cancer therapy, mutation in the tyrosine kinase (TK) domain of the KDR gene has not been reported. Here we detected the mutations and polymorphisms in the TK domain of KDR gene in human lung cancer patients and to give the basic evidence and clue for cancer prevention and target therapy. The entire sequence of exons 21, 22, 23 and 27 (which contain the coding sequence of tyrosine phosphorylation) in the TK domain of KDR gene in the patients with lung cancer and control healthy individuals were assayed by PCR and DNA sequencing. We also analyzed one non-coding single nucleotide polymorphisms (SNPs) in the KDR gene. No mutations were found in exon 22, 23 and 27. One heterozygous mutation of c.+2837 in exon 21 was found at a frequency of 2.08% (2/96) in the patients with lung cancer and none were detected in the healthy control individuals. The mutation was from a G to a A resulting in substitution of arginine with histidine residue. Our data suggested that we should focus on the mutation or SNP in the other regions or the expression levels of KDR gene, and the function of c.+2837 mutation of KDR gene may be needed further study in the future.

Background Immunohistochemistry (IHC) and fluorescent in situ hybridization are reliable methods for identifying c-Met protein expression or c-Met gene amplification. However, each technique requires a high-quality tissue sample, which might not be available. The aim of the present study was to investigate the correlation between the soluble c-Met level and tissue c-Met protein expression and the relationship between these markers and patient prognosis. Materials and Methods In 198 patients with advanced non–small-cell lung cancer, tumor tissue c-Met expression was determined using IHC according to the H score criteria. Positivity was defined as ≥ 50% of cells with strong staining (IHC 3+). The concentration of c-Met protein in paired plasma samples was measured using a human soluble c-Met quantitative enzyme-linked immunosorbent assay kit, and the predictive value was determined using receiver operating characteristic curve analysis. Results Of the 198 patients, 140 (70.7%) had tissue c-Met − findings and 58 (29.3%) tissue c-Met + findings. Receiver operating characteristic curve analysis showed 67.2% specificity and 65.0% sensitivity for predicting tissue c-Met positivity at a plasma c-Met cutoff of 766 ng/mL. The correlation between the soluble c-Met level and tissue c-Met protein expression was significant (Pearson's r = 0.309; P 766 ng/mL) had poorer overall survival than patients with low soluble c-Met levels (9.5 vs. 22.2 months; P P = .002). Conclusion A significant correlation was found between the plasma soluble c-Met levels and tissue c-Met protein expression in patients with advanced non–small-cell lung cancer. A high level of soluble c-Met was associated with a poor prognosis.

Objectives Most lung adenocarcinomas consist of mixtures of histological subtypes harboring different frequencies of driver gene mutations. However, little is known about intratumoral heterogeneity(ITH) within histologically heterogeneous primary lung cancers. Investigating key driver genes in respective morphological pattern is crucial to personalized treatment. Methods Morphologically different areas within the same surgically resected adenocarcinomas were extracted from tissues to analyze gene status in each growth pattern. Driver genes, epidermal growth factor receptor (EGFR), KRAS and EML4-ALK, were assessed by assays with different sensitivities. Results Seventy-nine consecutive eligible patients harboring a driver gene (EGFR = 65; KRAS = 10; EML4-ALK = 4) were enrolled. For EGFR mutations, ITH occurred in 13.3% (8/60) by direct sequencing (DS) and 1.7% (1/60) by amplification refractory mutation system (ARMS) (P = 0.016) among adenocarcinomas, but consistent within five adeno-squamous cell carcinomas by both methods. ITH among KRAS mutations were detected in 20% (2/10) by DS, whereas consistent (10/10) by high resolution melting. No discrepancies in EML4-ALK rearrangements existed according to fluorescence in situ hybridization. Conclusion Rare ITHs of EGFR/KRAS/EML4-ALK alterations within histologically heterogeneous primary lung adenocarcinomas existed by methods with higher sensitivity. Discrepancies might be due to abundance of mutant tumor cells and detection assays.

BACKGROUND: The hedgehog (Hh) pathway is involved in embryogenesis and organogenesis. GLI3 is one of the zinc-finger transcription factors in the Hh signaling pathway, which exist in both full-length (GLI3FL) and truncated (GLI3TR) forms. We investigated GLI3 expression in patients with non-small cell lung cancer (NSCLC). The role of GLI3 in lung carcinogenesis and its correlation with clinicopathological factors and overall survival (OS) in patients with NSCLC were explored. METHODS: GLI3FL and GLI3TR expression were analyzed immunohistochemically in 330 and 352 evaluable NSCLC tissues respectively. The association between GLI3FL and GLI3TR expression and clinicopathological parameters and OS were statistically analyzed. RESULTS: GLI3FL immunohistochemical staining could be observed in the cytoplasm, while GLI3TR staining could be observed in nucleus of malignant epithelial cells. High level expression of GLI3FL and GLI3TR were 52.7% and 45.2% respectively. GLI3FL was not significantly correlated with any clinicopathological parameter and survival. However, high-expression of GLI3TR was significantly associated with lymph node metastasis (P = 0.013) and poor OS (28.4 vs. 40.8 months, P = 0.010). In patients with adenocarcinoma of high and low GLI3TR expression, the median OS were 25.7 and 50.6 months respectively (P = 0.004). Multivariate analysis showed that GLI3TR expression (P = 0.036), tumor differentiation ( P< 0.001), disease stage ( P< 0.001) were independent prognostic factors for patients with NSCLC. CONCLUSION: Overexpression of GLI3TR in NSCLC, especially in adenocarcinoma, is associated with poor prognosis. GLI3TR expression is an independent prognostic factor in OS. GLI3TR may play an important role in the tumorigenesis of NSCLC.

Laminin 5 (Ln5) is an extracellular matrix protein that plays an important role in cell migration and tumor invasion. This study explored the expression of Ln5 and the role of its relationships with PTEN, phospho-EGFR (p-EGFR) and phospho-Akt (p-Akt) in the prognosis of patients with non-small cell lung cancer (NSCLC). The protein expression of Ln5, PTEN, p-EGFR and p-Akt was assessed by immunohistochemical analysis, and their relationships to prognosis were analyzed. Protein expression of Ln5, p-EGFR and p-Akt was detected in 61.2 (60/98), 60.2 (59/98) and 45.3% (43/95) of patients with NSCLC, respectively. Loss of PTEN expression was found in 67.7% of tumors (65/96). Ln5 expression was related to patient gender, histology and p-Akt expression (χ(2)=3.901, 4.549 and 6.985, respectively; P=0.048, 0.033 and 0.008, respectively). Patients with positive Ln5 expression had marginally poorer survival than Ln5-negative patients (median survival time 56.4 months vs. not reached; χ(2)=3.346; P=0.067). Overall survival was significantly different in patients with positive Ln5 expression combined with loss of PTEN, positive p-EGFR expression or positive p-Akt expression. Cox regression analysis showed that stage, co-expression of Ln5 and p-Akt, and PTEN were the three most independent prognostic factors for patients with NSCLC (χ(2)=27.906; P0.0005). The results highlight the complex relationships between extracellular matrix proteins and key signaling pathway molecules in tumorigenesis. Changes in the expression of Ln5 plus PTEN, p-EGFR or p-Akt define a distinct subset of lung cancers. Patients with such cancers have poorer survival and require early treatment that impacts survival.