Osimertinib after Chemoradiotherapy in Stage III EGFR-Mutated NSCLC.

BACKGROUND: Osimertinib is a recommended treatment for advanced non-small-cell lung cancer (NSCLC) with an epidermal growth factor receptor (EGFR) mutation and as adjuvant treatment for resected EGFR-mutated NSCLC. EGFR-tyrosine kinase inhibitors have shown preliminary efficacy in unresectable stage III EGFR-mutated NSCLC. METHODS: In this phase 3, double-blind, placebo-controlled trial, we randomly assigned patients with unresectable EGFR-mutated stage III NSCLC without progression during or after chemoradiotherapy to receive osimertinib or placebo until disease progression occurred (as assessed by blinded independent central review) or the regimen was discontinued. The primary end point was progression-free survival as assessed by blinded independent central review. RESULTS: A total of 216 patients who had undergone chemoradiotherapy were randomly assigned to receive osimertinib (143 patients) or placebo (73 patients). Osimertinib resulted in a significant progression-free survival benefit as compared with placebo: the median progression-free survival was 39.1 months with osimertinib versus 5.6 months with placebo, with a hazard ratio for disease progression or death of 0.16 (95% confidence interval [CI], 0.10 to 0.24; P<0.001). The percentage of patients who were alive and progression free at 12 months was 74% (95% CI, 65 to 80) with osimertinib and 22% (95% CI, 13 to 32) with placebo. Interim overall survival data (maturity, 20%) showed 36-month overall survival among 84% of patients with osimertinib (95% CI, 75 to 89) and 74% with placebo (95% CI, 57 to 85), with a hazard ratio for death of 0.81 (95% CI, 0.42 to 1.56; P = 0.53). The incidence of adverse events of grade 3 or higher was 35% in the osimertinib group and 12% in the placebo group; radiation pneumonitis (majority grade, 1 to 2) was reported in 48% and 38%, respectively. No new safety concerns emerged. CONCLUSIONS: Treatment with osimertinib resulted in significantly longer progression-free survival than placebo in patients with unresectable stage III EGFR-mutated NSCLC. (Funded by AstraZeneca; LAURA ClinicalTrials.gov number, NCT03521154.).

Tumor-intrinsic IFNα and CXCL10 are critical for immunotherapeutic efficacy by recruiting and activating T lymphocytes in tumor microenvironment.

Tumor immunotherapies targeting PD-(L)1 exhibit anti-tumor efficacy in only 10-30% of patients with various cancers. Literature has demonstrated that a "hot tumor" which contains high T lymphocytes in the tumor microenvironment exhibits a better response to immunotherapies than a "cold tumor." This study aimed to investigate whether tumor-intrinsic IFNα and CXCL10 determine the recruitment and activation of CD8+ T cells to become "hot tumor." In this study, we found that CXCL10 overexpressed in a variety of tumors including lung, colon, and liver tumors with a correlation with PD-L1. High PD-L1 and CXCL10 are associated with better survival rates in tumor patients receiving immunotherapies. IFNs-downstream transcriptional factor IRF-1 and STAT1 were correlated with PD-L1 and CXCL10 expression. We demonstrated that IRF-1 and STAT1 were both bound with the promoters of PD-L1 and CXCL10, sharing the same signaling pathway and determining IFNs-mediated PD-L1 and CXCL10 expression. In addition, IFNα significantly increased activation marker IFNγ in PBMCs, promoting M1 type monocyte differentiation, CD4+ T, and CD8+ T cell activation. Particularly, we found that CD8+ T lymphocytes abundantly expressed CXCR3, a receptor of CXCL10, by flow cytometry, indicating that tumor-intrinsic CXCL10 potentially recruited CD8+ T in tumor microenvironment. To demonstrate the hypothesis, immunotherapy-sensitive CT26 and immunotherapy-resistant LL/2 were used and we found that CT26 cells exhibited higher IFNα, IFNγ, CXCL10, and PD-L1 levels compared to LL/2, leading to higher IFNγ expression in mouse splenocytes. Moreover, we found that CD8+ T cells were recruited by CXCL10 in vitro, whereas SCH546738, an inhibitor of CXCR3, inhibited T cell migration and splenocytes-mediated anti-tumor effect. We then confirmed that CT26-derived tumor was sensitive to αPD-L1 immunotherapy and LL/2-tumor was resistant, whereas αPD-L1 significantly increased T lymphocyte activation marker CD107a in CT26-derived BALB/c mice. In conclusion, this study revealed that CXCL10 expression is correlated with PD-L1 in tumors, sharing the same signaling pathway and associating with better immunotherapeutic efficacy. Further evidence in the syngeneic tumor models demonstrated that immunotherapy-sensitive CT26 intrinsically exhibited higher IFNα and CXCL10 compared to immunotherapy-resistant LL/2 to recruit and activate CD8+ T cells in the tumor microenvironment, exhibiting "hot tumor" characteristic of sensitizing αPD-L1 immunotherapies.

The middle-aged and older Chinese adults' health using actigraphy in Taiwan (MOCHA-T): protocol for a multidimensional dataset of health and lifestyle.

BACKGROUND AND OBJECTIVES: Older adults keep transforming with Baby Boomers and Gen Xers being the leading older population. Their lifestyle, however, is not well understood. The middle-aged and older Chinese adults' health using actigraphy in Taiwan (MOCHA-T) collected both objective and subjective data to depict the health and lifestyle of this population. The objectives, design, and measures of the MOCHA-T study are introduced, and the caveats and future directions related to the use of the data are presented. METHODS: People aged 50 and over were recruited from the community, with a subset of women aged 45-49 invited to supplement data on menopause and aging. Four instruments (i.e., self-reported questionnaires, diary, wrist actigraphy recorder, and GPS) were used to collect measures of sociodemographic, health, psychosocial, behavioral, temporal, and spatial data. RESULTS: A total of 242 participants who returned the informed consent and questionnaires were recruited in the MOCHA-T study. Among them, 94.6%, 95.0%, and 25.2% also completed the diary, actigraphy, and GPS data, respectively. There was almost no difference in sociodemographic characteristics between those with and without a completed diary, actigraphy, and GPS data, except for age group and educational level for those who returned completed actigraphy data. CONCLUSION: The MOCHA-T study is a multidimensional dataset that allows researchers to describe the health, behaviors, and lifestyle patterns, and their interactions with the environment of the newer generation of middle-aged and older adults in Taiwan. It can be compared with other countries with actigraphy and GPS-based lifestyle data of middle-aged and older adults in the future.

Acetylation regulates the nucleocytoplasmic distribution and oncogenic function of karyopherin alpha 2 in lung adenocarcinoma.

Karyopherin subunit alpha 2 (KPNA2, importin α1) is a nucleoplasmic protein responsible for the nuclear import of proteins with classical nuclear localization signals. Aberrant nuclear accumulation of KPNA2 has been observed in numerous cancer tissues. AMP-activated protein kinase (AMPK) is involved in the phosphorylation and acetylation of KPNA2 in enterocytes. However, the impact of these post-translational modifications on modulating the nucleocytoplasmic distribution of KPNA2 and its oncogenic role remain unclear. Unlike nuclear accumulation of wild-type KPNA2, which promoted lung cancer cell migration, KPNA2 Lys22 acetylation-mimicking mutations (K22Q and K22Q/S105A) prevented nuclear localization of KPNA2 and reduced the cell migration ability. Cytosolic KPNA2 K22Q interacted with and restricted the nuclear entry of E2F transcription factor 1 (E2F1), an oncogenic cargo protein of KPNA2, in lung cancer cells. Intriguingly, the AMPK activator EX229 promoted the nuclear export of KPNA2 S105A. However, the CBP/p300 inhibitor CCS-1477 abolished this phenomenon, suggesting that CBP/p300-mediated acetylation of KPNA2 promoted KPNA2 nuclear export in lung cancer cells. Collectively, our findings suggest that the CBP/p300 positively regulates KPNA2 acetylation, which enhances its cytosolic localization and suppresses its oncogenic activity in lung cancer.

Radiotherapy enhances CXCR3highCD8+ T cell activation through inducing IFNγ-mediated CXCL10 and ICAM-1 expression in lung cancer cells.

Radiotherapy (RT) not only damages tumors but also induces interferon (IFN) expression in tumors. IFNs mediate PD-L1 to exhaust CD8+ T cells, but which also directly impact tumor cells and potentially activate anti-tumor immune surveillance. Little is known about the contradictory mechanism of IFNs in regulating CD8+ T-mediated anti-tumor activity in lung cancer. This study found that RT induced IFNs and CXCL9/10 expression in the RT-treated lung cancer cells. Specifically, RT- and IFNγ-pretreated A549 significantly activated CD8+ T cells, resulting in significant inhibition of A549 colony formation. RNAseq and consequent qPCR results revealed that IFNγ induced PD-L1, CXCL10, and ICAM-1, whereas PD-L1 knockdown activated CD8+ T cells, but ICAM-1 knockdown diminished CD8+ T cell activation. We further demonstrated that CXCR3 and CXCL10 decreased in the CD8+ T cells and nonCD8+ PBMCs, respectively, in the patients with lung cancer that expressed lower reactivation as co-cultured with A549 cells. In addition, inhibitors targeting CXCR3 and LFA-1 in CD8+ T cells significantly diminished CD8+ T cell activation and splenocytes-mediated anti-LL/2shPdl1. In conclusion, we validated that RT suppressed lung cancer and overexpress PD-L1, CXCL10, and ICAM-1, which exhibited different roles in regulating CD8+ T cell activity. We propose that CXCR3highCD8+ T cells stimulated by CXCL10 exhibit anti-tumor immunity, possibly by enhancing T cells-tumor cells adhesion through CXCL10/CXCR3-activated LFA-1-ICAM-1 interaction, but CXCR3lowCD8+ T cells with low CXCL10 in patients with lung cancer were exhausted by PD-L1 dominantly. Therefore, RT potentially activates CD8+ T cells by inducing IFNs-mediated CXCL10 and ICAM-1 expression in tumors to enhance CD8+ T-tumor adhesion and recognition. This study clarified the possible mechanisms of RT and IFNs in regulating CD8+ T cell activation in lung cancer.

Zinc deficiency associated with cutaneous toxicities induced by epidermal growth factor receptor tyrosine kinase inhibitor therapy in patients with lung adenocarcinoma.

PURPOSE: Cutaneous toxicities are common adverse effects following epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) therapy. Zinc deficiency causes diverse diseases, including skin toxicities. Therefore, this study aimed to investigate the role of zinc deficiency in patients with EGFR-TKI-induced skin toxicities. EXPERIMENTAL DESIGN: This retrospective study enrolled 269 patients with diverse skin disorders who visited our hospital between January 2016 and December 2017. The skin toxicity severities and plasma zinc levels of 101 EGFR-TKI-treated cancer patients were analysed and compared with those of 43 non-EGFR-TKI-treated cancer patients and 125 patients without cancer but presenting cutaneous manifestations. Additionally, the role of zinc in erlotinib-induced skin eruptions was established in a 14-day-murine model. Clinical features were further evaluated following systemic zinc supplementation in EGFR-TKI-treated cancer patients. RESULTS: EGFR-TKI-treated patients demonstrated severe cutaneous manifestations and a significant decrease in plasma zinc levels than those of the control groups. The serum zinc level and Common Terminology Criteria for Adverse Events (CTCAE) 5.0 grading of EGFR-TKI-induced skin toxicities showed a significant negative correlation (r = -0.29; p < 0.0001). Moreover, erlotinib treatment decreased the plasma zinc levels and induced periorificial dermatitis in rats confirming zinc deficiency following EGFR-TKI treatment. Zinc supplementation to the EGFR-TKI-treated cancer patients showed a significant decrease in the CTCEA grading (p < 0.0005 for mucositis and p < 0.0.0001 for all other cases) after 8 weeks. CONCLUSIONS: Skin impairment following EGFR-TKI therapy could be ameliorated through zinc supplementation. Thus, zinc supplementation should be considered for cancer patients undergoing EGFR-TKI therapy.

Methods for Collection of Extracellular Vesicles and Their Content RNA as Liquid Biopsy for Lung Cancer Detection: Application of Differential Centrifugation and Annexin A5 Coated Beads.

Extracellular vesicles (EVs) contain abundant extracellular RNA (exRNA), which can be a valuable source of liquid biopsy. However, as various RNA species exist in different types of EVs, lack of detailed characterization of these RNA species and efficient collection methods limits the clinical application of exRNA. In the present study, we measured two mRNAs, CK19 and PCTK1; one lncRNA, MALAT1; and two miRNAs, miR21 and miR155, in different EV fractions separated by differential centrifugation or captured by magnetic beads coated with annexin A5 (ANX beads). The results showed that in a cultured medium, the majority of mRNA and lncRNA exist in larger EVs, whereas miRNA exist in both large and small EVs from the differential centrifugation fractions. All these RNA species exist in ANX beads captured EVs. We then used ANX beads to capture EVs in plasma samples from non-small-cell lung cancer patients and age-matched healthy volunteers. We found that the ANX bead capturing could efficiently improve RNA detection from human plasma, compared with direct extraction of RNA from plasma. Using ANX-bead capturing and reverse transcription and quantitative PCR, we detected significantly higher levels of CK19 mRNA, MALAT1 lncRNA, and miR155 miRNA in the plasma of lung cancer patients. These facts suggested the collection methods strongly affect the results of exRNA measurement from EVs, and that ANX beads can be a useful tool for detecting exRNA from plasma samples in clinical application.

Nuclear accumulation of KPNA2 impacts radioresistance through positive regulation of the PLSCR1-STAT1 loop in lung adenocarcinoma.

Lung adenocarcinoma (ADC) is the predominant histological type of lung cancer, and radiotherapy is one of the current therapeutic strategies for lung cancer treatment. Unfortunately, biological complexity and cancer heterogeneity contribute to radioresistance development. Karyopherin α2 (KPNA2) is a member of the importin α family that mediates the nucleocytoplasmic transport of cargo proteins. KPNA2 overexpression is observed across cancer tissues of diverse origins. However, the role of KPNA2 in lung cancer radioresistance is unclear. Herein, we demonstrated that high expression of KPNA2 is positively correlated with radioresistance and cancer stem cell (CSC) properties in lung ADC cells. Radioresistant cells exhibited nuclear accumulation of KPNA2 and its cargos (OCT4 and c-MYC). Additionally, KPNA2 knockdown regulated CSC-related gene expression in radioresistant cells. Next-generation sequencing and bioinformatic analysis revealed that STAT1 activation and nuclear phospholipid scramblase 1 (PLSCR1) are involved in KPNA2-mediated radioresistance. Endogenous PLSCR1 interacting with KPNA2 and PLSCR1 knockdown suppressed the radioresistance induced by KPNA2 expression. Both STAT1 and PLSCR1 were found to be positively correlated with dysregulated KPNA2 in radioresistant cells and ADC tissues. We further demonstrated a potential positive feedback loop between PLSCR1 and STAT1 in radioresistant cells, and this PLSCR1-STAT1 loop modulates CSC characteristics. In addition, AKT1 knockdown attenuated the nuclear accumulation of KPNA2 in radioresistant lung cancer cells. Our results collectively support a mechanistic understanding of a novel role for KPNA2 in promoting radioresistance in lung ADC cells.

Circulating EGFR Mutations in Patients with Lung Adenocarcinoma by Circulating Tumor Cell Isolation Systems: A Concordance Study.

Background: We developed a hybrid platform using a negative combined with a positive selection strategy to capture circulating tumor cells (CTCs) and detect epidermal growth factor receptor (EGFR) mutations in patients with metastatic lung adenocarcinoma. Methods: Blood samples were collected from patients with pathology-proven treatment-naïve stage IV lung adenocarcinoma. Genomic DNA was extracted from CTCs collected for EGFR mutational tests. The second set of CTC-EGFR mutational tests were performed after three months of anti-cancer therapy. Results: A total of 80 samples collected from 28 patients enrolled between July 2016 and August 2018. Seventeen patients had EGFR mutations, including Exon 19 deletion (n = 11), L858R (n = 5), and de-novo T790 and L858R (n = 1). Concordance between tissue and CTCs before treatment was 88.2% in EGFR- mutant patients and 90.9% in non-mutant patients. The accuracy, sensitivity, specificity, positive predictive value, and negative predictive value of EGFR mutation tests for CTCs were 89.3%, 88.2%, 90.9%, 93.8%, and 83.3%, respectively. Conclusions: CTCs captured by a hybrid platform using a negative and positive selection strategy may serve as a suitable and reliable source of lung cancer tumor DNA for detecting EGFR mutations, including T790M.

Impact of tumor disappearance ratio on the prognosis of lung adenocarcinoma ≤2 cm in size: A retrospective cohort study.

BACKGROUND/PURPOSE: Lung cancer patients can have advanced-stages at diagnosis, even the tumor size is ≤2 cm. We aimed to study the relationship between image characteristics, clinical, and patholoigcal results. METHODS: We retrospectively enrolled patients with lung adenocarcinoma at Taichung Veterans General Hospital and Chang Gung Memorial Hospital from 2007 to 2015, who were diagnosed with treatment naïve primary tumor lesions at sizes less than 2 cm, as measured by computed tomography (CT) scans. The patient was analyzed for lymph node (LN) and distant metastasis evaluation, with clinicopathological characteristics, including tumor-disappearance ratio (TDR) (tumor diameter at the mediastinal/lung window) over chest CT scans, pathological diagnosis, disease-free survival (DFS), and overall survival (OS). RESULTS: Totally 280 patients were surveyed initially and showed significantly increase of clinical LN involvement and distant metastasis when TDR ≤75% compared with >75% (21.6% vs 0% for LN involvement; 27.1% vs 0% for distant metastasis; both p < 0.001). We included 199 patients having surgical treatment and follow-up for the survival analysis. With a TDR ≤75%, significantly worse DFS (HR, 19.23; 95% CI, 2.60-142.01; p = 0.004) and a trend of worse OS (HR, 4.97; 95% CI, 0.61-40.61; p = 0.134) were noted by Kaplan-Meier method. TDR ≤75% revealed more advanced pathological stage, and more tumors containing micropapillary or solid subtypes when diagnosed adenocarcinoma. CONCLUSION: For lung cancer patients with primary tumor ≤2 cm, TDR ≤75% was related to more advanced stages, the presence of micropapillary or solid components of adenocarcinoma subtypes, worse DFS, and a trend of worse OS.

The association between immune-related adverse events and survival outcomes in Asian patients with advanced melanoma receiving anti-PD-1 antibodies.

BACKGROUND: The association between immune-related adverse events (irAEs) and survival outcomes in patients with advanced melanoma receiving therapy with immune checkpoint inhibitors (ICIs) has not been well established, particularly in Asian melanoma. METHODS: We retrospectively reviewed 49 melanoma patients undergoing therapy with ICIs (anti-PD-1 monotherapy), and analyzed the correlation between irAEs and clinical outcomes including progression-free survival (PFS) and overall survival (OS). RESULTS: Overall, the patients who experienced grade 1-2 irAEs had longer PFS (median PFS, 4.6 vs. 2.5 months; HR, 0.52; 95% CI: 0.27-0.98; p = 0.042) and OS (median OS, 15.2 vs. 5.7 months; HR, 0.50; 95% CI: 0.24-1.02; p = 0.058) than the patients who did not experience irAEs. Regarding the type of irAE, the patients with either skin/vitiligo or endocrine irAEs showed better PFS (median PFS, 6.1 vs. 2.7 months; HR, 0.40, 95% CI: 0.21-0.74; p = 0.003) and OS (median OS, 18.7 vs. 4.5 months; HR, 0.34, 95% CI: 0.17-0.69, p = 0.003) than patients without any of these irAEs. CONCLUSIONS: Melanoma patients undergoing anti-PD-1 monotherapy and experiencing mild-to-moderate irAEs (grade 1-2), particularly skin (vitiligo)/endocrine irAEs had favorable survival outcomes. Therefore, the association between irAEs and the clinical outcomes in melanoma patients undergoing anti-PD-1 ICIs may be severity and type dependent.

Combined serum biomarkers in the noninvasive diagnosis of complicated parapneumonic effusions and empyema.

BACKGROUND: We previously demonstrated that the pleural levels of proteins (neutrophil gelatinase-associated lipocalin/NGAL, calprotectin, bactericidal permeability-increasing/BPI, azurocidin 1/AZU-1) were valuable markers for identifying complicated PPE (CPPE). Herein, this study was performed to evaluate whether these proteins are useful as serological markers for identifying CPPE and empyema. METHODS: A total of 137 participates were enrolled in this study. The levels of NGAL, calprotectin, BPI and AZU-1 were measured in serum and pleural fluid by enzyme-linked immunosorbent assay. We also characterized the diagnostic values of these markers between different groups. RESULTS: The serum levels of NGAL, calprotectin, and BPI in PPE patients were significantly higher than those in transudates, noninfectious exudates, and healthy controls. The area under the curve (AUC) values of NGAL, calprotectin, and BPI for distinguishing PPE from transudates or noninfectious exudates were around 0.861 to 0.953. In PPE group, serum NGAL and calprotectin levels were significantly elevated in patients with CPPE and empyema than in those with UPPE, whereas the serum BPI levels were similar between these two groups. In CPPE and empyema patients, the serum NGAL showed a positive correlation with the pleural fluid NGAL (r = 0.417, p <  0.01). When combined with serum CRP, the sensitivity and specificity of serum calprotectin for identifying CPPE and empyema were the highest at 73.52% and 80.55%, respectively. CONCLUSIONS: We concluded that serum calprotectin and NGAL were adjuvant serological markers for CPPE and empyema diagnosis. Patients present with pneumonia and pleural effusion signs in the chest x-ray and the combination of serum calprotectin and CRP constitutes a more highly sensitive and specific assay for identifying CPPE and empyema.

Golgi tethering factor golgin-97 suppresses breast cancer cell invasiveness by modulating NF-κB activity.

BACKGROUND: Golgin-97 is a tethering factor in the trans-Golgi network (TGN) and is crucial for vesicular trafficking and maintaining cell polarity. However, the significance of golgin-97 in human diseases such as cancer remains unclear. METHODS: We searched for a potential role of golgin-97 in cancers using Kaplan-Meier Plotter ( http://kmplot.com ) and Oncomine ( www.oncomine.org ) datasets. Specific functions of golgin-97 in migration and invasion were examined in golgin-97-knockdown and golgin-97-overexpressing cells. cDNA microarray, pathway analysis and qPCR were used to identify gene profiles regulated by golgin-97. The role of golgin-97 in NF-κB signaling pathway was examined by using subcellular fractionation, luciferase reporter assay, western blot analysis and immunofluorescence assay (IFA). RESULTS: We found that low expression of golgin-97 correlated with poor overall survival of cancer patients and was associated with invasiveness in breast cancer cells. Golgin-97 knockdown promoted cell migration and invasion, whereas re-expression of golgin-97 restored the above phenotypes in breast cancer cells. Microarray and pathway analyses revealed that golgin-97 knockdown induced the expression of several invasion-promoting genes that were transcriptionally regulated by NF-κB p65. Mechanistically, golgin-97 knockdown significantly reduced IκBα protein levels and activated NF-κB, whereas neither IκBα levels nor NF-κB activity was changed in TGN46- or GCC185-knockdown cells. Conversely, golgin-97 overexpression suppressed NF-κB activity and restored the levels of IκBα in golgin-97-knockdown cells. Interestingly, the results of Golgi-disturbing agent treatment revealed that the loss of Golgi integrity was not involved in the NF-κB activation induced by golgin-97 knockdown. Moreover, both TGN-bound and cytosolic golgin-97 inhibited NF-κB activation, indicating that golgin-97 functions as an NF-κB suppressor regardless of its subcellular localization. CONCLUSION: Our results collectively demonstrate a novel and suppressive role of golgin-97 in cancer invasiveness. We also provide a new avenue for exploring the relationship between the TGN, golgin-97 and NF-κB signaling in tumor progression.

Identification and Characterization of Potential Biomarkers by Quantitative Tissue Proteomics of Primary Lung Adenocarcinoma.

Lung cancer is the leading cause of cancer-related death worldwide. Both diagnostic and prognostic biomarkers are urgently needed to increase patient survival. In this study, we identified/quantified 1763 proteins from paired adenocarcinoma (ADC) tissues with different extents of lymph node (LN) involvement using an iTRAQ-based quantitative proteomic analysis. Based on a bioinformatics analysis and literature search, we selected six candidates (ERO1L, PABPC4, RCC1, RPS25, NARS, and TARS) from a set of 133 proteins that presented a 1.5-fold increase in expression in ADC tumors without LN metastasis compared with adjacent normal tissues. These six proteins were further verified using immunohistochemical staining and Western blot analyses. The protein levels of these six candidates were higher in tumor tissues compared with adjacent normal tissues. The ERO1L and NARS levels were positively associated with LN metastasis. Importantly, ERO1L overexpression in patients with early-stage ADC was positively correlated with poor survival, suggesting that ERO1L overexpression in primary sites of early-stage cancer tissues indicates a high risk for cancer micrometastasis. Moreover, we found that knockdown of either ERO1L or NARS reduced the viability and migration ability of ADC cells. Our results collectively provide a potential biomarker data set for ADC diagnosis/prognosis and reveal novel roles of ERO1L and NARS in ADC progression.

Predicting outcomes of EGFR-targeted therapy in non-small cell lung cancer patients using pleural effusions samples and peptide nucleic acid probe assay.

BACKGROUND: Mutation of epidermal growth factor receptor (EGFR) is a prediction marker of the response to tyrosine kinase inhibitor (TKI) drugs in non-small cell lung cancer (NSCLC) patients. As late stage lung cancer patients rarely undergo surgery, samples for EGFR mutation identification usually come from computed tomography (CT)-guided or endoscopic biopsies, which is invasive and costly. Pleural effusion may serve as a less invasive sample for EGFR mutation detection. METHODS: We designed a fluorophore-labeled peptide nucleic acid (PNA) probe assay for three types of EGFR mutations, including exon 19 deletions, L858R point mutations and T790M point mutations. The assay was applied in 39 pleural effusion samples from NSCLC patients. The correlation between detected EGFR status and clinical outcome were analyzed. RESULTS: In 15 paired samples, PNA probe assay in pleural effusion samples could detect all the mutations that were identified by conventional PCR plus Sanger sequencing in tissue biopsies. In addition, PNA probe assay detected three more T790M mutations. In all 39 pleural effusions, the PNA probe assay detected 27 having at least one of the three EGFR mutations. Among the patients before TKI treatment, those with a sensitizing mutation (either exon 19 deletion or L858R) but without T790M, had 94.1% response rate and longer progression-free survival (mean 10.8 months) than patients without detected mutation (mean 4.2 months) and patients with T790M (mean 1.7 months). CONCLUSIONS: Mutations detected in pleural effusions using PNA probe assay are highly associated with clinical outcome. This method appears to be a reliable way for the prediction of the efficacy of EGFR-targeted therapy.

In-depth proteomic analysis of six types of exudative pleural effusions for nonsmall cell lung cancer biomarker discovery.

Pleural effusion (PE), a tumor-proximal body fluid, may be a promising source for biomarker discovery in human cancers. Because a variety of pathological conditions can lead to PE, characterization of the relative PE proteomic profiles from different types of PEs would accelerate discovery of potential PE biomarkers specifically used to diagnose pulmonary disorders. Using quantitative proteomic approaches, we identified 772 nonredundant proteins from six types of exudative PEs, including three malignant PEs (MPE, from lung, breast, and gastric cancers), one lung cancer paramalignant PE, and two benign diseases (tuberculosis and pneumonia). Spectral counting was utilized to semiquantify PE protein levels. Principal component analysis, hierarchical clustering, and Gene Ontology of cellular process analyses revealed differential levels and functional profiling of proteins in each type of PE. We identified 30 candidate proteins with twofold higher levels (q<0.05) in lung cancer MPEs than in the two benign PEs. Three potential markers, MET, DPP4, and PTPRF, were further verified by ELISA using 345 PE samples. The protein levels of these potential biomarkers were significantly higher in lung cancer MPE than in benign diseases or lung cancer paramalignant PE. The area under the receiver-operator characteristic curve for three combined biomarkers in discriminating lung cancer MPE from benign diseases was 0.903. We also observed that the PE protein levels were more clearly discriminated in effusions in which the cytological examination was positive and that they would be useful in rescuing the false negative of cytological examination in diagnosis of nonsmall cell lung cancer-MPE. Western blotting analysis further demonstrated that MET overexpression in lung cancer cells would contribute to the elevation of soluble MET in MPE. Our results collectively demonstrate the utility of label-free quantitative proteomic approaches in establishing differential PE proteomes and provide a new database of proteins that can be used to facilitate identification of pulmonary disorder-related biomarkers.

A primer and probe set for detecting multiple types of EGFR exon 19 deletions.

EGFR exon 19 deletion is an important indicator for tyrosine kinase inhibitor treatment in non-small cell lung cancer. However, detection of exon 19 deletions faces a challenge: there are more than 30 types of mutations reported at the hotspot. Moreover, considering the application in body fluid samples, assays with high sensitivity and specificity are necessary for the detection of rare mutant alleles. Here, we describe a single tube reaction which could detect at least 29 types of exon 19 deletions with only an unlabeled peptide nucleic acid (PNA) clamp and a pair of DNA probes. The PNA clamp was used to inhibit amplification of wild-type templates; and the DNA probes were used to generate melting peaks for multiple types of mutations. Under optimal condition, the assay was able to detect as low as 0.01% mutant DNA in wild-type background, and had a limit of detection of 10 pg genomic DNA. Feasibility of the assay was tested in body fluid samples from lung cancer patients. The assay detected 100% and 60% of deletions in pleural effusions and plasma, respectively. We believe the present assay can be used in the clinical laboratories and has potential to be adapted for a microfluidic device.

Quantitative proteomics reveals a novel role of karyopherin alpha 2 in cell migration through the regulation of vimentin-pErk protein complex levels in lung cancer.

Karyopherin alpha 2 (KPNA2) is overexpressed in various human cancers and is associated with cancer invasiveness and poor prognosis. Herein, to understand the essential role of KPNA2 protein complexes in cancer progression, we applied stable isotope labeling with amino acids in cell culture (SILAC)-based quantitative proteomic strategy combined with immunoprecipitation (IP) to investigate the differential KPNA2 protein complexes in lung adenocarcinoma cell lines with different invasiveness potentials. We found that 64 KPNA2-interaction proteins displayed a 2-fold difference in abundance between CL1-5 (high invasiveness) and CL1-0 (low invasiveness) cells. Pathway map analysis revealed that the formation of complexes containing KPNA2 and cytoskeleton-remodeling-related proteins, including actin, beta tubulin, tubulin heterodimers, vimentin, keratin 8, keratin 18, and plectin, was associated with cancer invasiveness. IP demonstrated that the levels of KPNA2-vimentin-pErk complexes were significantly higher in CL1-5 cells than in CL1-0 cells. The KPNA2-vimentin-pErk complex was also up-regulated in the advanced stage compared with the early-stage lung adenocarcinoma tissues. Importantly, the levels of pErk as well as cell migration ability were significantly reduced in KPNA2-knockdown cells; however, migration was restored by treatment with pErk phosphatase inhibitors. Collectively, our results demonstrate the usefulness of a SILAC-based proteomic strategy for identifying invasiveness-associated KPNA2 protein complexes and provide new insight into the KPNA2-mediated modulation of cell migration.

Interactions between household air pollution and GWAS-identified lung cancer susceptibility markers in the Female Lung Cancer Consortium in Asia (FLCCA).

We previously carried out a multi-stage genome-wide association study (GWAS) on lung cancer among never smokers in the Female Lung Cancer Consortium in Asia (FLCCA) (6,609 cases, 7,457 controls) that identified novel susceptibility loci at 10q25.2, 6q22.2, and 6p21.32, and confirmed two previously identified loci at 5p15.33 and 3q28. Household air pollution (HAP) attributed to solid fuel burning for heating and cooking, is the leading cause of the overall disease burden in Southeast Asia, and is known to contain lung carcinogens. To evaluate the gene-HAP interactions associated with lung cancer in loci independent of smoking, we analyzed data from studies participating in FLCCA with fuel use information available (n = 3; 1,731 cases; 1,349 controls). Coal use was associated with a 30% increased risk of lung cancer (OR 1.3, 95% CI 1.0-1.6). Among the five a priori SNPs identified by our GWAS, two showed a significant interaction with coal use (HLA Class II rs2395185, p = 0.02; TP63 rs4488809 (rs4600802), p = 0.04). The risk of lung cancer associated with coal exposure varied with the respective alleles for these two SNPs. Our observations provide evidence that genetic variation in HLA Class II and TP63 may modify the association between HAP and lung cancer risk. The roles played in the cell cycle and inflammation pathways by the proteins encoded by these two genes provide biological plausibility for these interactions; however, additional replication studies are needed in other non-smoking populations.

Targeted proteomics pipeline reveals potential biomarkers for the diagnosis of metastatic lung cancer in pleural effusion.

The ability to discriminate lung cancer malignant pleural effusion (LC-MPE) from benign pleural effusion has profound implications for the therapy and prognosis of lung cancer. Here, we established a pipeline to verify potential biomarkers for this purpose. In the discovery phase, label-free quantification was performed for the proteome profiling of exudative pleural effusion in order to select 34 candidate biomarkers with significantly elevated levels in LC-MPE. In the verification phase, signature peptides for 34 candidates were first confirmed by accurate inclusion mass screening (AIMS). To quantify the candidates in PEs, multiple reaction monitoring mass spectrometry (MRM-MS) with stable isotope-labeled standards (SIS) peptides was performed for the 34 candidate biomarkers using the QconCAT approach for the generation of the SIS peptides. The results of the MRM assay were used to prioritize candidates based on their discriminatory power in 82 exudative PE samples. The five potential biomarkers (ALCAM, CDH1, MUC1, SPINT1, and THBS4; AUC > 0.7) and one three-marker panel (SPINT1/SVEP1/THBS4; AUC = 0.95) were able to effectively differentiate LC-MPE from benign PE. Collectively, these results demonstrate that our pipeline is a feasible platform for verifying potential biomarkers for human diseases.