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1.
Ann Surg Oncol ; 28(7): 3983-3993, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-33140254

RESUMO

BACKGROUND: Despite surgical resection, early lung adenocarcinoma has a recurrence rate of 20-50%. No clear predictive markers for recurrence of early lung adenocarcinoma are available. Targeted next-generation sequencing (NGS) is rarely used to identify recurrence-related genes. We aimed to identify genetic alterations that can predict recurrence, by comparing the molecular profiles of patient groups with and without recurrence. METHODS: Tissues from 230 patients with resected stage I-II lung adenocarcinoma (median follow-up: 49 months) were analyzed via targeted NGS for 207 cancer-related genes. The recurrence-free survival according to the number and type of mutation was estimated using the Kaplan-Meier method. Independent predictive biomarkers related to recurrence were identified using the Cox proportional hazards model. RESULTS: Recurrence was observed in 64 patients (27.8%). In multivariate analysis adjusted for age, sex, smoking history, stage, surgical mode, and visceral pleural invasion, the CTNNB1 mutation and fusion genes (ALK, ROS1, RET) were negative prognostic factors for recurrence in early-stage lung adenocarcinoma (HR 4.47, p = 0.001; HR 2.73, p = 0.009). EGFR mutation was a favorable factor (HR 0.51, p = 0.016), but the CTNNB1/EGFR co-mutations were negative predictors (HR 19.2, p < 0.001). TP53 mutation was a negative predictor compared with EGFR mutation for recurrence (HR 5.24, p = 0.02). CONCLUSIONS: Targeted NGS can provide valuable information to predict recurrence and identify patients at high recurrence risk, facilitating selection of the treatment strategy among close monitoring and adjuvant-targeted therapy. Larger datasets are required to validate these findings.


Assuntos
Adenocarcinoma de Pulmão , Neoplasias Pulmonares , Adenocarcinoma de Pulmão/genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Neoplasias Pulmonares/genética , Mutação , Recidiva Local de Neoplasia/genética , Recidiva Local de Neoplasia/cirurgia , Prognóstico
2.
Mol Cancer ; 17(1): 15, 2018 01 27.
Artigo em Inglês | MEDLINE | ID: mdl-29374476

RESUMO

Tumor cells shed an abundance of extracellular vesicles (EVs) to body fluids containing bioactive molecules including DNA, RNA, and protein. Investigations in the field of tumor-derived EVs open a new horizon in understanding cancer biology and its potential as cancer biomarkers as well as platforms for personalized medicine. This study demonstrates that successfully isolated EVs from plasma and bronchoalveolar lavage fluid (BALF) of non-small cell lung cancer (NSCLC) patients contain DNA that can be used for EGFR genotyping through liquid biopsy. In both plasma and BALF samples, liquid biopsy results using EV DNA show higher accordance with conventional tissue biopsy compared to the liquid biopsy of cfDNA. Especially, liquid biopsy with BALF EV DNA is tissue-specific and extremely sensitive compared to using cfDNA. Furthermore, use of BALF EV DNA also demonstrates higher efficiency in comparison to tissue rebiopsy for detecting p.T790 M mutation in the patients who developed resistance to EGFR-TKIs. These finding demonstrate possibility of liquid biopsy using EV DNA potentially replacing the current diagnostic methods for more accurate, cheaper, and faster results.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/metabolismo , DNA de Neoplasias , Vesículas Extracelulares/metabolismo , Genes erbB-1 , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Líquido da Lavagem Broncoalveolar , Carcinoma Pulmonar de Células não Pequenas/diagnóstico , DNA Tumoral Circulante , Resistencia a Medicamentos Antineoplásicos , Vesículas Extracelulares/ultraestrutura , Testes Genéticos , Técnicas de Genotipagem , Humanos , Biópsia Líquida , Neoplasias Pulmonares/patologia , Mutação
3.
BMC Cancer ; 18(1): 1236, 2018 Dec 10.
Artigo em Inglês | MEDLINE | ID: mdl-30526536

RESUMO

BACKGROUND: EGFR genotyping in pulmonary adenocarcinoma patients who develop pleural effusions is mostly performed using cytology or cell block slides with low sensitivity. Liquid biopsy using the supernatant of pleural effusions may be more effective because they contain many components released by cancer cells. Extracellular vesicles (EVs) are known to carry oncogenic double-stranded DNA that is considered a notable biomarker. Here, we investigate the efficiency of liquid biopsy using cell-free DNA (cfDNA) and extracellular vesicle-derived DNA (EV-derived DNA) from the supernatant of pleural effusions for EGFR genotyping in patients with pulmonary adenocarcinoma. METHODS: Fifty pleural effusion samples from patients with pulmonary adenocarcinoma were evaluated. The supernatant, after removing the cell pellet by centrifugation, was used for liquid biopsy, and EVs were isolated from the pleural effusion by ultracentrifugation. EV-derived DNA and cfDNA were extracted separately, and EGFR genotyping was performed by the PNA clamping method. RESULTS: Among 32 patients who were EGFR-tyrosine kinase inhibitor (TKI) naïve with a known tissue EGFR genotype, liquid biopsy using EV-derived DNA from the pleural effusion supernatant showed 100% matching results with tissue EGFR genotyping in 19 EGFR mutant cases and detected three additional EGFR mutations in patients with wild-type (WT) tissue. Liquid biopsy using cfDNA from pleural effusion supernatants missed two cases of tissue-based EGFR mutations and found two additional EGFR mutation cases. In 18 patients who acquired resistance to EGFR-TKI, EGFR genotyping using EV-derived DNA from the pleural effusion supernatant detected the T790 M mutation in 13 of 18 (72.2%) patients, and this mutation was detected in 11 (61.1%) patients using cfDNA. By contrast, only three patients were found to present the T790 M mutation when using cell block or cytology slides. CONCLUSIONS: Liquid biopsy using the supernatant of pleural effusions showed significantly improved results for EGFR genotyping compared to those using conventional cell block or cytology samples. Liquid biopsy using EV-derived DNA is promising for EGFR genotyping, including T790 M detection in pulmonary adenocarcinoma patients who develop pleural effusions.


Assuntos
Adenocarcinoma de Pulmão/diagnóstico , Biomarcadores Tumorais/análise , DNA Tumoral Circulante/análise , Derrame Pleural Maligno/diagnóstico , Adenocarcinoma de Pulmão/genética , Adulto , Idoso , Biomarcadores Tumorais/genética , Ácidos Nucleicos Livres/análise , Receptores ErbB/genética , Vesículas Extracelulares , Feminino , Genótipo , Humanos , Biópsia Líquida/métodos , Masculino , Pessoa de Meia-Idade , Derrame Pleural Maligno/genética
4.
J Korean Med Sci ; 33(1): e7, 2018 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-29215816

RESUMO

Human epidermal growth factor receptor 2 (HER2) mutation in non-small cell lung cancer (NSCLC) is an oncogenic driver that possibly becomes a druggable target to HER2-targeted therapy. The benefit of HER2-targeted therapy is much less defined especially in eastern populations. We provide evidence of clinical benefit of afatinib in a 50-year-old Asian woman with HER2-mutant NSCLC who previously failed cytotoxic chemotherapy and gefitinib treatment. Next-generation sequencing of the tumor tissue revealed a HER2 exon 20 mutation (c.2437A>G), which has never been reported. The patient was treated with afatinib for more than four months. She showed rapid radiologic response within a month, and maintained stable state until the last dose of afatinib.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Neoplasias Pulmonares/tratamento farmacológico , Quinazolinas/uso terapêutico , Radiossensibilizantes/uso terapêutico , Receptor ErbB-2/genética , Afatinib , Antineoplásicos/uso terapêutico , Carcinoma Pulmonar de Células não Pequenas/genética , Éxons , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Neoplasias Pulmonares/genética , Pessoa de Meia-Idade , Mutação , Análise de Sequência de DNA , Tomografia Computadorizada por Raios X , Resultado do Tratamento
5.
Clin Chem Lab Med ; 55(10): 1489-1495, 2017 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-28195541

RESUMO

BACKGROUND: Much attention has been focused on epidermal growth factor receptor (EGFR) mutation testing since the introduction of EGFR-tyrosine kinase inhibitors have improved survival in EGFR-positive lung cancer patients. Liquid biopsy using circulating tumor cells or cell-free DNA (cfDNA) has enabled less invasive testing, but requires a highly sensitive method. To date, liquid biopsy using bronchoalveolar lavage (BAL) fluid has rarely been used. METHODS: From 20 patients with lung adenocarcinoma, we isolated cfDNA from 20 samples of cell-free BAL fluid and 19 cell-free bronchial washing samples. cfDNA was examined for EGFR mutations using peptide nucleic acid (PNA)-mediated PCR clamping method. In cases where the results from the tumor biopsy and BAL-derived cfDNA test were not consistent, PANAMutyper™ R EGFR kit was used along with PNA clamping-assisted fluorescence melting curve analysis. RESULTS: We included 17 patients with advanced stage disease and three with non-advanced stage disease. Tumor biopsy detected EGFR mutations in 12 of the patients. One patient had a p.L858R mutation and a de novo p.T790M mutation. The results from PNA-mediated PCR clamping were 75.0% (9/12) concordant with the tumor biopsy results for EGFR mutation status. PANAMutyper with fluorescence melting curve analysis was performed in three cases, which detected EGFR mutations in two more patients (11/12, 91.7%). EGFR mutations were detected in the cfDNA extracted from two bronchial washing samples. CONCLUSIONS: cfDNA from BAL fluid could be used for molecular testing of EGFR mutations and identification of p.T790M mutations, with an easily applicable method.


Assuntos
Líquido da Lavagem Broncoalveolar/química , Receptores ErbB/genética , Neoplasias Pulmonares/patologia , Adulto , Idoso , DNA de Neoplasias/análise , Receptores ErbB/metabolismo , Feminino , Humanos , Neoplasias Pulmonares/metabolismo , Masculino , Pessoa de Meia-Idade , Mutação , Estadiamento de Neoplasias , Ácidos Nucleicos Peptídicos/metabolismo , Reação em Cadeia da Polimerase
6.
Biochem J ; 443(1): 49-56, 2012 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-22214184

RESUMO

TDP1 (tyrosyl-DNA phosphodiesterase 1), a member of the PLD (phospholipase D) superfamily, catalyses the hydrolysis of a phosphodiester bond between a tyrosine residue and the 3'-phosphate of DNA. We have previously identified and characterized the AtTDP gene in Arabidopsis thaliana, an orthologue of yeast and human TDP1 genes. Sequence alignment of TDP1 orthologues revealed that AtTDP has both a conserved C-terminal TDP domain and, uniquely, an N-terminal SMAD/FHA (forkhead-associated) domain. To help understand the function of this novel enzyme, we analysed the substrate saturation kinetics of full-length AtTDP compared with a truncated AtTDP mutant lacking the N-terminal FHA domain. The recombinant AtTDP protein hydrolysed a single-stranded DNA substrate with Km and kcat/Km values of 703±137 nM and (1.5±0.04)×10(9) M(-1)·min(-1) respectively. The AtTDP-(Δ1-122) protein (TDP domain) showed kinetic parameters that were equivalent to those of the full-length AtTDP protein. A basic amino acid sequence (RKKVKP) within the AtTDP-(Δ123-605) protein (FHA domain) was necessary for nuclear localization of AtTDP. Analysis of active-site mutations showed that a histidine and a lysine residue in each of the HKD motifs were critical for enzyme activity. Vanadates, inhibitors of phosphoryl transfer reactions, inhibited AtTDP enzymatic activity and retarded the growth of an Arabidopsis tdp mutant. Finally, we showed that expression of the AtTDP gene could complement a yeast tdp1Δrad1Δ mutant, rescuing the growth inhibitory effects of vanadate analogues and CPT (camptothecin). Taken together, the results of the present study demonstrate the structure-based function of AtTDP through which AtTDP can repair DNA strand breaks in plants.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Reparo do DNA , Diester Fosfórico Hidrolases/metabolismo , Folhas de Planta/enzimologia , Transporte Ativo do Núcleo Celular , Motivos de Aminoácidos , Sequência de Aminoácidos , Substituição de Aminoácidos , Arabidopsis/efeitos dos fármacos , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Camptotecina/farmacologia , Domínio Catalítico , Clorofila/metabolismo , Cinética , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Sinais de Localização Nuclear , Inibidores de Fosfodiesterase/farmacologia , Diester Fosfórico Hidrolases/química , Diester Fosfórico Hidrolases/genética , Folhas de Planta/efeitos dos fármacos , Folhas de Planta/genética , Folhas de Planta/crescimento & desenvolvimento , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Vanadatos/farmacologia
7.
Transl Lung Cancer Res ; 12(7): 1425-1435, 2023 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-37577304

RESUMO

Background: In our previous study, epidermal growth factor receptor (EGFR) genotyping using extracellular vesicles (EV)-derived DNA isolated from bronchoalveolar lavage fluid (BALF) was proven to be highly concordant with conventional tissue-based genotyping and its turn-around-time (TAT) was only 1-2 days. On this background, we prospectively validated the performance of EV-based BALF liquid biopsy for EGFR genotyping in the real practice of advanced non-small cell lung cancer (NSCLC) patients. Methods: After screening 120 newly diagnosed stage III-IV NSCLC patients, 51 cases were detected as EGFR-mutated by EV-based BALF EGFR genotyping and 40 patients were enrolled for gefitinib treatment. BALF EV were isolated by ultracentrifuge method and EGFR genotyping was performed with PCR-based PNA-clamping assisted fluorescence melting curve analysis. The objective response rate, progression-free survival (PFS), TAT, time to treatment initiation (TTI), and concordance rate were analyzed with clinical parameters. Results: There was only one false positive case among the 120 screened patients and the overall concordance rate between tissue biopsy and EV-based BALF liquid biopsy was 99.2% including the subtype of EGFR mutations. TAT for EV-based BALF EGFR genotyping was 1.9±1.1 days, while tissue-based TAT was 12.1±7.2 days (P<0.001). EGFR genotyping was determined even before obtaining histopathologic report in most cases. TTI in BALF EGFR genotyping was faster than tissue genotyping (7.8±6.5 vs. 13.8±12.9 days). Therapeutic outcomes of response rate and PFS were almost similar to tissue-based results. Conclusions: We demonstrated, for the first time, that EV-based BALF liquid biopsy should be an excellent platform for expeditious EGFR genotyping and rapid therapeutic intervention even before obtaining the result of histopathology in advanced NSCLC patients.

8.
Cancers (Basel) ; 14(11)2022 May 31.
Artigo em Inglês | MEDLINE | ID: mdl-35681723

RESUMO

To overcome the limitations of the tissue biopsy and plasma cfDNA liquid biopsy, we performed the EV-based BALF liquid biopsy of 224 newly diagnosed stage III-IV NSCLC patients and compared it with tissue genotyping and 110 plasma liquid biopsies. Isolation of EVs from BALF was performed by ultracentrifugation. EGFR genotyping was performed through peptide nucleic acid clamping-assisted fluorescence melting curve analysis. Compared with tissue-based genotyping, BALF liquid biopsy demonstrated a sensitivity, specificity, and concordance rates of 97.8%, 96.9%, and 97.7%, respectively. The performance of BALF liquid biopsy was almost identical to that of standard tissue-based genotyping. In contrast, plasma cfDNA-based liquid biopsy (n = 110) demonstrated sensitivity, specificity, and concordance rates of 48.5%, 86.3%, and 63.6%, respectively. The mean turn-around time of BALF liquid biopsy was significantly shorter (2.6 days) than that of tissue-based genotyping (13.9 days; p < 0.001). Therefore, the use of EV-based BALF shortens the time for confirmation of EGFR mutation status for starting EGFR-TKI treatment and can hence potentially improve clinical outcomes. As a result, we suggest that EV-based BALF EGFR testing in advanced lung NSCLC is a highly accurate rapid method and can be used as an alternative method for lung tissue biopsy.

9.
Cancers (Basel) ; 13(15)2021 Jul 29.
Artigo em Inglês | MEDLINE | ID: mdl-34359729

RESUMO

Extracellular vesicles (EVs) carry RNA, proteins, lipids, and diverse biomolecules for intercellular communication. Recent studies have reported that EVs contain double-stranded DNA (dsDNA) and oncogenic mutant DNA. The advantage of EV-derived DNA (EV DNA) over cell-free DNA (cfDNA) is the stability achieved through the encapsulation in the lipid bilayer of EVs, which protects EV DNA from degradation by external factors. The existence of DNA and its stability make EVs a useful source of biomarkers. However, fundamental research on EV DNA remains limited, and many aspects of EV DNA are poorly understood. This review examines the known characteristics of EV DNA, biogenesis of DNA-containing EVs, methylation, and next-generation sequencing (NGS) analysis using EV DNA for biomarker detection. On the basis of this knowledge, this review explores how EV DNA can be incorporated into diagnosis and prognosis in clinical settings, as well as gene transfer of EV DNA and its therapeutic potential.

10.
Cancers (Basel) ; 13(14)2021 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-34298845

RESUMO

Targeted NGS, widely applied to identify driver oncogenes in advanced lung adenocarcinoma, may also be applied to resected early stage cancers. We investigated resected EGFR-mutated lung adenocarcinoma mutation profiles to evaluate prognostic impacts. Tissues from 131 patients who had complete resection of stage I-IIIA EGFR-mutated lung adenocarcinoma were analyzed by targeted NGS for 207 cancer-related genes. Recurrence free survival (RFS) was estimated according to genetic alterations using the Kaplan-Meier method and Cox proportional regression analysis. The relapse rate was 25.2% (33/131). Five-year RFS of stages IA, IB, II, and IIIA were 82%, 75%, 35%, and 0%, respectively (p < 0.001). RFS decreased with the number of co-mutations (p = 0.025). Among co-mutations, the CTNNB1 mutation was associated with short RFS in a multivariate analysis (hazard ratio: 5.4, 95% confidence interval: 2.1-14.4; p = 0.001). TP53 mutations were associated with short RFS in stage IB-IIIA (p = 0.01). RFS was shorter with EGFR exon 19 deletion (19-del) than with mutation 21-L858R in stage IB-IIIA tumors (p = 0.008). Among 19-del subtypes, pL747_P753delinS (6/56, 8.9%) had shorter RFS than pE746_A750del (39/56, 69.6%), the most frequent subtype (p = 0.004).

11.
Transl Cancer Res ; 10(2): 1025-1033, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-35116429

RESUMO

BACKGROUND: Accumulating evidences indicate that AXL overexpression or activation is associated with cancer progression and acquired resistance to targeted anti-cancer drugs such as epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs). Despite recent development of several drugs that target multiple receptor tyrosine kinases (RTKs), drugs that selectively target AXL signaling are extremely rare. Short nucleic acid aptamers are non-immunogenic molecules with high binding affinity and specificity to their target molecules that could potentially be used as a novel cancer treatment. METHODS: Modified-DNA aptamers were selected on the basis of its ability to bind recombinant human AXL. AXL aptamers were selected for their inhibition of AXL and then selected aptamers were tested for their use to overcome acquired resistant to EGFR-TKI on a lung cancer cell with acquired resistance to erlotinib. RESULTS: These new AXL aptamers inhibited cell viability to an extent of 30-40% in HCC827/ER cells with acquired resistance to erlotinib. The possible mechanism of overcoming the acquired resistance may be by inhibiting the activation of Akt and Erk. Although, aptamers effectively decreased cell viability of erlotinib-resistant cell line, the combination of aptamers and erlotinib did not synergistically decrease the survival of the resistant cell line. CONCLUSIONS: We developed newly modified DNA aptamers that selectively bind to AXL receptors, and assessed their efficacy in a human lung cancer cell with acquired resistance to EGFR-TKI.

12.
Transl Lung Cancer Res ; 10(1): 104-116, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33569297

RESUMO

BACKGROUND: Extracellular vesicles (EVs) are membrane-bound and nanometer-sized particles released from most types of cells, containing double-stranded DNA reflecting mutational status of the parental tumor cells. Furthermore, epidermal growth factor receptor (EGFR) genotyping using EV-derived DNA (EV DNA) in bronchoalveolar lavage fluid (BALF) showed almost 100% sensitivity in patients with advanced non-small cell lung cancer (NSCLC). METHODS: We assessed the technical performance of DNA derived from BALF-EV (BALF EV DNA) in targeted next-generation sequencing (NGS) for detection and quantification of mutations compared with the matching tissue DNA in 20 lung adenocarcinomas. RESULTS: DNA yields, tumor purity, and depth of coverage were higher using the tissue DNA than using the BALF EV DNA. However, estimated library size was not significantly different between the two samples, and BALF EV DNA yielded longer fragments than tissue DNA. Overall mutation concordance between the two samples were 56% for nonsynonymous somatic mutations and increased to 81% for clinically significant mutations. By-variant sensitivity for clinically significant somatic mutations increased from 62% to 83% in the NGS of BALF EV DNA. Allele frequencies of EGFR and TP53 were higher in tissue DNA (10-25%) than in BALF EV DNA (<5%). Tumor mutation burden of BALF EV DNA correlated with that of tissue DNA. CONCLUSIONS: Our findings demonstrate, for the first time, that BALF EV DNA in patients with NSCLC can be a reliable DNA source for targeted NGS for the identification of actionable genetic alterations and that this approach has high clinical feasibility and utility.

13.
J Pathol Transl Med ; 54(3): 204-212, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32460474

RESUMO

With advances in target therapy, molecular analysis of tumors is routinely required for treatment decisions in patients with advanced non-small cell lung cancer (NSCLC). Liquid biopsy refers to the sampling and analysis of circulating cell-free tumor DNA (ctDNA) in various body fluids, primarily blood. Because the technique is minimally invasive, liquid biopsies are the future in cancer management. Epidermal growth factor receptor (EGFR) ctDNA tests have been performed in routine clinical practice in advanced NSCLC patients to guide tyrosine kinase inhibitor treatment. In the near future, liquid biopsy will be a crucial prognostic, predictive, and diagnostic method in NSCLC. Here we present the current status and future perspectives of liquid biopsy in NSCLC.

14.
J Pathol Transl Med ; 54(6): 453-461, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-33027851

RESUMO

Blood liquid biopsy has emerged as a way of overcoming the clinical limitations of repeat biopsy by testing for the presence of acquired resistance mutations to therapeutic agents. Despite its merits of repeatability and non-invasiveness, this method is currently only used as a supplemental test due to a relatively low sensitivity rate of 50%-60%, and cannot replace tissue biopsy. The circulating tumor DNAs used in blood liquid biopsies are passive products of fragmented DNA with a short half-life released following tumor cell death; the low sensitivity seen with liquid blood biopsy results from this instability, which makes increasing the sensitivity of this test fundamentally difficult. Extracellular vesicles (EVs) are ideal carriers of cancer biomarkers, as cancer cells secret an abundance of EVs, and the contents of tumor cell-originated EVs reflect the molecular and genetic composition of parental cells. In addition, EV-derived DNAs (EV DNAs) consist of large-sized genomic DNAs and tumor-specific oncogenic mutant DNAs. For these reasons, liquid biopsy using EV DNA has the potential to overcome issues arising from tissue shortages associated with small biopsies, which are often seen in lung cancer patients, and the biopsy product can be used in other diagnostic methods, such as epidermal growth factor receptor (EGFR) mutation testing and next-generation sequencing (NGS). A higher sensitivity can be achieved when EV DNAs obtained from bronchoalveolar lavage fluid (BALF) are used rather than those from blood. BALF, when obtained close to the tumor site, is a promising liquid biopsy tool, as it enables the gathering of both cellular and non-cellular fractions of the tumor microenvironment, and provides increased diagnostic sensitivity when compared to blood.

15.
Korean J Intern Med ; 35(3): 692-702, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32066220

RESUMO

BACKGROUND/AIMS: We performed a large-scale, retrospective, nationwide, cohort study to investigate the risk factors for lung cancer among never-smoking Korean females. METHODS: The study data were collected from a general health examination and questionnaire survey of eligible populations conducted between January 1, 2003 and December 31, 2004; the data were acquired from the tailored big data distribution service of the National Health Insurance Service. After a 1-year clearance period, 5,860,922 of 6,318,878 never-smoking female participants with no previous history of lung cancer were investigated. After a median follow-up of 11.4 years, 43,473 (0.74%) participants were defined as "newly diagnosed lung cancer". RESULTS: After adjusting for all variables at baseline, the variables older age, lower body mass index (BMI), less exercise, frequent alcohol drinking, meat-based diet, rural residence, and previous history of cancer were associated with a higher incidence of lung cancer. Low BMI (< 18.5 kg/m2: hazard ratio [HR], 1.33; 95% confidence interval [CI], 1.27 to 1.40) was a significant independent risk factor; as BMI decreased, HR increased. Negative associations between BMI and lung-cancer development were also observed after controlling for age (p for trend < 0.001). Drinking alcohol one to two times a week (HR, 1.25; 95% CI, 1.21 to 1.28) and eating a meat-based diet (HR, 1.08; 95% CI, 1.01 to 1.15) were associated with lung-cancer incidence. CONCLUSION: Modifiable baseline characteristics, such as BMI, exercise, alcohol consumption, and diet, are risk factors for lung-cancer development among never- smoking females. Thus, lifestyle modifications may help prevent lung cancer.


Assuntos
Neoplasias Pulmonares , Fumar , Idoso , Consumo de Bebidas Alcoólicas/efeitos adversos , Consumo de Bebidas Alcoólicas/epidemiologia , Índice de Massa Corporal , Estudos de Coortes , Feminino , Humanos , Incidência , Neoplasias Pulmonares/epidemiologia , República da Coreia/epidemiologia , Estudos Retrospectivos , Fatores de Risco , Fumar/efeitos adversos , Fumar/epidemiologia
16.
Transl Lung Cancer Res ; 8(6): 1051-1060, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32010582

RESUMO

BACKGROUND: Extracellular vesicles (EV) have been proven to contain double-stranded DNA reflecting the mutational status of the parental tumor cells in non-small cell lung cancer (NSCLC), which can be translated into clinically useful EV-based liquid biopsy for Epidermal growth factor receptor (EGFR) genotyping using bronchoalveolar lavage fluid (BALF) obtained from tumor site. METHODS: Patients subjected for an initial lung cancer work-up underwent bronchoscopy and BALF was obtained from tumor site. After isolating EVs from BALF by ultracentrifugation, EV-derived DNA (EV DNA) was extracted for subsequent EGFR genotyping performed through peptide nucleic acid (PNA)-mediated Real-Time PCR. The sensitivity, specificity, and concordance rate of BALF EV-based EGFR genotyping were calculated in comparison to tissue genotyping. RESULTS: The average sensitivity and specificity of BALF EV-based EGFR genotyping were 76% and 87%, respectively, while the sensitivity significantly increased as the stage progressed. Especially, in stage IV, BALF EV-based EGFR typing identified all tissue-proven EGFR mutant cases (n=31) and detected 6 additional mutant cases. The concordance rate was 79% in stage I, 100% in stage II, 74% in stage III, and 92% in stage IV. As TNM stage advanced, especially in the presence of metastasis, concordance rate significantly increased (P<0.05). CONCLUSIONS: The use of BALF for the collection of EV DNA in lung cancer patients resulted in a highly accurate diagnosis. The establishment of a fast and reliable method to identify target genes using EV DNA illustrated that it can overcome the problems of low sensitivity and instability in using cell-free DNA (cfDNA).

17.
Cell Oncol (Dordr) ; 42(4): 449-458, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-30838525

RESUMO

PURPOSE: Despite the development of molecular targeted therapies, few advances have been made in the treatment of lung squamous cell carcinoma (SCC). SOX2 amplification is one of the most common genetic alterations in SCC. Here, we investigated the effects of THZ1, a potent cyclin-dependent kinase 7 (CDK7) inhibitor that plays a key role in gene transcription, in SCC. METHODS: Lung SCC-derived cell viabilities were assessed using a CCK-8 assay. SOX2 expression and RNAPII-CTD phosphorylation levels after THZ1 treatment were determined by Western blotting. The effect of SOX2 suppression using shRNA was assessed by flow cytometry. Gene expression patterns after THZ1 treatment of lung SCC-derived cells were identified using microarray-based mRNA profiling. RESULTS: We found that THZ1 treatment led to suppression of cell growth and apoptotic cell death in SOX2-amplified SCC-derived cells only, whereas the modest growth-inhibitory effect of cisplatin did not differ according to SOX2 amplification status. We also found that THZ1 decreased the phosphorylation of the carboxyl-terminal domain of RNA polymerase II and the expression of several genes. Specifically, we found that the expression of transcription-associated genes, including SOX2, was down-regulated by THZ1 in SOX2-amplified SCC cells. This inhibition of SOX2 expression resulted in suppression of the growth of these cells. CONCLUSIONS: From our data, we conclude that THZ1 may effectively control the proliferation and survival of SOX2-amplified SCC cells through a decrease in global transcriptional activity, suggesting that CDK7 inhibition leading to transcription suppression may be a promising therapeutic option for lung SCC with a SOX2 amplification.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Quinases Ciclina-Dependentes/antagonistas & inibidores , Amplificação de Genes , Neoplasias Pulmonares/tratamento farmacológico , Terapia de Alvo Molecular , Fatores de Transcrição SOXB1/genética , Apoptose/efeitos dos fármacos , Carcinoma Pulmonar de Células não Pequenas/enzimologia , Carcinoma Pulmonar de Células não Pequenas/genética , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Quinases Ciclina-Dependentes/metabolismo , Regulação para Baixo/efeitos dos fármacos , Amplificação de Genes/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Neoplasias Pulmonares/enzimologia , Neoplasias Pulmonares/genética , Fenilenodiaminas/farmacologia , Fenilenodiaminas/uso terapêutico , Fosforilação/efeitos dos fármacos , Pirimidinas/farmacologia , Pirimidinas/uso terapêutico , RNA Polimerase II/metabolismo , Fatores de Transcrição SOXB1/metabolismo , Transcrição Gênica/efeitos dos fármacos , Quinase Ativadora de Quinase Dependente de Ciclina
18.
Mol Cells ; 26(2): 200-5, 2008 Aug 31.
Artigo em Inglês | MEDLINE | ID: mdl-18596410

RESUMO

The mitogen-activated protein kinase (MAPK) signaling pathway is activated in response to extracellular stimuli and regulates various activities in eukaryotic cells. Following exposure to stimuli, MAPK is known to be activated via dual phosphorylation at a conserved TxY motif in the activation loop; both threonine and tyrosine residues are phosphorylated by an upstream kinase. However, the mechanism underlying dual phosphorylation is not clearly understood. In the budding yeast Saccharomyces cerevisiae, the Hog1 MAPK mediates the high-osmolarity glycerol (HOG) signaling pathway. Tandem mass spectrometry and phosphospecific immunoblotting were performed to quantitatively monitor the dynamic changes occurring in the phosphorylation status of the TxY motif of Hog1 on exposure to osmotic stress. The results of our study suggest that the tyrosine residue is preferentially and dynamically phosphorylated following stimulation, and this in turn leads to the dual phosphorylation. The tyrosine residue was hyperphosphorylated in the absence of a threonine residue; this result suggests that the threonine residue is critical for the control of signaling noise and adaptation to osmotic stress.


Assuntos
Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Sequência de Aminoácidos , Pressão Osmótica , Fosforilação , Saccharomyces cerevisiae/metabolismo , Espectrometria de Massas por Ionização por Electrospray/métodos , Espectrometria de Massas em Tandem/métodos , Treonina/metabolismo , Tirosina/metabolismo
19.
Mol Cells ; 26(1): 41-7, 2008 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-18596411

RESUMO

Mitogen-activated protein kinase (MAPK) signaling is a crucial component of eukaryotic cells; it plays an important role in responses to extracellular stimuli and in the regulation of various cellular activities. The signaling cascade is evolutionarily conserved in the eukaryotic kingdom from yeast to human. In response to a variety of extracellular signals, MAPK activity is known to be regulated via phosphorylation of a conserved TxY motif at the activation loop in which both threonine and tyrosine residues are phosphorylated by the upstream kinase. However, the mechanism by which both residues are phosphorylated continues to remain elusive. In the budding yeast, Saccharomyces cerevisiae, Fus3 MAPK is involved in the mating signaling pathway. In order to elucidate the functional mechanism of MAPK activation, we quantitatively profiled phosphorylation of the TxY motif in Fus3 using mass spectrometry (MS). We used synthetic heavy stable isotope-labeled phosphopeptides and nonphosphopeptides corresponding to the proteolytic TxY motif of Fus3 and accompanying data-dependent tandem MS to quantitatively monitor dynamic changes in the phosphorylation events of MAPK. Phosphospecific immunoblotting and the MS data suggested that the tyrosine residue is dynamically phosphorylated upon stimulation and that this leads to dual phosphorylation. In contrast, the magnitude of threonine phosphorylation did not change significantly. However, the absence of a threonine residue leads to hyperphosphorylation of the tyrosine residue in the unstimulated condition, suggesting that the threonine residue contributes to the control of signaling noise.


Assuntos
Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Cromatografia Líquida , Eletroforese em Gel Bidimensional , Eletroforese em Gel de Poliacrilamida , Immunoblotting , Imunoprecipitação , Fragmentos de Peptídeos/metabolismo , Fosforilação , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Tirosina/metabolismo
20.
Clin Lung Cancer ; 19(5): e775-e781, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-30149884

RESUMO

INTRODUCTION: HER2 mutations are found in 2% to 4% of non-small-cell lung cancer cases and are usually mutually exclusive with other genetic alterations. We screened a large cohort of patients from multiple institutions in Korea, described the characteristics of HER2-mutant cases, and reported on several patients who were treated with pan-HER inhibitors. PATIENTS AND METHODS: The study population consisted of 360 patients diagnosed with adenocarcinoma from 4 institutions in Korea from June 2015 to September 2016. Tissue specimens from all participants were screened by direct sequencing, and next-generation sequencing was conducted only on specimens that were positive in direct sequencing. HER2-targeted therapy, either poziotinib or afatinib, was orally administrated. RESULTS: Next-generation sequencing was conducted in 129 patients, and finally 29 (8.1%) patients with HER2 mutation were identified. Most patients were female (58.6%), had never smoked (70.0%), and had stage IV non-small-cell lung cancer (55.2%). For all patients, the histologic type was adenocarcinoma, with no coexisting EGFR or ALK alterations. The most common type of HER2 mutation (48.3%) was c.2326_2327insTGT in exon 20. A partial response was observed in 2 patients who received poziotinib and 1 patient who received afatinib. The main toxicities of the pan-HER inhibitors were nausea, diarrhea, and mucositis. CONCLUSION: HER2 mutation was estimated at a frequency of approximately 8.1% in Korean patients with adenocarcinoma in the absence of known driver mutations. Because some of the HER2-mutant cases responded to poziotinib or afatinib, further studies are warranted.


Assuntos
Adenocarcinoma/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Neoplasias Pulmonares/tratamento farmacológico , Mutação , Quinazolinas/uso terapêutico , Receptor ErbB-2/genética , Adenocarcinoma/genética , Adenocarcinoma/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Feminino , Seguimentos , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Masculino , Pessoa de Meia-Idade , Prognóstico , Estudos Retrospectivos , Taxa de Sobrevida
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