Lung Cancer Biopsies Research Articles

Abstract 4463: Tumor-derived cell-free DNA from bronchoalveolar lavage fluid (BALF): A potential liquid biopsy analysis in lung cancer patients

Abstract Background: Bronchoalveolar lavage (BAL) is the most common technique for sampling the components of the alveolar space. Here, we evaluated the potential use of BAL fluid (BALF) in liquid biopsy in lung cancer. Methods: This study enrolled 56 lung cancer patients. And 57 BALF (separated supernatant and precipitate) samples and 56 peripheral blood lymphocytes samples were collected. 57 formalin-fixed Paraffin-embedded (FFPE) tissues were also obtain. We utilized a 1021-gene NGS panel in matched tissue DNA, BALF supernatant cfDNA and BALF precipitate DNA to identify somatic mutations with white blood cell DNA as a germline control. Results: Mutations were identified in 52 (~91.22%) BALF precipitate samples and 55 (~96.49%) BALF supernatant samples, comparing to 53 (92.98%) in tumor samples. EGFR mutations were observed in 22 BALF precipitate samples and 24 supernatant samples. Among these, 23 were also identified in matched tumor samples. One BALF precipitate samples specific and two BALF supernatant samples specific EGFR mutations were confirmed by Cobas assay. In addition, gene fusions including ALK, ROS1 and RET were found in 6 tumors, 4 BALF precipitate samples and 5 supernatant samples, respectively. In total, there were 195 mutations identified in the tumor samples, of which 147 (~75.4%) mutations were detected in the matched BALF precipitate samples, 168 (~86.1%) mutations were detected in the matched BALF supernatant samples and 145 (~74.4%) mutations were shared in three types of samples. Tumor mutation burden (TMB) were calculated, and the consistency between tissue and BALF precipitate was 90% and the consistency between tissue and BALF supernatant was 94%. Conclusions: Liquid biopsy using BALF shown high potential to profiling genetic alterations of patients with lung cancer, which might be implemented and standardized into clinical use. Citation Format: Yuhua Gong, Xinyu Zhang, Chun Li, Weiran Wang, Maosong Ye, Yancheng Zhao, Xin Yi, Yaping Xu, Qin Hu, Yanfang Guan, Ling Yang, Xuefeng Xia, Min Zhou, Jian'an Huang, Hua Zhang, Tao Ren, Qian Shen, Kai Wang, Yingyong Hou, Xin Zhang. Tumor-derived cell-free DNA from bronchoalveolar lavage fluid (BALF): A potential liquid biopsy analysis in lung cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4463.

The ADAM17 protease promotes tobacco smoke carcinogen-induced lung tumorigenesis.

Lung cancer is the leading cause of cancer-related mortality, with most cases attributed to tobacco smoking, in which nicotine-derived nitrosamine ketone (NNK) is the most potent lung carcinogen. The ADAM17 protease is responsible for the ectodomain shedding of many pro-tumorigenic cytokines, growth factors and receptors, and therefore is an attractive target in cancer. However, the role of ADAM17 in promoting tobacco smoke carcinogen-induced lung carcinogenesis is unknown. The hypomorphic Adam17ex/ex mice-characterized by reduced global ADAM17 expression-were backcrossed onto the NNK-sensitive pseudo-A/J background. CRISPR-driven and inhibitor-based (GW280264X, and ADAM17 prodomain) ADAM17 targeting was employed in the human lung adenocarcinoma cell lines A549 and NCI-H23. Human lung cancer biopsies were also used for analyses. The Adam17ex/ex mice displayed marked protection against NNK-induced lung adenocarcinoma. Specifically, the number and size of lung lesions in NNK-treated pseudo-A/J Adam17ex/ex mice were significantly reduced compared with wild-type littermate controls. This was associated with lower proliferative index throughout the lung epithelium. ADAM17 targeting in A549 and NCI-H23 cells led to reduced proliferative and colony-forming capacities. Notably, among select ADAM17 substrates, ADAM17 deficiency abrogated shedding of the soluble IL-6 receptor (sIL-6R), which coincided with the blockade of sIL-6R-mediated trans-signaling via ERK MAPK cascade. Furthermore, NNK upregulated phosphorylation of p38 MAPK, whose pharmacological inhibition suppressed ADAM17 threonine phosphorylation. Importantly, ADAM17 threonine phosphorylation was significantly upregulated in human lung adenocarcinoma with smoking history compared with their cancer-free controls. Our study identifies the ADAM17/sIL-6R/ERK MAPK axis as a candidate therapeutic strategy against tobacco smoke-associated lung carcinogenesis.

Exo-miRNAs as a New Tool for Liquid Biopsy in Lung Cancer.

Lung cancer is the predominant cause of cancer-related deaths. The high mortality rates are mainly due to the lack of diagnosis before the cancer is at a late stage. Liquid biopsy is a promising technique that could allow early diagnosis of lung cancer and better treatment selection for patients. Cell-free microRNAs have been detected in biological fluids, such as serum and plasma, and are considered interesting biomarkers for lung cancer screening and detection. Exosomes are nanovesicles of 30–150 nm and can be released by different cell types within the tumor microenvironment. Their exosomal composition reflects that of their parental cells and could be potentially useful as a biomarker for lung cancer diagnosis. This review summarizes the state-of-the-art of circulating microRNAs (miRNAs) in lung cancer, focusing on their potential use in clinical practice. Moreover, we describe the importance of exosomal miRNA cargo in lung cancer detection and their potential role during lung carcinogenesis. Finally, we discuss our experience with the analysis of circulating exosomal miRNAs in the bioMILD screening trial.

The predictive and prognostic significance of liquid biopsy in advanced epidermal growth factor receptor-mutated non-small cell lung cancer: A prospective study

ObjectiveTo determine the predictive and prognostic roles of three blood-based biomarkers: circulating tumour DNA (ctDNA), circulating tumour cells (CTC) and carcinoembryonic antigen (CEA), in patients with advanced epidermal growth factor receptor-mutated (EGFR+) lung cancer. Materials and methodsWe recruited 28 patients with 103 serial blood samples. We performed mutational analyses for EGFR mutations using droplet digital PCR (ddPCR) on ctDNA. We evaluated the accuracy of EGFR mutation detection in ctDNA compared with tissue biopsy. We also quantified CTCs, ctDNA and CEA in serially collected blood samples, and evaluated the baseline and changes in these blood-based biomarkers with clinical outcomes. ResultsEGFR mutation detection in plasma was highly concordant as compared with tissue biopsy. Detectable baseline ctDNA was associated with higher disease burden (p < 0.01). Early disappearance of ctDNA at 4 weeks was associated with radiological response at 12 weeks of treatment (p = 0.01) and improved progression free survival (PFS) (HR 5.47, 95%CI 1.32–22.72, p = 0.02) and overall survival (OS) (HR 5.46, 95%CI 1.28–23.22, p = 0.02). A decrease in CTC count at 4 weeks was associated with improved PFS (HR 3.81, 95%CI 1.13–12.79, p = 0.03) but not OS. 85% of patients with radiological progression had a ctDNA rise compared with 22% of patients with stable disease (p=0.01). ctDNA rise was seen on average 170 days prior to radiological progression. There is a significant association between the rise of CEA level with radiological progression (p=0.001). ConclusionEarly change in ctDNA, CTC and CEA levels may be long-term predictors of treatment benefit and failure prior to availability of radiological response data.

Identifying actionable somatic mutations in lung cancer using cell-free DNA from bronchial washing fluid.

8528 Background: Bronchial washing is the most common technique for sampling the components of the alveolar space. Here, we evaluated the potential use of bronchial washing fluid (BWF) in liquid biopsy in lung cancer. Methods: This study enrolled 65 lung cancer patients. BWF (separated supernatant and precipitate) samples, peripheral blood lymphocytes (PBL) and formalin-fixed paraffin-embedded tissues were obtained and subjected to next-generation sequencing using a 1021-gene panel. Results: Mutations were identified in 58 (89.2%) of BWF precipitate (BWFp) samples and 64 (98.5%) of BWF supernatant (BWFs) samples, comparing with 61 (93.8%) of tumor tissues. In total, 461 somatic mutations were identified in tissues, of which 331 (71.8%) and 381 (82.6%) were detected in the matched BWFp and BWFs samples. In addition, there were 44.6% of patients carrying actionable variants identified in tissue DNA, including EGFR, ALK, ROS1, RET, etc. (Table). Similarly, there were 40.0% of BWFp samples and 44.6% of BWFs samples identified actionable variants. Moreover, tumor mutation burden (TMB) was also calculated. Nearly 9% of BWFp samples and 23% of BWFs samples were TMB-H (more than 9 mutations per megabase), comparing with 20% of tissues. Significantly, the combined results of three types of samples showed that, 49.2% of patients carrying actionable variants and 24.6% of patients with TMB-H, which suggested more patients benefit from targeted therapy or immunotherapy. Conclusions: In summary, liquid biopsy using BWF showed high potential to identify actionable mutations and to calculate TMB grade of patients with lung cancer, which might be implemented and standardized into clinical use. [Table: see text]

Defining tissue requirements for a reliable detection of tumor mutational burden (TMB) in clinical FFPE samples.

e14265 Background: In the context of immuno-oncology related cancer treatment, tumor mutational burden (TMB) is currently explored as a predictive biomarker for several human malignancies. Several sequencing assays are increasingly commercially available. Established methodologies require rather large amounts of DNA input (in the range of 100 ng) which, however, are frequently not available from small biopsies. We aim to investigate how tissue size and DNA input influence TMB scores. Methods: DNA from 20 specimens (12 biopsies of non-small cell lung cancer (NSCLC); 8 surgical resection specimens from NSCLC, colorectal cancer and endometrial carcinomas) was manually extracted by using the Qiagen GeneRead DNA FFPE kit. Cases were selected to provide a wide range of relative tumor cell content (from &lt; 10% to &gt; 50%) and to include microsatellite-stable and –instable (MSI) cases. Samples were analyzed in triplicates from predefined numbers of unstained sections of a standardized tissue size. DNA quantification was done fluorometrically and by qPCR. Up to 40 ng of DNA were analyzed with the QIASeq TMB Panel (incl. MSI primer boosters; Qiagen). Sequencing was done on an Illumina NextSeq platform, TMB scores and MSI status were determined by using the CLC workbench 5.0.1 (Qiagen). Results: Biopsy samples generated less numbers of DNA molecules (as detected by unique molecular identifiers, UMIs) and less overall reads compared to resection samples. UMI coverage was &gt; 500x in all samples with &gt; 15 ng DNA input. TMB scores were highly reproducible among all samples with &gt; 15 ng DNA but differed significantly among samples with limited DNA input. Interestingly, TMB scores were inversely correlated with DNA input among those samples with &lt; 15ng. Thus, valid TMB scores could also be obtained from only one slice of 1 cm2 tissue from tumor resections or 3 slices of an endoscopic biopsy (each of 5µm thickness). All pre-characterized MSI carcinomas could be detected correctly. Conclusions: We provide first evidence that TMB can be reliably determined also in small biopsies yielding limited DNA content. However, false high TMB scores can occur in samples with &lt; 15ng DNA input. Our results contribute to the definition of minimum requirements (tissue size and DNA input) for valid TMB measurement on clinical FFPE samples.

Efficacy of tyrosine kinase inhibitors (TKIs) based on the ALK resistance mutations on amplicon-based liquid biopsy in ALK positive non-small cell lung cancer (NSCLC) patients (pts).

3055 Background: Acquired ALK resistance mutations (mut.) are the main mechanism of tyrosine kinase inhibitor (TKI) resistance (30-50%). While next-generation TKIs are more active on mut. than earlier TKIs, compound ALK resistance are associated with failure to next-generation TKIs. We evaluated the clinical utility of detecting ALK resistance mutations in blood to predict TKI efficacy. Methods: ALK positive advanced NSCLC pts were prospectively enrolled between Oct. 2015 and Aug. 2018 in 8 French institutions. Prospective samples were collected; ctDNA was analyzed by amplicon-based Inivata InVisionFirst-Lung. Results: A total of 101 pts with advanced ALK positive NSCLC were enrolled and 328 samples collected. In samples collected at TKI failure (N=74), we detected 9 single and 7 complex (≥2) ALK resistance mut. (22%), associated with EML4-ALK variant 3 (38%) vs. variant 2 (13%) vs. variant 1 (none); 30% had other somatic mut. (mainly TP53 and KRAS, PI3KCA, MET, etc.). No mutations were detected in 48% of samples (ctDNA neg). ALK mut. were more frequent after 2nd/3rd generation TKI (43% post-lorlatinib (7), 29% post-2nd gen. (31), 11% post-crizotinib (36)). ALKG1202R was the most common, as single (n=3) or complex mut. (n=4). The median overall survival (mOS) was 100.4 mo. (95% CI 41.9-158.9) and the median progression free-survival (mPFS) to subsequent line was 2.8 mo. (0.7-4.9). Patients with ctDNA neg had mOS of 105 mo. (39.3-172.1) vs. 58.5 mo. (33.1-84.0) if ≥1 ALK mut. vs. 44.1 mo. (20.0-68.2) if others ( P=0.001). Pts with the complex ALK mut. had worse OS compared to singles ALK mut. (mOS 26.9 mo. vs. 58.5 mo., P=0.001); ALK complex mut. were associated with poor efficacy to subsequent therapy (PFS &lt;3 mo. in 57%; no cases with PFS &gt;6 mo.) vs. single mut., with longer PFS (PFS &gt;6 mo. in 56%). Detectable ALKG1202R mut. were associated with shorter median OS (58.3 mo.; 7.9-109.1) vs. overall population; 86% of cases developed rapid PD (PFS &lt;3mo.) to subsequent therapy with only one durable response to lorlatinib (PFS &gt;6mo.). Conclusions: The absence of ctDNA mutations at TKI failure was associated with prolonged OS, whereas complex ALK mutations at TKI failure may predict resistance to subsequent therapy. Larger and specifically designed studies should be performed to validate these findings.

Glypican-1 in serum-derived exosomes as a potential biomarker in liquid biopsy of non-small cell lung cancer

Glypican-1 in serum-derived exosomes as a potential biomarker in liquid biopsy of non-small cell lung cancer

Lung Cancer and Liquid Biopsy: Realities and Challenges in Routine Clinical Practice

Lung Cancer and Liquid Biopsy: Realities and Challenges in Routine Clinical Practice

204P - Diagnostic value of concomitant use of radial probe endobronchial ultrasound with guide sheath and transbronchial biopsy in lung cancer

204P - Diagnostic value of concomitant use of radial probe endobronchial ultrasound with guide sheath and transbronchial biopsy in lung cancer

26P - Potentialities of liquid biopsy in advanced non-small cell lung cancer (aNSCLC): Early evaluation of sentinel mutations in plasma and outcome of patients treated with immunotherapy

26P - Potentialities of liquid biopsy in advanced non-small cell lung cancer (aNSCLC): Early evaluation of sentinel mutations in plasma and outcome of patients treated with immunotherapy

A combined gene expression tool for parallel histological prediction and gene fusion detection in non-small cell lung cancer

Accurate histological classification and identification of fusion genes represent two cornerstones of clinical diagnostics in non-small cell lung cancer (NSCLC). Here, we present a NanoString gene expression platform and a novel platform-independent, single sample predictor (SSP) of NSCLC histology for combined, simultaneous, histological classification and fusion gene detection in minimal formalin fixed paraffin embedded (FFPE) tissue. The SSP was developed in 68 NSCLC tumors of adenocarcinoma (AC), squamous cell carcinoma (SqCC) and large-cell neuroendocrine carcinoma (LCNEC) histology, based on NanoString expression of 11 (CHGA, SYP, CD56, SFTPG, NAPSA, TTF-1, TP73L, KRT6A, KRT5, KRT40, KRT16) relevant genes for IHC-based NSCLC histology classification. The SSP was combined with a gene fusion detection module (analyzing ALK, RET, ROS1, MET, NRG1, and NTRK1) into a multicomponent NanoString assay. The histological SSP was validated in six cohorts varying in size (n = 11–199), tissue origin (early or advanced disease), histological composition (including undifferentiated cancer), and gene expression platform. Fusion gene detection revealed five EML4-ALK fusions, four KIF5B-RET fusions, two CD74-NRG1 fusion and three MET exon 14 skipping events among 131 tested cases. The histological SSP was successfully trained and tested in the development cohort (mean AUC = 0.96 in iterated test sets). The SSP proved successful in predicting histology of NSCLC tumors of well-defined subgroups and difficult undifferentiated morphology irrespective of gene expression data platform. Discrepancies between gene expression prediction and histologic diagnosis included cases with mixed histologies, true large cell carcinomas, or poorly differentiated adenocarcinomas with mucin expression. In summary, we present a proof-of-concept multicomponent assay for parallel histological classification and multiplexed fusion gene detection in archival tissue, including a novel platform-independent histological SSP classifier. The assay and SSP could serve as a promising complement in the routine evaluation of diagnostic lung cancer biopsies.

Real-World Data of the Correlation between EGFR Determination by Liquid Biopsy in Non-squamous Non-small Cell Lung Cancer (NSCLC) and the EGFR Profile in Tumor Biopsy.

EGFR-mutated non-small cell lung cancer (NSCLC) has significant improved outcomes when treated with EGFR-tyrosine kinase inhibitors (TKI). Thus, EGFR-mutational status should be assessed at diagnosis and in the course of treatment with TKI. However, tissue samples are not always evaluable, and molecular profiling has been increasingly performed in cell-free tumor DNA (ctDNA) from blood samples. Our objective is to evaluate the reliability of ctDNA profiling in plasma samples in a real-world setting. We retrospectively analyzed the patients diagnosed with non-squamous NSCLC from May 2016 to December 2017 at Hospital Universitario Doctor Peset who had been tested for EGFR mutations in tissue and plasma samples. Both samples were sent to an external laboratory to perform the analysis by the cobas® EGFR assay. Percentage of agreement and concordance were calculated by kappa statistic. Of 102 patients reviewed, 89 were eligible. The overall EGFR mutation frequency was 18.6% for the evaluable tissue samples and 19.6% for evaluable plasma samples. Mutation status concordance between matched samples was 87.4%. Cohen's kappa index (κ) = 0.6 (sensitivity 70.6%, specificity 91.7%, positive predictive value 66.7%, negative predictive value 93%). When concordance was stablished only in stage IV tumors κ = 0.7, suggesting a higher agreement in advanced disease. This real-world data suggest that plasma is a feasible sample for ctDNA EGFR mutation assessment. Results of ctDNA molecular profiling are reliable when using a validated technique such as the cobas® EGFR assay, especially in patients that cannot undergo a tissue biopsy.

Lung cancer and residential radon in never-smokers: A pooling study in the Northwest of Spain

BackgroundUsing a pooled case-control study design, including only never-smokers, we have assessed the association of residential radon exposure with the subsequent occurrence of lung cancer. We also investigated whether residential radon poses a different risk specifically for adenocarcinoma. MethodsWe pooled individual data from different case-control studies conducted in recent years in Northwestern Spain which investigated residential radon and lung cancer. All participants were never-smokers. Cases had a confirmed biopsy of primary lung cancer. Hospital controls were selected at pre-surgery units, presenting for non-complex surgical procedures. They were interviewed using a standardized instrument. Residential radon was measured using alpha track detectors at the Galician Radon Laboratory at the University of Santiago de Compostela. ResultsA total of 1415 individuals, 523 cases and 892 controls were included. We observed an odds ratio of 1.73 (95%CI: 1.27–2.35) for individuals exposed to ≥ 200 Bq/m3 compared with those exposed to ≤100 Bq/m3. Lung cancer risk for adenocarcinoma was 1.52 (95%CI: 1.14–2.02) using the same categories for radon exposure. ConclusionsResidential radon is a clear risk factor for lung cancer in never-smokers. Our data suggest that radon exposure is associated with all histological types of lung cancer and also with adenocarcinoma, which is currently the most frequent histological type for this disease.

Diagnostic value of rapid on-site evaluation during transbronchial biopsy for peripheral lung cancer.

An increasing number of pulmonary lesions-particularly, peripheral lung lesions-are identified with current technological advancements. Notably, the yield of traditional bronchoscopy for the diagnosis of peripheral lung lesions is low. This study evaluated the diagnostic value of rapid on-site evaluation (ROSE) during transbronchial biopsy for peripheral lung cancer. This study included 641 patients who underwent transbronchial biopsy for suspected lung cancer at the Respiratory Department of Tianjin Medical University General Hospital between January 2012 and December 2016. Based on whether ROSE was used, patients were assigned to the ROSE group (353 patients) or non-ROSE group (288 patients). In the ROSE group, several air-dried smears were processed with Diff-Quik staining; the remaining samples were placed in 10% formalin. Diagnostic yields for central and peripheral lung cancer were compared between the two groups. In addition, ROSE results were compared with final diagnoses. Diagnostic yield for peripheral lung cancer, stratified by pathology, was significantly higher in the ROSE group than in the non-ROSE group (42.9% vs. 30.7%, P < 0.05). The sensitivity, specificity, positive predictive value, negative predictive value and diagnostic accuracy of ROSE for peripheral pulmonary lesions were 90.0%, 89.5%, 94.0%, 82.8% and 89.8%, respectively. Conformance was high between ROSE and final pathologic evaluations during transbronchial biopsy (Kappa = 0.780, P = 0.035). There were no procedure-related deaths. ROSE during conventional transbronchial biopsy improves diagnostic yield, stratified by pathology, for patients with peripheral lung cancer via live feedback. Moreover, ROSE diagnosis correlates with final cytopathological diagnosis.

Accuracy of Biopsy needle in Computed Tomography Guided Biopsy Using a Self-Made Gelatin Phantom

The purpose of this study was to analyze the actual reflection of the biopsy needle when performing computed tomography (CT) guided biopsy in lung cancer. CT used in the experiment was a 128-channel dual source SOMATOM Definition flash. A 21-gauge biopsy needle was inserted into the self-made gelatin phantom at 10 mm intervals from 20 mm to 80 mm on the Y -axis of the CT scanner. At each insertion depth, the CT axial scan was performed with 0.6 mm, 1.0 mm, and 2.0 mm slice thickness. We examined 30 times for each slice thickness for a total of 270 times. These acquired images were used to compare actual and measured insertion lengths. As a result, 40 mm depth was measured as 4.00 ± 0.01 in 1.0 mm slice thickness (p > 0.05) while the remaining slice thickness was considered smaller or larger than the actual value. A 1.0 mm slice thickness was closer to the actual measurement value than the other two slice thicknesses (p < 0.05). Therefore, the accuracy of CT-guided biopsy in lung cancer is increased by positioning the lesion at the isocenter at a slice thickness of 1.0 mm.

Single tube liquid biopsy for advanced non-small cell lung cancer.

The need for a liquid biopsy in non-small cell lung cancer (NSCLC) patients is rapidly increasing. We studied the relation between overall survival (OS) and the presence of four cancer biomarkers from a single blood draw in advanced NSCLC patients: EpCAMhigh circulating tumor cells (CTC), EpCAMlow CTC, tumor-derived extracellular vesicles (tdEV) and cell-free circulating tumor DNA (ctDNA). EpCAMhigh CTC were detected with CellSearch, tdEV in the CellSearch images and EpCAMlow CTC with filtration after CellSearch. ctDNA was isolated from plasma and mutations present in the primary tumor were tracked with deep sequencing methods. In 97 patients, 21% had ≥2 EpCAMhigh CTC, 15% had ≥2 EpCAMlow CTC, 27% had ≥18 tdEV and 19% had ctDNA with ≥10% mutant allele frequency. Either one of these four biomarkers could be detected in 45% of the patients and all biomarkers were present in 2%. In 11 out of 16 patients (69%) mutations were detected in the ctDNA. Two or more unfavorable biomarkers were associated with poor OS. The presence of EpCAMhigh CTC and elevated levels of tdEV and ctDNA was associated with a poor OS; however, the presence of EpCAMlow CTC was not. This single tube approach enables simultaneous analysis of multiple biomarkers to explore their potential as a liquid biopsy.

Competitive evolution of NSCLC tumor clones and the drug resistance mechanism of first-generation EGFR-TKIs in Chinese NSCLC patients

PurposeAlthough many studies have reported on the resistance mechanism of first-generation EGFR TKIs (1st EGFR TKIs) treatment, large-scale dynamic ctDNA mutation analysis based on liquid biopsy for non-small cell lung cancer (NSCLC) in the Chinese population is rare. Using in-depth integration and analysis of ctDNA genomic mutation data and clinical data at multiple time points during the treatment of 53 NSCLC patients, we described the resistance mechanisms of 1st EGFR TKIs treatment more comprehensively and dynamically. The resulting profile of the polyclonal competitive evolution of the tumor provides some new insights into the precise treatment of NSCLC. Experimental designA prospective study was conducted in patients with advanced NSCLC with acquired resistance to erlotinib, gefitinib or icotinib. By liquid biopsy, we detected mutations in 124 tumor-associated genes in the context of drug resistance. These 124 genes covered all tumor therapeutic targets and related biological pathways. During the entire course of treatment, the interval between two liquid biopsies was two months. ResultsUnlike the common mutations tested in tissue samples, our data showed a higher coverage of tumor heterogeneity (32.65%), more complex patterns of resistance and some new resistance mutation sites, such as EGFR p.V769M and KRAS p.A11V. The major resistance-associated mutations detected were still EGFR p.T790M (45.28%), other point mutations in EGFR (33.9%), and KRAS and NRAS mutations (15.09%). These mutation ratios might be considered as a preliminary summary of the characteristics of Chinese patients. In addition, starting from the two baseline mutations of the EGFR gene (19del vs. L858R), we first described the detailed mutation profile of the EGFR gene. Although there was no significant difference in the number of patients with EGFR p.19del and EGFR p.L858R baseline mutations (24% vs. 16%, P = 0.15), patients from the EGFR p.19del baseline group were much more likely to develop EGFR p.T790M resistance mutations (62.1% vs. 19.3%, P = 0.007). Through careful integration of gene mutation information and clinical phenotype information, an interesting phenomenon was found. Although the variant allele fraction (VAF) of the EGFR p.T790M mutation was significantly linearly correlated with that of the EGFR drug-sensitive mutation (r = 0.68, P = 0.00025), neither VAF was associated with the tumor volume at the advanced stage. It was shown that other tumor clones might contribute more to the resistance to 1st EGFR TKIs treatment than tumor clones carrying the EGFR p.T790M mutation when resistance developed. By further analysis, we found that, in some patients, when the primary tumor clones detected were those carrying EGFR−/− mutations (both types the EGFR p.19del/p.L858R and EGFR p.T790M mutation types were missing), most of them showed a poor prognosis and ineffective late treatment, indicating that EGFR−/− played a more important role than EGFR p.T790M in the process of NSCLC drug resistance in these patients. From the perspective of the clonal evolution of NSCLC tumor cells, these phenomena could be explained by the competitive evolution between different tumor clones. In addition, two new mutations, KRAS p.A11V and EGFR p.V769M, emerged significantly during drug resistance in NSCLC patients and had shown obvious competitive clonal evolution characteristics. Combined with clear clinical drug resistance phenotypic information, we believed that these two new mutations might be related to new drug resistance mechanisms and deserve further study. We have also seen an interesting phenomenon. In some patients undergoing 1st EGFR TKIs treatment, the EGFR p.T790M mutation appeared, disappeared, and reappeared, and this spatial and temporal diversity of the EGFR p.T790M mutation was regulated by targeted drug and chemotherapy and was correlated with the individual tumor mutation profile. ConclusionsThe constitution and competitive evolution of the tumor clones have a decisive influence on treatment and can be regulated by targeted drugs and chemotherapy. Additionally, EGFR p.T790M spatial and temporal diversity during treatment warrants more attention, and this spatial and temporal diversity may be useful for the choice of treatment strategies for certain NSCLC patients. Through longitudinal cfDNA sample analysis, the resistance mechanism and dynamic clinical features of Chinese NSCLC patients are systematically established as reliable and meaningful to understand acquired resistance and make further personalized treatment decisions dynamically. Two new potential drug resistance-associated mutations in EGFR and KRAS have been found and are worthy of further study. Finally, our research shows that the evolutionary process of tumor cloning can be artificially regulated and intervened, possibly providing a new way to treat tumors.

Next-generation sequencing of circulating tumor DNA for detection of gene mutations in lung cancer: implications for precision treatment.

BackgroundLung cancer remains a major global health problem, which causes millions of deaths annually. Because the prognosis is mainly determined by the stage of lung cancer, precise early diagnosis is of great significance to improve the survival and prognosis. Circulating tumor DNA (ctDNA) has been recognized as a sensitive and specific biomarker for the detection of early- and late-stage lung cancer, and next-generation sequencing (NGS) of ctDNA has been accepted as a noninvasive tool for early identification and monitoring of cancer mutations. This study aimed to assess the value of NGS-based ctDNA analysis in detecting gene mutations in lung cancer patients.MethodsA total of 101 subjects with pathological diagnosis of lung cancer were enrolled, and blood samples were collected. ctDNA samples were prepared and subjected to NGS assays.ResultsThere were 31 cases harboring 40 gene mutations, and EGFR was the most frequently mutated gene (27.72%). In addition, there were seven cases with double mutations and one case with triple mutations, with EGFR p.T790M mutation exhibiting the highest frequency.ConclusionOur findings demonstrate that NGS of ctDNA is effective in detecting gene mutations in lung cancer patients, and may be used as a liquid biopsy for lung cancer, which facilitates the development of precision treatment regimens for lung cancer.

The Utility of Computed Tomography-Guided Biopsy for Lung Cancer

The Utility of Computed Tomography-Guided Biopsy for Lung Cancer