Targeting WEE1 enhances the antitumor effect of KRAS-mutated non-small cell lung cancer harboring TP53 mutations.

The clinical development of Kirsten rat sarcoma virus (KRAS)-G12C inhibitors for the treatment of KRAS-mutant lung cancer is limited by the presence of co-mutations, intrinsic resistance, and the emergence of acquired resistance. Therefore, innovative strategies for enhancing apoptosis in KRAS-mutated non-small cell lung cancer (NSCLC) are urgently needed. Through CRISPR-Cas9 knockout screening using a library of 746 crRNAs and drug screening with a custom library of 432 compounds, we discover that WEE1 kinase inhibitors are potent enhancers of apoptosis, particularly in KRAS-mutant NSCLC cells harboring TP53 mutations. Mechanistically, WEE1 inhibition promotes G2/M transition and reduces checkpoint kinase 2 (CHK2) and Rad51 expression in the DNA damage response (DDR) pathway, which is associated with apoptosis and the repair of DNA double-strand breaks, leading to mitotic catastrophe. Notably, the combined inhibition of KRAS-G12C and WEE1 consistently suppresses tumor growth. Our results suggest targeting WEE1 as a promising therapeutic strategy for KRAS-mutated NSCLC with TP53 mutations.

Focal adhesion kinase-YAP signaling axis drives drug-tolerant persister cells and residual disease in lung cancer.

Targeted therapy is effective in many tumor types including lung cancer, the leading cause of cancer mortality. Paradigm defining examples are targeted therapies directed against non-small cell lung cancer (NSCLC) subtypes with oncogenic alterations in EGFR, ALK and KRAS. The success of targeted therapy is limited by drug-tolerant persister cells (DTPs) which withstand and adapt to treatment and comprise the residual disease state that is typical during treatment with clinical targeted therapies. Here, we integrate studies in patient-derived and immunocompetent lung cancer models and clinical specimens obtained from patients on targeted therapy to uncover a focal adhesion kinase (FAK)-YAP signaling axis that promotes residual disease during oncogenic EGFR-, ALK-, and KRAS-targeted therapies. FAK-YAP signaling inhibition combined with the primary targeted therapy suppressed residual drug-tolerant cells and enhanced tumor responses. This study unveils a FAK-YAP signaling module that promotes residual disease in lung cancer and mechanism-based therapeutic strategies to improve tumor response.

Astrocyte-induced mGluR1 activates human lung cancer brain metastasis via glutamate-dependent stabilization of EGFR.

There are limited methods to stably analyze the interactions between cancer cells and glial cells in vitro, which hinders our molecular understanding. Here, we develop a simple and stable culture method of mouse glial cells, termed mixed-glial culture on/in soft substrate (MGS), which serves well as a platform to study cancer-glia interactions. Using this method, we find that human lung cancer cells become overly dependent on metabotropic glutamate receptor 1 (mGluR1) signaling in the brain microenvironment. Mechanistically, interactions with astrocytes induce mGluR1 in cancer cells through the Wnt-5a/prickle planar cell polarity protein 1 (PRICKLE1)/RE1 silencing transcription factor (REST) axis. Induced mGluR1 directly interacts with and stabilizes the epidermal growth factor receptor (EGFR) in a glutamate-dependent manner, and these cells then become responsive to mGluR1 inhibition. Our results highlight increased dependence on mGluR1 signaling as an adaptive strategy and vulnerability of human lung cancer brain metastasis.

The role of APOBEC3B in lung tumor evolution and targeted cancer therapy resistance.

In this study, the impact of the apolipoprotein B mRNA-editing catalytic subunit-like (APOBEC) enzyme APOBEC3B (A3B) on epidermal growth factor receptor (EGFR)-driven lung cancer was assessed. A3B expression in EGFR mutant (EGFRmut) non-small-cell lung cancer (NSCLC) mouse models constrained tumorigenesis, while A3B expression in tumors treated with EGFR-targeted cancer therapy was associated with treatment resistance. Analyses of human NSCLC models treated with EGFR-targeted therapy showed upregulation of A3B and revealed therapy-induced activation of nuclear factor kappa B (NF-κB) as an inducer of A3B expression. Significantly reduced viability was observed with A3B deficiency, and A3B was required for the enrichment of APOBEC mutation signatures, in targeted therapy-treated human NSCLC preclinical models. Upregulation of A3B was confirmed in patients with NSCLC treated with EGFR-targeted therapy. This study uncovers the multifaceted roles of A3B in NSCLC and identifies A3B as a potential target for more durable responses to targeted cancer therapy.

A novel approach for the non-invasive diagnosis of pulmonary nodules using low-depth whole-genome sequencing of cell-free DNA.

Background: Differentiating between benign and malignant pulmonary nodules is a diagnostic challenge, and inaccurate detection can result in unnecessary invasive procedures. Cell-free DNA (cfDNA) has been successfully utilized to detect various solid tumors. In this study, we developed a genome-wide approach to explore the characteristics of cfDNA sequencing reads obtained by low-depth whole-genome sequencing (LD-WGS) to diagnose pulmonary nodules. Methods: LD-WGS was performed on cfDNA extracted from 420 plasma samples from individuals with pulmonary nodules that were no more than 30 mm in diameter, as determined by computed tomography (CT). The sequencing read distribution patterns of cfDNA were analyzed and used to establish a model for distinguishing benign from malignant pulmonary nodules. Results: We proposed the concept of weighted reads distribution difference (WRDD) based on the copy number alterations (CNAs) of cfDNA to construct a benign and malignant diagnostic (BEMAD) algorithm model. In a training cohort of 360 plasma samples, the model achieved an average area under the receiver operating characteristic (ROC) curve (AUC) value of 0.84 in 10-fold cross-validation. The model was validated in an independent cohort of 60 plasma samples, obtaining an AUC value of 0.87. The BEMAD model could distinguish benign from malignant nodules at a sensitivity of 74% and a specificity of 86%. Furthermore, analysis of the critical features of the cfDNA using the BEMAD model identified repeat regions that were associated with microsatellite instability, which is an important indicator of tumorigenesis. Conclusions: This study provides a novel non-invasive diagnostic approach to discriminate between benign and malignant pulmonary nodules to avoid unnecessary invasive procedures.

Deficiency of the splicing factor RBM10 limits EGFR inhibitor response in EGFR-mutant lung cancer.

Molecularly targeted cancer therapy has improved outcomes for patients with cancer with targetable oncoproteins, such as mutant EGFR in lung cancer. Yet, the long-term survival of these patients remains limited, because treatment responses are typically incomplete. One potential explanation for the lack of complete and durable responses is that oncogene-driven cancers with activating mutations of EGFR often harbor additional co-occurring genetic alterations. This hypothesis remains untested for most genetic alterations that co-occur with mutant EGFR. Here, we report the functional impact of inactivating genetic alterations of the mRNA splicing factor RNA-binding motif 10 (RBM10) that co-occur with mutant EGFR. RBM10 deficiency decreased EGFR inhibitor efficacy in patient-derived EGFR-mutant tumor models. RBM10 modulated mRNA alternative splicing of the mitochondrial apoptotic regulator Bcl-x to regulate tumor cell apoptosis during treatment. Genetic inactivation of RBM10 diminished EGFR inhibitor-mediated apoptosis by decreasing the ratio of (proapoptotic) Bcl-xS to (antiapoptotic) Bcl-xL isoforms of Bcl-x. RBM10 deficiency was a biomarker of poor response to EGFR inhibitor treatment in clinical samples. Coinhibition of Bcl-xL and mutant EGFR overcame the resistance induced by RBM10 deficiency. This study sheds light on the role of co-occurring genetic alterations and on the effect of splicing factor deficiency on the modulation of sensitivity to targeted kinase inhibitor cancer therapy.

Methylation of Tumor Suppressive miRNAs in Plasma from Patients With Pancreaticobiliary Diseases.

BACKGROUND/AIM: We previously reported the usefulness of aberrant methylation of tumor suppressive miRNAs in bile to discriminate pancreaticobiliary cancers (PBCs) from benign pancreaticobiliary diseases (BD). Here we performed a methylation analysis of plasma miRNAs to identify miRNAs specific for PBCs. PATIENTS AND METHODS: Plasma was collected from 80 patients with pancreatic cancer (PC); 18 with biliary tract cancer (BTC) and 28 with BD. Sequences encoding 3 tumor suppressive miRNAs (miR-200a, -200b, and -1247) were PCR amplified and sequenced, and their methylation rates were determined. RESULTS: The methylation rate of miR-1247 was significantly higher in patients with BTC than in those with BD, and tended to be higher in patients with PC than in those with BD. Furthermore, it was significantly higher in three patients with stages I/II BTC than in those with BD. CONCLUSION: Methylation of miR-1247 in plasma may be useful to distinguish BTC from BD.

Trametinib overcomes KRAS-G12V-induced osimertinib resistance in a leptomeningeal carcinomatosis model of EGFR-mutant lung cancer.

Leptomeningeal carcinomatosis (LMC) occurs frequently in non-small cell lung cancer (NSCLC) harboring epidermal growth factor receptor (EGFR) mutations and is associated with acquired resistance to EGFR tyrosine kinase inhibitors (EGFR-TKIs). However, the mechanism by which LMC acquires resistance to osimertinib, a third-generation EGFR-TKI, is unclear. In this study, we elucidated the resistance mechanism and searched for a novel therapeutic strategy. We induced osimertinib resistance in a mouse model of LMC using an EGFR-mutant NSCLC cell line (PC9) via continuous oral osimertinib treatment and administration of established resistant cells and examined the resistance mechanism using next-generation sequencing. We detected the Kirsten rat sarcoma (KRAS)-G12V mutation in resistant cells, which retained the EGFR exon 19 deletion. Experiments involving KRAS knockdown in resistant cells and KRAS-G12V overexpression in parental cells revealed the involvement of KRAS-G12V in osimertinib resistance. Cotreatment with trametinib (a MEK inhibitor) and osimertinib resensitized the cells to osimertinib. Furthermore, in the mouse model of LMC with resistant cells, combined osimertinib and trametinib treatment successfully controlled LMC progression. These findings suggest a potential novel therapy against KRAS-G12V-harboring osimertinib-resistant LMC in EGFR-mutant NSCLC.

Androgen replacement therapy for cancer-related symptoms in male: result of prospective randomized trial (ARTFORM study).

BACKGROUND: Hypogonadism associated with cancer is reported to cause cachexia and a variety of physical and psychological symptoms. This study aims to evaluate whether androgen replacement therapy can improve cancer-related symptoms in male advanced cancer patients. METHODS: An investigator-initiated, prospective, and randomized controlled study was conducted. Patients with low serum testosterone levels (total or free testosterone levels were <2.31 ng/mL or <11.8 pg/mL, respectively) were randomly assigned to the control or testosterone enanthate administration (testosterone group) groups. Testosterone enanthate was injected into the muscle tissue at a dose of 250 mg every 4 weeks (baseline, week 4, and week 8). Differences in quality of life questionnaires and cachexia-related serum protein levels between groups were assessed. RESULTS: This study enrolled and randomized 106 and 81 patients, respectively. Moreover, 41 and 40 patients were in the control and testosterone groups, respectively. Although no significant differences in the change of subscales and total scores in Functional Assessment of Anorexia/Cachexia Treatment were noted from the baseline between the two groups, the testosterone group showed a significantly better change in the 'unhappiness' item of the Edmonton Symptom Assessment System at week 12 compared with baseline versus the control group (-1.4 and 0.0 points, respectively; mean, P = 0.007). No significant differences exist in the change of serum interleukin-6 and insulin-like growth factor-1 levels at week 12 from the baseline between the control and testosterone groups. Consequently, the testosterone group significantly inhibited the change in serum tumour necrotic factor-α level at week 12 from the baseline compared with the control group (+0.4 and +0.1 pg/mL, respectively; mean, P = 0.005). CONCLUSIONS: Although testosterone enanthate did not improve most of the items in health-related quality of life questionnaires, testosterone enanthate induced a significantly better change in the 'unhappiness' item at week 12 compared with the control. Testosterone enanthate may be a potential treatment option for male advanced cancer patients.

Single-cell lineages reveal the rates, routes, and drivers of metastasis in cancer xenografts.

Detailed phylogenies of tumor populations can recount the history and chronology of critical events during cancer progression, such as metastatic dissemination. We applied a Cas9-based, single-cell lineage tracer to study the rates, routes, and drivers of metastasis in a lung cancer xenograft mouse model. We report deeply resolved phylogenies for tens of thousands of cancer cells traced over months of growth and dissemination. This revealed stark heterogeneity in metastatic capacity, arising from preexisting and heritable differences in gene expression. We demonstrate that these identified genes can drive invasiveness and uncovered an unanticipated suppressive role for KRT17 We also show that metastases disseminated via multidirectional tissue routes and complex seeding topologies. Overall, we demonstrate the power of tracing cancer progression at subclonal resolution and vast scale.

Osimertinib Overcomes Alectinib Resistance Caused by Amphiregulin in a Leptomeningeal Carcinomatosis Model of ALK-Rearranged Lung Cancer.

INTRODUCTION: Leptomeningeal carcinomatosis (LMC) occurs frequently in anaplastic lymphoma kinase (ALK)-rearranged NSCLC and develops acquired resistance to ALK tyrosine kinase inhibitors (ALK TKIs). This study aimed to clarify the resistance mechanism to alectinib, a second-generation ALK TKI, in LMC and test a novel therapeutic strategy. METHODS: We induced alectinib resistance in an LMC mouse model with ALK-rearranged NSCLC cell line, A925LPE3, by continuous oral alectinib treatment, established A925L/AR cells. Resistance mechanisms were analyzed using several assays, including Western blot and receptor tyrosine kinase array. We also measured amphiregulin (AREG) concentrations in cerebrospinal fluid from patients with ALK-rearranged NSCLC with alectinib-refractory LMC by enzyme-linked immunosorbent assay. RESULTS: A925L/AR cells were moderately resistant to various ALK TKIs, such as alectinib, crizotinib, ceritinib, and lorlatinib, compared with parental cells in vitro. A925L/AR cells acquired the resistance by EGFR activation resulting from AREG overexpression caused by decreased expression of microRNA-449a. EGFR TKIs and anti-EGFR antibody resensitized A925L/AR cells to alectinib in vitro. In the LMC model with A925L/AR cells, combined treatment with alectinib and EGFR TKIs, such as erlotinib and osimertinib, successfully controlled progression of LMC. Mass spectrometry imaging showed accumulation of the EGFR TKIs in the tumor lesions. Moreover, notably higher AREG levels were detected in cerebrospinal fluid of patients with alectinib-resistant ALK-rearranged NSCLC with LMC (n = 4), compared with patients with EGFR-mutated NSCLC with EGFR TKI-resistant LMC (n = 30), or patients without LMC (n = 24). CONCLUSIONS: These findings indicate the potential of novel therapies targeting both ALK and EGFR for the treatment of ALK TKI-resistant LMC in ALK-rearranged NSCLC.

CIC-DUX4 oncoprotein drives sarcoma metastasis and tumorigenesis via distinct regulatory programs.

Transcription factor fusion genes create oncoproteins that drive oncogenesis and represent challenging therapeutic targets. Understanding the molecular targets by which such fusion oncoproteins promote malignancy offers an approach to develop rational treatment strategies to improve clinical outcomes. Capicua-double homeobox 4 (CIC-DUX4) is a transcription factor fusion oncoprotein that defines certain undifferentiated round cell sarcomas with high metastatic propensity and poor clinical outcomes. The molecular targets regulated by the CIC-DUX4 oncoprotein that promote this aggressive malignancy remain largely unknown. We demonstrated that increased expression of ETS variant 4 (ETV4) and cyclin E1 (CCNE1) occurs via neomorphic, direct effects of CIC-DUX4 and drives tumor metastasis and survival, respectively. We uncovered a molecular dependence on the CCNE-CDK2 cell cycle complex that renders CIC-DUX4-expressing tumors sensitive to inhibition of the CCNE-CDK2 complex, suggesting a therapeutic strategy for CIC-DUX4-expressing tumors. Our findings highlight a paradigm of functional diversification of transcriptional repertoires controlled by a genetically aberrant transcriptional regulator, with therapeutic implications.

Development of an LC-MS/MS-based method for quantitation of osimertinib in human plasma and cerebrospinal fluid.

Aim: The transitivity of osimertinib to cerebrospinal fluid (CSF) is of clinical concern. A quantitative LC-MS/MS method for the determination of osimertinib in human plasma and CSF was developed to evaluate its transitivity. Results: The calibration range was 40-1000 nM in plasma and 0.8-100 nM in CSF. Accuracy and precision were within 15%. Osimertinib in the CSF but not in plasma strongly adsorbed onto the storage container. The mean adsorbed loss of osimertinib was 45.5% in CSF. Nonspecific binding in CSF was decreased by protein addition (mean loss = 5.8%). Conclusion: A robust validated method was developed for the quantification of osimertinib in human plasma and CSF.

Epithelial-to-Mesenchymal Transition Is a Mechanism of ALK Inhibitor Resistance in Lung Cancer Independent of ALK Mutation Status.

Mutations in the ALK gene are detectable in approximately 40% of ALK-rearranged lung cancers resistant to ALK inhibitors. Although epithelial-to-mesenchymal transition (EMT) is a mechanism of resistance to various targeted drugs, its involvement in ALK inhibitor resistance is largely unknown. In this study, we report that both ALK-mutant L1196M and EMT were concomitantly detected in a single crizotinib-resistant lesion in a patient with ALK-rearranged lung cancer. Digital PCR analyses combined with microdissection after IHC staining for EMT markers revealed that ALK L1196M was predominantly detected in epithelial-type tumor cells, indicating that mesenchymal phenotype and ALK mutation can coexist as independent mechanisms underlying ALK inhibitor-resistant cancers. Preclinical experiments with crizotinib-resistant lung cancer cells showed that EMT associated with decreased expression of miR-200c and increased expression of ZEB1 caused cross-resistance to new-generation ALK inhibitors alectinib, ceritinib, and lorlatinib. Pretreatment with the histone deacetylase (HDAC) inhibitor quisinostat overcame this resistance by reverting EMT in vitro and in vivo. These findings indicate that HDAC inhibitor pretreatment followed by a new ALK inhibitor may be useful to circumvent resistance constituted by coexistence of resistance mutations and EMT in the heterogeneous tumor. SIGNIFICANCE: These findings show that dual inhibition of HDAC and ALK receptor tyrosine kinase activities provides a means to circumvent crizotinib resistance in lung cancer.

Development and validation of a method for gefitinib quantification in dried blood spots using liquid chromatography-tandem mass spectrometry: Application to finger-prick clinical blood samples of patients with non-small cell lung cancer.

A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for the quantification of gefitinib in dried blood spots (DBSs). Gefitinib was extracted with methanol from DBS of 3 mm in diameter and detected using a triple quadrupole mass spectrometer. The method was validated by evaluating its precision, accuracy, selectivity, carryover, matrix effect, recovery, and stability. For clinical validation, paired finger-prick DBS and plasma concentrations were compared for 10 patients with non-small cell lung cancer (NSCLC) taking gefitinib. The calibration linear range was 37.5-2400 ng/mL (coefficient of determination [R2] = 0.99), encompassing the therapeutic concentrations of gefitinib. The accuracy and precision were within 15% of the quality control (QC) concentrations of 80, 200, and 2000 ng/mL. The lower limit of quantification was determined to be 40 ng/mL. Gefitinib was stable in DBSs for up to 5 months at room temperature and -20 °C, and at 40 °C for 24 h. A good correlation was observed between the gefitinib levels measured by the DBS method and plasma concentrations (R2 = 0.99). This method provides a simple, fast, and accurate approach to the quantitative analysis of gefitinib in finger-prick DBSs. The method would be useful for minimally invasive evaluation of the clinical gefitinib blood concentration.

Standard-dose osimertinib for refractory leptomeningeal metastases in T790M-positive EGFR-mutant non-small cell lung cancer.

BACKGROUND: Osimertinib demonstrated promising efficacy for refractory leptomeningeal metastases (LM) in preclinical data and a clinical study at 160 mg, but there is limited data for the standard 80 mg dose. METHODS: T790M-positive patients with suspected LM after classical epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) failure were enroled. RESULTS: We investigated 13 patients (5 definitive and 8 possible LM cases). In two of the five definitive cases with T790M in and outside the central nervous system (CNS), osimertinib was effective for both lesions, with cerebrospinal fluid (CSF) clearance of cancer cells and sensitive/T790M mutations. In three definitive cases with extra-CNS T790M without CSF T790M, cancer cells and sensitive mutations in the CSF persisted after osimertinib initiation. The median progression-free survival of all 13 patients was 7.2 months. Osimertinib was generally well-tolerated despite poor performance status, but interstitial lung disease (grade 2) was confirmed in one patient. Based on 25 samples from 13 patients, the osimertinib CSF penetration rate was 2.5±0.3%. CONCLUSIONS: Osimertinib 80 mg is a useful therapeutic option for refractory LM after classical EGFR-TKI failure. It appears more effective in CSF T790M-positive cases.

Androgen replacement therapy for cancer-related symptoms in male advanced cancer patients: study protocol for a randomised prospective trial (ARTFORM study).

Recent studies reveal that hypogonadism with low serum androgen levels is associated with advanced cancer and induction of most cancer-related symptoms. We designed an ARTFORM study to evaluate the efficacy of androgen replacement therapy in male advanced cancer patients. The ARTFORM study is an investigator-initiated, randomised controlled trial comparing intramuscle injection of testosterone enanthate with non-administration in male advanced cancer patients with non-curative locally advanced or metastatic lesions. Serum total and free testosterone levels are measured and patients with low testosterone level are randomised. The primary endpoint is the difference in validated health-related quality of life questionnaires at week 12. Trial registration of the ARTFORM study is assigned to University hospital Medical Information Network, Center identifier UMIN 000010939. J. Med. Invest. 64: 202-204, August, 2017.

Programmed death-ligand 1 expression and T790M status in EGFR-mutant non-small cell lung cancer.

BACKGROUND: Differential biology and prognosis between T790M+ and T790M- populations imply immunological differences also. METHODS: We retrospectively analyzed programmed death-ligand 1 (PD-L1) expression and T790M status in rebiopsied samples of epidermal growth factor receptor (EGFR)-mutant non-small cell lung cancer (NSCLC). PD-L1 immunohistochemistry was performed using the SP142 antibody for tumour cell (TC) and tumour-infiltrating immune cell (IC) and the 28-8 antibody for TC. PD-L1+ was defined as TC or IC ≥1%. RESULTS: We investigated 67 available rebiopsied histologic samples in 47 patients. Using the SP142, prevalence of PD-L1 any+, moderate+, and strong+ in T790M+ vs. T790M- samples were 31% vs. 61%, 8% vs. 15%, and 0% vs. 2%, respectively, representing PD-L1+ prevalence of T790M+ samples was significantly lower than that of T790M- (p=0.0149). Prevalence of any TC+/IC+ in T790M+ vs. T790M- samples were TC: 31% vs. 51% (p=0.0997) and IC: 8% vs. 27% (p=0.0536), respectively. Using the 28-8, median percentage of PD-L1+ in T790M+ samples was 1.9 (range, 0-27.2), whereas T790M- was 4.1 (range, 0-89.8) (p=0.0801). Prevalence of PD-L1+ ≥1%, ≥5%, and ≥10% in T790M+ vs. T790M- samples were 77% vs. 83% (p=0.5476), 31% vs. 49% (p=0.1419), and 12% vs. 27% (p=0.1213), respectively. In 9 of 11 patients receiving multiple rebiopsies, T790M and/or PD-L1 expression revealed temporal dynamism. Survival curves according to PD-L1 expression/T790M status suggested better prognosis in PD-L1-/T790M+ population. CONCLUSIONS: T790M+ status was correlated to lower PD-L1 expression. PD-L1 expression might have a prognostic value and interaction with T790M mutation in EGFR-mutant NSCLC.

Rebiopsy of Histological Samples in Pretreated Non-small Cell Lung Cancer: Comparison Among Rebiopsy Procedures.

AIM: The aim of the present study was to compare successful rate, failure reasons, and complications among procedures of histological rebiopsy. PATIENTS AND METHODS: We retrospectively reviewed medical records of histologically rebiopsied cases with non-small cell lung cancer. RESULTS: One hundred and eleven histological rebiopsies were performed in: 86 (77%) lung; 11 (10%) lymph node; 5 (5%) pleura; 4 (4%) liver; 2 (2%) muscle; 2 (2%) adrenal gland; and 1 (1%) rib. Successful rate by computed tomography-guided biopsy (CTGB), transbronchial biopsy (TBB), and ultrasound-guided biopsy were 86% (48/56), 90% (28/31), and 100% (24/24), respectively. Reasons for rebiopsy failure by CTGB were no/insufficient malignant cells (n=5) and pneumothorax (n=3), and those by TBB were no/insufficient malignant cells (n=2) and bleeding (n=1). Severe complications (≥grade 3): one grade 3 pneumothorax and one grade 4 air embolization were observed in two (2%, 2/111) cases receiving CTGB. CONCLUSION: Rebiopsy of histological samples can be highly successful and feasible by optimal procedural selection.