Tatsuo Ohira

Detection of the T790M mutation of EGFR in plasma of advanced non–small cell lung cancer patients with acquired resistance to tyrosine kinase inhibitors (West Japan oncology group 8014LTR study)

Introduction Next-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) have been developed to overcome resistance to earlier generations of such drugs mediated by a secondary T790M mutation of EGFR, but the performance of a second tumor biopsy to assess T790M mutation status can be problematic. Methods We developed and evaluated liquid biopsy assays for detection of TKI-sensitizing and T790M mutations of EGFR by droplet digital PCR (ddPCR) in EGFR mutation–positive non–small cell lung cancer (NSCLC) patients with acquired EGFR-TKI resistance. Results A total of 260 patients was enrolled between November 2014 and March 2015 at 29 centers for this West Japan Oncology Group (WJOG 8014LTR) study. Plasma specimens from all subjects as well as tumor tissue or malignant pleural effusion or ascites fluid from 41 patients were collected after the development of EGFR-TKI resistance. All plasma samples were genotyped successfully and the results were reported to physicians within 14 days. TKI-sensitizing and T790M mutations were detected in plasma of 120 (46.2%) and 75 (28.8%) patients, respectively. T790M was detected in 56.7% of patients with plasma positive for TKI-sensitizing mutations. For the 41 patients with paired samples obtained after acquisition of EGFR-TKI resistance, the concordance for mutation detection by ddPCR in plasma compared with tumor tissue or malignant fluid specimens was 78.0% for TKI-sensitizing mutations and 65.9% for T790M. Conclusions Noninvasive genotyping by ddPCR with cell-free DNA extracted from plasma is a promising approach to the detection of gene mutations during targeted treatment.

High-quality 3-dimensional image simulation for pulmonary lobectomy and segmentectomy: results of preoperative assessment of pulmonary vessels and short-term surgical outcomes in consecutive patients undergoing video-assisted thoracic surgery

OBJECTIVESnThe aim of this study was to evaluate the effectiveness of 3-dimensional computed tomography (3D-CT) software in short-term surgical outcomes and the assessment of variations of pulmonary vessel branching patterns on performing video-assisted thoracic surgery (VATS).nnnMETHODSnThe study included 179 consecutive patients who had undergone VATS anatomical lung resection, of which 172 were lobectomies (96%) and 7 were segmentectomies (4%), from May 2011 through January 2013. There were 124 patients (69%) in whom 3D-CT was performed and 55 patients (31%) who had not undergone 3D-CT. Observed actual pulmonary vessel branching patterns by intraoperative findings or footage were compared with the 3D image findings. Various surgical outcomes, including the occurrence of postoperative complications, in this study defined as those of Grade 2 or above under the Clavien-Dindo classification system, and total operative time, were retrieved from available clinical records.nnnRESULTSnAmong the 124 patients with preoperative 3D imaging, there were 5 (4%) conversions from VATS to thoracotomy. The incidence rate of patients with postoperative complications was 8% (n = 10), and there were no 30-day or 90-day mortalities. Pulmonary artery (PA) branches were precisely identified for 97.8% (309 of 316) of branches on 3D images, and the sizes of the seven undetected branches (five in the right upper lobe, two in the left upper lobe) ranged from 1 to 2 mm. The 3D images accurately revealed 15 cases (12%) of anomalous or unusual PA branches and 5 cases (4%) of variant pulmonary veins. Multivariate logistic regression analysis of the association with postoperative complications and operative time in 165 lung cancer patients demonstrated that male gender was the only statistically significant independent predictor of complications (risk ratio: 5.432, P = 0.013), and patients without 3D imaging tended to have operative complications (risk ratio: 2.852, P = 0.074), whereas conducting the 3D-CT (risk ratio: 2.282, P = 0.021) as well as intraoperative bleeding amount (risk ratio: 1.005, P = 0.005) had significant association with operative time.nnnCONCLUSIONSnHigh-quality 3D-CT images clearly revealed the anatomies of pulmonary vessels, which could play important roles in safe and efficient VATS anatomical resection.

Association between high-resolution computed tomography findings and the IASLC/ATS/ERS classification of small lung adenocarcinomas in Japanese patients

OBJECTIVESnThe detection rate of small pulmonary nodules has recently increased and new techniques have been developed to improve diagnostic yield. The IASLC/ATS/ERS classification demonstrated a difference in prognosis depending on the histological subtypes of lung adenocarcinoma. We evaluated the association between high-resolution computed tomography (HRCT) findings and the classification of these tumors.nnnMETHODSnWe reviewed the data of 220 lung adenocarcinoma (≤3 cm) patients who received complete resection with lymph node dissection in our hospital. From the HRCT findings, the tumors were classified into the following 3 categories: pure-solid nodules, part-solid nodules, or pure ground-glass opacity (GGO) nodules. Pathological invasion factor (PIF) was evaluated by the degree of blood vessel invasion, lymphatic permeation, and visceral pleural invasion.nnnRESULTSnThe tumors were classified as pure GGO nodules in 16 patients, part-solid nodules in 91, and pure-solid nodules in 113 from the HRCT findings. Tumors were diagnosed as noninvasive or minimally invasive adenocarcinomas (NMIADs) in 44 patients, and invasive adenocarcinomas (IADs) in 176. Lymph node metastasis was present in 31 patients (14.1%) and PIF in 101 (45.9%). All pure-solid nodules were IADs with a high PIF frequency (75.2%) or with lymph node metastasis (26.5%). All pure GGO nodules were NMIADs or lepidic-predominant adenocarcinomas. Among the part-solid nodules, IAD was detected in 67.0% of the patients and PIF in 16.5%. The consolidation/tumor (C/T) ratio and consolidation size were associated with IAD (optimal cut-off values: 0.4 and 8mm, respectively) and PIF (0.8 and 15 mm, respectively).nnnCONCLUSIONSnThe HRCT findings correlated with the IASLC/ATS/ERS classification and were useful for evaluating the histological nature of the tumors. Most pure-solid tumors had the potential for high-grade malignancy, including PIF and lymph node metastasis. For part-solid tumors, the C/T ratio and consolidation size were important for predicting PIF and for diagnosing IAD according to this classification.

Tumor volume determines the feasibility of cell‐free DNA sequencing for mutation detection in non–small cell lung cancer

Next‐generation sequencing (NGS) and digital PCR technologies allow analysis of the mutational profile of circulating cell‐free DNA (cfDNA) in individuals with advanced lung cancer. We have now evaluated the feasibility of cfDNA sequencing for mutation detection in patients with non‐small cell lung cancer at earlier stages. A total of 150 matched tumor and serum samples were collected from non‐small cell lung cancer patients at stages IA–IIIA. Amplicon sequencing with DNA extracted from tumor tissue detected frequent mutations in EGFR (37% of patients), TP53 (39%), and KRAS (10%), consistent with previous findings. In contrast, NGS of cfDNA identified only EGFR, TP53, and PIK3CA mutations in three, five, and one patient, respectively, even though adequate amounts of cfDNA were extracted (median of 4936 copies/mL serum). Next‐generation sequencing showed a high accuracy (98.8%) compared with droplet digital PCR for cfDNA mutation detection, suggesting that the low frequency of mutations in cfDNA was not due to a low assay sensitivity. Whereas the yield of cfDNA did not differ among tumor stages, the cfDNA mutations were detected in seven patients at stages IIA–IIIA and at T2b or T3. Tumor volume was significantly higher in the cfDNA mutation‐positive patients than in the negative patients at stages T2b–T4 (159.1 ± 58.0 vs. 52.5 ± 9.9 cm3, P = 0.014). Our results thus suggest that tumor volume is a determinant of the feasibility of mutation detection with cfDNA as the analyte.

A proteomic profiling of laser-microdissected lung adenocarcinoma cells of early lepidic-types

BackgroundIn the new pathologic classification of lung adenocarcinoma proposed by IASLC/ATS/ERS in 2011, lepidic type adenocarcinomas are constituted by three subtypes; adenocarcinoma in situ (AIS), minimally invasive adenocarcinoma (MIA) and lepidic predominant invasive adenocarcinoma (LPIA). Although these subtypes are speculated to show sequential progression from preinvasive lesion to invasive lung cancer, changes of protein expressions during these processes have not been fully studied yet. This study aims to glimpse a proteomic view of the early lepidic type lung adenocarcinomas.MethodsA total of nine formalin-fixed and paraffin-embedded (FFPE) lepidic type lung adenocarcinoma tissues were selected from our archives, three tissues each in AIS, MIA and LPIA. The tumor and peripheral non-tumor cells in these FFPE tissues were collected with laser microdissection (LMD). Using liquid chromatography-tandem mass spectrometry (MS/MS), protein compositions were compared with respect to the peptide separation profiles among tumors collected from three types of tissues, AIS, MIA and LPIA. Proteins identified were semi-quantified by spectral counting-based or identification-based approach, and statistical evaluation was performed by pairwise G-tests.ResultsA total of 840 proteins were identified. Spectral counting-based semi-quantitative comparisons of all identified proteins through AIS to LPIA have revealed that the protein expression profile of LPIA was significantly differentiated from other subtypes. 70 proteins including HPX, CTTN, CDH1, EGFR, MUC1 were found as LPIA-type marker candidates, 15 protein candidates for MIA-type marker included CRABP2, LMO7, and RNPEP, and 26 protein candidates for AIS-type marker included LTA4H and SOD2. The STRING gene set enrichment resulted from the protein-protein interaction (PPI) network analysis suggested that AIS was rather associated with pathways of focal adhesion, adherens junction, tight junction, that MIA had a strong association predominantly with pathways of proteoglycans in cancer and with PI3K-Akt. In contrast, LPIA was associated broadly with numerous tumor-progression pathways including ErbB, Ras, Rap1 and HIF-1 signalings.ConclusionsThe proteomic profiles obtained in this study demonstrated the technical feasibility to elucidate protein candidates differentially expressed in FFPE tissues of LPIA. Our results may provide candidates of disease-oriented proteins which may be related to mechanisms of the early-stage progression of lung adenocarcinoma.

High-Speed 3-Dimensional Imaging in Robot-Assisted Thoracic Surgical Procedures

We used a high-speed 3-dimensional (3D) image analysis system (SYNAPSE VINCENT, Fujifilm Corp, Tokyo, Japan) to determine the best positioning of robotic arms and instruments preoperatively. The da Vinci S (Intuitive Surgical Inc, Sunnyvale, CA) was easily set up accurately and rapidly for this operation. Preoperative simulation and intraoperative navigation using the SYNAPSE VINCENT for robot-assisted thoracic operations enabled efficient planning of the operation settings. The SYNAPSE VINCENT can detect the tumor location and depictxa0surrounding tissues quickly, accurately, and safely. This system is also excellent for navigational and educational use.

Survival outcomes for oligometastasis in resected non-small cell lung cancer.

Background We investigated the factors associated with post-recurrence survival and the treatment for non-small-cell lung cancer patients with postoperative distant recurrence, especially oligometastasis. Methods We reviewed the data of 272 patients with distant recurrence who underwent resection of non-small-cell lung cancer from January 2000 through December 2011. Results The type of distant recurrence was classified as oligometastasis (nu2009=u200976, 28%) or polymetastasis (nu2009=u2009196, 72%). Forty-seven (62%) patients with oligometastasis received local therapy (surgery 5, radiotherapy 9, sequential local and systemic therapy 28, chemoradiotherapy 5). Multivariate analysis revealed older age, non-adenocarcinoma, shorter disease-free interval, no pulmonary metastasis, liver metastases, bone metastases, and polymetastasis had significant associations with unfavorable post-recurrence survival. Subgroup analysis of patients with oligometastasis showed histology and disease-free interval had a great impact on survival. Smoking history and histology were associated with survival in patients with lung oligometastasis, whereas systemic treatment and longer disease-free interval were related to increased post-recurrence survival in those with brain oligometastasis. Conclusions This study showed that an oligometastatic state per se was a significant favorable factor. Optimization of personalized systemic treatment and adding local treatment are important in the management of patients with non-small-cell lung cancer and oligometastasis.

Prognostic Factors for Survival After Recurrence in Patients With Completely Resected Lung Adenocarcinoma: Important Roles of Epidermal Growth Factor Receptor Mutation Status and the Current Staging System

UNLABELLEDnEpidermal growth factor receptor (EGFR) status and pathological stage (p-stage) were shown to be essential prognostic factors for estimating survival after recurrence of lung adenocarcinoma. In patients with EGFR mutations, those with early p-stage tumors showed better survival after disease recurrence than those with advanced p-stage tumors. The EGFR mutation status and p-stage could also prompt the design of clinical trials on adjuvant therapy for patients after complete surgical resection.nnnBACKGROUNDnThe current staging system and epidermal growth factor receptor (EGFR) mutation status are key factors for predicting survival. However, the significance of these factors as predictors of survival after disease recurrence (PRS) has not been sufficiently elucidated. The objective of this study was to investigate the clinicopathological factors, particularly the EGFR mutation status and pathological stage (p-stage), which affect PRS in patients with completely resected lung adenocarcinoma.nnnPATIENTS AND METHODSnWe retrospectively reviewed the data of 198 consecutive lung adenocarcinoma patients with disease recurrence who previously underwent complete surgical resection in our hospital.nnnRESULTSnOf the 198 patients, 117 were examined for EGFR mutations (mutants). Mutants were detected in 57 patients (28.7%). The patients with mutants had a significantly better 3-year PRS (3y-PRS) rate (68.6%) than those with an EGFR wild type (WT) status (51.7%) or an unknown (UN) status (27.0%). The 3y-PRS rates for p-stage I to II (p-I-II) and p-stage III (p-III) were 52.5% and 29.3%, respectively. Multivariate survival analysis showed that the EGFR mutation status and p-stage had significant associations with favorable PRS. The 3y-PRS rate for mutants/p-I-II (81.4%) was significantly better than that for mutants/p-III (48.0%). Conversely, there was no significant difference between mutants/p-III and WT/UN/p-I-II (3y-PRS: 40.7%) or between mutants/p-III and WT/UN/p-III (3y-PRS: 24.4%).nnnCONCLUSIONnEGFR status and p-stage were shown to be essential prognostic factors for estimating PRS. In patients with mutants, those with early p-stage tumors showed better PRS than those with advanced p-stage tumors.

Repression of Smad3 by Stat3 and c-Ski/SnoN induces gefitinib resistance in lung adenocarcinoma

Cancer-associated inflammation develops resistance to the epidermal growth-factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) in non-small cell lung cancers (NSCLCs) harboring oncogenic EGFR mutations. Stat3-mediated interleukin (IL)-6 signaling and Smad-mediated transforming growth factor-β (TGF-β) signaling pathways play crucial regulatory roles in cancer-associated inflammation. However, mechanisms how these pathways regulate sensitivity and resistance to EGFR-TKI in NSCLCs remain largely undetermined. Here we show that signal transducer and activator of transcription (Stat)3 represses Smad3 in synergy with the potent negative regulators of TGF-β signaling, c-Ski and SnoN, whereby renders gefitinib-sensitive HCC827xa0cells resistant. We found that IL-6 signaling via phosphorylated Stat3 induced gefitinib resistance as repressing transcription of Smad3, whereas TGF-β enhanced gefitinib sensitivity as activating transcription of Smad3 in HCC827xa0cells with gefitinib-sensitizing EGFR mutation. Promoter analyses showed that Stat3 synergized with c-Ski/SnoN to repress Smad2/3/4-induced transcription of the Smad3 gene. Smad3 was found to be an apoptosis inducer, which upregulated pro-apoptotic genes such as caspase-3 and downregulated anti-apoptotic genes such as Bcl-2. Our results suggest that derepression of Smad3 can be a therapeutic strategy to prevent gefitinib-resistance in NSCLCs with gefitinib-sensitizing EGFR mutation.

Factors Influencing the Diagnostic Accuracy of Identifying the Histologic Type of Non–Small-Lung Cancer With Small Samples

BACKGROUNDnThe pathologic diagnosis has become a greater consideration in decision-making regarding the treatment options for lung cancer. Therefore, the accurate diagnosis of the tumor histologic type is essential, even when only small biopsy or cytology samples are available. However, the risk of a misdiagnosis with smaller biopsy samples is greater. The factors underlying the increased risk of a misdiagnosis in small samples are unknown. The aim of the present study was to identify the clinical and pathologic factors (other than immunohistochemical staining) that influence the pathologic diagnostic accuracy in small biopsy and cytological lung samples obtained by bronchoscopy.nnnPATIENTS AND METHODSnWe performed transbronchial lung biopsy or brushing and lavage to determine the preoperative diagnosis of 126 of 299 surgically resected lung cancer specimens. We assessed the diagnostic accuracy of the preoperative transbronchoscopic examination findings against that of the surgically resected lung specimens.nnnRESULTSnOn univariate analysis, the mean pathologic tumor size in the noncorresponding cases was larger than that in corresponding cases. Vascular invasion was also more prevalent in the noncorresponding cases. The tumor differentiation grade in the noncorresponding cases was poorer than in the corresponding cases. The noncorresponding cases were at a more progressed stage. On multivariate analysis, the pathologic tumor size and tumor differentiation grade were associated with the noncorresponding cases.nnnCONCLUSIONnWe found a larger tumor size and poor differentiation grade were indicative of lung cancer tissue with a greater content of heterogeneous cells. Therefore, a possibility exists of a false diagnosis using only these factors. Thus, treatment decisions should be made considering the pathologic diagnosis and other relevant factors.