Association of frequent hypermethylation with high grade histological subtype in lung adenocarcinoma.

Lung adenocarcinoma is classified morphologically into five histological subtypes according to the WHO classification. While each histological subtype correlates with a distinct prognosis, the molecular basis has not been fully elucidated. Here we conducted DNA methylation analysis of 30 lung adenocarcinoma cases annotated with the predominant histological subtypes and three normal lung cases using the Infinium BeadChip. Unsupervised hierarchical clustering analysis revealed three subgroups with different methylation levels: high-, intermediate-, and low-methylation epigenotypes (HME, IME, and LME). Micropapillary pattern (MPP)-predominant cases and those with MPP components were significantly enriched in HME (p = 0.02 and p = 0.03, respectively). HME cases showed a significantly poor prognosis for recurrence-free survival (p < 0.001) and overall survival (p = 0.006). We identified 365 HME marker genes specifically hypermethylated in HME cases with enrichment of "cell morphogenesis" related genes; 305 IME marker genes hypermethylated in HME and IME, but not in LME, with enrichment "embryonic organ morphogenesis"-related genes; 257 Common marker genes hypermethylated commonly in all cancer cases, with enrichment of "regionalization"-related genes. We extracted surrogate markers for each epigenotype and designed pyrosequencing primers for five HME markers (TCERG1L, CXCL12, FAM181B, HOXA11, GAD2), three IME markers (TBX18, ZNF154, NWD2) and three Common markers (SCT, GJD2, BARHL2). DNA methylation profiling using Infinium data was validated by pyrosequencing, and HME cases defined by pyrosequencing results also showed the worse recurrence-free survival. In conclusion, lung adenocarcinomas are stratified into subtypes with distinct DNA methylation levels, and the high-methylation subtype correlated with MPP-predominant cases and those with MPP components and showed a poor prognosis.

Genetic alterations in squamous cell lung cancer associated with idiopathic pulmonary fibrosis.

Patients with idiopathic pulmonary fibrosis (IPF) are at higher risk of developing lung cancers including squamous cell lung carcinoma (SCC), which typically carries a poor prognosis. Although the molecular basis of cancer development subsequent to IPF has not been fully investigated, we recently reported two epigenetic phenotypes characterized by frequent and infrequent DNA hypermethylation in SCC, and an association of the infrequent hypermethylation phenotype with IPF-associated SCCs. Here, we conducted targeted exon sequencing in SCCs with and without IPF using the Human Lung Cancer Panel to investigate the genetic basis of IPF-associated SCC. SCCs with and without IPF displayed comparable numbers of total mutations (137 ± 22 vs 131 ± 27, P = .5), nonsynonymous mutations (72 ± 14 vs 69 ± 16, P = .5), indels (3.0 ± 3.5 vs 3.0 ± 3.9, P = 1) and synonymous mutations (62 ± 9.1 vs 60 ± 12, P = .5). Signature 1 was the predominant signature in SCCs with and without IPF. SETD2 and NFE2L2 mutations were significantly associated with IPF (44% vs 13%, P = .03 for SETD2; 38% vs 10%, P = .04 for NFE2L2). MYC amplification, assessed by copy number variant analysis, was also significantly associated with IPF (18.8% vs 0%, P = .04). Mutations in TP53 and CDKN2A were observed relatively frequently in SCCs with frequent hypermethylation (P = .02 for TP53 and P = .06 for CDKN2A). Survival analysis revealed that the SETD2 mutation was significantly associated with worse prognosis (P = .04). Collectively, we found frequent involvement of SETD2 and NFE2L2 mutations and MYC amplification in SCCs with IPF, and an association of a SETD2 mutation with poorer prognosis.

A low DNA methylation epigenotype in lung squamous cell carcinoma and its association with idiopathic pulmonary fibrosis and poorer prognosis.

Patients with idiopathic pulmonary fibrosis (IPF) have higher risk of developing lung cancer, for example, squamous cell carcinoma (SCC), and show poor prognosis, while the molecular basis has not been fully investigated. Here we conducted DNA methylome analysis of lung SCC using 20 SCC samples with/without IPF, and noncancerous lung tissue samples from smokers/nonsmokers, using Infinium HumanMethylation 450K array. SCC was clustered into low- and high-methylation epigenotypes by hierarchical clustering analysis. Genes hypermethylated in SCC significantly included genes targeted by polycomb repressive complex in embryonic stem cells, and genes associated with Gene Ontology terms, for example, "transcription" and "cell adhesion," while genes hypermethylated specifically in high-methylation subgroup significantly included genes associated with "negative regulation of growth." Low-methylation subgroup significantly correlated with IPF (78%, vs. 17% in high-methylation subgroup, p = 0.04), and the correlation was validated by additional Infinium analysis of SCC samples (n = 44 in total), and data from The Cancer Genome Atlas (n = 390). The correlation between low-methylation subgroup and IPF was further validated by quantitative methylation analysis of marker genes commonly hypermethylated in SCC (HOXA2, HOXA9 and PCDHGB6), and markers specifically hypermethylated in high-methylation subgroup (DLEC1, CFTR, MT1M, CRIP3 and ALDH7A1) in 77 SCC cases using pyrosequencing (p = 0.003). Furthermore, low-methylation epigenotype significantly correlated with poorer prognosis among all SCC patients, or among patients without IPF. Multivariate analysis showed that low-methylation epigenotype is an independent predictor of poor prognosis. These may suggest that lung SCC could be stratified into molecular subtypes with distinct prognosis, and low-methylation lung SCC that significantly correlates with IPF shows unfavorable outcome.

High expression of CXCL14 is a biomarker of lung adenocarcinoma with micropapillary pattern.

Lung adenocarcinoma with micropapillary pattern (MPP) has an aggressive malignant behavior. Limited resection should be avoided because of its high recurrence rate. If adenocarcinoma with MPP is diagnosed preoperatively, the selection of proper treatment is possible. To explore a preoperative biomarker for diagnosing MPP, we undertook RNA sequencing analysis of 25 clinical samples as the training set, including 6 MPP, 16 other adenocarcinoma subtypes, and 3 normal lung tissues. Unsupervised hierarchical clustering analysis suggested a presence of subgroup with MPP showing different gene expression phenotype. We extracted differentially expressed genes with high expression levels in MPP samples, and chose VSIG1, CXCL14, and BAMBI as candidate biomarkers for MPP. Reverse transcription-quantitative PCR analysis confirmed a significantly higher expression of VSIG1 (P = .03) and CXCL14 (P = .02) in MPP than others. In a validation set of 4 MPP and 4 non-MPP samples, CXCL14 expression was validated to be significantly higher in MPP than in non-MPP (P = .04). Comparing a total of 10 MPP and 20 non-MPP samples, the area under the curve of CXCL14 to distinguish MPP from others was 0.89. The threshold value was 0.0116, corresponding to sensitivity 80% and specificity 90%. In immunostaining of CXCL14, the staining score was significantly higher in MPP cases than others, where not only the MPP component but also other components showed heterogeneous staining in adenocarcinoma tissues with MPP. Moreover, a higher staining score of CXCL14 was significantly associated with poorer prognosis in all patients (P = .01) or within cases in stage I-III (P = .01). In summary, we identified CXCL14 as a possible diagnostic biomarker of MPP.

Surgical outcomes of pulmonary metastasis from hepatopancreatobiliary carcinomas: a comparison with pulmonary metastasis from colorectal carcinomas.

OBJECTIVES: Surgical indications for pulmonary metastasis from hepatopancreatobiliary (HPB) carcinomas remain controversial. METHODS: Between 2000 and 2015, 25 patients with pulmonary metastasis from HPB carcinomas and 145 with that from colorectal carcinomas underwent metastasectomies in our institution. The primary diseases were hepatocellular carcinoma (HCC) in 8 patients, pancreatic carcinoma (PC) in 12 and biliary tract carcinoma (BTC) in 5. All patients had a sufficient pulmonary reserve, controlled primary disease and no evidence of other metastatic disease. Perioperative factors were investigated retrospectively to analyze the overall survival (OS), pulmonary metastasis-free survival (PmFS) after pulmonary metastasectomy and disease-free interval between surgery for primary disease and the development of pulmonary metastasis (DFI). RESULTS: Complete resection was performed in all patients with lobectomy in 3, segmentectomy in 5 and partial resection in 17. The respective 1-, 2- and 5-year OS rates after metastasectomy were 82.6%, 69.8% and 69.8% in HPB patients and 98.3%, 92.4% and 78.0% in colorectal carcinoma patients (p = 0.351). The 2-year PmFS of HPB patients was 80.0%, versus 60.6% for colorectal carcinoma patients (p = 0.265). The DFI was 41.4 months for HPB patients and 34.5 months for colorectal carcinoma patients (p = 0.273). CONCLUSIONS: Metastasectomy for pulmonary metastasis from HPB may be performed in carefully selected patients.

Keys to successful induction chemoradiotherapy followed by surgery for stage III/N2 non-small cell lung cancer.

Surgical intervention after induction chemoradiation is designed as curative treatment for resectable stage III/N2 non-small cell lung cancer. However, there is no definitive evidence to support this approach, possibly because successful treatment requires certain "arts", such as proper patient selection, an appropriate induction regimen, and choice of the best surgical procedure. We review the previous reports and discuss our own experience to explore the appropriate strategy for patients with resectable stage III/N2 disease, and to identify the factors associated with successful surgical intervention. Among the studies reviewed, the complete resection rate among intention-to-treat cases was correlated well with the 5-year survival rate, whereas the pneumonectomy rate was correlated inversely with the 5-year survival rate. The clinical response rate and downstaging after induction treatment were not associated with survival. Based on these findings, we conclude that complete resection with the avoidance of pneumonectomy is important when selecting candidates for multimodal treatment including radical surgery.

Does segmentectomy really preserve the pulmonary function better than lobectomy for patients with early-stage lung cancer?

PURPOSE: Recently, segmentectomy has been considered as an alternative to lobectomy in early peripheral non-small lung cancer (NSCLC); however, controversy has remained regarding the long-term functional advantage after segmentectomy. The aim of this study was to analyze the postoperative lung function after segmentectomy and lobectomy for non-small cell lung cancer. METHODS: Patients with p-T1aN0M0 NSCLC who had undergone segmentectomy (n = 37) or lobectomy (n = 33) were retrospectively analyzed. The ratios of postoperative to preoperative forced vital capacity (FVC) and forced expiratory volume in 1 s (FEV1) were defined as the recovery rates. The radiological lung volume and weight were evaluated before and more than 6 months after surgery, and the postoperative values were compared with the predicted values that were calculated from the preoperative values, subtracting the resected lobes or segments. RESULTS: The clinical characteristics, including the preoperative lung function showed no significant differences between the groups. No statistical differences were recognized in the trend lines for recovery ratios of FVC and FEV1.0 (P = 0.96 and P = 0.33). The recovery ratios for radiologic lung volume and weight showed no significant differences (P = 0.46 and P = 0.22). The postoperative lung volume and weight were almost the same as the predicted values after segmentectomy, whereas those after lobectomy were significantly higher than the predicted values. CONCLUSIONS: No functional advantage for segmentectomy was observed during long-term follow-up, possibly due to compensatory lung growth after lobectomy.

High-grade fetal adenocarcinoma of the lung is a tumour with a fetal phenotype that shows diverse differentiation, including high-grade neuroendocrine carcinoma: a clinicopathological, immunohistochemical and mutational study of 20 cases.

AIMS: High-grade fetal adenocarcinoma (H-FLAC) is a rare variant of pulmonary adenocarcinoma; this study aims to elucidate its clinicopathological features and genetic abnormalities. METHODS AND RESULTS: Clinicopathological, immunohistochemical and mutational analyses were performed on 20 surgically resected lung cancers that showed H-FLAC histology in various proportions. These tumours predominantly occurred in elderly males and in 10 patients who were heavy smokers. Four cases were pure H-FLAC, and 16 cases were mixed H-FLAC, which were found to be combined with conventional-type adenocarcinoma (15 cases), large-cell neuroendocrine carcinoma (three cases), small-cell carcinoma (one case), enteric adenocarcinoma (two cases), choriocarcinoma (two cases), and a solid-clear cell pattern (seven cases). The fetal phenotype and diverse differentiation were supported by the immunoexpression of α-fetoprotein (95%), thyroid transcription factor-1 (TTF-1) (50%), neuroendocrine markers (30-45%), proneural markers (50-69%), and CDX2 (40%). Except for TTF-1 expression (pure H-FLACs, 0%; mixed H-FLACs, 63%), there were no significant differences in histological or immunohistochemical findings between pure and mixed H-FLACs. EGFR, KRAS, BRAF and PIK3CA mutations were identified in 20%, 0%, 0% and 7% of the tumours, respectively. CONCLUSIONS: Lung adenocarcinomas with H-FLAC features possess the potential for multidirectional differentiation, and are not strongly associated with known major driver gene mutations.

Elastography for Predicting and Localizing Nodal Metastases during Endobronchial Ultrasound.

BACKGROUND: Elastography is a relatively new technology that can generate images reflective of tissue stiffness (elasticity). Neoplastic tissue is usually stiffer than normal structures. OBJECTIVES: The aim of this study was to evaluate the feasibility and utility of elastography when combined with convex-probe endobronchial ultrasound (CP-EBUS) for predicting and localizing metastatic lymph nodes during endobronchial ultrasound with transbronchial needle aspiration (EBUS-TBNA). METHODS: Consecutive results of endobronchial elastography of lymph nodes performed using EBUS- TBNA were prospectively collected and retrospectively analyzed. Elastography images were acquired as JPEG images and also recorded as video clips. Stiff area ratios [(stiff areas as blue pixels) / (lymph node areas as region of interest pixels)] for each lymph node determined by elastography were collated with the results of pathological diagnosis. We also performed elastography of surgically resected lymph nodes and compared image findings with pathological sections. RESULTS: We evaluated 49 lymph nodes in 21 patients by CP-EBUS. There were 16 metastatic nodes (10 lung cancer metastases and 6 metastases from extrathoracic malignancies). Mean stiff area ratios were significantly greater for metastatic lymph nodes (0.478) than for benign nodes (0.216; p = 0.0002). Using a cutoff value of 0.311 for stiff area ratios, the sensitivity and specificity for predicting metastatic disease were 0.81 and 0.85, respectively. The stiff area was histologically compatible with metastatic distribution in surgically resected lymph nodes. CONCLUSIONS: Endobronchial elastography is feasible for lymph nodes when combined with CP-EBUS. Stiff area ratios are useful for predicting metastatic lymph nodes, which may be an efficient guide for TBNA.

Sarcomatous Components May Predict Prognosis in Patients with Pulmonary Pleomorphic Carcinoma.

OBJECTIVES AND METHODS: Pulmonary pleomorphic carcinoma often presents with clinically aggressive behavior. We retrospectively reviewed 16 patients who underwent surgery from 1999 to 2013. RESULTS: Sarcomatous components included spindle cell type in six (38%) patients, giant cell type in three (19%) patients, and mixed cell type in seven (44%) patients. The 5-year overall survival rate was 40.6%. On univariate analysis, spindle cell type, pN0, and small tumor diameter were associated with better prognosis. The 5-year survival of six patients with spindle cell type was 83.3%. CONCLUSIONS: Despite the small sample size, we suggest that patients with spindle cell type may have favorable prognoses.

[Surgical treatment of primary chest wall tumor].

BACKGROUND: Primary chest wall tumors are uncommon and there is limited information in the literature regarding treatment strategies for these tumors. METHODS: We retrospectively reviewed 14 patients who were referred for surgical resection for a primary chest wall tumor. RESULTS: Except for neurogenic tumors, 14 primary chest wall tumors were resected among 3,260 surgical cases during a 13-year period in our institution. Complete resection was attempted for all 14 patients;8 had benign tumors and 6 had malignant tumors. Tumor pathology was extremely varied as they arose from all anatomic structures of the chest wall. Chest wall reconstruction was performed for 7 patients;2 patients underwent an additional extended resection because their tumors were diagnosed as malignant during or after surgery;and only 1 patient with a primitive neuroectodermal tumor died of recurrence after surgery. CONCLUSIONS: The data and results for primary chest tumors are limited due to the uncommon nature of this entity and the extremely variable histology. In general, a preoperative diagnosis is difficult and a definitive diagnosis can only be made during or after surgery. Wide radical resection of these tumors should be attempted, particularly if malignancy is diagnosed.

[Epithelioid cell granuloma of the liver mimicking a primary phrenic tumor].

We here report an epithelioid cell granuloma of the liver, imaging of which mimicked a phrenic tumor. A 75-year-old woman was admitted to the hospital for evaluation of an abnormal shadow in the left lower lung field on a chest X-ray. Surgery was performed for a suspected schwannoma arising from the phrenic nerve or a primary diaphragmatic tumor based on chest computed tomography(CT) and magnetic resonance imaging (MRI). The intra-operative findings showed that the tumor did not originate from the diaphragm but from the left lobe of the liver with feeding vessels from the liver. Securing a sufficient margin, the tumor was surgically resected as a primary liver tumor. Histologically, the tumor was diagnosed as an epithelioid cell granuloma of the liver. It is sometimes difficult to discriminate organs from which tumors developing around the diaphragm because of the difficulty to perform biopsy or the presence of many candidate organs neighboring the diaphragm.

A bronchial mucous gland adenoma harboring GNAS R201C mutation.

We present a case of bronchial mucous gland adenoma (MGA) and discuss the results of its cytomorphological and cytogenetic examination serving as a basis for the differential diagnosis. To our best knowledge, this is a first report that demonstrate a GNAS gene (R201C) mutation in mucous gland adenoma, which may play an important role in MGA tumorigenesis, as is the case in other mucinous-type epithelial neoplasms of various organs.

Near-Infrared-Guided Pulmonary Segmentectomy After Endobronchial Indocyanine Green Injection.

BACKGROUND: The aim of this study is to prospectively determine the feasibility and safety of near-infrared fluorescence-guided pulmonary segmentectomy after endobronchial indocyanine green (ICG) injection using virtual bronchoscopy. METHODS: Fifteen patients who underwent pulmonary segmentectomy were prospectively enrolled. Using preoperative computed tomography datasets a bronchial road map was created to determine the bronchus for ICG injection. Immediately after intubation ICG was injected into the target bronchi using an ultrathin bronchoscope. During the operation a near-infrared thoracoscope was used to detect ICG fluorescence and determine the intersegmental plane. The assessment points were (1) whether the ICG demarcation lines corresponded to the intersegmental lines expected from the pulmonary veins, (2) whether it was possible for the planned segmentectomy to be completed by electrocautery and 1 or fewer uses of an automated suturing device according to the demarcation plane, (3) whether any surgical complications occurred intraoperatively or (4) in the 1 month after surgery, and (5) whether the target lesion was removed completely with sufficient surgical margin to evaluate the feasibility and safety of this procedure. RESULTS: In 13 cases (87%) a segmentectomy was completed in the planned way with sufficient surgical margins. The failure in 2 cases was due to a technical issue in the bronchial injection. No complications developed intraoperatively. Recurrent air leakage occurred in 1 case. No procedure-related adverse event was noted postoperatively. CONCLUSIONS: Near-infrared-guided pulmonary segmentectomy with endobronchial ICG injection using virtual bronchoscopy was safe and feasible, and minor technical revision can make this procedure more reliable.

Emergency surgery for hemothorax due to a ruptured pulmonary arteriovenous malformation.

Pulmonary arteriovenous malformation (PAVM) is a potential cause of hemothorax. The risk of PAVM rupture is reported to be higher during pregnancy for several reasons, including increased body fluid and a change in hormonal conditions. A 34-year-old pregnant woman suddenly felt right chest pain and dyspnea in the 28th week of gestation. Chest X-ray and computed tomography showed massive right pleural effusion. Her vital signs gradually deteriorated with hemorrhagic shock, necessitating emergency surgery. During exploratory thoracoscopy, active bleeding from the middle lobe was noticed and gauze packing was required to maintain her blood pressure. Following conversion to major thoracotomy, wedge resection of the middle lobe was performed with a linear stapler, and finally, her general condition became stable. Her postoperative course was uneventful. A histological examination of the resected specimen confirmed the diagnosis of ruptured PAVM. Her baby was successfully delivered at the 38th week of gestation.

Is needle biopsy a risk factor of pleural recurrence after surgery for non-small cell lung cancer?

BACKGROUND: Tumor seeding, whereby malignant cells are deposited along the needle tract, is considered to be a potential hazard of needle biopsies. The aim of this study is to elucidate the relationship between needle biopsies for lung tumor, such as a preoperative computed tomography-guided needle biopsy (PCTGNB) or an intraoperative fine-needle aspiration biopsy (IFNAB), and ipsilateral pleural recurrence (PR) after lung cancer surgery. METHODS: Between 2008 and 2017, 1,047 patients with non-small cell lung cancer (NSCLC) underwent curative lung resection in our institution. They were divided into two groups: those in whom the first recurrent site was the ipsilateral pleural cavity (PR group) and the others (control group). Risk factors of PR were investigated retrospectively. RESULTS: Recurrence was observed in 191 patients (18.2%), 25 of whom were categorized to the PR group (17 malignant effusion, 10 dissemination). Pathological tumor [2-4], lymph nodes [1-2], pleural, lymphatic and vascular invasion (each ≥1) factors and patients who underwent PCTGNB were more frequently observed in the PR group than in the control group (each P<0.01) whereas the proportion of patients who underwent IFNAB was not significant. A multivariate analysis identified pathological lymph node factor and the frequency of PCTGNB as independent risk factors for PR with hazard ratios of 7.33 (95% CI, 2.93-19.8; P<0.01) and 6.92 (95% CI, 2.25-17.8; P<0.01), respectively. CONCLUSIONS: PCTGNB is a risk factor of PR but IFNAB is not. Indications for PCTGNB should be carefully determined.

Impact of free tumor clusters on prognosis after resection of pulmonary adenocarcinoma.

OBJECTIVE: Pulmonary adenocarcinoma with a micropapillary component (MPC) has aggressive malignant behavior even if resectable. The aim of this study was to determine clinicopathologic features of patients who underwent surgery for pulmonary adenocarcinoma harboring MPCs, with particular focus on coexistent free tumor clusters (FTCs). METHODS: We retrospectively reviewed 444 patients with pulmonary adenocarcinoma who underwent surgery from March 2007 to July 2013. An MPC was defined as a >5% micropapillary pattern. We also defined FTCs to be a group of more than 3 small clusters containing <20 nonintegrated micropapillary tumor cells that were spreading within air spaces, >3 mm apart from the main tumor. The clinicopathologic characteristics of patients with and without FTCs were retrospectively investigated in MPC-positive patients. RESULTS: MPCs were identified in 67 patients (15.1%), 31 of whom (46.3%) were positive for FTCs. The distance between the furthest edge of FTCs and main tumors did not exceed the diameter of the main tumor in each case (average, 7.3 mm). Locoregional recurrences were frequently observed in FTC-positive patients. FTC-positive patients experienced a significantly lower 5-year recurrence-free survival rate compared with FTC-negative/MPC-positive patients (20.4% vs 52.2%, P < .001). Recurrence-free survival of FTC-negative and -positive patients was equivalent to that of patients with p-T2 and p-T3 MPC-negative adenocarcinoma, respectively. CONCLUSIONS: Coexistence of FTCs resulted in a further negative impact on postoperative prognosis among MPC-positive adenocarcinomas and should be considered for upstaging the p-T factor and during evaluation of surgical margins.