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.

Endobronchial ultrasound-guided transbronchial needle aspiration in patients with previously treated lung cancer.

PURPOSE: The sampling and accurate diagnosis of lymph nodes during the clinical history of lung cancer are essential for selecting the appropriate treatment strategies. This study aims to evaluate the feasibility of endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) in patients with previously treated lung cancer. METHODS: Patients who underwent EBUS-TBNA after treatment for lung cancer were retrospectively reviewed. We classified the patients into two groups; Group 1 (G1): Indicated to have a recurrence of new lesions after radical surgery or chemo/radiotherapy with a curative intent; and Group 2 (G2): Indicated to have residual tumor cells after undergoing primary treatment for chemo/radiotherapy or re-staging after induction therapy prior to surgery. RESULTS: Seventy previously treated lung cancer cases (G1, n = 52; G2, n = 18) were enrolled. Thirty-two cases (61.5%) had recurrent disease in G1, and 9 cases (50.0%) had nodal metastasis in G2. The diagnostic accuracy was 95.2% in G1 and 88.9% in G2. Twenty-four cases were examined for epidermal growth factor receptor (EGFR) mutations, and 9 (37.5%) cases had mutations, including two cases with a T790M mutation. Furthermore, in one case, a re-biopsy revealed that the initial adenocarcinoma had transformed into small cell lung cancer. CONCLUSION: Performing EBUS-TBNA during lung cancer treatment showed a high diagnostic yield. Samples obtained by EBUS-TBNA were helpful in determining when to perform repeat biomarker testing as well as for making pathological re-evaluations.

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.

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.

Can modified frailty index predict postoperative complication after lung cancer surgery?

PURPOSE: The impact of the modified frailty index (mFI) on postoperative complications after lung cancer surgery was investigated. METHODS: Patients who underwent lung cancer surgery in 2017 were included. 30-day postoperative mortality and morbidity were evaluated according to their Clavien-Dindo classification. mFI values are presented as the sum of values of 11 included items. Logistic regression was used to assess the effect of mFI on postoperative severe complication incidence. RESULTS: Among 190 patients considered, severe postoperative complications (Grade 3 or more) were observed in 30 (16%). No patients died within 30 days of surgery. The incidence of severe complications was 3.6% in patients with mFI of 0, 16.2% in patients with mFI of 1, 23.4% in patients with mFI of 2, and 31.6% in patients with mFI of 3 or more, and was correlated with the grade of mFI. Univariate and multivariate analyses showed that the high mFI was significantly predictive of postoperative complications. Frail patients of mFI ≥ 2 were at 3.0-fold greater risk of severe complications than non-frail patients of mFI 0 or 1. CONCLUSION: mFI was associated with morbidity after lung cancer surgery. Preoperative frailty assessment and appropriate intervention to frail patients would be required to improve postoperative outcomes.

What are the risk factors for postoperative home oxygen therapy in patients with lung cancer?

Background: Home oxygen therapy (HOT) is used to treat chronic respiratory diseases and is sometimes required in patients with lung cancer after radical surgery. We aimed to identify the risk factors for postoperative home-based oxygen therapy in patients with lung cancer. Methods: Patients who underwent surgery for primary lung cancer at Chiba University Hospital between January 2019 and March 2021 were included. Patients who did not undergo complete resection, died in hospital after surgery, or used oxygen therapy preoperatively were excluded. Eligible patients were divided into HOT and non-HOT groups. They were retrospectively analyzed for risk factors for postoperative HOT using medical records in a multivariate analysis. Results: A total of 410 patients were included in this study, 24 (5.9%) of whom required HOT after surgery. The HOT group comprised significantly more men, heavy smokers, and patients with pulmonary comorbidities, low percent forced expiratory volume, percent forced vital capacity, predicted postoperative forced expiratory volume in 1 s, and postoperative pulmonary complications on univariate analysis. In a multivariate analysis, independent risk factors for postoperative HOT were pulmonary comorbidities [odds ratio (OR): 5.94; 95% confidence interval (CI): 1.64-21.5; P=0.002) and postoperative pulmonary complications (OR: 5.39; 95% CI: 2.14-13.5; P<0.001). The postoperative HOT application rate was calculated according to a formula developed for this purpose. Conclusions: Comorbid pulmonary diseases and postoperative pulmonary complications were significantly associated with postoperative HOT in patients with lung cancer.

Transbronchial real-time lung tumor localization with folate receptor-targeted near-infrared molecular imaging: A proof of concept study in animal models.

OBJECTIVE: The diagnostic yield of bronchoscopy is not satisfactory, even with recent navigation technologies, especially for tumors located outside of the bronchial lumen. Our objective was to perform a preclinical assessment of folate receptor-targeted near-infrared imaging-guided bronchoscopy to detect peribronchial tumors. METHODS: Pafolacianine, a folate receptor-targeted molecular imaging agent, was used as a near-infrared fluorescent imaging agent. An ultra-thin composite optical fiberscope was used for laser irradiation and fluorescence imaging. Subcutaneous xenografts of KB cells in mice were used as folate receptor-positive tumors. Tumor-to-background ratio was calculated by the fluorescence intensity value of muscle tissues acquired by the ultra-thin composite optical fiberscope system and validated using a separate spectral imaging system. Ex vivo swine lungs into which pafolacianine-laden KB tumors were transplanted at various sites were used as a peribronchial tumor model. RESULTS: With the in vivo murine model, tumor-to-background ratio observed by ultra-thin composite optical fiberscope peaked at 24 hours after pafolacianine injection (tumor-to-background ratio: 2.56 at 0.05 mg/kg, 2.03 at 0.025 mg/kg). The fluorescence intensity ratios between KB tumors and normal mouse lung parenchyma postmortem were 6.09 at 0.05 mg/kg and 5.08 at 0.025 mg/kg. In the peribronchial tumor model, the ultra-thin composite optical fiberscope system could successfully detect fluorescence from pafolacianine-laden folate receptor-positive tumors with 0.05 mg/kg at the carina and those with 0.025 mg/kg and 0.05 mg/kg in the peripheral airway. CONCLUSIONS: Transbronchial detection of pafolacianine-laden folate receptor-positive tumors by near-infrared imaging was feasible in ex vivo swine lungs. Further in vivo preclinical assessment is needed to confirm the feasibility of this technology.

Preclinical feasibility of bronchoscopic fluorescence-guided lung sentinel lymph node mapping.

OBJECTIVE: Lung sentinel lymph node mapping, where peritumorally injected material is tracked through the lymphatics, aims to find the first potential sites of nodal metastasis. We sought to evaluate the preclinical feasibility of bronchoscopic fluorescence-guided sentinel lymph node mapping. METHODS: Healthy Yorkshire pigs were used; sentinel lymph node mapping was performed with indocyanine green. The primary fluorescence imaging method was an ultrathin composite fiberscope placed in the bronchoscope working channel. Secondary methods used a fluorescence thoracoscope placed in the trachea (rigid bronchoscopy) and pretracheal fascial plane (mediastinoscopy) to validate ultrathin composite fiberscope settings for sentinel lymph node detection. A tracheostomy was created, and the pig was placed in a lateral decubitus position. Transbronchial intraparenchymal indocyanine green injection was performed primarily in the right lower lobe. Ultrathin composite fiberscope and rigid bronchoscopy were performed with (n = 6) or without (n = 2) mediastinoscopy, with the former group guiding dose and ultrathin composite fiberscope optimization. Fluorescent targets were interrogated by endobronchial ultrasound before ultrathin composite fiberscope-guided transbronchial needle aspiration. Specimen fluorescence was documented before creating cytological smears. Pigs were killed postprocedure for nodal dissection. RESULTS: A total of 100 µL of 10 mg/mL indocyanine green generated strong transbronchial fluorescence with low risk of indocyanine green contamination. Fluorescence was detectable by 10 minutes postinjection. There was concordance among ultrathin composite fiberscope, rigid bronchoscopy, and mediastinoscopy. Except for 1 pig with airway contamination, ultrathin composite fiberscope-guided endobronchial ultrasound transbronchial needle aspiration obtained fluorescent material in all pigs. Specimen fluorescence was associated with specimen adequacy. CONCLUSIONS: Bronchoscopic fluorescence-guided sentinel lymph node mapping was feasible, with specimen fluorescence providing real-time feedback on sentinel lymph node biopsy success. If translated to clinical practice, attention must be paid to minimizing indocyanine green leakage.

Postoperative pain and quality of life after lung cancer surgery: a prospective observational study.

BACKGROUND: We aimed to identify the factors associated with postoperative pain, quality of life, and development of chronic pain after lung cancer surgery, including pain sensation threshold, fentanyl sensitivity, and surgical procedures. METHODS: We conducted a single-center prospective observational study involving lung cancer patients. Brief pain inventory, including nine items concerning pain and quality of life, was investigated at 1 week, 1 month, and 3 months postoperatively. Pain sensation threshold and fentanyl sensitivity were assessed preoperatively. RESULTS: Of the 146 patients who were enrolled, 100 who met our criteria were analyzed. Thoracoscopic surgery was performed in 42 patients and minimally invasive thoracotomy in 58 patients. Pain sensation threshold and fentanyl sensitivity were normally distributed among the patients and were not significantly associated with brief pain inventory scores at each postoperative time-point. The average pain score 1 week after the operation was significantly higher in the thoracotomy group than in the thoracoscopic surgery group (P<0.050). The worst pain scores did not differ between the groups at all the examination periods. Pain sensation threshold, fentanyl sensitivity, and surgical procedures were not related to the incidence of post-thoracotomy pain syndrome. CONCLUSIONS: Individual pain sensation threshold and fentanyl sensitivity were not associated with subjective postoperative pain score, quality of life score, or development of post-thoracotomy pain syndrome.

Repeated photodynamic therapy mediates the abscopal effect through multiple innate and adaptive immune responses with and without immune checkpoint therapy.

In combination with immune checkpoint inhibitors, photodynamic therapy can induce robust immune responses capable of preventing local tumor recurrence and delaying the growth of distant, untreated disease (ie. the abscopal effect). Previously, we found that repeated photodynamic therapy (R-PDT) using porphyrin lipoprotein (PLP) as a photosensitizer, without the addition of an immune checkpoint inhibitor, can induce the abscopal effect. To understand why PLP mediated R-PDT alone can induce the abscopal effect, and how the addition of an immune checkpoint inhibitor can further strengthen the abscopal effect, we investigated the broader immune mechanisms facilitated by R-PDT and combination R-PDT + anti-PD-1 monoclonal antibody (αPD-1) in a highly aggressive, subcutaneous AE17-OVA mesothelioma dual tumor-bearing C57BL/6 mice. We found a 46.64-fold and 61.33-fold increase in interleukin-6 (IL-6) after R-PDT and combination R-PDT + αPD-1 relative to PBS respectively, suggesting broad innate immune activation. There was a greater propensity for antigen presentation in the spleen and distal, non-irradiated tumor draining lymph nodes, as dendritic cells and macrophages had increased expression of MHC class II, CD80, and CD86, after R-PDT and combination R-PDT + αPD-1. Concurrently, there was a shift in the proportions of CD4+ T cell subsets in the spleen, and an increase in the frequency of CD8+ T cells in the distal, non-irradiated tumor draining lymph nodes. While R-PDT had an acceptable safety profile, combination R-PDT + αPD-1 induced 1.26-fold higher serum potassium and 1.33-fold phosphorus, suggestive of mild laboratory tumor lysis syndrome. Histology revealed an absence of gross inflammation in critical organs after R-PDT and combination R-PDT + αPD-1 relative to PBS-treated mice. Taken together, our findings shed light on how the abscopal effect can be induced by PDT and strengthened by combination R-PDT + αPD-1, and suggests minimal toxicities after R-PDT.

Assessment of effectiveness and safety of thrombolytic therapy to pulmonary emboli by endobronchial ultrasound-guided transbronchial needle injection.

Objective: Endobronchial ultrasound-guided transbronchial needle injection (EBUS-TBNI) may effectively treat acute pulmonary embolisms (PEs). Here, we assessed the effectiveness of clot dissolution and safety of tissue plasminogen activator (t-PA) injection using EBUS-TBNI in a 1-week survival study of a porcine PE model. Methods: Six pigs with bilateral PEs were used: 3 for t-PA injection using EBUS-TBNI (TBNI group) and 3 for systemic administration of t-PA (systemic group). Once bilateral PEs were created, each 25 mg of t-PA injection using EBUS-TBNI for bilateral PEs (a total of 50 mg t-PA) and 100 mg of t-PA systemic administration was performed on day 1. Hemodynamic parameters, blood tests, and contrast-enhanced computed tomography scans were carried out at several time points. On day 7, pigs were humanely killed to evaluate the residual clot volume in the pulmonary arteries. Results: The average of percent change of residual clot volumes was significantly lower in the TBNI group than in the systemic group (%: systemic group 36.6 ± 22.6 vs TBNI group 9.6 ± 6.1, P < .01) on day 3. Considering the elapsed time, the average decrease of clot volume per hour at pre-t-PA to post t-PA was significantly greater in the TBNI group than in the systemic group (mm3/hour: systemic 68.1 ± 68.1 vs TBNI 256.8 ± 148.1, P < .05). No hemorrhage was observed intracranially, intrathoracically, or intraperitoneally on any contrast-enhanced computed tomography images. Conclusions: This study revealed that t-PA injection using EBUS-TBNI is an effective and safe way to dissolve clots.

Prediction of Nodal Metastasis in Lung Cancer Using Deep Learning of Endobronchial Ultrasound Images.

Endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) is a valid modality for nodal lung cancer staging. The sonographic features of EBUS helps determine suspicious lymph nodes (LNs). To facilitate this use of this method, machine-learning-based computer-aided diagnosis (CAD) of medical imaging has been introduced in clinical practice. This study investigated the feasibility of CAD for the prediction of nodal metastasis in lung cancer using endobronchial ultrasound images. Image data of patients who underwent EBUS-TBNA were collected from a video clip. Xception was used as a convolutional neural network to predict the nodal metastasis of lung cancer. The prediction accuracy of nodal metastasis through deep learning (DL) was evaluated using both the five-fold cross-validation and hold-out methods. Eighty percent of the collected images were used in five-fold cross-validation, and all the images were used for the hold-out method. Ninety-one patients (166 LNs) were enrolled in this study. A total of 5255 and 6444 extracted images from the video clip were analyzed using the five-fold cross-validation and hold-out methods, respectively. The prediction of LN metastasis by CAD using EBUS images showed high diagnostic accuracy with high specificity. CAD during EBUS-TBNA may help improve the diagnostic efficiency and reduce invasiveness of the procedure.

The combination of endobronchial elastography and sonographic findings during endobronchial ultrasound-guided transbronchial needle aspiration for predicting nodal metastasis.

BACKGROUND: During endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA), the sonographic findings of B-mode imaging, as well as endobronchial elastography, can be obtained noninvasively and used for the prediction of nodal metastasis. METHODS: Patients with lung cancer or suspected lung cancer who underwent EBUS-TBNA were recorded prospectively and reviewed retrospectively. Both the B-mode sonographic and elastographic findings were independently evaluated for each lymph node. The sonographic features were classified according to previously published criteria. If oval shape, indistinct margins, homogenous echogenicity, and the absence of coagulation necrosis sign were all observed by B-mode imaging, then the lymph node was judged to be benign by sonographic imaging. In addition, if the stiffer area comprised more than 31% of the entire lymph node area, then the lymph node was judged to be malignant by elastography. We compared the results of these imaging-based predictions with the pathological diagnoses. RESULTS: The prevalence of nodal metastasis was 78/228 (34.2%). B-mode sonography predicted 95.8% of benign lymph nodes, and elastography predicted 72.1% of malignant lymph nodes. By combining the two modalities, 59 of 71 (83.1%) lymph nodes judged as malignant by both analyses were pathologically proven to be malignant, and 101 of 105 (96.2%) lymph nodes judged as benign by both analyses were pathologically proven to be benign. CONCLUSION: The combination of elastography and sonographic findings showed good sensitivity and a high negative predictive value, which may facilitate selecting the most suspicious lymph nodes for biopsy. KEY POINTS: Significant findings of the study. The combination of endobronchial elastography and sonography resulted in a higher diagnostic yield than either modality alone for predicting benign and malignant lymph nodes in patients with lung cancer. WHAT THIS STUDY ADDS: The combination of endobronchial elastography and sonography will help clinicians identify the most suspicious lymph nodes for puncturing during EBUS-TBNA, which may improve the efficiency of EBUS-TBNA.

Intracardiac Tumors With Extracardiac Extension Diagnosed by Endoscopic Ultrasound With Bronchoscope-Guided Fine-Needle Aspiration.

Obtaining biopsy specimens for pathologic diagnosis of primary cardiac tumors is challenging because of their anatomic location and the risk of tumor embolization. Due to the difficulty of histologic diagnosis and limited treatment strategies, it is not uncommon for patients to be treated based on radiologic findings alone. However, a firm pathologic diagnosis may permit more appropriate treatment selection, especially for those with primary cardiac lymphoma. Endoscopic ultrasound with bronchoscope-guided fine-needle aspiration is a minimally invasive modality for sampling mediastinal lymph nodes and mediastinal lesions adjacent to the esophagus. In this case report we present 2 patients with cardiac tumors that were successfully and safely diagnosed by endoscopic ultrasound with bronchoscope-guided fine-needle aspiration.