Development of a Novel One-Step Automated Rapid in situ Hybridization for Anaplastic Lymphoma Kinase Rearrangement Using Non-Contact Alternating-Current Electric-Field Mixing.

Echinoderm microtubule-associated protein-like 4 and anaplastic lymphoma kinase (ALK) fusion gene rearrangement is a key driver mutation in non-small cell lung cancer (NSCLC). Although Break-Apart ALK fluorescence in situ hybridization (FISH) is a reliable diagnostic method for detecting ALK gene rearrangement, it is also costly and time-consuming to use as a routine screening test. Our aim was to evaluate the clinical utility of a novel one-step, automated, rapid FISH (Auto-RaFISH) method developed to facilitate hybridization. This method takes advantage of the non-contact mixing effect of an alternating-current electric field. Ten representative specimens from 85 patients diagnosed at multiple centers with primary lung cancer with identified ALK-FISH status were collected. The specimens were all tested using FISH, RaFISH, and Auto-RaFISH. With both RaFISH protocols, the ALK test was completed within 4.5 h, as compared to the 20 h needed for the standard protocol. We found 100% agreement between the standard FISH, RaFISH, and new Auto-RaFISH based on the ALK status, and all methods stained equally well. These findings suggest that Auto-RaFISH could potentially serve as an automated clinical tool for prompt determination of ALK status in NSCLC.

Reagent-saving immunohistochemistry for HER2 using non-contact alternating current electric field mixing.

AIMS: Human epidermal growth factor receptor 2 (HER2)-targeted agents are an effective approach to treating patients with HER2-positive breast cancer. However, the lack of survival benefit in HER2-negative patients, as well as the toxic effects and high cost of the drugs, highlight the need for accurate and prompt assessment of HER2 status. Our aim was to evaluate the clinical utility of a novel reagent-saving immunohistochemistry method (AC-IHC) that saves HER2 antibody by taking advantage of the non-contact mixing effect in microdroplets subjected to an alternating current electric field. METHODS: Ninety-five specimens were used from patients diagnosed with primary breast cancers identified immunohistochemically as HER2 0/1+, 2+ or 3+ using ASCO/CAP guideline-certified standard IHC. The specimens were all tested using the conventional IHC method (1:50 antibody dilution) as well as AC-IHC (1:50 dilution) and reagent-saving AC-IHC (1:100 dilution). RESULTS: The reagent-saving AC-IHC produced stable results with less non-specific staining using smaller amounts of labelled antibody. Moreover, the staining and accuracy of HER2 status evaluated with the reagent-saving AC-IHC method was equal to that achieved with standard IHC. CONCLUSIONS: These results suggest reagent-saving AC-IHC could be used as a clinical tool for accurate and stable HER2 IHC, even when reagent concentrations vary.

Lung Cancer Surgery in Partial Anomalous Pulmonary Venous Connection Patients.

We report pulmonary resections for lung cancers in 2 patients with partial anomalous pulmonary venous connection (PAPVC) identified preoperatively. In case 1, right upper lobectomy was performed as the definitive operation for both lung cancer and PAPVC in the same lobe. In case 2, because lung cancer and PAPVC existed in different lobes, cardiac catheterization was performed to evaluate the need for correction of the PAPVC. Then, left lower lobectomy was safely performed without correcting the PAPVC located in the left upper lobe. The treatment plan for patients with PAPVC who require pulmonary resection should be carefully considered.

Novel method for rapid fluorescence in-situ hybridization of ALK rearrangement using non-contact alternating current electric field mixing.

Echinoderm microtubule-associated protein-like 4 gene and anaplastic lymphoma kinase gene (EML4-ALK) rearrangement is a key driver mutation in non-small cell lung cancer (NSCLC). Although Break-Apart ALK fluorescence in situ hybridization (FISH) is a reliable diagnostic method for detecting ALK gene rearrangement, it is too costly and time-consuming for use as a routine screening test. Our aim was to evaluate the clinical utility of a novel rapid FISH (RaFISH) method developed to facilitate hybridization. RaFISH takes advantage of the non-contact mixing effect of an alternating current (AC) electric field. Eighty-five specimens were used from patients diagnosed with NSCLC identified immunohistochemically as ALK 0, (1/2+) or (3+). With RaFISH, the ALK test was completed within 4.5 h, as compared to 20 h needed for the standard FISH. Although RaFISH produced results more promptly, the staining and accuracy of the ALK evaluation with RaFISH was equal to the standard. We found 97.6% agreement between FISH and RaFISH based on the status of the ALK signals. These results suggest RaFISH could be used as a clinical tool to promptly determine ALK status.

Rapid Immunohistochemistry With Thyroid Transcription Factor-1 for Pulmonary Adenocarcinoma.

BACKGROUND: Intraoperative pathologic diagnosis of solitary pulmonary tumors to differentiate between metastatic and primary lung cancer is extremely important to determine the appropriate range of excision. Accurate intraoperative pathologic evaluation may be often difficult, however, and needs additional immunohistochemical (IHC) evaluation to support the diagnosis. Although conventional IHC is a powerful tool for diagnosis, its clinical use is limited intraoperatively because of time constraints. To address this issue, we developed a device that enables complete and rapid IHC (R-IHC) analyses within 20 minutes. We aimed to evaluate the discriminative ability of the R-IHC with anti-thyroid transcription factor-1 (TTF-1) antibody, which is a highly specific IHC marker for primary lung adenocarcinoma. METHODS: A total of 61 pulmonary tumors that were resected at our institute from May 2011 to September 2013 were retrospectively examined. The samples were sectioned, labeled with anti-TTF-1 antibody using the R-IHC method, and pathologically evaluated. The standard used for evaluation was conventional IHC with TTF-1. RESULTS: With the R-IHC procedure, analyses were completed within 20 minutes, with a diagnostic accuracy of 96.7% (59 of 61). Among the 47 primary lung adenocarcinomas, the R-IHC detected 31 (66%) tumors that were positive for TTF-1, with a positive predictive value of 100% (31 of 31). CONCLUSIONS: Our newly developed method of R-IHC with anti-TTF-1 antibody was useful for diagnosing and differentiation of solitary pulmonary tumors. This technology may prove to be an important supplement to standard intraoperative pathologic diagnosis in routine practice.

Management of Intersegmental Plane on Pulmonary Segmentectomy Concerning Postoperative Complications.

BACKGROUND: For primary lung cancer, anatomic pulmonary segmentectomy may have a functional advantage over lobectomy; however, persistent air leak or delayed pneumothorax after segmentectomy is prevalent and increases surgical morbidity. To prevent pulmonary complications after pulmonary segmentectomy, we used 2 methods for the involved intersegmental plane: coverage using polyglycolic acid mesh and fibrin glue (mesh cover) or closing it by suturing the pleural edge (pleural suture). We aimed to compare postoperative pulmonary function and complications between the 2 groups. METHODS: A total of 133 patients who underwent pulmonary segmentectomy for stage IA non-small cell lung cancer were analyzed retrospectively. A pulmonary function test, including vital capacity and forced expiratory volume in 1 second, was performed preoperatively and at 1 and 6 months postoperatively. Propensity score analysis generated 2 matched pairs of 46 patients in the pleural suture and mesh cover groups. RESULTS: In each group, there was no significant difference in the recovery rate of vital capacity and forced expiratory volume in 1 second at 1 and 6 months postoperatively. Compared with the pleural suture group, the mesh cover group had higher incidence of prolonged air leak (8.7% versus 0%; p = 0.042), delayed pneumothorax (10.9% versus 2.2%; p = 0.051). On logistic regression analysis, management of intersegmental plane by either mesh cover or pleural suture was the only independent factor related to pulmonary complications (prolonged air leak or delayed pneumothorax) after pulmonary segmentectomy (odds ratio: 5.26, p = 0.047; odds ratio: 13.39, p = 0.018, respectively). CONCLUSIONS: Pleural suturing of the involved intersegmental plane during pulmonary segmentectomy appeared to be an acceptable method to reduce postoperative pulmonary complications.

New PET/CT criterion for nodal staging in non-small cell lung cancer: measurement of the ratio of section area of standard uptake values ≥2.5/lymph node section area.

BACKGROUND: The aim of the present study was to use surgical and histological results to develop a simple noninvasive technique for improving nodal staging using preoperative PET/CT in patients with resectable non-small cell lung cancer (NSCLC). METHODS: Preoperative PET/CT findings (163 patients) and pathological diagnoses after surgical resection were evaluated. Using PET/CT images, lymph node section area (SA), the maximum standardized uptake value (SUVmax), SA of SUV ≥2.5 and ≥3.0 were drawn freehand and measured using caliper software. Receiver operating characteristic (ROC) curves were then used to analyze those data. RESULTS: Based on ROC analyses, the cut-off values for SA of SUV ≥2.5, SA of SUV ≥3.0, SUV ≥2.5 SA/node SA and SUV ≥3.0 SA/node SA for diagnosis of lymph node metastasis were 200 mm2, 30 mm2, 1.0 and 0.4. SUV ≥2.5 SA/node SA ≥1.0 had the highest negative predictive value, and the sensitivity, specificity, positive predictive value, negative predictive value and accuracy of nodal staging were 61.1, 73.4, 36.7, 88.2 and 70.9%. CONCLUSIONS: When diagnosing nodal staging based a lymph node SUV ≥2.5 SA/node SA ratio of ≥1.0, it can be an effective criterion for use to determine surgical indications.

Thymic carcinoma with myasthenia gravis: Two case reports.

INTRODUCTION: Myasthenia gravis (MG) has been reported to correlate with earlier-stage thymoma, and theoretically does not accompany thymic carcinoma. However, we encountered two cases of thymic carcinoma with MG. PRESENTATION OF CASES: Case 1 involved a 54-year-old man who had been diagnosed with MG based on symptoms and detection of anti-acetylcholine receptor antibody (ARAB). Computed tomography (CT) revealed an anterior mediastinal tumor 30mm in diameter. Prednisolone (PSL) and tacrolimus were administered without surgery at that time. Six years after diagnosis of MG, he was admitted to our hospital and underwent extended thymectomy. Pathological examination revealed type B2-B3 thymoma according to World Health Organization criteria, comprising 80% of the tumor with small cell carcinoma as 20%. Case 2 involved a 51-year-old woman. She had been diagnosed with MG based on eyelid ptosis and detection of ARAB. Ten years after diagnosis of MG, diaphragm elevation was detected on chest X-ray. CT revealed an anterior mediastinal tumor, 47mm in diameter. We suspected tumor invasion to the right phrenic nerve, right atrium, and superior vena cava. We therefore performed extended thymectomy after preoperative radiotherapy (40Gy). Pathological examination revealed squamous cell carcinoma. DISCUSSION: Most cases of thymic carcinomas appear to arise de novo, but appearance in thymomas has been described. In both our cases, MG was treated with pharmacotherapy alone without extended thymectomy, and thymic carcinoma was considered to have developed from the thymoma during long-term follow-up. CONCLUSION: Thymic carcinoma can accompany MG.

Effect of early mobilization on discharge disposition of mechanically ventilated patients.

[Purpose] The purpose of this study was to clarify the benefits of early mobilization for mechanically ventilated patients for their survival to discharge to home from the hospital. [Subjects and Methods] Medical records were retrospectively analyzed of patients who satisfied the following criteria: age ≥ 18 years; performance status 0-2 and independent living at their home before admission; mechanical ventilation for more than 48 h; and survival after mechanical ventilation. Mechanically ventilated patients in the early mobilization (EM) group (n = 48) received mobilization therapy, limb exercise and chest physiotherapy, whereas those in the control group (n = 60) received bed rest alone. Univariate and multivariate logistic regression analyses were performed to identify clinical variables associated with discharge disposition. [Results] Early mobilization was a positive independent factor and the presence of neurological deficits was a negative factor contributing to discharge to home. Among patients surviving mechanical ventilation without neurological deficits, the rate of discharge to home was significantly higher among patients in the EM group that in the control group (76% vs. 40%). [Conclusion] Early mobilization can improve the rate of discharge to home of patients requiring mechanical ventilation because of non-neurological deficits.

[A case of complete resection of non-squamous non-small-cell lung cancer after induction chemotherapy using carboplatin, paclitaxel, and bevacizumab].

Since locally advanced non-small-cell lung cancer(NSCLC)treated by surgical therapy alone has a poor prognosis, the efficacy of preoperative chemotherapy for improving postoperative survival in patients with NSCLC is controversial. A 69-year-old female was referred to our hospital for Stage IIIA(cT3bulkyN2M0)adenocarcinoma of the right lung. Computed tomography(CT)revealed a small nodule, 21mm in diameter, in the right S6, with isolated pulmonary metastasis in the same lobe and bulky subcarinal lymph node swelling. She received two courses of a combination of carboplatin(CBDCA AUC5), paclitaxel(PTX 200mg/m2)and bevacizumab(15mg/kg)as induction therapy, and showed no serious adverse effects. CT after induction therapy revealed a minor radiographic response. She underwent right lower lobectomy with node dissection 2a in 2011. An intercostal muscle flap was used for the bronchial stump. Histopathological examination revealed adenocarcinoma pT1aN0M0 of Stage I A, and showed that the pulmonary metastasis had disappeared even though the effect of induction chemotherapy was Ef2. Induction chemotherapy with carboplatin, paclitaxel and bevacizumab may be useful for treatment of locally advanced lung cancer.

The development of a knowledge base for basic active structures: an example case of dopamine agonists.

BACKGROUND: Chemical compounds affecting a bioactivity can usually be classified into several groups, each of which shares a characteristic substructure. We call these substructures "basic active structures" or BASs. The extraction of BASs is challenging when the database of compounds contains a variety of skeletons. Data mining technology, associated with the work of chemists, has enabled the systematic elaboration of BASs. RESULTS: This paper presents a BAS knowledge base, BASiC, which currently covers 46 activities and is available on the Internet. We use the dopamine agonists D1, D2, and Dauto as examples and illustrate the process of BAS extraction. The resulting BASs were reasonably interpreted after proposing a few template structures. CONCLUSIONS: The knowledge base is useful for drug design. Proposed BASs and their supporting structures in the knowledge base will facilitate the development of new template structures for other activities, and will be useful in the design of new lead compounds via reasonable interpretations of active structures.

Predictive activity profiling of drugs by topological-fragment-spectra-based support vector machines.

Aiming at the prediction of pleiotropic effects of drugs, we have investigated the multilabel classification of drugs that have one or more of 100 different kinds of activity labels. Structural feature representation of each drug molecule was based on the topological fragment spectra method, which was proposed in our previous work. Support vector machine (SVM) was used for the classification and the prediction of their activity classes. Multilabel classification was carried out by a set of the SVM classifiers. The collective SVM classifiers were trained with a training set of 59,180 compounds and validated by another set (validation set) of 29,590 compounds. For a test set that consists of 9,864 compounds, the classifiers correctly classified 80.8% of the drugs into their own active classes. The SVM classifiers also successfully performed predictions of the activity spectra for multilabel compounds.

Classification of dopamine antagonists using TFS-based artificial neural network.

In the former work, the authors proposed the Topological Fragment Spectral (TFS) method as a tool for the description of the topological structure profile of a molecule. This paper describes the TFS-based artificial neural network (TFS/ANN) approach for the classification and the prediction of pharmacological active classes of chemicals. Dopamine antagonists of 1227 that interact with different types of receptors (D1, D2, D3, and D4) were used for the training. The TFS/ANN successfully classified 89% of the drugs into their own active classes. Then, the trained model was used for predicting the class of unknown compounds. For the prediction set of 137 drugs that were not included in the training set, the TFS/ANN model predicted 111 (81%) drugs of them into their own active classes correctly.

MolSpace: a computer desktop tool for visualization of massive molecular data.

The authors have developed a software tool, MolSpace, to visualize massive molecular datasets. MolSpace can project a set of massive multivariate data onto a visual space (two- or three-dimensional space) by means of principal component analysis. MolSpace allows users not only to draw a scatter diagram of the data but also to display their two- or three-dimensional molecular structures as the objects in that space. With a probe (a molecular object) the user can navigate vast data spaces, thus facilitating understanding of the data structure. In addition, partial space searching is also available that is based on similarity searching techniques. It is possible to interrogate a three-dimensional structure of a chemical compound that corresponds to each object on the space in real time. The detail of the system is discussed with an illustrative example.