Hiroaki Toba

Aberrant DNA methylation of some tumor suppressor genes in lung cancers from workers with chromate exposure

Our previous studies revealed a variety of genetic changes in lung cancers from chromate‐exposed workers (chromate lung cancer). In the present study, we examined epigenetic changes in chromate lung cancers. Nested‐methylation‐specific PCR was employed in studying the methylation of CpG islands in the APC, MGMT, hMLH1 genes in 36 chromate lung cancers and 25 nonchromate lung cancers. Methylation in chromate lung cancers was detected at 86% for APC, 20% for MGMT, and 28% for hMLH1. Whereas, it occurred at lower frequencies in nonchromate lung cancers, particularly in APC (44%) and hMLH1 (0%) genes. Our previous study showed that methylation of p16 gene in chromate lung cancer and nonchromate lung cancer was 33% and 26%, respectively. The mean methylation index (MI), a reflection of the overall methylation status, was significantly higher in chromate lung cancers than nonchromate lung cancers (0.41 vs. 0.21, P = 0.001). Methylation of multiple genes (particularly hMLH1, p16, and APC genes) had experienced more than 15 yr of chromate exposure in chromate lung cancer (MI: <15 yr; 0.19, ≥15 yr, 0.42). There is a significant correlation of p16 and hMLH1 methylation with the expressional decrease or loss of the corresponding gene products (P = 0.037 and 0.024) respectively, and an inverse correlation between APC and MGMT methylation (P = 0.014). This study provides a novel evidence for the chromium carcinogenesis that chromate lung cancer is linked to the progressive methylation of some tumor suppressor genes, which may be related to genomic instability.

18F-fluorodeoxyglucose positron emission tomography/computed tomography is useful in postoperative follow-up of asymptomatic non-small-cell lung cancer patients

OBJECTIVES Postoperative follow-up and surveillance after curative resection for non-small-cell lung cancer (NSCLC) patients are generally performed. However, there is no consensus on the best programme at this time. The aim of this study was to evaluate the diagnostic capability of (18)F-fluorodeoxyglucose positron emission tomography/computed tomography (FDG-PET/CT) in postoperative NSCLC patients without clinical and radiological evidence of recurrence, as a follow-up and surveillance programme. METHODS Between January 2005 and April 2010, a total of 101 NSCLC patients underwent potentially curative operations and follow-up FDG-PET/CT was performed in patients without clinical and radiological evidence of recurrence at least once a year in principle. A total of 233 FDG-PET/CT studies were entered and retrospectively reviewed. RESULTS Eighteen (18%) asymptomatic patients had recurrent diseases and 22 recurrent sites were confirmed. Of 22 recurrent sites, recurrence was diagnosed by histological examination in 9 (41%) sites and by imaging examination in 13 (59%) sites. FDG-PET/CT correctly diagnosed recurrence in 17 of the 18 (94%) patients and 21 of the 22 (95%) recurrent sites. The sensitivity, specificity, positive predictive value, negative predictive value and accuracy were 94.4, 97.6, 89.5, 98.8 and 97.0%, respectively. On the other hand, in 3 patients, other diseases were detected and treated appropriately. Post-recurrence therapies were performed in all patients with recurrence, but 4 (22%) patients died of the original diseases. The median post-recurrence survival was 25.2 months, and the 1- and 2-year post-recurrence survival rates were 83.3 and 69.6%, respectively. CONCLUSIONS FDG-PET/CT is a useful tool that has high capability to detect recurrences in asymptomatic NSCLC patients after a potentially curative operation. However, a large-scale multi-institutional randomized control trial may be needed to ascertain the benefit of surveillance with FDG-PET/CT.

Aberrant methylation of tumour-related genes in thymic epithelial tumours

BACKGROUND Thymoma is an uncommon neoplasm derived from epithelial cells of the thymus. Few studies have addressed the genetic alterations that occur in the tumourigenesis of thymoma. METHODS We examined aberrant DNA methylation of DAP-K, p-16, MGMT and HPP1 genes in 26 thymomas and 6 thymic carcinoma to clarify the association between aberrant DNA methylation and clinicopathological features. RESULTS Fifteen (47%) of 32 thymic epithelial tumours showed aberrant methylation. Aberrant methylation was more frequent in thymic carcinoma (86%) than in thymoma (29%). Moreover, the frequency of tumours with methylation of multiple genes in thymic carcinoma was higher than in thymoma (60% vs 20%). In thymoma, the frequency of tumour methylation, including the type A tumour component (28%), was lower than that of tumours with type B tumour component (42%). MGMT methylation was detected in 23% of thymoma and in 83% of thymic carcinoma. The frequency of methylation of the MGMT gene in both tumours was high compared with the other 3 genes. CONCLUSIONS Aberrant DNA methylation was more frequent in thymic carcinoma than in thymoma, and the frequency of DNA methylation in thymic epithelial tumours is roughly parallel to their malignant behaviour.

Fluoroscopy-assisted thoracoscopic resection after computed tomography-guided bronchoscopic metallic coil marking for small peripheral pulmonary lesions

OBJECTIVES To re-evaluate the efficacy of fluoroscopy-assisted thoracoscopic resection after computed tomography (CT)-guided bronchoscopic metallic coil marking (FATS-CM), which was our original method for small peripheral pulmonary lesions. METHODS Fifty-eight patients with 63 lesions underwent FATS-CM. A metallic coil was installed in the bronchus nearest to the lesion under CT fluoroscopic guidance with ultrathin bronchoscopy. The virtual bronchoscopic navigation (VBN) system was used in 14 cases. Afterwards, we basically performed wide wedge resection (WWR) using a C-arm-shaped roentgenographic fluoroscope during thoracoscopic surgery initially, and then the final procedure was determined by intraoperative histological diagnosis. Moreover, we prospectively treated ground-glass opacity (GGO) lesions of <20 mm diameter according to our treatment protocol from September 2004. RESULTS We could install coils in the objective bronchi in all cases. The average time required for the marking procedure was 38.9 (15-120) min. Pneumothorax was recognized in 1 (1.7%) case as a complication, but no fatal complications occurred. We could also install coils for each lesion in 4 cases (9 lesions) with multiple lesions. In 14 cases with the VBN system, the examination time and CT number were significantly reduced (P < 0.05 and <0.001, respectively), compared with those of 40 cases without the VBN system. The average interval between the CM and the operation was 5.6 (0-30) days. We never experienced a case of migration preoperatively. Sixty-two (98.4%) lesions were definitively identified, and WWRs were performed using three trocars in 58 (92.1%) cases during thoracoscopic surgery. Lobectomy was initially performed in only 1 case owing to coil migration. Thirty-seven of 40 cases (92.5%) were in line with the treatment protocol. There were no local-regional recurrences in all cases undergoing WWR. CONCLUSIONS We could prospectively show that our method was suitable to perform WWR with a sufficient margin for small GGO lesions of <20 mm. Moreover, we reconfirmed that the advantages of our method were safety, permitting flexibility in scheduling operations and a high ability to deal with multiple lesions. Additionally, our method became a minimally invasive and mature technique by using a new VBN system.

18F-fluorodeoxyglucose positron emission tomography/computed tomography and the relationship between fluorodeoxyglucose uptake and the expression of hypoxia-inducible factor-1α, glucose transporter-1 and vascular endothelial growth factor in thymic epithelial tumours.

OBJECTIVES The objective of this study was to evaluate the usefulness of (18)F-fluorodeoxyglucose positron emission tomography/computed tomography (FDG-PET/CT) and the relationships among the expressions of hypoxia-inducible factor-1α (HIF-1α), glucose transporter-1 (Glut-1) and vascular endothelial growth factor (VEGF), histological type, other clinical factors and FDG uptake in thymic epithelial tumours. METHODS Thirty-three patients who underwent FDG-PET/CT before treatment were reviewed. All types of tumours were reclassified into three subgroups: low-risk thymomas (types A, AB and B1), high-risk thymomas (types B2 and B3) and thymic carcinomas. Tumour contour, pattern of FDG uptake, tumour size and maximum standardized uptake value (SUVmax) were obtained. Expressions of HIF-1α, Glut-1 and VEGF were analysed immunohistochemically, and these expressions were evaluated using grading scales. RESULTS FDG uptake was visually recognized in all (100%) tumours. A homogeneous pattern of FDG uptake was increasingly observed in the order of low-risk thymomas to high-risk thymomas to thymic carcinomas (P = 0.016). SUVmax for thymic carcinomas was significantly higher than that for thymomas (P = 0.008). With the optimal cut-off value of SUVmax of 5.6, the sensitivity, specificity and accuracy for diagnosing thymic carcinoma were 0.75, 0.80 and 0.79, respectively. Regarding the mean scoring of HIF-1α, Glut-1 and VEGF, increasing trends were observed in the order of low-risk thymomas to high-risk thymomas to thymic carcinomas. Tumour size revealed a significant correlation with SUVmax (r = 0.60, P < 0.001), and the expression of HIF-1α showed a moderate association, but the expression of Glut-1 showed no correlation with SUVmax. Regarding correlations between the expression of the three markers, there were moderate associations between HIF-1α and Glut-1, and HIF-1α and VEGF, and a significant correlation between Glut-1 and VEGF (r = 0.60, P < 0.001). In type B1 thymoma, HIF-1α and Glut-1 were partly expressed in non-neoplastic immature lymphocytes. CONCLUSIONS FDG-PET/CT should be performed in patients with tumours in the anterior mediastinum because the pattern of FDG uptake and SUVmax are useful in the differential diagnosis of thymic epithelial tumours. Furthermore, the expressions of HIF-1α, Glut-1 and VEGF might be associated with malignancy of thymic epithelial tumours. In contrast, FDG uptake might be dependent on tumour size rather than Glut-1 overexpression.

Methylation and expression profiles of MGMT gene in thymic epithelial tumors

OBJECTIVES A key challenge in diagnosis and treatment of thymic epithelial tumors (TET) is in improving our understanding of the genetic and epigenetic changes of these relatively rare tumors. METHODS Methylation specific PCR (MSP) and immunohistochemistry were applied to 66 TET to profile the methylation status of DNA repair gene O6-methylguanine DNA methyltransferase (MGMT) and its protein expression in TET to clarify the association between MGMT status and clinicopathological features, response to chemotherapy and overall survival. RESULTS MGMT methylation was significantly more frequent in thymic carcinoma than in thymoma (17/23, 74% versus 13/44, 29%; P<0.001). Loss of expression of MGMT protein was significantly more frequent in thymic carcinoma than in thymoma (20/23, 87% versus 10/44, 23%; P<0.0001). There is a significant correlation between of MGMT methylation and loss of its protein expression (P<0.0003). MGMT methylation and loss of expression were significantly more frequent in advanced thymic epithelial tumors (III/IV) than in early tumors (I/II). CONCLUSION MGMT methylation plays a soul role in development of TET, especially in thymic carcinoma. Therefore, translation of our results from basic molecular research to clinical practice may have important implication for considering MGMT methylation as a marker and a target of future therapies in TET.

Functional evaluation of pallid mice with genetic emphysema.

Studies on pallid mice models of genetic emphysema have conventionally focused on morphological or biochemical evaluations. However, it is important to consider the functional aspects. We evaluated the exercise capacity and respiratory function in male pallid mice and male C57BL/6J mice at 3, 6, 12, and 15 months of age. The functional evaluations were conducted using a treadmill and a pulmonary function analysis device. The morphology of the lungs was analyzed on the basis of mean linear intercept (Lm) values. The body weights of the pallid mice at 12 and 15 months were significantly lower than those of the age-matched C57BL/6J mice. The pallid mice showed deterioration in exercise capacity from 6 months, as indicated by the trends in running distance. At 6, 12, and 15 months, the pallid mice showed significantly higher pulmonary compliance and significantly lower forced expiratory volume in 20 ms (FEV20 ms)/vital capacity (VC) values in comparison with the corresponding values for the C57BL/6J mice. In the morphological analysis of the pallid mice, emphysema was detected from 12 months, and the mice showed a significantly larger Lm at 12 months. The exercise capacity and lung function in the pallid mice significantly deteriorated from 6 months, at which time no pathological changes in the lung were detected. The deterioration in the exercise capacity and pulmonary function preceded the microscopic morphological changes.

Computed tomography lymphography by transbronchial injection of iopamidol to identify sentinel nodes in preoperative patients with non–small cell lung cancer: A pilot study

OBJECTIVE The objective of the present study was to assess the safety and feasibility of computed tomography lymphography by transbronchial injection of a water-soluble extracellular computed tomography contrast agent. METHODS From April 2010 to May 2011, patients with clinical stage I non-small cell lung cancer who were candidates for lobectomy were enrolled in the present study. An ultrathin bronchoscope was inserted to the target bronchus under the guidance of virtual bronchoscopic navigation images. Computed tomography images of the chest were obtained 0.5 and 5 minutes after 2 or 3 mL of iopamidol was injected through a microcatheter. Sentinel nodes were identified when the maximum computed tomography attenuation value of the lymph nodes on the postcontrast computed tomography images increased by 30 Hounsfield units or more compared with the precontrast images. Patients underwent lobectomy with standard lymph node dissection. RESULTS The ultrathin bronchoscope could access the targeted bronchus, and iopamidol was delivered into the peritumoral area in all 13 patients without any complications. Sentinel nodes were identified in 12 (92.3%) of the 13 patients. The average number of sentinel nodes was 1.5 (range, 1-2). Pathologic examination revealed metastatic lymph nodes in 2 patients. Metastatic nodes were included with the sentinel nodes. CONCLUSIONS Computed tomography lymphography by transbronchial injection of iopamidol was a safe and feasible method to identify the sentinel nodes in patients with clinical stage I non-small cell lung cancer.

5-Aminolevulinic acid-induced fluorescence diagnosis of pleural malignant tumor

BACKGROUND It is known that endogenously synthesized protoporphyrin IX (PpIX) following the administration of 5-aminolevulinic acid (5-ALA) is an effective photosensitizer for photodynamic diagnosis (PDD). We tested in vivo and in vitro susceptibility of human lung cancer and mesothelioma cells to photodynamic diagnosis (PDD) using 5-aminolevulinic acid (5-ALA) as a photosensitizer. METHODS Human lung cancer cell lines A549, Ma44-3, FT821 and human mesothelioma cell lines MSTO-211H, NCI-H290, Y-MESO-14 were incubated with 0.03% 5-ALA for 4 h. After incubation, protoporphyrin IX (PpIX) fluorescence was detected using a fluorescence microscope. Pleural carcinosis was induced in severe combined immunodeficiency disease mice using the previous cell lines to test the efficacy of PDD in vivo. The mice were sacrificed 4 h after oral administration of 400 mg/kg of 5-ALA. We counted the visible tumors under white light then fluorescence light. RESULTS In vitro, clear red fluorescence was observed in all cell lines. The mean fluorescence intensity was stronger in A549 and FT821 cells than Ma44-3 cells (165.59±26.49, 157.62±18.93 vs. 104.01±17.58). Also, MSTO-211H and NCI-H290 cells had stronger fluorescence intensity than Y-MESO-14 cells (142.51±26.85, 165.16±12.91 vs. 92.31±8.69). In vivo, the tumor detection rate of fluorescence diagnosis was 1.1-4.5 times higher than that of white light. The mean number of metastases detected by the PDD was significantly higher than that of white light for FT821 (p=0.004), Ma44-3 (p=0.006) and Y-MESO-14 cell lines (p=0.005), but not for A549, NCI-H290 and MSTO-211H cell lines. Small lesions were detected by fluorescence diagnosis even though the lesions were invisible macroscopically under white light. CONCLUSION Our results suggest the possibility of clinical application of fluorescence diagnosis with intrapleural malignant tumors.

Late pulmonary metastases from malignant melanoma of the left planta

We report a patient treated for malignant melanoma of the left planta who developed pulmonary metastases after a disease-free interval of 10 years. Metastatectomy was performed after observing progress for 12 months. However, 18 months later, pretracheal and right axillary lymph node metastases occurred. Because pulmonary metastasis from malignant melanoma may occur after 10 years or more, we observe patients carefully. If it does occur, an operation should be considered under limited indications. We should also recognize that recurrence may occur within a short interval after metastatectomy as well.