Koichiro Kenzaki

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.

Triterpenes augment the inhibitory effects of anticancer drugs on growth of human esophageal carcinoma cells in vitro and suppress experimental metastasis in vivo

The antineoplastic effects of combinations of anticancer drugs (5‐fluorouracil, irinotecan and cisplatin) and triterpenes (ursolic acid, betulinic acid, oleanolic acid and a Japanese apricot extract (JAE) containing triterpenes) on esophageal squamous carcinoma cells were examined by the WST‐8 (2‐(2‐methoxy‐ 4‐nitrophenyl)‐3‐(4‐nitrophenyl)‐5‐(2,4‐disulfophenyl)‐2H‐tetrazolium, monosodium salt) assay in vitro and by an animal model in vivo. Triterpenes and JAE showed additive and synergistic cytotoxic effects, respectively, on esophageal squamous carcinoma cells (YES‐2cells) by combinational use of 5‐fluorouracil. JAE and 5‐fluorouracil induced cell cycle arrest at G2/M phase and at S phase, respectively, and caused apoptosis in YES‐2 cells. A new animal model of esophageal cancer causing tumor colonization of the peritoneal cavity and producing bloody ascites was made by injecting YES‐2 cells into the peritoneal cavity of a severe combined immunodeficiency mouse. In this model, 5‐fluorouracil inhibited colonization of tumor cells in the peritoneum. The addition of JAE to 5‐fluorouracil augmented the suppression of experimental metastasis of the peritoneum. The numbers of peritoneal nodules of more than 2 mm in diameter in mice treated with 5‐fluorouracil and JAE were less than those in mice treated with 5‐fluorouracil alone or JAE alone. These results suggest that triterpenes, especially JAE, are effective supplements for enhancing the chemotherapeutic effect of 5‐fluorouracil on esophageal cancer.

The balance of VEGF-C and VEGFR-3 mRNA is a predictor of lymph node metastasis in non-small cell lung cancer

A positive association between vascular endothelial growth factor-C (VEGF-C) expression and lymph node metastasis has been reported in several cancers. However, the relationship of VEGF-C and lymph node metastasis in some cancers, including non-small cell lung cancer (NSCLC), is controversial. We evaluated the VEGF-C and vascular endothelial growth factor receptor-3 (VEGFR-3) expression in NSCLC samples from patients who had undergone surgery between 1998 and 2002 using real-time quantitative RT–PCR and immunohistochemical staining. We failed to find a positive association between VEGF-C and VEGFR-3 mRNA expression and lymph node metastasis in NSCLC. An immunohistological study demonstrated that VEGF-C was expressed not only in cancer cells, but also in macrophages in NSCLC, and that VEGFR-3 was expressed in cancer cells, macrophages, type II pneumocytes and lymph vessels. The VEGF-C/VEGFR-3 ratio of the node-positive group was significantly higher than that of the node-negative group. Immunohistochemical staining showed that VEGFR-3 was mainly expressed in cancer cells. The immunoreactivity of VEGF-C and VEGFR-3 was roughly correlated to the mRNA levels of VEGF-C and VEGFR-3 in real-time PCR. VEGF-C mRNA alone has no positive association with lymph node metastasis in NSCLC. The VEGF-C/VEGFR-3 ratio was positively associated with lymph node metastasis in NSCLC. This suggests that VEGF-C promotes lymph node metastasis while being influenced by the strength of the VEGF-C autocrine loop, and the VEGF-C/VEGFR-3 ratio can be a useful predictor of lymph node metastasis in NSCLC.

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.

Photodynamic therapy using a second generation photosensitizer, Talaporfin.

BACKGROUND Photodynamic therapy (PDT) using Talaporfin is an attractive treatment for central-type early lung cancer. It was noted that some patients had altered levels of arterial oxygen saturation as indicated by pulse oximeter (SpO2) and arterial oxygen saturation levels in blood gas analysis (SaO2) during PDT. The present experiments were designed to provide an explanation for these findings. METHODS The influence of Talaporfin on SpO2 using in vitro and in vivo experiments, and clinically, was examined. RESULTS Our in vitro and in vivo experiments showed a linear relationship between Talaporfin concentration in the plasma and the SpO2 level (R=0.9957 and 0.9837). The apparent SpO2 level decreased according to the increase of Talaporfin concentration in the plasma. In two patients with PDT, SpO2 levels at 4-6h and 24h after Talaporfin administration were 93% and 97%, respectively. CONCLUSIONS Talaporfin raised blood absorbance at 660nm with a pulse oximeter. It appeared that the presence of the drug falsely decreased the level of SpO2 since SpO2 did not actually change.

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.

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.