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AimsMalignant pleural mesothelioma with heterologous elements (such as osseous, cartilaginous or rhabdomyoblastic differentiation) is very rare. We tried to differentiate such mesothelioma cases from extraskeletal pleural osteosarcoma, which is very challenging.MethodsWe compared 10 malignant pleural mesotheliomas (three biphasic and seven sarcomatoid types) with two pleural osteosarcomas using clinicopathological and immunohistochemical methods, and also fluorescence in situ hybridisation (FISH) to examine for homozygous deletion of p16.ResultsThe median age was 72 years for mesotheliomas, and 69 years for osteosarcoma. For mesothelioma, eight cases were male and two were female. Growth was diffuse in all mesothelioma cases except case 10, where it was localised, as it was for the two osteosarcomas. Among mesothelioma cases, 80% displayed osteosarcomatous and 60% chondromatous elements, while 10% exhibited rhabdomyoblastic ones. Immunohistochemical labelling for calretinin and AE1/AE3 was present in 8/10 and 7/10 mesotheliomas, respectively, but in only one osteosarcoma. Loss of methylthioadenosine phosphorylase was seen in 5/7 mesotheliomas. FISH analysis revealed homozygous deletion of p16 in 5/8 mesothelioma and 2/2 osteosarcoma. Median survival was 6.5 months after biopsy or surgical operation in mesothelioma, and 12 months after operation in osteosarcoma.ConclusionsAlthough median survival was longer for osteosarcoma than for malignant mesothelioma, we could not differentiate mesothelioma from pleural osteosarcoma on the combined basis of clinicopathological and immunohistochemical data, and FISH analysis. However, diffuse growth was more frequent in mesothelioma than in osteosarcoma.

The report of patients with lung cancer who could receive PDT combined with other modalities like surgery and chemotherapy is relatively rare. Combination of PDT and surgery is useful for multiple lung cancers (MPLC) or minimally invasive procedure to reduce resection line to peripheral site of superficial invasion in advanced lung cancer. Also, PDT combined with chemotherapy for advanced lung cancer with central airway stenosis seems to be usefull for local control and improvement of patient’s QOL. MPLCs were noted in 22 (34.4%) of 64 patients treated with PDT using Laserphyrin (synchronous:10, and metachronous: 12). Among them, 10 patients (synch:2, meta:8) underwent surgery (lobectomy: 9, pneumonectomy: 1) for peripheral-type lung cancer as their first primary lesion followed by PDT for central type early stage lung cancer. CRs were achieved after PDT in all patients, and all patients were alive. We performed induction PDT to reduce resection line for 28 patents. Among them, histological type revealed squamous cell ca. in 24. Superficial invasion was recognized in trachea: 2, carina: 3, main bronchus or 2nd carina: 22. Reduction surgery could be successfully performed in 23 of 28 patients (82%). PDT combined with chemotherapy was performed for 12 consecutive patients with 13 advanced NSCLC whose stages were IIIA–IV. The median stenosis rates before treatment, 1 week, and 1 month after treatment were 60%, 15%, and 15%, respectively. All patients improved symptoms and QOL after treatment. The mean survival time was 5.9 months, and the overall one-year survival rate was 30.0%.PDT combined with other modalities may be a promising strategy in lung cancer treatment.

Pulmonary mucoepidermoid carcinoma (PMEC) are rare, accounting for 0.1–0.2% of all malignant lung tumors. Furthermore, endobronchial lesions are rare and are more commonly found in the segmental or lobar bronchi. We present, to the best of our knowledge, the first case of successful treatment with photodynamic therapy (PDT) for PMEC. A 77-year-old male presented with cough and hemosputum for 4 months. Chest computed tomography showed a mass in the right intermediate bronchus. Endobronchial biopsy revealed a diagnosis of PMEC. An optimal surgical technique to preserve respiratory function was desirable as most of the tumor emerged from the bronchial glands in the central airways and was of low-grade type. Hence, PDT was performed. Repeat bronchoscopies were performed 5 years after the PDT and showed no evidence of tumor recurrence. PDT is more likely to be effective for low-grade PMECs that are visible on bronchoscopy.

Intractable advanced lung cancer can be treated palliatively with photodynamic therapy (PDT) combined with chemotherapy to remove central and peripheral (lobar or segmental bronchi) bronchial stenosis and obstruction. We present data for 12 (eight men, four women) consecutive patients with 13 advanced non-small cell lung carcinomas in whom curative operations were contraindicated, who underwent PDT combined with chemotherapy for local control of the intraluminal lesions. The mean age was 73.3 years (range, 58–80 years), and the stages of cancer were IIA–IV. The median stenosis rates before treatment, one week post-treatment, and one month post-treatment were 60% (range, 30%–100%), 15% (range, 15%–99%), and 15% (range 15%–60%), respectively. The mean and median survival times were 9.3 and 5.9 months, respectively. The overall 1-year survival rate was 30.0%. No PDT-related morbidity or mortality occurred. In this single-institution study, all patients experienced improved symptoms and quality of life at one week after treatment, furthermore, an objective response was evidenced by the substantial increase in the openings of the bronchial lumen and prevention of obstructive pneumonia. Therefore, PDT with chemotherapy was useful and safe for the treatment of bronchial obstruction.

BACKGROUND: The reported age-specific survival rates of lung cancer patients have been largely inconsistent. Management strategies for younger patients and treatment outcomes are not well characterized. METHODS: Out of the 4,697 lung cancer patients with treatment history at Tokyo Medical University Hospital between January 2000 and December 2014, 266 patients were

症例は59歳男性，持続する両側頚部腫脹を自覚し受診．頚胸部CTを施行し，両側顎下腺腫脹，縦隔リンパ節の腫大を認めた．頚部針生検施行したが，結果は炎症性唾液腺組織であり，悪性細胞は認めなかった．肺癌，悪性リンパ腫等を疑い胸腔鏡下縦隔リンパ節生検を施行したが，悪性所見を認めず炎症性腫大であった．シェーグレン症候群，ミクリッツ病を疑い採血，顎下腺組織免疫染色を追加し，リンパ球は著名なIgG4陽性形質細胞浸潤を認めた．また，dry eye，dry mouthも認め，血清IgG 1,618 mg/dl，IgG4 487 mg/dl，抗SS-Aと抗SS-B，抗核抗体は陰性であった．以上よりシェーグレン症候群ではなくミクリッツ病と診断した．現在は，プレドニゾロン40 mg/dayより開始し漸減中であるが，唾液腺，縦隔リンパ節は縮小し，dry eye，dry mouthの改善も認めた．

Endoscopic tumor ablation is a valuable option for inoperable postoperative laryngotracheal intraluminal invasion of well-differentiated thyroid carcinoma (DTC).To investigate whether DTC invasion to the laryngotracheal mucosa can be controlled by 'simple' tumor ablation considering its relatively slow-growing nature.Twenty-two consecutive patients underwent endoscopic tumor ablation caused by DTC for local control of intraluminal lesions with no significant extrinsic laryngotracheal compression in symptomatic or asymptomatic patients in whom radical operations were contraindicated. Debulking by Nd:YAG laser was followed by electrocoagulation and microwave coagulation for the residual tumor base.The critical complication, post-treatment supraglottic stenosis, was managed by prophylactic minitracheotomy. During the follow-up period of up to 125 months, 6 of 22 patients died (median survival 50 months), mainly of lung metastases, but all had a patent airway at death. Post-surgical extraluminal lesion growth is indolent and since relapse of the intraluminal lesion is the main cause of symptoms, satisfactory local control could be obtained by re-ablation of the mucosal lesion every few years. Lesions requiring retreatment within 1 year after initial treatment usually have high-grade malignancy, causing extrinsic compression, and prognosis is unfavorable.

Background Video-assisted thoracic surgery (VATS) is widely used for the treatment of spontaneous pneumothorax, and the recurrence rate is high. The goal of the study was to examine the use of polyglycolic acid (PGA) sheets, together with platelet-rich plasma (PRP) from autologous blood for the prevention of postoperative recurrence of spontaneous pneumothorax. Materials and Methods We performed a retrospective study of 65 patients who underwent VATS for spontaneous pneumothorax from March 2008 to November 2011. The patients were divided into groups: without reinforcement (Group A, n = 33) and with reinforcement of the visceral pleura around the staple lines with the PGA sheet and PRP (Group B, n = 32). The postoperative follow-up period was 18 months. Results Chest tubes were used for 3.4 and 3.1 days in Groups A and B, respectively, with no significant difference between the groups. However, the recurrence rate (18.2%; 6 cases) in Group A was significantly higher than that in Group B ( p = 0.02). The recurrence rates in patients younger than 25 years in Group A and Group B were 26.1 and 0.0%, respectively ( p = 0.03). In Group A, the mean age with recurrence (18.3 years old) was significantly lower than the mean age without recurrence ( p = 0.03). Conclusion These results suggest that the use of PGA sheets and PRP might be effective for the prevention of postoperative recurrence of spontaneous pneumothorax.

Malignant mesothelioma (MM) is associated with asbestos exposure and the presence of SV40 viral sequences. Recently, we reported that SV40 infection of human mesothelial cells (HM) causes aberrant methylation of the tumor suppressor gene (TSG) RASSF1A. We investigated methylation of 12 genes by methylation-specific PCR in 63 MMs, six MM cell lines, and two foci of SV40-infected HM. Methylation percentages of the tested genes ranged from 3 to 65%. The frequencies of HPP1, RASSF1A, Cyclin D2, and RRAD methylation, and the value of the methylation index, were significantly higher in SV40 sequence-positive MMs than in SV40-negative MMs. Methylation of TMS1 and HIC-1 was associated with shortened survival. SV40-infected HM showed progressive aberrant methylation of seven genes (RASSF1A, HPP1, DcR1, TMS1, CRBP1, HIC-1, and RRAD) during serial passage. Our results demonstrate a relationship between SV40 and methylation of multiple genes in MM, indicating that the virus plays a role in the pathogenesis of MM.

Purpose: Aberrant methylation of the 5′ gene promoter regions is an epigenetic phenomenon that is the major mechanism for silencing of tumor suppressor genes in many cancer types. The aims of our study were (a) to compare the methylation profiles of the major forms of hematological malignancies and (b) to determine the methylation profile of monoclonal gammopathy of undetermined significance (MGUS) and compare it with that of multiple myeloma (MM). Experimental Design: We compared the aberrant promoter methylation profile of 14 known or suspected tumor suppressor genes in leukemias (n = 48), lymphomas (n = 42), and MMs (n = 40). We also examined the methylation profile of MGUS (n = 20), a premalignant plasma cell dyscrasia. The genes studied represent five of the six “hallmarks of cancer.” Results: Peripheral blood lymphocytes (n = 14) from healthy volunteers were negative for methylation of all genes, and methylation percentages in 41 nonmalignant tissues (peripheral blood mononuclear cells, bone marrows, and lymph nodes) from hematological patients were low (0–9%) for all 14 genes, confirming that methylation was tumor specific. Ten of the genes were methylated at frequencies of 29–68% in one or more tumor types, and the methylation indices (an indicator of overall methylation) varied from 0.25 to 0.34. With two exceptions, the methylation patterns of leukemias and lymphomas were similar. However, the pattern of MMs varied from the other tumor types for six genes. In general, the methylation pattern of MGUS was similar to that of MM, although the methylation frequencies were lower (the methylation index of MGUS was 0.15, and that of MM was 0.3). However, the methylation frequencies of six genes were significantly higher in MGUS than in control tissues. The relatively high frequencies of methylation in MGUS are consistent with it being a premalignant condition. Conclusions: The three major forms of lymphoid/hematopoietic malignancies show overlapping but individual patterns of methylation.

Despite major improvements in patient management, the prognosis for patients with lung cancer remains dismal. As our knowledge of the molecular biology of cancers has increased, new targets for therapeutic interventions have been identified. In this article, we discuss some of the more recent developments in this field. They include revisiting some of the established concepts, such as retinoid metabolism and the inhibition of cyclooxygenase-2 metabolism. In addition, newer targets, such as transforming growth factor-beta signaling, Janus-activated kinase/signal transducers and activators of transcription pathway, and cell invasion are discussed. These studies demonstrate that multiple, often overlapping, mechanisms of disruption are present in lung cancer cells, presenting a plethora of molecular targets.

HIN-1 (high in normal-1) is a putative cytokine with growth inhibitory activities and is downregulated by aberrant methylation in breast cancers. We studied HIN-1 methylation status in many types of adult and pediatric malignancies and cell lines. We examined the expression of HIN-1 mRNA in 52 cell lines and the promoter methylation status in the cell lines and in over 800 primary tumors representing 17 tumor types using methylation specific PCR. Promoter methylation was observed in 73% of breast cancer, 67% of nonsmall cell lung cancer (NSCLC), 30% of small cell lung cancer (SCLC) and 57% of malignant mesothelioma (MM) cell lines, and methylation was completely correlated with loss of expression. Expression negative cell lines restored HIN-1 expression after treatment with 5-aza-2'-deoxycytidine. Promoter methylation of HIN-1 was found in 90% of retinoblastomas, 73% of Wilms' tumors, 61% of rhabdomyosarcomas, 57% of breast cancers, 52% of prostate cancers, 40% of MMs, 28% of NSCLCs and 27% of lymphomas. Methylation frequencies in colorectal cancers, cervical cancers, bronchial carcinoids, SCLCs, neuroblastomas, osteosarcomas, leukemia, medulloblastomas and bladder cancers were lower (4-21%), while hepatoblastomas lacked methylation. HIN-1 methylation was rarely detected in nonmalignant tissues (8 of 165, 5%). Aberrant methylation of HIN-1 with loss of expression is a common event and may contribute to the pathogenesis of many types of human malignancies.

症例は63歳男性, 結核性胸膜炎の既往がある.胸部X線写真, CT上右膿胸腔の増大を認めたため, 肥厚した胸膜を用いた肋膜外air-plombage術を施行した.術1年後の呼吸機能は, 若干の改善を示したのみであったが, 血液ガス分析では, 著明な改善が認められた.肋膜外air-plombage 術は, 肋間筋を胸壁に残すことにより胸郭の変形が少なく, 骨性胸郭が維持され, 術後長期に肋骨骨折を起こしにくいと考えられる.また, 本症例のように壁側胸膜の十分に肥厚している場合は, 肋膜外剥離腔と膿胸腔の完全な隔絶が得られやすく, 新たな術式の一つとなる可能性が示唆された.

肺癌切除例89例を対象に腫瘍の捺印細胞標本を用い, 増殖細胞核抗原 (PCNA) およびp53の免疫染色を行いそれぞれの発現率と予後の関連について検討した.1) PCNA発現率が25%未満の群は25%以上の群に比較し予後が良かった (p

To define the clinicopathological features and discuss the optimal management of carcinoma showing thymus-like differentiation (CASTLE). We retrospectively analyzed six patients with CASTLE. The subjects comprised two men and four women (average age at initial diagnosis, 61 years, range 47–75 years). Preoperative biopsy yielded a correct diagnosis in two patients. Five patients underwent surgery and one was treated with radiation therapy alone. Four had extrathyroidal invasion and three had lymph node metastasis. During the clinical course, tracheal invasion was detected in five patients, the upper extent of the tumor being the lower half of the first tracheal ring. Two of these patients underwent tracheal sleeve resection. Two patients received postoperative radiotherapy for nodal metastasis, and one, after palliative surgery. The median follow-up period was 67 months (range 38–129). Recurrence was found 10 years post-therapy in the patient treated with radiation therapy only, resulting in death soon after. Although local recurrence was not found in the remaining five patients, new pulmonary metastases were diagnosed in the patient who underwent non-curative surgery. CASTLE can be diagnosed preoperatively by core needle biopsy and CD5 staining. Curative resection with neck dissection followed by radiotherapy can yield a good outcome. Larynx-sparing complete resection may be more feasible for CASTLE, even though it has a higher incidence of tracheal invasion than differentiated thyroid carcinoma.

肺気腫症例に対し胸腔鏡下の炭酸ガスレーザー照射が有効であるかどうかを検討した.4例の肺気腫症例に対し胸腔鏡下に炭酸ガスレーザー照射を行い, 手術前後の呼吸機能および運動機能を比較検討した.術前右心カテーテル検査では肺高血圧症が全例に認められた.術中の肉眼的所見では照射により気腫化部分の縮小が認められた.全例とも2日目に胸腔ドレーンを抜管でき, 術後40日以内に退院した.術後合併症は皆無であり, 胸腔鏡下炭酸ガスレーザー照射は安全に施行できた.手術前後の胸部X線写真およびCTの比較では1例に気腫化部分の明らかな縮小を認めた。1秒量は術前0.35±0.11に比し術後0.42±0.21とやや増加し, 2例では運動機能の改善と自覚症状の改善を見た.他の2例では自覚症状に変化を認めなかった.胸腔鏡下炭酸ガスレーザー照射は一部の肺気腫症例に対して有効であった.

Patients with large cell neuroendocrine carcinoma (LCNEC) of the lung are considered to have poor prognosis. However, the benefit of adjuvant chemotherapy for these patients has not been established. In this study, we retrospectively evaluated the efficacy of perioperative chemotherapy for patients with completely resected LCNEC in a single-center setting. From 1999 through 2007, 45 patients with surgically resected LCNEC or mixed LCNEC containing at least one portion of the neuroendocrine differentiation or morphology in non-small cell lung carcinoma were enrolled as participants of this study. Survival rates were calculated by the Kaplan-Meier method. Differences between survival curves were computed with the log-rank test. For multivariate analysis, the Cox's proportional hazards regression model was used to evaluate variables that were significant predictors of survival. Of 1397 patients undergoing surgical resection for primary lung cancer from 1999 to 2007, 45 (3.2%) were classified as LCNEC. Thirty-six (80%) patients were men, and nine (20%) were women. Twenty-four (92%) of 26 patients were present or past smokers. Twenty-three (41%) of 45 patients received perioperative chemotherapy, including seven induction chemotherapies and 16 adjuvant chemotherapies. Survival of patients who underwent perioperative adjuvant chemotherapy was significantly higher than that of those who received surgery alone (P = 0.04). The 5-year survival rate of patients who underwent perioperative adjuvant chemotherapy was 87.5%, whereas that of patients who underwent surgery alone was 58.5%. Even in stage I cases, perioperative adjuvant chemotherapy still favors survival compared with surgery alone. In the Cox proportional hazard multivariate analysis, surgery with or without chemotherapy showed an independent prognostic influence on overall survival (P = 0.0457). Patients who received surgery alone were 9.5 times more likely to die than patients who underwent surgery plus chemotherapy. In conclusion, perioperative chemotherapy will be needed to improve survival in patients with LCNEC. As the population of LCNEC is small, it has been difficult to conduct randomized controlled trials to show the survival benefit of adjuvant chemotherapy. This should be, therefore, evaluated further in prospective multi-institutional phase II trials.

The therapeutic strategies for non-small cell lung carcinoma (NSCLC) with N1 and N2 disease differ remarkably. Debate exists about the definition of the borderline between N1 and N2 stations. This study evaluated the prognostic effect of N1 disease, especially focused on the significance of the main bronchial node (No. 10) vs N2 disease.The records of 1601 patients who underwent complete pulmonary resection for NSCLC were reviewed to examine the clinical features of lymph nodal involvement.There were 1086 patients (67.8%) with pN0 disease, 202 (12.6%) with pN1, and 274 (17.1%) with pN2 disease; overall 5-year survival rates were 74.7%, 56.1% and 28.9%, respectively (p0.001). Overall 5-year survival rates were 60.2% in hilar N1 and 49.6% in intralobar N1. Overall 5-year survival rates were 58.6% in N1 without node 10 and 35.1% in N1 with node 10. A significant difference was observed between N0 and N1 without node 10 (p0.001), and N1 without node 10 and N1 with node 10 (p = 0.033); however, the difference between N1 with node 10 and N2 was not significant. The status of node 10 involvement was an independent prognostic factor of pN1 patients, as well as age and gender.Patients with node 10-positive N1 disease have an unfavorable prognosis, and the disease behaves like N2 disease. The definition of clear borderline between N1 and N2 is mandatory to achieve a uniform classification map. This study offers further information for clinical and therapeutic purposes.

Non-small cell lung cancer (NSCLC) constitutes approximately 85% of all lung cancers, with patients having a poor prognosis. Approximately one third of NSCLC patients present with early-stage disease in which potentially curative resection and multi-modality therapy. Although adjuvant chemotherapy is the standard practice for patients with stages I-III breast and colorectal cancer, the therapeutic efficacy of adjuvant chemotherapy, following complete surgical resection of early stage NSCLC, has not been fully established. Several prospective randomized trials for patients with early stage NSCLC (stages I-IIIA) have confirmed a survival benefit with cisplatin-based adjuvant chemotherapy, as demonstrated in the 1995 meta-analysis performed by the NSCLC Collaborative Group. Studies from Japan have reported that adjuvant therapy with uracil-tegaful (UFT) afforded an improvement of 4% in the 5-year survival rate and a relative risk reduction of 26% in mortality at 5 years among patients with T1-2N0 (stage I) disease. In particular, the Japan Lung Cancer Research Group has demonstrated an improvement in the 5-year survival rate of 11%, favoring chemotherapy with UFT in the subset of patients with T2N0 (stage IB) disease. Two published meta-analyses based on abstracts have estimated a relative risk reduction in mortality of 11-13% at 5 years. The Lung Adjuvant Cisplatin Evaluation (LACE), which was based on a pooled analysis of five randomized trials, has demonstrated that cisplatin-based adjuvant chemotherapy improved survival in patients with completely resected NSCLC. This benefit depended on stage, being greatest in patients with stage II or IIIA disease. This analysis has suggested that platinum-based adjuvant chemotherapy may have no benefit for patients with stage IA and only a marginal benefit for patients with stage IB. Thus, the information available at the current time supports the administration of adjuvant chemotherapy for patients who have undergone complete resection of stages IB-IIIA NSCLC. Further research is needed to define the role of adjuvant platinum-based chemotherapy and its use, in conjunction with chest radiotherapy as the treatment for patients with resected stages IB and IIIA NSCLC.

Bronchiolitis obliterans (BO) is the main cause of late mortality among long-term survivors of lung transplantation. Chemokine-chemokine receptor (CCR) interaction and subsequent recruitment of infiltrating cells to the graft are early events in the development of chronic rejection of transplanted lungs. The present study investigated whether blockade of chemokine receptors CCR1 and CCR5 with Met-regulated-on-activation, normal T cells expressed and secreted (RANTES), an amino-terminal modified derivative of RANTES/CCL5, affects the development of BO in murine model and we sought to determine the expression of RANTES/CCL5 and their relationship with extracellular signal-regulated kinase (ERK).BALB/c mouse tracheas were heterotopically transplanted into C57Black6 recipients and treated for 21 days with either Met-RANTES at 20 microg/day or vehicle. Animals were killed at 21 days after transplantation for histologic examination of ERK expression.RANTES/CCL5 was highly expressed in allografts compare to isografts. Met-RANTES treatment ameliorated fibrous airway obliteration in a mouse model of BO and decreased ERK expression.Blockade of chemokine receptors by Met-RANTES ameliorated airway obliteration and decreased ERK expression. These findings suggest that chemokine receptors CCR1 and CCR5 play significant roles in the development of chronic rejection and ERK may be a new molecular target for chronic rejection.

We report the case of a patient with hypereosinophilia and invasive thymoma harboring probable clonal proliferation of CD4+, CD8+, and CD25+ T-lymphocytes. A 64-year-old woman had eosinophilia (14.1 x 10(9)/L) and an anterior mediastinal tumor with elevated levels of serum immunoglobulin E (609.8 mg/dL) and interleukin 5 (239 pg/mL). Bone marrow aspirate showed marked infiltration by morphologically normal eosinophils with a normal karyotype but no FIP1L1-PDGFRA fusion gene. Flow cytometric analysis revealed an increasing number of CD3+/CD25+ lymphocytes in the peripheral blood, and the resected thymoma had infiltrated lymphocytes with CD4/CD8/CD25 antigens. Moreover, the thymoma had T-cell receptor rearrangements with a cytogenetically clonal nature, ie, t(2;4)(p22;q26). Although the number of patients with thymoma showing hypereosinophilia is small, this case suggests that a subset of patients with thymoma may have clonal expansion of T-lymphocytes with abnormal phenotypes that affect clinical manifestations, including hypereosinophilia.

We report a new method for collecting and establishing small airway epithelial cells (SAEC). This method enables the evaluation of the cytokine responsiveness of SAEC, which is still unknown. In this study we evaluated intercellular adhesion molecule-1 (ICAM-1) expression on SAEC stimulated with several inflammatory cytokines and compared it with that on large airway epithelial cells (LAEC).LAEC and SAEC were treated with IFN-gamma, TNF-alpha, IL-1beta, or their combination. ICAM-1 expression under various conditions was quantified by flow cytometry. Furthermore, immunocytochemical staining was performed to determine intranuclear displacement of signal transducer and activator transcription 1 (Stat1) during ICAM-1 expression by various cytokine stimulations.1) ICAM-1 expression on both LAEC and SAEC was significantly increased by IFN-gamma stimulation alone and synergistically enhanced by IFN-gamma plus TNF-alpha or IL-1beta stimulation, 2) intranuclear displacement of Stat1 in SAEC by the stimulation with IFN-gamma plus TNF-alpha or IL-1beta was recognized earlier in comparison with that by IFN-gamma stimulation alone.The previously unknown peripheral cytokine responsiveness and its mechanisms of SAEC were revealed by this study, which contributes to the understanding of chronic lung allograft rejection recognized around small airways.

We investigated aberrant methylation in 101 prostate cancers(PCa) and 32 histologically normal prostate tissues. We focused on genes largely in the apoptotic pathway. Methylation frequencies of the genes were Reprimo, 54%; TMS1, 47%; DcR1, 45%; RRAD, 37%; DcR2, 37%; CRBP1, 34%; HPP1, 32%; RIZ1, 31%; DRM/Gremlin, 21%; SOCS1, 20%; DR4, 5%; DR5, 1%. Methylation of Reprimo and TMS1 correlate with preoperative serum prostate-specific antigen. Methylation of TMS1, DcR1, DcR2, and CRBP1 correlate with Gleason score. Methylation of TMS1 and unmethylation of both DcR1 and DcR2 correlate with poorer disease free survival by univariate and multivariate analyses. Our data suggest that methylation of multiple genes may be involved in pathogenesis and correlate with prognosis of PCa.

Caveolin-1 (CAV1), an essential structural constituent of caveolae that plays an important role in cellular processes such as transport and signaling, has been implicated in the development of human cancers. However, it is unclear whether CAV1 is acting like an oncogene or tumor suppressor gene. We found that CAV1 expression was reduced or absent in 95% of small cell lung cancers (SCLCs; n = 21 lines), whereas it was retained in 76% of non-small cell lung cancers (NSCLCs; n = 25 lines) compared with normal human lung epithelial cultures, where it was abundantly expressed. CAV1 expression was tightly linked to the ability to grow attached to the plastic cell culture surface, whereas CAV1-nonexpressing lung cancers of both SCLC and NSCLC type grew as suspension cultures. In addition, attached lung cancer cultures expressed phosphorylated focal adhesion kinase, whereas suspension cultures did not. Lack of CAV1 expression was tightly associated with CAV1 promoter methylation (P < 0.0001) such that CAV1 methylation was found in 93% of SCLCs (n = 15) and 9% of NSCLCs (n = 11), whereas 5-aza-2′deoxycytidine treatment restored CAV1 expression in SCLCs. Exogenous CAV1 expression in SCLCs significantly inhibited soft-agar colony formation but did not lead to attachment. By contrast, CAV1 knockdown in NSCLCs mediated by small interfering RNA against CAV1 led to inhibition of cellular proliferation and soft-agar and liquid colony formation. Importantly, CAV1 knockdown led to reduced phospho-focal adhesion kinase and RalA, but not RalB, levels in NSCLC cells. These results suggest different roles for CAV1 in SCLC, where CAV1 acts like a tumor suppressor gene, and NSCLC, where it appears required for survival and growth.

TNF-related apoptosis-inducing ligand (TRAIL) selectively induces programmed cell death (apoptosis) in various cancer cells but not in normal cells. TRAIL is known to bind to 4 different receptors, 2 proapoptotic (DR4 and DR5), and 2 potentially antiapoptotic receptors lacking death domains (DcR1 and DcR2). Aberrant promoter methylation and resultant silencing of tumor suppressor genes play an important role in the pathogenesis of many tumor types. Recently aberrant methylation of TRAIL decoy receptors was reported in pediatric tumor cell lines and neuroblastomas. We examined the methylation and expression status of TRAIL receptor genes in cancers of breast, lung, mesothelioma, prostate, bladder, cervix, ovary, brain and in hematopoietic malignancies. Aberrant methylation of DcR1 or DcR2 was present in 70% of primary breast cancers, 31% of primary lung cancers, in 63% of primary malignant mesothelioma (MM), in 60% of prostate cancer, in 42% of bladder cancer, in 100% of cervical cancer, in 43% of ovarian cancer, in 41% of lymphoma, in 26% of leukemia and in 56% of multiple myeloma. Methylation of DR4 and DR5 was rare in all the tumor types examined. Methylation of all the 4 receptors was rare in non malignant tissues. In cell lines, aberrant methylation of DcR1 was present in 11 of 23 (48%) breast, 10 of 27 (37%) lung and 3 of 7 (43%) MM, whereas aberrant methylation of DcR2 was present in 17 of 23 (74%) breast, 13 of 27 (48%) lung and 5 of 7 (71%) MM. The concordance between loss of gene expression and aberrant methylation ranged from 70-100%. Treatment with 5-aza-2'-deoxycytidine restored DcR1 and DcR2 expression in 9 methylated cell lines confirming that aberrant methylation was the cause for silencing of DcR1 and DcR2 expression. Our results demonstrate that DcR1 and DcR2 genes are frequently methylated in various tumor types, and that the role of decoy receptors in tumor pathogenesis needs to be re-evaluated.

Alterations of the noncoding displacement (D) loop of mitochondrial DNA are present in many cancers. These alterations include numerical changes in a homopolymeric C tract (PCT) at positions 303-309 and single base substitutions (SBS). We determined the frequency of D-loop alterations in lung cancer cell lines and tumors, and related them to clinicopathologic features.We sequenced the entire D-loop of 28 lung cancer cell lines [12 small cell lung cancer (SCLC) and 16 non-small cell lung cancer (NSCLC)] and matched B-lymphoblastoid cell lines. In 55 resected NSCLCs and corresponding nonmalignant lungs we determined the length of the PCT.In nonmalignant cell lines and tissues the most frequent PCT repeat number was seven (36 of 83; 43%) with a range of six to nine. Alterations, often multiple, were present in 17 of 28 (61%) of the cell lines, including 8 of 12 SCLC (67%) and 9 of 16 NSCLC (56%) lines. They consisted of SBS in 8 of 28 lines (29%), all of which were homoplasmic, and PCT changes in 14 of 28 (50%) lines, 8 of which were homoplasmic. Of interest, 95% (40 of 42) of the SBS were present within the two hypervariable regions in the D-loop. Because SBS were more frequent if PCT changes were present, only the PCT number was determined in resected samples. PCT changes were present in 11 of 55 (20%) of the NSCLC tumors. Changes were never noted in tumors when the PCT number in the nonmalignant tissue was seven, and only two tumor cell lines had changes when the PCT number in the matched lymphoblastoid cell line was seven. These changes were higher in squamous cell carcinomas (8 of 25; 32%) than in adenocarcinomas (3 of 30; 10%; P = 0.04) and in large tumors (T3 and T4; 7 of 20; 35%) compared with smaller tumors (T1 and T2; 4 of 35; 11%; P = 0.04). Smoking history, gender, age, and stage were not related to frequency of PCT change.Our findings indicate that D-loop alterations are frequent in lung cancers and their cell lines, and that these changes are weakly associated with certain clinical parameters. In tumors PCT changes were only present when the corresponding nonmalignant lung demonstrated a variation from the most common repeat number of seven.

To determine the role of methylation of HIC1, a candidate tumor suppressor gene on 17p13.3, in various types of pediatric tumors.We examined the methylation status of the HIC1 promoter by methylation specific PCR in 157 pediatric tumors and 27 nonmalignant tissues. We correlated methylation with mRNA expression by reverse transcription-PCR in eight tumor-derived cell lines.HIC1 methylation was frequent in medulloblastomas (80%, 12 of 15), retinoblastomas (67%, 6 of 9), rhabdomyosarcomas (59%, 13 of 22), germ cell tumors (55%, 6 of 11), and neurouroblastic tumors (36%, 14 of 39); neuroblastomas (43%, 12 of 28); ganglioneuromas (17%, 1 of 6); and ganglioneuroblastomas (20%, 1 of 5). In contrast, a low incidence of methylation was observed in osteosarcomas (17%, 2 of 12), Ewing's tumors (9%, 1 of 11), Wilms' tumors (3%, 1 of 31), and hepatoblastomas (0%, 0 of 7). HIC1 methylation was more frequent in the aggressive alveolar subtype of rhabdomyosarcomas (100%, 8 of 8) than the embryonal subtype (33%, 4 of 12; P0.005) and was rare in the nonmalignant tissues examined. Methylation was also demonstrated by sequencing in nine randomly selected tumor samples. Seven of eight pediatric tumor cell lines examined were methylated and showed loss or reduced HIC1 mRNA. Expression was strongly induced in all cell lines by treatment with the demethylating agent 5-aza 2'deoxycytidine.Our data suggest that aberrant methylation of HIC1 may play a role in the pathogenesis of specific pediatric tumors.

14-3-3σ gene has been shown to be responsible for G2 cell cycle checkpoint control by p53 in response to DNA damage in human cells. In order to increase the potential utility of 14-3-3σ gene as a molecular marker in tumor analysis and prognosis, we established and validated a quantitative real-time MSP assay and correlated our findings with the standard MSP assay.We examined the expression of 14-3-3 σ gene by reverse transcription PCR (RT-PCR) in breast and lung cancer cell lines and control non-malignant tissue samples. To elucidate the mechanism of gene silencing, we studied the methylation patterns in cell lines, tumors and non-malignant control tissues of breast and lung using previously reported MSP assay. For fluorescence based quantitative Real-Time PCR assay, we designed primers and probe specific to 14-3-3σ gene, validated the assay in cell lines and non-malignant control tissues of breast and lung and extended the study to primary tumors and corresponding non-malignant tissues.The concordances between the standard MSP assay and the real-time assay were 95-100%. The overall concordances between standard MSP and real-time assay in 60 cell lines were 97%. By real-time assay, the differences in methylation frequencies between malignant and non-malignant breast and between malignant and non-malignant lung tissues; between NSCLC and SCLC cell lines; between MSP (-) and MSP (+) samples and between MSP (+) and MSP (++) samples were statistically significant. The mean real-time values for MSP (-), MSP (+) and MSP (++) samples were 2, 28 and 53 respectively.We conclude that promoter methylation is a valid pathway for silencing of 14-3-3σ gene in primary breast and lung carcinomas. The real-time assay to distinguish the extent and degree of methylation of 14-3-3σ gene among malignant and non-malignant tissues would potentially enhance the utility of this marker in breast and lung cancer analysis and prognosis.

Methylation of the promoter regions of CpG-rich sites in genes is the major mechanism for the silencing of many genes in tumors. Methylation of the key apoptosis-related gene caspase 8 (CASP8) has been reported in some childhood tumors and in neuroendocrine lung tumors. We examined the methylation status of 181 pediatric tumors and found frequent methylation in rhabdomyosarcomas (83%), medulloblastomas (81%), retinoblastomas (59%), and neuroblastomas (52%). Methylation frequencies were low in Wilms' tumors (19%) and absent in hepatoblastomas, acute leukemias, osteosarcomas, Ewing's sarcomas, and ganglioneuromas and in normal tissues. Methylation of CASP8 and the tumor suppressor gene RASSF1A were highly significantly correlated in all tumor types by both the chi(2) and the Fisher's exact tests (P0.0001 for both tests). Because the region of the gene examined by us and others is not located in the promoter region and lacks features of a CpG island, we explored the relationship between methylation and gene silencing in detail using 23 pediatric tumor cell lines. Studies included relating the methylation of the region to gene expression at mRNA and protein levels, enzymatic assays of gene function, clonal analysis of PCR amplicons of the region, and exposure to a demethylating agent. These studies indicated that methylation correlated with the loss of gene function in most cases; however, other mechanisms of gene inactivation were present in some cases. Posttranscriptional inactivation of the closely related gene caspase 10 was present in many cell lines. Our results suggest that deregulation of the death receptor pathway to apoptosis is frequent in many types of pediatric tumors and their cell lines.

Expression of the cadherin family member CDH13 (H-cadherin) is reduced in several human tumors, and it has been hypothesized that this gene functions as a tumor suppressor gene. Previously, we reported that the 5' region of CDH13 is frequently methylated in breast and lung cancers. Here we confirmed the promoter activity of 5' region of CDH13 by luciferase assay and examined its aberrant methylation in colorectal cancers, cell lines, and adenomas. Methylation status was investigated by methylation-specific PCR (MSP) and by bisulfite DNA sequencing of cloned DNA of PCR amplicons. In cell lines, we examined the correlation between methylation status and mRNA expression by reverse transcription-PCR. Aberrant methylation of CDH13 was present in 7 of 13 (54%) cell lines, and expression was absent in 6 of 13 (46%) cell lines. CDH13 expression was present in six cell lines that showed only the unmethylated form by MSP and in one cell line that showed both the methylated and unmethylated forms. Treatment with 5-aza-2'-deoxycytidine restored CDH13 expression in methylated cell lines. In surgically resected samples, 17 of 35 (49%) cases of primary colorectal cancer, 2 of 33 (6%) cases of corresponding nonmalignant colorectal mucosa, and 8 of 19 (42%) adenomas were methylated. Sequence data after bisulfite treatment indicated that primary cancers and two cell lines with loss of expression were highly methylated compared with nonmalignant colorectal epithelial cells, especially at the attachment sites of primers for MSP, although there was heterogeneity in methylation status. Our results suggest that CDH13 expression is frequently silenced by aberrant methylation in colorectal cancers and adenomas and that methylation of CDH13 commences at an early stage of multistep colorectal tumorigenesis.

Inactivation of the p16 and ESR1 tumor suppressor genes by promoter lesion methylation has been reported in many tumor types, including lung cancer. We examined the blood of 95 non-small cell lung cancer patients (66 cases of adenocarcinoma, 23 of squamous cell carcinoma and 6 of large cell carcinoma) and 30 controls consisting of normal subjects and benign disease patients to determine the methylation ratios of p16 and ESR1 using real-time PCR. For both genes, there was a statistically significant difference in the methylation ratio between non-small cell lung cancer patients and controls (p16; p

e17505 Background: Patients with large cell neuroendocrine carcinoma of the lung (LCNEC) are considered to have poor prognosis. This study was undertaken to evaluate the efficacy of perioperative c...

7514 Background: Total number of lymph-nodes has recently proven prognostic in early breast and colorectal cancer. In this study we retrospectively evaluated the prognostic impact of the number of resected and involved lymph-nodes on the survival of stage I-III NSCLC. Methods: A series of 928 consecutive NSCLC pts who underwent complete lobectomy, bilobectomy or pneumonectomy with lymph-nodes dissection from 1/2000 to 11/2007 at Tokyo Medical University was eligible. Log rank and Cox proportional hazard model was used to estimate survival rates and relative risks. Results: Demographics are as follows: median age: 65.0 (22–87yrs), sex: 547 males and 381 females, median follow-up time: 2.5 yrs, clinical stage: 765 stage I, 84 stage II and 76 stage III, histology: 684 adenocarcinoma, 182 squamous cell carcinoma, and 62 others, operation: 870 lobectomy, 42 bilobectomy and 16 pneumonectomy, mean number of resected LN: 15 (1–49), mean number of involved LN: 0.9 (0–22). We observed a statistically significant increasing trend in overall survival (OS) between 0–3 and 4 and more of number of involved LN (P No significant financial relationships to disclose.

7199 Background: Aberrant methylation of CpG islands in promoter regions of tumor cells is one of the major mechanisms for silencing of tumor suppressor genes. The RAS association domain family 1A (RASSF1A) gene was isolated from the 3p21.3 region homozygously deleted in lung cancer cell lines, and it was shown to be inactivated by hypermethylation of the promoter region in lung cancers. In this study, we investigated the clinicopathological significances of RASSF1A methylation in the development and/or progression of small-sized (less than 2.0cm) lung adenocarcinoma. It is important to identify a marker for high-risk early stage patients who should benefit from new investigational adjuvant therapies. Methods: Surgically resected specimens from 77 cases of small-sized primary lung adenocarcinoma. We determined the frequency of aberrant promoter methylation of the RASSF1A genes in small-sized adenocarcinoma. Aberrant promoter methylation was examined using methylation-specific PCR (MSP). Results: Twenty-five of 77 (32.5%) tumors showed RASSF1A methylation. RASSF1A methylation was dominantly detected in smoker (P < 0.03). There was no significant correlation of RASSF1A methylation with gender, age, T stage, N stage and pathological stage. RASSF1A methylation correlated with adverse survival by univariate analysis (P < 0.005) as well as multivariate analysis (P = 0.0062; RR 4.251; 95% C.I., 1.507–11.993). Furthermore, RASSF1A promoter hypermethylation in resected stage I small-sized lung adenocarcinoma was associated with impaired patient survival (P < 0.01). Conclusions: Aberrant promoter methylation of the RASSF1A was present in 25 of 77 (32.5%) of small-sized lung adenocarcinoma by MSP assay. These results indicated that epigenetic inactivation of RASSF1A plays an important role in the progression of small-sized lung adenocarcinoma, and that RASSF1A hypermethylation appears to be a useful molecular marker for the prognosis of patients with small-sized and stage I lung adenocarcinoma. RASSF1A is a potential tumor suppressor gene that undergoes epigenetic inactivation in lung adenocarcinoma through hypermethylation of its promoter region. RASSF1A methylation was significantly related to unfavorable prognosis in small-sized lung adenocarcinoma. No significant financial relationships to disclose.