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T. Shirakusa



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    P2.01 - Poster Session 2 - Cancer Biology (ID 145)

    • Event: WCLC 2013
    • Type: Poster Session
    • Track: Biology
    • Presentations: 1
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      P2.01-005 - Diagnostic application of 9p21 Homozygous and Heterozygous Deletion Detected by Fluorescence in Situ Hybridization (FISH) and p16 Real-Time PCR in Malignant Mesothelioma (ID 1162)

      09:30 - 09:30  |  Author(s): T. Shirakusa

      • Abstract

      Background
      Background: Detection of homozygous deletion of the 9p21 locus, the site of the CDKN2A/p16 gene, is useful in differentiating reactive mesothelial hyperplasia (RMH) from malignant pleural mesothelioma (MPM) in pleural effusion cytology, biopsy specimens, and surgical specimens. However, the cutoff value for 9p21 FISH varies. Furthermore, 9p21 FISH reveals not only homozygous deletion, but also heterozygous deletion, in MPM tissue samples, and only two studies have evaluated heterozygous deletion. The aim of this study was to establish a FISH cutoff value for 9p21 homozygous deletion and apply it to a clinical diagnosis. In addition, we investigated the significance of 9p21 heterozygous deletion in MPM.

      Methods
      Design: Ninety-five histopathological (49 MPM + 47 RMH) and 35 cytological (15 MPM + 20 RMH) cases were studied. Cutoff values were evaluated as mean + 3SD based on the results of RMH 9p21 FISH. The copy number of the p16 gene was analyzed in 22 MPM tissue samples and 17 RMH by real-time PCR, using RSP6 as a control gene, and samples were divided into three groups (1.0: no deletion, 0.5: one allele deletion, and 0: two allele deletions). Methylation-specific PCR (MSP) was performed in two heterozygous deletion dominant cases. Overall survival was evaluated using Kaplan-Meier survival analysis and log-rank test.

      Results
      Results: In MPM, 44/49 (90%) cases were homozygous deletion-positive (>10% cutoff value), and 12/49 (24%) cases were heterozygous deletion-positive (>46.6% cutoff value) by p16 FISH. None of RMH cases were deletion-positive in tissue samples. Real-time PCR was performed for 22 cases of MPM and 17 cases of RMH. The p16 copy number in RMH was 0.83-1.28 (average 1.06). In MPM, 16/22 (73%) revealed a two-allele deletion pattern for p16. Two cases of homozygous deletion-negative MPM by FISH also had no deletion pattern by real-time PCR. One case of heterozygous deletion-dominant MPM (over 55%) revealed a two-allele deletion of p16 by real-time PCR, and two cases showed a one-allele deletion of p16. These cases had no methylation by MSP. In 25 cases of MPM for which follow-up was available, the overall survival rate was significantly lower in homozygous deletion-positive MPM (21/25 cases) than homozygous deletion-negative MPM (4/25 cases) (p= 0.01).

      Conclusion
      Conclusion: 9p21 FISH was useful for differentiating MPM from RMH, and p16 homozygous deletion in MPM has the potential to be a prognostic factor. Moreover, a combination of p16 FISH, p16 real-time PCR and MSP is useful for discriminating heterozygous deletion from homozygous deletion in MPM. The presence of heterozygous deletion suggest the possibility of a p16-independent tumorgenesis pathway in MPM.

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    P3.06 - Poster Session 3 - Prognostic and Predictive Biomarkers (ID 178)

    • Event: WCLC 2013
    • Type: Poster Session
    • Track: Biology
    • Presentations: 1
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      P3.06-011 - The impact of E-cadherin transcriptional factors on the survival of pulmonary pleomorphic carcinoma (ID 1161)

      09:30 - 09:30  |  Author(s): T. Shirakusa

      • Abstract

      Background
      Pleomorphic carcinoma of the lung is a rare epithelial tumor, and its clinicopathological characteristics and prognostic factors are still controversial. Up-regulation of E-cadherin repressors is known to increase tumor cell invasion and motility in non-small cell lung cancer cells. The aim of this study was to clarify the clinicopathological characteristics and prognostic factors, such as E-cadherin repressors (e.g., Zinc finger E-box-binding homeobox [ZEB], Snail, and Twist), in pleomorphic carcinoma.

      Methods
      We reviewed 2,328 cases of resected lung cancers that occurred between 1988 and 2011 and identified 62 patients with pulmonary pleomorphic carcinoma. Immunohistochemical staining for ZEB, Snail, and Twist was performed, and nuclear expression was estimated by the percentage of positive cells. The patients were divided into two groups; a diffuse expression group (≥75%) or a focal expression group (<75%). Clinicopathological variables and the expression of E-cadherin repressors were investigated retrospectively to analyze prognostic factors for the disease-specific survival rate of 42 patients after lung resection.

      Results
      The TNM pathological stages of pleomorphic carcinoma were classified as follows: 7 (11.2%) cases with stage IA, 15 (24.1%) with stage IB, 3 (4.8%) with stage IIA, 20 (32.2%) with stage IIB, 10 (16.1%) with stage IIIA, 4 (6.4%) with stage IIIB, 3 (4.8%) with stage IV. The 5-year disease-specific survival rate after pulmonary resection was 68.3%. Forty-seven (75.8%) tumors contained at least 10% spindle and/or giant cells, and the other fifteen (24.1%) consisted entirely of spindle cells and giant cells. An adenocarcinoma component was found in 34 (54.8%) cases, a squamous cell carcinoma component in 7 (11.2%) cases, an adenosquamous cell carcinoma component in 4 (6.4%) cases, and a large cell carcinoma component in 2 (3.2%) cases. The pleomorphic carcinoma patients were divided into five groups according to the predominant epithelial component, and there were no significant differences in the disease-specific survival rate between the groups. Those with a predominance of spindle-cell or giant-cell components also showed no difference in disease-specific survival. Nuclear ZEB-1 expression was positive only in the pleomorphic component. Diffuse expression of ZEB1 was found in seven patients. Snail and Twist were positive in epithelial and pleomorphic components, but at various levels; however, expression tended to be higher in the pleomorphic component. Thirteen patients had diffuse expression of Snail, and eight had diffuse expression of Twist. Using multivariate analysis, lymph node metastasis, pleural invasion, and diffuse expression of ZEB1 in the pleomorphic component all predicted poorer disease-specific survival (p=0.007, 0.022, and 0.016, respectively). Nuclear ZEB1 expression was higher in cases with lymphatic permeation compared to those without lymphatic permeation, but this association was not statistically significant (p=0.107).

      Conclusion
      Several unfavorable prognostic factors for pulmonary plemorphic carcinoma have been reported, such as massive necrosis, TNM stages, lymph nodes metastasis, pleural invasion, and complete resection. Our current study showed that lymph node metastasis, pleural invasion, and diffuse expression of ZEB1 in the pleomorphic component suggested a worse prognosis for pulmonary pleomorphic carcinoma. ZEB1 may promote the aggressiveness and invasiveness of this malignant tumor.