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Akira Tangoku



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    MA20 - Thymic Tumors: From Molecular to Clinical Results and New Challenges in Other Rare Thoracic Tumors (ID 149)

    • Event: WCLC 2019
    • Type: Mini Oral Session
    • Track: Thymoma/Other Thoracic Malignancies
    • Presentations: 2
    • Now Available
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      MA20.02 - GAD1 Expression and Its Methylation Become Indicators of Malignant Behavior in Thymic Epithelial Tumor (Now Available) (ID 2370)

      11:30 - 13:00  |  Author(s): Akira Tangoku

      • Abstract
      • Presentation
      • Slides

      Background

      Genome-wide screening for aberrantly methylated CpG islands was performed in 7 thymic carcinoma (TC) samples and 8 type-B3 thymoma samples using HumanMethylation450 K BeadChip (Illumina, Santa Clara, CA, USA) analysis. We identified 93 genes as commonly hypermethylated in TC comparing to type-B3 thymoma. GAD1 (glutamic acid decarboxylase 1) was one of the most significant hypermethylated genes in TC. GAD1 catalyzes the production ofγ-aminobutyric acid (GABA). Some recent reports showed that GAD1 expression is significantly increased in neoplastic tissues. However, the underlying mechanism of elevated GAD1 remains elusive. In this study, we examine mRNA and protein expressions and DNA methylation of GAD1 in thymic epithelial tumors (TETs).

      Method

      In total, 95 thymic tumor samples (A; 9, AB; 11, B1; 19, B2; 21, B3; 14, carcinoma; 21) and 22 paired normal tissues were obtained from patients with histologically proven TET, who underwent surgery at the Tokushima University Hospital (Tokushima, Japan) between 1990 and 2016. The methylation status of thymic epithelial tumor samples was validated by pyrosequencing. The expression status was analyzed by quantitative polymerase chain reaction (qPCR) and immunohistochemistry (IHC).

      Result

      The previous study (Oncogene 2015, 1–14) showed that the key locus responsible for GAD1 reactivation was mapped to DNA methylation-sensitive CTCF-binding site (CTCF-BS3) within the third intron of GAD1. We targeted this region for pyrosequencing, which confirmed that DNA methylation of GAD1 in TC was significantly higher than in thymoma (32.8% versus 4.0%, P<0.001). It revealed a high degree of both sensitivity and specificity for discriminating TCs and thymomas (AUC=0.936). There was no significant difference in the methylation rate between thymoma and normal thymus (P = 0.917); however, the DNA methylation rate in TC was higher than in normal thymus (P=0.015). qPCR revealed that GAD1 mRNA expression levels in TC were higher than in thymoma (qPCR; 2.03 vs 0.38, P < 0.001). IHC showed statically different GAD1 expression between TC and thymoma (93.75% vs 28.98%, P < 0.001). There was a slight positive correlation between the mRNA expression levels and methylation levels (Spearman’s rank correlation coefficient, ρ = 0.427); however, no differences of DNA methylation and expression of GAD1 was observed among subtype of thymoma according to WHO histologic classification.

      Conclusion

      TC had frequent DNA methylation of CTCF-binding site 3 in GAD1, and high levels of mRNA and protein of GAD1. GAD1 may resent an epigenetic therapeutic target in TC.

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      MA20.03 - DNA Methylation of MT1A and NPTX2 Genes Predict Malignant Behavior of Thymic Epithelial Tumors (Now Available) (ID 3031)

      11:30 - 13:00  |  Author(s): Akira Tangoku

      • Abstract
      • Presentation
      • Slides

      Background

      Our previous studies showed that DNA methylation of cancer-related genes, such as DAP-K, p-16, MGMT, HPP1 was higher in thymic carcinoma (TC) than in thymoma (Lung Cancer 64:155-, 2009, 83:279-,2013). Genome-wide screening for aberrantly methylated CpG islands was performed in 7 TC samples and 8 type-B3 thymoma samples using HumanMethylation 450 K BeadChip (Illumina, Santa Clara, CA, USA) analysis. We identified 93 genes as commonly hypermethylated in TC comparing to B3 thymoma.We chose 2 candidate cancer-related genes; MT1A and NPTX2. MT1A which is an isozyme of metallothioneins is related to metabolism of trace elements, such as zinc, copper. DNA methylation of MT1A was higher in malignant melanoma than in normal melanocytes. NPTX2 has studied as synapse-related proteins. DNA methylation of NPTX2 was higher in some cancers than in normal tissues.

      Method

      In total, 48 thymic tumor samples (thymoma;31, carcinoma; 17) and 22 paired normal tissues were obtained from patients with histologically proven thymic epithelial tumor (TET), who underwent surgery at Tokushima University Hospital (Tokushima, Japan) between 1990 and 2016. The methylation status of TET samples was validated by pyrosequencing. The expression of mRNA in MT1A and NPTX2 genes was validated by RT-PCR (SYBR® Green method).

      Result

      DNA methylation of MT1A gene was significantly higher in TC compared to thymoma (26.4% versus 9.5%, P<0.01). It revealed high degrees of sensitivity and specificity for discriminating TCs and thymomas (AUC=0.903). Although DNA methylation was significantly higher in TC than in normal thymus, there was no significant difference between DNA methylation of thymoma and normal thymus. No differences of MT1A DNA methylation was observed among subtype of thymoma according to WHO histologic classification. In MT1A gene, there was no correlation has observed between DNA methylation and mRNA expression.

      On the other hand, NPTX2 gene was also significantly higher in TC compared to thymoma (38.0% vs 17.5%, P<0.01). It revealed high degrees of sensitivity and specificity for discriminating TCs and thymomas (AUC=0.765). Although DNA methylation was significantly higher in TC than in normal thymus, there was no significant difference between DNA methylation of thymoma and normal thymus. No differences of NPTX2 DNA methylation was observed among subtype of thymoma according to WHO histologic classification. There was no correlation has observed between DNA methylation and mRNA expression in all TETs, there was a reverse correlation in thymic carcinomas. The mean value of the frequency of the DNA methylation of was MT1A and NPTX2 divided into higher and lower level groups. A significant difference was observed in the relapse-free survival between the higher and lower level groups (p=0.015, p=0.042).

      Conclusion

      DNA methylation of MT1A and NPTX2 was significantly higher in TC compared to thymoma and normal thymus.

      Epigenetic alteration may be related to progression and malignancy in TET. 

      In NPTX2 gene, there was a reverse correlation between DNA methylation and mRNA expression in thymic carcinomas. It may act as tumor suppressor gene.

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