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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
- Moderators:Kazuya Kondo, Edith Michelle Marom
- Coordinates: 9/10/2019, 11:30 - 13:00, San Francisco (2009)
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): Hiromitsu Takizawa
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): Hiromitsu Takizawa
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.
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.
P2.03 - Biology (ID 162)
- Event: WCLC 2019
- Type: Poster Viewing in the Exhibit Hall
- Track: Biology
- Presentations: 1
- Now Available
- Coordinates: 9/09/2019, 10:15 - 18:15, Exhibit Hall
P2.03-22 - Chromate Exposure Induces DNA Hypermethylation of the Mismatch Repair Gene MLH1 in Lung Cancer (Now Available) (ID 2156)
10:15 - 18:15 | Author(s): Hiromitsu Takizawa
Hexavalent chromium is recognized as a human carcinogen. To elucidate the role of chromate on carcinogenesis, we have investigated molecular features of LC from chromate workers (chromate LC). Chromate LC frequently had the microsatellite instability (MSI), and that the MSI was associated with repression of MLH1, which is one of the essential DNA mismatch repair (MMR) proteins. In the present study, we investigated methylation status of the promoter region of MLH1 determined quantitatively by bisulfite-pyrosequencing in the paired tumorous/ non-tumorous sample sets of chromate and non-chromate LCs. Moreover, we analyzed three DNA double-strand break (DSB) repair genes (MRE11, RAD50, and DNA-PKcs) as possible targets of MSI by fragment length polymorphism analysis.Method
Thirty-two lung tumor samples were obtained from chromate workers with LC during surgery or at autopsy at 5 hospitals between August 1975 and October 1997. Thirty-one tumors were obtained from LC patients without chromate exposure during surgery at Tokushima University Hospital as a control group (non-chromate LC).
DNA was extracted and bisulfite conversion of DNA was conducted using the EpiTect Bisulfite Kit (QIAGEN). PCR primers and sequencing primer for quantification of methylation level in region -209 nucleotides (nt) to -181 nt from the transcription start site in the MLH1. Pyrosequencing of 5 CpG sites in MLH1 was performed with sequencing primers using a PyroMark 24 Pyrosequencing System, version 2.0.6 (QIAGEN). Regions encompassing mononucleotide repeated sequences of genes were amplified using nested-PCR procedure. Fragments were separated by automated capillary electrophoresis in an ABI Prism 3130/3130xl Genetic Analyzer (Applied Biosystems) and electropherograms were analyzed using the GeneMapper software (Applied Biosystems).Result
The mean methylation level of tumorous tissue was 21.1±15.7% and was significantly higher than that of non-tumorous tissue, 10.9±9.4% (P = 0.004) in chromate LC. In non-chromate LC, there was no significant difference between tumorous and non-tumorous tissues: 3.9±5.1% in tumorous tissue versus 4.9±4.1% in non-tumorous tissue. The mean methylation level of tumorous tissues was significantly higher in chromate LC than in non-chromate LC (P < 0.001). The mean methylation level of non-tumorous tissues tended to be higher in the chromate LC than in non-chromate LC (P = 0.062). There was a significant positive correlation between the methylation level and chromate exposure period in tumorous tissue of chromate LC (r = 0.481, P = 0.017). The methylation level was significantly higher in LCs with reduced expression of MLH1 than in LCs with normal expression of MLH1(P = 0.019). The incidence of mutations at mononucleotide repeats was observed in 50.0% of chromate LC and 28.6% of non-chromate LC in MRE11, and 17.4% of chromate LC and 0.0% of non-chromate LC in RAD50, and in 53.8% of chromate LC and 46.2% of non-chromate LC in DNA-PKcs. The incidence of mutation of mononucleotide tended to be higher in chromate LC than in non-chromate LC in MRE11. In RAD50, the mutation was significantly higher frequency in chromate LC than in non-chromate LC.Conclusion
These results suggest that chromate exposure might induce MLH1 hypermethylation in LC, which is possible cause of carcinogenesis.