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Assessment of LINE-1 and Alu hypomethylation level in colorectal cancer and precancerous lesion to disclose their clinicopathologic and prognostic implication

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의과대학 의학과
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서울대학교 대학원
Colorecral cancerAdjuvant therapyLong interspersed element-1 (LINE-1)AluDNA methylationMSIpyrosequencingprognosis
학위논문 (박사)-- 서울대학교 대학원 : 의학과 병리학전공, 2015. 8. 강경훈.
Introduction: Recent studies have tried to understand the comprehensive tumorigenesis of Colorectal cancers (CRCs) which include the genetic and epigenetic pathways. Even though the epigenetic pathways are more actively studied than before, the researches related to the global DNA hypomethylation in CRCs has not been done much so far.
Genomic hypomethylation in carcinomas is related with the hypomethylation of repetitive DNA elements which comprise 45% of the human genome. In association with tumorigenesis, CpG sites in the repetitive DNA elements tend to undergo demethylation, leading to generalized diffuse genomic hypomethylation and consequentially genomic instability, activation of transposable element and oncogene. Recent studies have demonstrated that a higher degree of Long interspersed nuclear element-1 (LINE-1) hypomethylation is related to poor prognosis of CRCs.
In this study, I have analyzed LINE-1 and Alu methylation level in Korean CRCs and precancerous lesions based on the results of previous studies. In particular, the relation between LINE-1 and Alu methylation and clinical prognosis in MSI positive CRC, and the relation between LINE-1 hypomethylation and clinical prognosis in stage III or high-risk stage II colorectal cancers treated with adjuvant combination therapy of 5-fluorouracil, leucovorin and oxaliplatin (FOLFOX) were elucidated.

Methods: Analysis was done in 767 CRCs, and of those 207 Microsatellite instability positive (MSI+) CRCs and 154 precancerous lesions for their methylation levels of LINE-1 and Alu repetitive DNA elements using pyrosequencing assay. The correlation of the tumoral LINE-1 and Alu methylation change with clinicopathological information including survival data was analyzed.
I also analyzed 427 resected stage III or high-risk stage II CRCs for their status in L1 methylation, CpG island methylator phenotype (CIMP), microsatellite instability (MSI), and KRAS/BRAF mutation. Four CpG sites were assayed for L1 methylation using pyrosequencing. The average methylation level of L1 methylation on combined two CpG sites (CpG sites 2 and 3) was obtained for each CRC case.

Results: The hypomethylation of LINE-1 and Alu did not have the value as a prognostic factor when the analysis was done for the whole colorectal cancer samples. However in MSI+ CRCs, univariate survival analysis showed that low Alu methylation status (<18.60%) and low LINE-1 methylation status (<53.00%) were significantly associated with shorter overall survival time (log-rank test, P = 0.009 and P < 0.001, respectively). Multivariate analysis using nine parameters (Alu methylation status, LINE-1 methylation status, age, disease stage [tumor, node, metastasis staging system], differentiation, Crohn-like lymphoid reaction, KRAS/BRAF mutation status, CIMP status, and peritumoral lymphocytic infiltration), which were significantly prognostic in MSI+ CRCs, revealed that low LINE-1 methylation status was an independent prognostic factor of MSI+ CRCs (P = 0.009), whereas low Alu methylation status was not.
L1 hypomethylation was closely associated with nodal metastasis but did not show any association with age of onset, gender, tumor subsite, tumor differentiation, mucinous histology, lymphatic emboli, venous invasion, perineural invasion, T stage, and KRAS/BRAF mutation in patients with stage III or high-risk stage II CRCs treated with adjuvant FOLFOX. Multivariate analysis revealed that L1 hypomethylation as well as mucinous histology, T stage, N stage, lymphatic emboli, and KRAS mutation was an independent prognostic parameter heralding poor prognosis in stage III or high-risk stage II CRCs treated with adjuvant FOLFOX.

Conclusions: Clinical outcomes of MSI+ CRCs depend on LINE-1 methylation status, suggesting that lower LINE-1 methylation status serves as a significant prognostic parameter of adverse prognosis in MSI+ CRCs.
Tumoral L1 hypomethylation correlated independently with poor prognosis in patients with stage III or high-risk CRCs treated with adjuvant FOLFOX, and could be utilized as biomarker identifying a subset which might be resistant against adjuvant FOLFOX.
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