Methylation Levels of LINE-1 As a Useful Marker for Venous Invasion in Both FFPE and Frozen Tumor Tissues of Gastric Cancer.
Author(s): Min J, Choi B, Han TS, Lee HJ, Kong SH, Suh YS, Kim TH, Choe HN, Kim WH, Hur K, Yang HK
Publication: Mol Cells, 2017, Vol. 40(5), Page 346-354
PubMed ID: 28535662 PubMed Review Paper? No
Purpose of Paper
This paper compared long interspersed nuclear element-1 (LINE-1) methylation in formalin-fixed paraffin-embedded (FFPE) and frozen gastric cancer specimens, and investigated if LINE-1 methylation was associated with clinical features or patient characteristics.
Conclusion of Paper
LINE-1 methylation levels were significantly lower in frozen than FFPE specimens and while CpG3 was hypomethylated compared to other sites in frozen specimens, it was hypermethylated compared to CpG1 and CpG2 in FFPE specimens. In frozen specimens, LINE-1 methylation was significantly different in cancer specimens compared to normal mucosa specimens, in males compared to females, in differentiated versus undifferentiated tumors, and in the presence of lymphatic or venous invasion. In FFPE specimens, significant differences in LINE-1 methylation were limited to the presence of venous invasion, the location of the tumor within the stomach, and patient age. LINE-1 methylation was not significantly affected by Lauren classification, perineural invasion, tumor node metastasis (TNM) stage, or microsatellite instability in either frozen or FFPE specimens.
Studies
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Study Purpose
This study compared LINE-1 hypomethylation in 25 FFPE and 41 frozen gastric cancer specimens and investigated if LINE-1 methylation was associated with clinical features or patient characteristics. Case-matched normal mucosa specimens were also available for the 41 gastric cancer specimens that were snap-frozen in liquid nitrogen, but normal adjacent sampless were not available for FFPE specimens. Details of specimen procurement and processing were not provided. FFPE slides (unspecified number and thickness) were deparaffinized in xylene and DNA was extracted using the QIAamp DNA Mini Kit. DNA was extracted from frozen specimens using QuickGene DNA tissue kit with QG-Mini80. DNA was bisulfate modified with the EpiTect Bisulfite Kit and then stored at -20˚C until pyrosequencing of LINE-1.
Summary of Findings:
LINE-1 methylation levels were significantly lower in frozen than FFPE specimens. CpG3 was hypomethylated compared to CpG1 (P=0.031), CpG2 (P<0.001), and CpG4 (P<0.001) in frozen specimens. However in FFPE specimens, CpG3 was hypermethylated compared to CpG1 (P=0.031) and CpG2 (P<0.001) while CpG4 was hypermethylated compared to all other sites (P<0.001). In frozen specimens, LINE-1 hypomethylation was observed in gastric cancer specimens in comparison to normal mucosa specimens (P<0.001 all four sites), in males compared to females (P=0.021 for CpG1, P=0.007 for CpG2, P<0.001 for CpG3 and P=0.008 for CpG4), in differentiated versus undifferentiated tumors (P=0.017 for CpG1, P=0.012 for CpG2, P=0.036 for CpG3, and P=0.021 for CpG4), and with the presence of lymphatic invasion (P=0.041 for CpG1, P=0.020 for CpG2, P=0.020 for CpG3, and P=0.029 for CpG4) or venous invasion (P=0.037 for CpG1, P=0.016 for CpG2, P=0.016 for CpG3 and P=0.012 for CpG4). In FFPE specimens, significant effects were limited to the presence of venous invasion (not significant for CpG1, P=0.042 for CpG2, P=0.007 for CpG3 and P=0.025for CpG4). In addition, LINE-1 hypomethylation of FFPE specimens was significantly affected by location of the tumor in the upper versus middle third of the stomach (P=0.014 for CpG1, not significant for other sites) and middle versus lower third of the stomach (P=0.002 for CpG1, P=0.008 for CpG2, not significant for CpG3 or CpG4), and patient age (≤57 versus >57 years, P=0.043 for CpG1, not significant for other sites). LINE-1 in frozen specimens was not significantly affected by patient age (≤61 versus >61), Lauren classification, perineural invasion, TNM stage, or microsatellite instability. LINE-1 methylation in FFPE specimens was comparable in high tumor (T) stage without lymph node (LN) metastasis (T3/4N0) and low T stage with more than seven regional LN metastasis (T1N3) tumors and was unaffected by patient sex, tumor differentiation, Lauren classification, lymphatic invasion, perineural invasion, TNM stage, or microsatellite invasion.
Biospecimens
Preservative Types
- Frozen
- Formalin
Diagnoses:
- Neoplastic - Normal Adjacent
- Neoplastic - Carcinoma
Platform:
Analyte Technology Platform DNA Bisulfite conversion assay DNA Next generation sequencing Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Preaquisition Patient gender Female
Male
Preaquisition Patient age ≤61
>61
≤57
>57
Biospecimen Preservation Type of fixation/preservation Snap frozen
Formalin (buffered)
Preaquisition Prognostic factor Differentiated versus undifferentiated tumor
Presence or absence of lymphatic invasion
Presence or absence of venous invasion
Presence or absence of perineural invasion
Stage II
Stage III
Stage IV
Microsatellite stable or instable
Preaquisition Diagnosis/ patient condition Tumor
Normal