NIH, National Cancer Institute, Division of Cancer Treatment and Diagnosis (DCTD) NIH - National Institutes of Health National Cancer Institute DCTD - Division of Cancer Treatment and Diagnosis

Methodological and Biological Factors Influencing Global DNA Methylation Results Measured by LINE-1 Pyrosequencing Assay in Colorectal Tissue and Liquid Biopsy Samples.

Author(s): Szigeti KA, Barták BK, Nagy ZB, Zsigrai S, Papp M, Márkus E, Igaz P, Takács I, Molnár B, Kalmár A

Publication: Int J Mol Sci, 2022, Vol. 23, Page

PubMed ID: 36232908 PubMed Review Paper? No

Purpose of Paper

This paper compared LINE-1 methylation in matched frozen and formalin-fixed paraffin-embedded (FFPE) tissue; between tissue either collected endoscopically during a colonoscopy or collected surgically; in matched EDTA buffy coat and Roche plasma specimens; in buffy coats subjected to a 1 or 2 h proteinase K digestion; in buffy coat from blood stored at room temperature (Roche and EDTA tubes) or 4°C (EDTA tubes) for up to 6 h; in blood collected before, during, and after exercise; and in tissue and blood specimens from healthy volunteers and those with colorectal tumors. The potential influence of patient age, diagnosis, and MTHFR mutations on LINE-1 methylation of tissue was also investigated.  

Conclusion of Paper

LINE-1 methylation was significantly lower in FFPE than matched frozen colorectal tissue,  higher when extracted DNA was stored at -20°C for 2 years compared to 2 days, and a trend toward lower LINE-1 methylation was observed when the specimen was collected during surgery compared to when collected by endoscopy. While LINE-1 methylation was not correlated with patient age and was comparable in tissue specimens from men <40 and >40 years of age, it was higher in tissue specimens from females <40 years of age than >40 years of age. LINE-1 methylation was significantly higher in plasma isolated from Roche cell-free DNA (cfDNA) tubes than when DNA was extracted from EDTA buffy coats and significantly lower in buffy coat specimens subjected to a 1 h proteinase K step than in standard buffy coat specimens. LINE-1 methylation of DNA from buffy coat was not significantly affected by storage of EDTA or Roche blood specimens at room temperature for up to 6 h or storage of EDTA blood at 4°C for up to 6 h. Only non-significant changes in LINE-1 methylation were observed in cfDNA from blood collected during and following exercise compared to specimens collected during the rest period (post-exercise). LINE-1 methylation patterns in tissue specimens differed between non-tumorous and tumorous colorectal specimens but were similar among buffy coats from healthy controls and patients with colorectal cancers.

Studies

  1. Study Purpose

    This study compared LINE-1 methylation in matched frozen and FFPE specimens, between specimens collected endoscopically during a colonoscopy and those collected during surgical resection, and before and after storage of extracted DNA at -20°C for 2 days or 2 years. The potential influence of patient age, diagnosis, and MTHFR mutations on LINE-1 methylation was also investigated.  Tissue specimens were collected during colonoscopy and included 45 healthy, 23 normal adjacent tissue specimens to a colorectal adenoma, 37 colorectal adenomas, 24 normal adjacent tissues to a colorectal carcinoma, 36 colorectal carcinomas (CRC), and 15 inflammatory bowel disease specimens that were flash frozen; tissue specimens that included 9 healthy tissues, 12 adenomas, and 4 colorectal carcinoma specimens that were formalin-fixed and paraffin-embedded were also analyzed. The effects of collection method (surgery versus endoscopy) was investigated using 36 CRC specimens and 24 normal tissues adjacent to a colorectal carcinoma that were obtained endoscopically during colonoscopy and 21 CRC normal adjacent tissues to a CRC and 21 CRC specimens collected during the subsequent resection. No further details of specimen processing were provided. DNA was extracted from frozen and FFPE specimens using the High Pure PCR Template Preparation Kit and quantified by spectrophotometer using the Qubit dsDNA HS Assay Kit. DNA was bisulfite converted using the Zymo EZ DNA Methylation Direct Kit, and LINE-1 methylation was assessed using the Pyromark Q24 CpG LINE-1 Kit. To investigate the effect of storing extracted DNA, DNA was stored at -20°C and reanalyzed after 2 days and 2 years.

    Summary of Findings:

    LINE-1 methylation was significantly lower in FFPE than matched frozen colorectal tissue (tumorous and non-tumorous) specimens (73.0%±5.3% versus 76.1%±2.8%, P<0.01), and a trend toward lower LINE-1 methylation was observed when the specimen was collected via surgery compared to endoscopy (71.4%±6.5% versus 72.1%±6.9%). Following storage of extracted DNA from tissue specimens at -20°C for 2 years, LINE-1 methylation increased 3.2% (P<0.01), but only a non-significant trend toward decreased methylation was observed following storage at -20°C for 2 days, and there was no effect of storing bisulfite converted methylated and unmethylated DNA standards for up to 2 years. LINE-1 methylation was not correlated with patient age (ρ=-0.18, P=0.25) and was comparable in tissue specimens from men <40 and >40 years of age but was higher in tissue specimens from females <40 years of age than >40 years of age (78.1% versus 76.8%, P<0.05).  LINE-1 methylation was significantly elevated in CpG position 1 compared to mean methylation (P<0.01) and CpG positions 2 and 3 (P<0.01, both) when assessed in DNA from biopsies of non-tumorous colorectal specimens (healthy, normal adjacent to a colorectal carcinoma, normal adjacent to a colorectal adenoma, and inflammatory bowel disease specimens), but LINE-1 methylation of all CpG positions were comparable to mean methylation in tumorous biopsy specimens.   LINE-1 methylation was lower in adenoma specimens that were heterozygous for either of the MTHFR mutations (C677T or A1298C) than in wildtype adenoma specimens (P<0.05, both), but no difference in LINE-1 methylation was observed between MTHFR wildtype and heterozygous healthy tissue or colorectal carcinoma specimens.

    Biospecimens
    Preservative Types
    • Frozen
    • Formalin
    Diagnoses:
    • Neoplastic - Carcinoma
    • Neoplastic - Normal Adjacent
    • Irritable Bowel Syndrome
    • Normal
    • Neoplastic - Benign
    Platform:
    AnalyteTechnology Platform
    DNA Real-time qPCR
    DNA Bisulfite conversion assay
    Pre-analytical Factors:
    ClassificationPre-analytical FactorValue(s)
    Preaquisition Patient age <40 years
    1-78 years
    >40 years
    Storage Storage duration 0 days
    2 days
    2 years
    Preaquisition Surgical procedure type Endoscopic
    Surgical
    Preaquisition Diagnosis/ patient condition Healthy
    Colorectal adenoma
    Colorectal carcinoma
    Inflammatory bowel disease
  2. Study Purpose

    This study compared LINE-1 methylation in matched EDTA buffy coat and Roche plasma specimens; in buffy coats subjected to a 1 or 2 h proteinase K digestion; in buffy coat from blood stored at room temperature (Roche and EDTA tubes) or 4°C (EDTA tubes) for up to 6 h; in blood collected before, during, and after exercise; and in specimens from healthy volunteers and those with colorectal tumors. Blood was collected from 19 healthy volunteers, 10 patients with colorectal adenoma, and 10 patients with colorectal carcinoma into K3EDTA Vacuettes; blood was also collected from 4 healthy volunteers into Roche Cell-Free DNA Collection Tubes. Additionally, K3EDTA blood was collected from five healthy athletes before, during, and after a twenty-five minute run on a treadmill.  To investigate the effects of storage, blood was stored in EDTA tubes for 0, 3, and 6 h at room temperature or 4°C and Roche tubes were stored for 0, 3, and 6 h at room temperature before processing. Buffy coat was isolated by centrifugation at 1350 x g for 12 min with a 0, 1, or 2 h proteinase K digestion step. Plasma was isolated by dual centrifugation at 1350 x g for 12 min. DNA was extracted from buffy coat using the High Pure PCR Template Preparation Kit and from plasma using the Quick-cfDNA Serum & Plasma Kit. DNA was quantified by spectrophotometer using the Qubit dsDNA HS Assay Kit. DNA was bisulfite converted using the Zymo EZ DNA Methylation Direct Kit, and LINE-1 methylation was assessed using the Pyromark Q24 CpG LINE-1 Kit.

    Summary of Findings:

    LINE-1 methylation was significantly higher in plasma isolated from blood collected in Roche cfDNA tubes than in DNA from EDTA buffy coats (+2.1%, P<0.05). LINE -1 methylation was significantly lower in buffy coat specimens subjected to a 1 h proteinase K step than in standard buffy coat specimens (-0.4%, P<0.05), but no difference was noted when the proteinase K digestion was extended to 2 h. LINE-1 methylation of DNA from buffy coat was not significantly affected by storage of EDTA or Roche blood at room temperature or 4°C for up to 6 h. Similarly, LINE-1 methylation of cfDNA was nonsignificantly reduced after blood was stored in EDTA tubes for up to 6 h at either temperature (-0.1 to -1.6%) or in Roche tubes for 6 h at room temperature (-0.7%), but increased nonsignificantly when blood was stored in Roche tubes for up to 3 h at room temperature (1.4%).  A nonsignificant decrease in LINE-1 methylation was observed in cfDNA isolated from blood collected during and following exercise compared to specimens collected during the rest period (79.7% and 79.4%, respectively, versus 80.7%).  In contrast, LINE-1 methylation was elevated at CpG position 1 compared to mean methylation, position 2, and position 3 (P<0.01, all) in DNA from buffy coat of both healthy volunteers and patients with colorectal tumors. LINE-1 methylation was lower at CpG position 2 and 3 compared to mean methylation (P<0.01, all) in DNA from buffy coat isolated from both healthy volunteers and patients with colorectal tumors.

    Biospecimens
    Preservative Types
    • Streck/BCT
    • None (Fresh)
    Diagnoses:
    • Neoplastic - Carcinoma
    • Normal
    • Neoplastic - Benign
    Platform:
    AnalyteTechnology Platform
    DNA Real-time qPCR
    DNA Bisulfite conversion assay
    Pre-analytical Factors:
    ClassificationPre-analytical FactorValue(s)
    Preaquisition Diagnosis/ patient condition Healthy
    Colorectal tumors
    Biospecimen Acquisition Type of collection container/solution K3EDTA Vacuette
    Roche Cell-Free DNA Collection Tube
    Biospecimen Acquisition Time of biospecimen collection Before exercise
    During exercise
    After exercise
    Biospecimen Aliquots and Components Blood and blood products Buffy coat
    Plasma
    Storage Storage duration 0 h
    3 h
    6 h
    Storage Storage temperature 4°C
    Room temperature
    Analyte Extraction and Purification Protein digestion None
    1 h
    2 h
    Biospecimen Aliquots and Components Centrifugation Centrifugation delays investigated

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