Improved results of LINE-1 methylation analysis in formalin-fixed, paraffin-embedded tissues with the application of a heating step during the DNA extraction process

Wen, Xianyu,Jeong, Seorin,Kim, Younghoon,Bae, Jeong Mo,Cho, Nam Yun,Kim, Jung Ho,Kang, Gyeong Hoon BioMed Central 2017 CLINICAL EPIGENETICS Vol.9 No.-

<P><B>Background</B></P><P>Formalin-fixed, paraffin-embedded (FFPE) tissues are important resources for profiling DNA methylation changes and for studying a variety of diseases. However, formalin fixation introduces inter-strand crosslinking, which might cause incomplete bisulfite conversion of unmethylated cytosines, which might lead to falsely elevated measurements of methylation levels in pyrosequencing assays. Long interspersed nucleotide element-1 (LINE-1) is a major constituent of repetitive transposable DNA elements, and its methylation is referred to correlates with global DNA methylation. To identify whether formalin fixation might impact the measured values of methylation in LINE-1 repetitive elements and whether prolonged heat-induced denaturation of DNA might reduce the artificial increases in measured values caused by formalin fixation, we analyzed paired fresh-frozen (FF) and FFPE xenograft tissue samples for their methylation levels in LINE-1 using a pyrosequencing assay. To further confirm the effect of a heating step in the measurement of LINE-1 or single gene methylation levels, we analyzed FFPE tissue samples of gastric cancer and colorectal cancer for their methylation status in LINE-1 and eight single genes, respectively.</P><P><B>Results</B></P><P>Formalin fixation led to an increase in the measured values of LINE-1 methylation regardless of the duration of fixation. Prolonged heating of the DNA at 95 °C for 30 min before bisulfite conversion was found (1) to decrease the discrepancy in the measured values between the paired FF and FFPE tissue samples, (2) to decrease the standard deviation of the measured value of LINE-1 methylation levels in FFPE tissue samples of gastric cancer, and (3) to improve the performance in the measurement of single gene methylation levels in FFPE tissue samples of colorectal cancer.</P><P><B>Conclusions</B></P><P>Formalin fixation leads to artificial increases in the measured values of LINE-1 methylation, and the application of prolonged heating of DNA samples decreases the discrepancy in the measured values of LINE-1 methylation between paired FF and FFPE tissue samples. The application of prolonged heating of DNA samples improves bisulfite conversion-based measurement of LINE-1 or single gene methylation levels in FFPE tissue samples.</P><P><B>Electronic supplementary material</B></P><P>The online version of this article (doi:10.1186/s13148-016-0308-0) contains supplementary material, which is available to authorized users.</P>

Ferroelectric Properties of MBi 4 Ti 4 O 15 (M = Sr, Pb, Ca) Thin Films

XIANYU, WEN,WON, TAE-YOUNG,LEE, WAN Taylor Francis 2004 Integrated ferroelectrics Vol.65 No.1

<P>Among the ferroelectric bismuth layered perovskites belonging to Aurivillius family, the MBi 4 Ti 4 O 15 (MBT15, M=Sr, Ca, Pb) thin films have been fabricated by a chemical solution deposition technique, and their structures and ferroelectric properties were investigated in this work. The SrBi 4 Ti 4 O 15 (SBT15) film fabricated on IrO 2 presented the highest remanent polarization (P r ) among the films in this series. It demonstrated a saturated hysteresis loop at 5 V with P r of 19&mgr;C/cm 2 and coercive field (P s ) of 116 kV/cm. The PbBi 4 Ti 4 O 15 (PBT15) thin films were selectively controlled in c-axis and off-c-axis orientation on Pt layer by adjusting the annealing condition. The off-c-axis oriented PbBi 4 Ti 4 O 15 film demonstrated considerably higher P r (8.7&mgr;C/cm 2 ) than that of c-axis oriented film (3.7&mgr;C/cm 2 ). Regardless of grain orientation, however, PBT15 films were not fatigued up to 10 10 cycles under 9V application. The CaBi 4 Ti 4 O 15 (CBT15) thin films were also produced on Pt and IrO 2 electrode, respectively, by controlling the annealing condition. The CBT15 film on IrO 2 presented higher P r (∼⃒10.8&mgr;C/cm 2 ) than that of film (∼⃒6.7&mgr;C/cm 2 ) on Pt.†Present address: Samsung Advanced Institute of Technology, P.O. Box 111, Suwon 440-600, Korea</P>

Fibroblast Growth Factor Receptor 1 (FGFR1) Amplification Detected by Droplet Digital Polymerase Chain Reaction (ddPCR) Is a Prognostic Factor in Colorectal Cancers

배정모,Xianyu Wen,김태신,곽윤진,조남윤,이혜승,강경훈 대한암학회 2020 Cancer Research and Treatment Vol.52 No.1

Purpose The purpose of this study was to reveal the clinicopathological characteristics and prognostic implications associated with fibroblast growth factor receptor 1 (FGFR1) amplification in colorectal cancers (CRCs). Materials and Methods We measured the copy number of FGFR1 by droplet digital polymerase chain reaction (ddPCR), and analyzed the FGFR1 expression by immunohistochemistry, in 764 surgically resected CRCs (SNUH2007 dataset, 384 CRCs; SNUH Folfox dataset, 380 CRCs). Results CRCs with ! 3.3 copies of the FGFR1 gene were classified as FGFR1 amplified. FGFR1 amplification was found in 10 of the 384 CRCs (2.6%) in the SNUH2007 dataset, and in 28 of the 380 CRCs (7.4%) in the SNUH Folfox dataset. In the SNUH2007 dataset, there was no association between the FGFR1 copy number status and sex, gross appearance, stage, or differentiation. High FGFR1 expression was associated with female sex and KRASmutation. At the molecular level, FGFR1 amplification was mutually exclusive with BRAF mutation, microsatellite instability, and MLH1 methylation, in both SNUH2007 and SNUH Folfox datasets. Survival analysis revealed that FGFR1 amplification was associated with significantly worse clinical outcome compared with no FGFR1 amplification, in both SNUH2007 and SNUH Folfox datasets. Within the SNUH2007 dataset, CRC patients with high FGFR1 expression had an inferior progression-free survival compared with those with low FGFR1 expression. The FGFR inhibitor, PD173074, repressed the proliferation of a CRC cell line overexpressing FGFR1, but not of cells with FGFR1 amplification. Conclusion FGFR1 amplification measured by ddPCR can be a prognostic indicator of poor clinical outcome in patients with CRCs.

Associations and prognostic implications of Eastern Cooperative Oncology Group performance status and tumoral <i>LINE-1</i> methylation status in stage III colon cancer patients

Chen, Duo,Wen, Xianyu,Song, Young Seok,Rhee, Ye-Young,Lee, Tae Hun,Cho, Nam Yun,Han, Sae-Won,Kim, Tae-You,Kang, Gyeong Hoon BioMed Central 2016 CLINICAL EPIGENETICS Vol.8 No.-

<P><B>Background</B></P><P>Low methylation status of <I>LINE-1</I> in tumors is associated with poor survival in patients with colon cancer. Eastern Cooperative Oncology Group performance status (ECOG-PS) is a method to assess the functional status of a patient. We retrospectively evaluated the relationship between ECOG-PS and <I>LINE-1</I> methylation in colorectal cancers (CRCs) and their prognostic impact in CRC or colon cancer patients receiving adjuvant 5-fluorouracil/leucovorin/oxaliplatin (FOLFOX).</P><P><B>Results</B></P><P><I>LINE-1</I> methylation and microsatellite instability were analyzed in stage III or high-risk stage II CRCs (<I>n</I> = 336). <I>LINE-1</I> methylation levels were correlated with clinicopathological features, including PS and recurrence-free survival (RFS). The association between the tumoral <I>LINE-1</I> methylation level and PS was observed (OR = 2.56, <I>P</I> < 0.001). Differences in <I>LINE-1</I> methylation levels in cancer tissue between the PS 0 and 1 groups were significant in patients older than 60 years (<I>P</I> = 0.001), the overweight body mass index group (<I>P =</I> 0.005), and the stage III disease group (<I>P</I> = 0.008). Prognostic significances of <I>LINE-1</I> methylation status or combined PS and <I>LINE-1</I> methylation statuses were identified in stage III colon cancers, not in stage III and high-risk stage II CRCs. Low <I>LINE-1</I> methylation status was closely associated with a shorter RFS time. The difference between PS(0)/<I>LINE-1</I>(high) and PS(≥1)/<I>LINE-1</I>(low) was significant, which suggests that colon cancer patients with concurrent PS(≥1)/<I>LINE-1</I> (low) have a higher recurrence rate.</P><P><B>Conclusions</B></P><P>PS was associated with <I>LINE-1</I> methylation in CRC tissue. <I>LINE-1</I> methylation was associated with RFS in stage III colon cancer patients who were treated with adjuvant FOLFOX chemotherapy. Combined PS and <I>LINE-1</I> methylation status might serve as a useful predictor of cancer recurrence.</P><P><B>Electronic supplementary material</B></P><P>The online version of this article (doi:10.1186/s13148-016-0203-8) contains supplementary material, which is available to authorized users.</P>

Distinct clinical outcomes of two CIMP-positive colorectal cancer subtypes based on a revised CIMP classification system

Bae, Jeong Mo,Kim, Jung Ho,Kwak, Yoonjin,Lee, Dae-Won,Cha, Yongjun,Wen, Xianyu,Lee, Tae Hun,Cho, Nam-Yun,Jeong, Seung-Yong,Park, Kyu Joo,Han, Sae Won,Lee, Hye Seung,Kim, Tae-You,Kang, Gyeong Hoon Nature Publishing Group 2017 The British journal of cancer Vol.116 No.8

<P><B>Background:</B></P><P>Colorectal cancer (CRC) is a heterogeneous disease in terms of molecular carcinogenic pathways. Based on recent findings regarding the multiple serrated neoplasia pathway, we revised an eight-marker panel for a new CIMP classification system.</P><P><B>Methods:</B></P><P>1370 patients who received surgical resection for CRCs were classified into three CIMP subtypes (CIMP-N: 0–4 methylated markers, CIMP-P1: 5–6 methylated markers and CIMP-P2: 7–8 methylated markers). Our findings were validated in a separate set of high-risk stage II or stage III CRCs receiving adjuvant fluoropyrimidine plus oxaliplatin (<I>n</I>=950).</P><P><B>Results:</B></P><P>A total of 1287/62/21 CRCs cases were classified as CIMP-N/CIMP-P1/CIMP-P2, respectively. CIMP-N showed male predominance, distal location, lower T, N category and devoid of <I>BRAF</I> mutation, microsatellite instability (MSI) and <I>MLH1</I> methylation. CIMP-P1 showed female predominance, proximal location, advanced TNM stage, mild decrease of CK20 and CDX2 expression, mild increase of CK7 expression, <I>BRAF</I> mutation, MSI and <I>MLH1</I> methylation. CIMP-P2 showed older age, female predominance, proximal location, advanced T category, markedly reduced CK20 and CDX2 expression, rare <I>KRAS</I> mutation, high frequency of CK7 expression, <I>BRAF</I> mutation, MSI and <I>MLH1</I> methylation. CIMP-N showed better 5-year cancer-specific survival (CSS; HR=0.47; 95% CI: 0.28–0.78) in discovery set and better 5-year relapse-free survival (RFS; HR=0.50; 95% CI: 0.29–0.88) in validation set compared with CIMP-P1. CIMP-P2 showed marginally better 5-year CSS (HR=0.28, 95% CI: 0.07–1.22) in discovery set and marginally better 5-year RFS (HR=0.21, 95% CI: 0.05–0.92) in validation set compared with CIMP-P1.</P><P><B>Conclusions:</B></P><P>CIMP subtypes classified using our revised system showed different clinical outcomes, demonstrating the heterogeneity of multiple serrated precursors of CIMP-positive CRCs.</P>

Reply to ‘Comment on ‘Distinct clinical outcomes of two CIMP-positive colorectal cancer subtypes based on a revised CIMP classification system”

Bae, Jeong Mo,Kim, Jung Ho,Kwak, Yoonjin,Lee, Dae-Won,Cha, Yongjun,Wen, Xianyu,Lee, Tae Hun,Cho, Nam-Yun,Jeong, Seung-Yong,Park, Kyu Joo,Han, Sae Won,Lee, Hye Seung,Kim, Tae-You,Kang, Gyeong Hoon Nature Publishing Group 2018 The British journal of cancer Vol. No.

Adverse prognostic impact of the CpG island methylator phenotype in metastatic colorectal cancer

Cha, Yongjun,Kim, Kyung-Ju,Han, Sae-Won,Rhee, Ye Young,Bae, Jeong Mo,Wen, Xianyu,Cho, Nam-Yun,Lee, Dae-Won,Lee, Kyung-Hun,Kim, Tae-Yong,Oh, Do-Youn,Im, Seock-Ah,Bang, Yung-Jue,Jeong, Seung-Yong,Park, Nature Publishing Group 2016 The British journal of cancer Vol. No.

<P><B>Background:</B></P><P>The association between the CpG island methylator phenotype (CIMP) and clinical outcomes in metastatic colorectal cancer remains unclear. We investigated the prognostic impact of CIMP in patients with metastatic colorectal cancer treated with systemic chemotherapy.</P><P><B>Methods:</B></P><P>Eight CIMP-specific promoters (<I>CACNA1G</I>, <I>IGF2</I>, <I>NEUROG1</I>, <I>RUNX3</I>, <I>SOCS1</I>, <I>CDKN2A</I>, <I>CRABP1</I>, and <I>MLH1</I>) were examined. The CIMP status was determined by the number of methylated promoters as high (⩾5), low (1–4), and negative (0).</P><P><B>Results:</B></P><P>A total of 153 patients were included (men/women, 103/50; median age, 61 years; range, 22–80 years). The CIMP status was negative/low/high in 77/ 69/7 patients, respectively. Overall survival (OS) was significantly different among the three CIMP groups, with median values of 35.7, 22.2, and 9.77 months for the negative, low, and high groups, respectively (<I>P</I><0.001). For patients treated with fluoropyrimidine and oxaliplatin first-line chemotherapy (<I>N</I>=128), OS and progression-free survival (PFS) were significantly different among the three CIMP groups; the median OS was 37.9, 23.8, and 6.77 months for the negative, low, and high groups, respectively <I>(P</I><0.001), while the median PFS was 9.97, 7.87, and 1.83 months, respectively (<I>P</I>=0.002). Response rates were marginally different among the three CIMP groups (53.4% <I>vs</I> 45.1% <I>vs</I> 16.7%, respectively; <I>P</I>=0.107). For patients treated with fluoropyrimidine and irinotecan second-line chemotherapy (<I>N</I>=86), only OS showed a difference according to the CIMP status, with median values of 20.4, 13.4, and 2.90 months for the negative, low, and high groups, respectively (<I>P</I><0.001).</P><P><B>Conclusions:</B></P><P>The CIMP status is a negative prognostic factor for patients with metastatic colorectal cancer treated with chemotherapy.</P>