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  • 1
    Online Resource
    Online Resource
    Identification and validation of candidate epigenetic biomarkers in lung adenocarcinoma
    Springer Science and Business Media LLC ; 2016
    In:  Scientific Reports Vol. 6, No. 1 ( 2016-10-26)
    Details
    In: Scientific Reports, Springer Science and Business Media LLC, Vol. 6, No. 1 ( 2016-10-26)
    Abstract: Lung cancer is the number one cause of cancer-related deaths worldwide. DNA methylation is an epigenetic mechanism that regulates gene expression, and disease-specific methylation changes can be targeted as biomarkers. We have compared the genome-wide methylation pattern in tumor and tumor-adjacent normal lung tissue from four lung adenocarcinoma (LAC) patients using DNA methylation microarrays and identified 74 differentially methylated regions (DMRs). Eighteen DMRs were selected for validation in a cohort comprising primary tumors from 52 LAC patients and tumor-adjacent normal lung tissue from 32 patients by methylation-sensitive high resolution melting (MS-HRM) analysis. Significant increases in methylation were confirmed for 15 DMRs associated with the genes and genomic regions: OSR1 , SIM1 , GHSR , OTX2 , LOC648987 , HIST1H3E , HIST1H3G/HIST1H2BI , HIST1H2AJ/HIST1H2BM, HOXD10 , HOXD3 , HOXB3/HOXB4 , HOXA3 , HOXA5 , Chr1(q21.1).A, and Chr6(p22.1). In particular the OSR1 , SIM1 and HOXB3/HOXB4 regions demonstrated high potential as biomarkers in LAC. For OSR1 , hypermethylation was detected in 47/48 LAC cases compared to 1/31 tumor-adjacent normal lung samples. Similarly, 45/49 and 36/48 LAC cases compared to 3/31 and 0/31 tumor-adjacent normal lung samples showed hypermethylation of the SIM1 and HOXB3/HOXB4 regions, respectively. In conclusion, this study has identified and validated 15 DMRs that can be targeted as biomarkers in LAC.
    Type of Medium: Online Resource
    ISSN: 2045-2322
    URL: Article
    DOI: 10.1038/srep35807
    Language: English
    Publisher: Springer Science and Business Media LLC
    Publication Date: 2016
    detail.hit.zdb_id: 2615211-3
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  • 2
    Online Resource
    Online Resource
    Quality assessment of DNA derived from up to 30 years old formalin fixed paraffin embedded (FFPE) tissue for PCR-based methylation analysis using SMART-MSP and MS-HRM
    Springer Science and Business Media LLC ; 2009
    In:  BMC Cancer Vol. 9, No. 1 ( 2009-12)
    Details
    In: BMC Cancer, Springer Science and Business Media LLC, Vol. 9, No. 1 ( 2009-12)
    Abstract: The High Resolution Melting (HRM) technology has recently been introduced as a rapid and robust analysis tool for the detection of DNA methylation. The methylation status of multiple tumor suppressor genes may serve as biomarkers for early cancer diagnostics, for prediction of prognosis and for prediction of response to treatment. Therefore, it is important that methodologies for detection of DNA methylation continue to evolve. Sensitive Melting Analysis after Real Time - Methylation Specific PCR (SMART-MSP) and Methylation Sensitive - High Resolution Melting (MS-HRM) are two methods for single locus DNA methylation detection based on HRM. Methods Here, we have assessed the quality of DNA extracted from up to 30 years old Formalin Fixed Paraffin Embedded (FFPE) tissue for DNA methylation analysis using SMART-MSP and MS-HRM. The quality assessment was performed on DNA extracted from 54 Non-Small Cell Lung Cancer (NSCLC) samples derived from FFPE tissue, collected over 30 years and grouped into five years intervals. For each sample, the methylation levels of the CDKN2A ( p16 ) and RARB promoters were estimated using SMART-MSP and MS-HRM assays designed to assess the methylation status of the same CpG positions. This allowed for a direct comparison of the methylation levels estimated by the two methods for each sample. Results CDKN2A promoter methylation levels were successfully determined by SMART-MSP and MS-HRM in all 54 samples. Identical methylation estimates were obtained by the two methods in 46 of the samples. The methylation levels of the RARB promoter were successfully determined by SMART-MSP in all samples. When using MS-HRM to assess RARB methylation five samples failed to amplify and 15 samples showed a melting profile characteristic for heterogeneous methylation. Twenty-seven of the remaining 34 samples, for which the methylation level could be estimated, gave the same result as observed when using SMART-MSP. Conclusion MS-HRM and SMART-MSP can be successfully used for single locus methylation studies using DNA derived from up to 30 years old FFPE tissue. Furthermore, it can be expected that MS-HRM and SMART-MSP will provide similar methylation estimates when assays are designed to analyze the same CpG positions.
    Type of Medium: Online Resource
    ISSN: 1471-2407
    URL: Article
    DOI: 10.1186/1471-2407-9-453
    Language: English
    Publisher: Springer Science and Business Media LLC
    Publication Date: 2009
    detail.hit.zdb_id: 2041352-X
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  • 3
    Online Resource
    Online Resource
    Competitive amplification of differentially melting amplicons (CADMA) enables sensitive and direct detection of all mutation types by high-resolution melting analysis
    Hindawi Limited ; 2012
    In:  Human Mutation Vol. 33, No. 1 ( 2012-01), p. 264-271
    Details
    In: Human Mutation, Hindawi Limited, Vol. 33, No. 1 ( 2012-01), p. 264-271
    Type of Medium: Online Resource
    ISSN: 1059-7794
    URL: Article
    DOI: 10.1002/humu.21598
    Language: English
    Publisher: Hindawi Limited
    Publication Date: 2012
    detail.hit.zdb_id: 1498165-8
    SSG: 12
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  • 4
    Online Resource
    Online Resource
    The Challenges of Comparing a Clinically Validated Test to Other Methods
    Elsevier BV ; 2013
    In:  The Journal of Molecular Diagnostics Vol. 15, No. 4 ( 2013-07), p. 535-537
    Details
    In: The Journal of Molecular Diagnostics, Elsevier BV, Vol. 15, No. 4 ( 2013-07), p. 535-537
    Type of Medium: Online Resource
    ISSN: 1525-1578
    URL: Article
    DOI: 10.1016/j.jmoldx.2013.02.007
    RVK:
    XA 55072
    Language: English
    Publisher: Elsevier BV
    Publication Date: 2013
    detail.hit.zdb_id: 2032654-3
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  • 5
    Online Resource
    Online Resource
    Increased sensitivity of KRAS mutation detection by high-resolution melting analysis of COLD-PCR products
    Hindawi Limited ; 2010
    In:  Human Mutation Vol. 31, No. 12 ( 2010-12), p. 1366-1373
    Details
    In: Human Mutation, Hindawi Limited, Vol. 31, No. 12 ( 2010-12), p. 1366-1373
    Type of Medium: Online Resource
    ISSN: 1059-7794
    URL: Article
    DOI: 10.1002/humu.21358
    Language: English
    Publisher: Hindawi Limited
    Publication Date: 2010
    detail.hit.zdb_id: 1498165-8
    SSG: 12
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  • 6
    Online Resource
    Online Resource
    Abstract 4918: Quality assessment of DNA derived from up to 30 year old archival tissues from lung cancer patients for PCR-based methylation analysis using SMART-MSP and MS-HRM
    American Association for Cancer Research (AACR) ; 2010
    In:  Cancer Research Vol. 70, No. 8_Supplement ( 2010-04-15), p. 4918-4918
    Details
    In: Cancer Research, American Association for Cancer Research (AACR), Vol. 70, No. 8_Supplement ( 2010-04-15), p. 4918-4918
    Abstract: Background: The High-Resolution Melting (HRM) technology has recently been introduced as a rapid and robust analysis tool for the detection of DNA methylation. The methylation status of multiple tumor suppressor genes may serve as biomarkers for early cancer diagnostics, for prediction of prognosis and for prediction of response to treatment. Therefore, it is important that methodologies for detection of DNA methylation continue to evolve. Sensitive Melting Analysis after Real Time - Methylation Specific PCR (SMART-MSP) and Methylation Sensitive - High Resolution Melting (MS-HRM) are two methods for single locus DNA methylation detection based on HRM. Methods: Here, we have assessed the quality of DNA extracted from 30-years-old Formalin Fixed Paraffin Embedded (FFPE) tissue for DNA methylation analysis using SMART-MSP and MS-HRM. The quality assessment was performed on DNA extracted from 54 Non-Small Cell Lung Cancer (NSCLC) samples derived from FFPE tissue, collected over 30 years and grouped into five-years-intervals. For each sample, the methylation level of the CDKN2A (p16) and RARB promoters were estimated using SMART-MSP and MS-HRM assays designed to assess the methylation status of the same CpG positions. This allowed for a direct comparison of the methylation level estimated by the two methods for each sample. Results: The methylation level of the CDKN2A promoter was successfully determined by SMART-MSP and MS-HRM in all of the 54 samples. The same methylation estimates were obtained by the two methods in 46 of samples. The methylation level of the RARB promoter was successfully determined by SMART-MSP in all of samples. When using MS-HRM to assess RARB methylation five samples failed to amplify and 15 samples showed a melting profile characteristic for heterogeneous methylation. Out of the remainder 34 samples, for which the methylation level could be estimated, 27 gave the same result as observed when using SMART-MSP. Conclusion: MS-HRM and SMART-MSP can be successfully used for single locus methylation studies of DNA derived from up to 30-years-old FFPE tissue samples. Furthermore, it can be expected that MS-HRM and SMART-MSP will provide similar methylation estimates when assays are designed to analyze the same CpG positions. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4918.
    Type of Medium: Online Resource
    ISSN: 0008-5472 , 1538-7445
    URL: Article
    DOI: 10.1158/1538-7445.AM10-4918
    RVK:
    XA 36000
    RVK:
    XA 10000
    Language: English
    Publisher: American Association for Cancer Research (AACR)
    Publication Date: 2010
    detail.hit.zdb_id: 2036785-5
    detail.hit.zdb_id: 1432-1
    detail.hit.zdb_id: 410466-3
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  • 7
    Online Resource
    Online Resource
    Abstract 2115: Increased sensitivity of KRAS mutation detection by High-Resolution Melting analysis of COLD-PCR products
    American Association for Cancer Research (AACR) ; 2010
    In:  Cancer Research Vol. 70, No. 8_Supplement ( 2010-04-15), p. 2115-2115
    Details
    In: Cancer Research, American Association for Cancer Research (AACR), Vol. 70, No. 8_Supplement ( 2010-04-15), p. 2115-2115
    Abstract: Much effort has been put into the development of sophisticated technologies enabling the detection of clinically significant low-level tumor specific KRAS mutations. The constitutively active KRAS oncogene harbors a mutation hotspot in codon 12 and 13 of exon 2. These mutations are predictors of response to treatment with the monoclonal antibodies panitumumab and cetuximab targeting the epidermal growth factor receptor (EGFR). Therefore, it is crucial to develop robust and sensitive assays targeting these mutations. Coamplification at lower denaturation temperature-PCR (COLD-PCR) is a new form of PCR that selectively amplifies mutation containing amplicons based on the lower melting temperature of heteroduplexes versus homoduplexes. We have developed an assay based on real-time COLD-PCR and high-resolution melting (HRM) targeting codon 12 and 13 for simultaneous detection of the various mutations found in this mutation hotspot. By using mutation containing cell-lines serially diluted into wild-type DNA we have compared the sensitivity of HRM after standard PCR with the sensitivity of HRM after COLD-PCR. We have also analyzed 60 colorectal cancer samples for which KRAS mutation status previously has been evaluated by the DxS kit which is approved by the U.S. Food and Drug Administration. Replacing standard PCR by COLD-PCR increased the sensitivity of the assay by up to 50 fold depending on the specific mutation. Furthermore, we have obtained very similar results when using our COLD-PCR assay as we obtained when using the DxS kit and confirmed the HRM results with Sanger sequencing. In conclusion, applying COLD-PCR to HRM analysis is a simple way of increasing the sensitivity of KRAS mutation detection without adding to the complexity and cost of the experiments. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2115.
    Type of Medium: Online Resource
    ISSN: 0008-5472 , 1538-7445
    URL: Article
    DOI: 10.1158/1538-7445.AM10-2115
    RVK:
    XA 36000
    RVK:
    XA 10000
    Language: English
    Publisher: American Association for Cancer Research (AACR)
    Publication Date: 2010
    detail.hit.zdb_id: 2036785-5
    detail.hit.zdb_id: 1432-1
    detail.hit.zdb_id: 410466-3
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  • 8
    Online Resource
    Online Resource
    Evaluation of BRAF Mutation Testing Methodologies in Formalin-Fixed, Paraffin-Embedded Cutaneous Melanomas
    Elsevier BV ; 2013
    In:  The Journal of Molecular Diagnostics Vol. 15, No. 1 ( 2013-01), p. 70-80
    Details
    In: The Journal of Molecular Diagnostics, Elsevier BV, Vol. 15, No. 1 ( 2013-01), p. 70-80
    Type of Medium: Online Resource
    ISSN: 1525-1578
    URL: Article
    DOI: 10.1016/j.jmoldx.2012.08.003
    RVK:
    XA 55072
    Language: English
    Publisher: Elsevier BV
    Publication Date: 2013
    detail.hit.zdb_id: 2032654-3
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