Laser Capture Microdissection

Figure 1. Multiple selected regions containing desired specific cell populations for LCM.

For good-quality genome-wide DNA methylation input of a pure population of target cells is required. LCM is an accurate method for isolating these specific target cells out of complex heterogeneous tissue.

For LCM, 16 micron thick sections of FFPE tissue are cut and mounted on membranes fixed on glass slides. The sections are then dewaxed, dehydrated and stained with Hematoxylin. After digital imaging of the stained slides, regions of interest are annotated by a pathologist. The annotated regions are cut by a laser beam using an LCM system and collected in tubes (Figure 2).

After the extraction of genomic DNA from the micro dissected tissues by proteinase K, the concentrations of the extracted DNA are determined, and the samples are ready for MeD-seq analysis.

Figure 2. Isolation of tumor tissue using LCM

Publications about our techniques

Boers R, Boers J, de Hoon B, et al. Genome Res. 2018;28(1):88-99. doi:10.1101/gr.222885.117

Genome-wide DNA methylation profiling using the methylation-dependent restriction enzyme LpnPI Genome Res. 2018 28(1):88-99

DOI: 10.1101/gr.222885.117

Vos M, Boers R, Vriends ALM, et al. PLoS One. 2020;15(1):e0228014. Published 2020 Jan 23. doi:10.1371/journal.pone.0228014

MicroRNA expression and DNA methylation profiles do not distinguish between primary and recurrent well-differentiated liposarcoma

Bonde J, Floore A, Ejegod D, Vink FJ, Hesselink A, van de Ven PM, Valenčak AO, Pedersen H, Doorn S, Quint WG, Petry KU, Poljak M, Stanczuk G, Cuschieri K, de Sanjosé S, Bleeker M, Berkhof J, Meijer CJLM, Heideman DAM. Int J Cancer. 2020 Sep 30. doi: 10.1002/ijc.33320.

Methylation markers FAM19A4 and miR124-2 as triage strategy for primary human papillomavirus screen positive women: A large European multicenter study.

DOI: 10.1002/ijc.33320.

Leeman A, Jenkins D, Del Pino M, Ordi J, Torné A, Doorbar J, Meijer CJLM, van Kemenade FJ, Quint WGV. Cancer Med. 2020 Apr;9(7):2454-2461. doi: 10.1002/cam4.2855. Epub 2020 Feb 5.

Expression of p16 and HPV E4 on biopsy samples and methylation of FAM19A4 and miR124-2 on cervical cytology samples in the classification of cervical squamous intraepithelial lesions.

DOI: 10.1002/cam4.2855.

van der Zee RP, Richel O, van Noesel CJM, Ciocănea-Teodorescu I, van Splunter AP, Ter Braak TJ, Nathan M, Cuming T, Sheaff M, Kreuter A, Meijer CJLM, Quint WGV, de Vries HJC, Prins JM, Steenbergen RDM. Clin Infect Dis. 2020 Apr 8:ciaa397. doi: 10.1093/cid/ciaa397. Online ahead of print.

Cancer risk stratification of anal intraepithelial neoplasia in HIV-positive men by validated methylation markers associated with progression to cancer.

DOI: 10.1093/cid/ciaa397.

van Baars R, van der Marel J, Snijders PJ, Rodriquez-Manfredi A, ter Harmsel B, van den Munckhof HA, Ordi J, del Pino M, van de Sandt MM, Wentzensen N, Meijer CJ, Quint WG. Int J Cancer. 2016 Jan 15;138(2):463-71. doi: 10.1002/ijc.29706. Epub 2015 Aug 14.

CADM1 and MAL methylation status in cervical scrapes is representative of the most severe underlying lesion in women with multiple cervical biopsies.

DOI: 10.1002/ijc.29706

van der Zee RP, Richel O, van Noesel CJM, Novianti PW, Ciocanea-Teodorescu I, van Splunter AP, Duin S, van den Berk GEL, Meijer CJLM, Quint WGV, de Vries HJC, Prins JM, Steenbergen RDM. Clin Infect Dis. 2019 Mar 19;68(7):1110-1117. doi: 10.1093/cid/ciy601.

Host Cell Deoxyribonucleic Acid Methylation Markers for the Detection of High-grade Anal Intraepithelial Neoplasia and Anal Cancer.

DOI: 10.1093/cid/ciy601.

Leeman A, Del Pino M, Marimon L, Torné A, Ordi J, Ter Harmsel B, Meijer CJLM, Jenkins D, Van Kemenade FJ, Quint WGV. Int J Cancer. 2019 Jan 1;144(1):160-168. doi: 10.1002/ijc.31787. Epub 2018 Nov 18.

Reliable identification of women with CIN3+ using hrHPV genotyping and methylation markers in a cytology-screened referral population.

DOI: 10.1002/ijc.31787

Floore A, Hesselink A, Oštrbenk A, Alcaniz E, Rothe B, Pedersen H, Torres Hortal M, Doorn S, Quint W, Petry KU, Poljak M, Cuschieri K, Bonde J, de Sanjosé S, Bleeker M, Heideman D. J Clin Lab Anal. 2019 May;33(4):e22854. doi: 10.1002/jcla.22854. Epub 2019 Feb 13.

Intra- and inter-laboratory agreement of the FAM19A4/mir124-2 methylation test: Results from an international study.

DOI: 10.1002/jcla.22854

Vink FJ, Meijer CJLM, Clifford GM, Poljak M, Oštrbenk A, Petry KU, Rothe B, Bonde J, Pedersen H, de Sanjosé S, Torres M, Del Pino M, Quint WGV, Cuschieri K, Boada EA, van Trommel NE, Lissenberg-Witte BI, Floore AN, Hesselink AT, Steenbergen RDM, Bleeker MCG, Heideman DAM. Int J Cancer. 2020 Aug 15;147(4):1215-1221. doi: 10.1002/ijc.32614. Epub 2019 Sep 9.

FAM19A4/miR124-2 methylation in invasive cervical cancer: A retrospective cross-sectional worldwide study.

DOI: 10.1002/ijc.32614

Leeman A, Ebisch RMF, Kasius A, Bosgraaf RP, Jenkins D, van de Sandt MM, de Strooper LMA, Heideman DAM, Snijders PJF, Massuger LFAG, Bekkers RLM, Meijer CJLM, van Kemenade FJ, Quint WGV, Melchers WJG. Gynecol Oncol. 2018 Nov;151(2):311-318. doi: 10.1016/j.ygyno.2018.09.006. Epub 2018 Sep 13.

Defining hrHPV genotypes in cervical intraepithelial neoplasia by laser capture microdissection supports reflex triage of self-samples using HPV16/18 and FAM19A4/miR124-2 methylation.

DOI: 10.1016/j.ygyno.2018.09.006.