Onkogēnu un audzēju supresorgēnu kopiju skaita variācija un metilācijas statuss krūts audzēju audos
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Date
2021
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Rīgas Stradiņa universitāte
Rīga Stradiņš University
Rīga Stradiņš University
Abstract
Krūts audzēji ir ļoti heterogēni, un to nosaka dažādi ģenētiski faktori un notikumi - kā, piemēram, gēnu delēcija, duplikācija un metilācija. Šie faktori ir svarīgi prognozes noteikšanai un terapijas izvēlei. Tamdēļ CNV - kopiju skaita variācija un metilācijas statuss un tā asociācija ar kopējo dzīvildze/izdzīvotību tika pētīts.
Tika izdalīts DNS no 451 svaigi saldētas secīgas krūts audzēju biopsijas un no 22 veselu krūts audu paraugiem (paraugi no RSU Onkoloģijas institūta kolekcijas, ievākti no 2012. līdz 2019. gadam), tika izmantota uz kollonām bāzēta DNS izdalīšanas metode (Qiagen). Kopiju skaita variācija un metilācijas statuss tika noteikti ar Multiplex Ligation-dependant Probe Amplification (MLPA) ar Breast tumor probemix un Metylation-Specific MLPA (MS-MLPA) ar Tumor suppressor mix 1 probemix, ievērojot ražotāj prtokolu un norādes (MRC-Holland). Dati tika apstrādāti datorprogramā Coffalyser. Datu vizualizācija un statistiskā analīze tika veikta ar datorprogrammu R Bioconductor.
47 gēnu kopiju skaita variācija (21 onkogēns, 26 audzēju nomācējgēni) un 26 audzēju nomācējgēnu metilācijas statuss tika analizēti 451 secīgos krūts audzēju paraugos un 22 veselu krūts audu paraugos. 118 (26,2%) paraugos netika konstatētas ne kopiju skaita izmaiņas, ne metilācijas izmaiņas. Visbiežāk delēcijas notika audzēju supresorgēnos CDH1 (23,1%), VHL (8,9%) BRCA1 (8,4%). Starp onkogēniem MYC (19,4%), MTDH (15,0%), PRDM14 (10,6%) visbiežāk novēro duplikācijas. RASSF1 (41,9%), APC (18,0%) un CDH13 (11,2%) gēni visbiežāk tika metilēti. Korelācija starp kopiju skaita variāciju, metilācijas statusu un to asociāciju ar kopējo izdzīvotību tika analizēti.
Kopiju skaita variācija ir biežs notikums krūts audzēju paraugos un tika novērots73,8% gadījumu. Tika novērota audzēju supresorgēnu inaktivācija ģenētisku un epiģenētisku notikumu dēļ. Kopiju skaita variācija un metilācijas statuss ietekmē kopējo izdzīvotību.
Breast cancers are very heterogeneous, that is determined by various genetic events such as gene deletion, duplication and methylation. It is important in prognosis and therapy determination. Therefor CNV (copy number variation) and methylation status association with survival was studied. DNA was purified from 451 frozen sequential breast cancer biopsies and 22 healthy breast tissue samples (from RSU Institute of Oncology collection, obtained from 2012-2019), using spin column-based DNA purification (Qiagen). Copy number variation and methylation status were detected by Multiplex Ligation-dependant Probe Amplification (MLPA) with Breast Tumor probemix and Metylation-Specific MLPA (MS-MLPA) with Tumor suppressor mix 1 probemix according to manufacturers instructions (MRC-Holland) and analysed in Coffalyser software. Heat maps were built and statistical analysis performed by R Bioconductor. CNV of 47 genes (21 oncogenes; 27 tumor suppressor genes) and methylation status of 26 tumor suppressor genes analysed in 451 consecutive breast cancer samples and 22 controls. 118 (26,2%) samples had neither CNV, nor methylation status changes. Most frequent deletions were in tumor suppressors CDH1 (23,1%), VHL (8,9%) BRCA1 (8,4%). Among oncogenes most commonly MYC (19,4%), MTDH (15,0%), PRDM14 (10,6%) were duplicated. The RASSF1 (41,9%), APC (18,0%) and the CDH13 (11,2%) genes were methylated most frequently. Correlation between CNV, methylation status and overall survival was determined. CNV is common event in breast cancer tissue samples and happens in 73,8% of cases. Inactivation of tumor suppressor genes by genetic and epigenetic mechanisms were observed. CNV and methylation status influences overall survival.
Breast cancers are very heterogeneous, that is determined by various genetic events such as gene deletion, duplication and methylation. It is important in prognosis and therapy determination. Therefor CNV (copy number variation) and methylation status association with survival was studied. DNA was purified from 451 frozen sequential breast cancer biopsies and 22 healthy breast tissue samples (from RSU Institute of Oncology collection, obtained from 2012-2019), using spin column-based DNA purification (Qiagen). Copy number variation and methylation status were detected by Multiplex Ligation-dependant Probe Amplification (MLPA) with Breast Tumor probemix and Metylation-Specific MLPA (MS-MLPA) with Tumor suppressor mix 1 probemix according to manufacturers instructions (MRC-Holland) and analysed in Coffalyser software. Heat maps were built and statistical analysis performed by R Bioconductor. CNV of 47 genes (21 oncogenes; 27 tumor suppressor genes) and methylation status of 26 tumor suppressor genes analysed in 451 consecutive breast cancer samples and 22 controls. 118 (26,2%) samples had neither CNV, nor methylation status changes. Most frequent deletions were in tumor suppressors CDH1 (23,1%), VHL (8,9%) BRCA1 (8,4%). Among oncogenes most commonly MYC (19,4%), MTDH (15,0%), PRDM14 (10,6%) were duplicated. The RASSF1 (41,9%), APC (18,0%) and the CDH13 (11,2%) genes were methylated most frequently. Correlation between CNV, methylation status and overall survival was determined. CNV is common event in breast cancer tissue samples and happens in 73,8% of cases. Inactivation of tumor suppressor genes by genetic and epigenetic mechanisms were observed. CNV and methylation status influences overall survival.
Description
Medicīna
Medicine
Veselības aprūpe
Health Care
Medicine
Veselības aprūpe
Health Care
Keywords
Krūts vēzis, kopiju skaita variācija, metilācija, MLPA, Breast cancer, CNV, methylation, MLPA