Molekuláris vizsgálatok paraffinos szövetmintából: korlátok és lehetőségek

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1 Molekuláris vizsgálatok paraffinos szövetmintából: korlátok és lehetőségek Group of Molecular Cytogenetics and Cellular Oncology Károly Szuhai, M.D., Ph.D

2 The OMICs What can happen GENOME What appears to be happening BIOINFORMATICS TRANSCRIPTOME What makes it happen PROTEOME SYSTEMS BIOLOGY What actually happens METABOLOME PHENOTYPE Adapted from: Ahn et al., PLoS Med, 26. Károly Szuhai 2 MPT, Siófok, 21

3 Biomarker Discovery and Validation GENOME DNA Specific tests Mutation detection Clonality test FISH (cell based) Pathogen detection Genome-wide screening Array-CGH/SNP-array Whole genome sequencing TRANSCRIPTOME Specific tests Chimera detection Mutation detection MRD RNA Transcriptome-wide Expression profiling Array-chip Next generation sequencing Expression Mutation Splice variants Personalized medicine PROTEOME Proteins Specific tests Immunohistochemistry Proteome-wide MALDI MALDI-TOF, Functional screening kinome profiling TMA research discovery tool Károly Szuhai 3 MPT, Siófok, 21

4 FFPE-DNA & array-cgh I Sezary-Syndrome-array-CGH van Dekken et al., Genes Chromosomes Cancer, 26 Knijnenburg et al., Cytometry, 27, Oosting et al., Gen Res, out of 43 FFPE-GEJ tumor samples informative Vermeer et al., Cancer Res, 28 Károly Szuhai 4 MPT, Siófok, 21

5 FFPE-DNA & array-cgh I Sezary-Syndrome-array-CGH van Beers et al., Br J Cancer, van Dekken et al., Genes Chromosomes Cancer, 26 Knijnenburg et al., Cytometry, 27, Oosting et al., Gen Res, out of 43 FFPE-GEJ tumor samples informative Vermeer et al., Cancer Res, 28 Károly Szuhai 5 MPT, Siófok, 21

6 Nem jeleníthető meg a kép. Lehet, hogy nincs elegendő memória a megnyitásához, de az sem kizárt, hogy sérült a kép. Indítsa újra a számítógépet, és nyissa meg újból a fájlt. Ha továbbra is a piros x ikon jelenik meg, törölje a képet, és szúrja be ismét. FFPE-DNA & array-cgh II Rare chondrosarcoma Dediff., clear cell, mesench. Primary angiosarcoma of bone cases >1 EuroBoNet institutions Decalcified samples EDTA formic acid Pearson, Bland-Altman correlation 2 ng DNA loaded per sample (Nanodrop) Agilent 4x44k oligo array FFPE chemical labeling kit from Agilent/Kreatech 5ng DNA FFPE RP kit (Invitrogen) 5 and 15 ng de Jong et al., submitted Verbeke et al., submitted Károly Szuhai 6 MPT, Siófok, 21

7 FFPE-DNA & array-cgh II Rare chondrosarcoma Dediff., clear cell, mesench. Primary angiosarcoma of bone cases >1 EuroBoNet institutions Decalcified samples EDTA formic acid Pearson, Bland-Altman correlation 2 ng DNA loaded per sample (Nanodrop) Agilent 4x44k oligo array FFPE chemical labeling kit from Agilent/Kreatech 5ng DNA FFPE RP kit (Invitrogen) 5 and 15 ng de Jong et al., submitted Verbeke et al., submitted Károly Szuhai 7 MPT, Siófok, 21

8 FFPE-DNA & array-cgh II Rare chondrosarcoma Dediff., clear cell, mesench. Primary angiosarcoma of bone cases >1 EuroBoNet institutions Decalcified samples EDTA formic acid Pearson, Bland-Altman correlation 2 ng DNA loaded per sample (Nanodrop) Agilent 4x44k oligo array FFPE chemical labeling kit from Agilent/Kreatech 5ng DNA FFPE RP kit (Invitrogen) 5 and 15 ng de Jong et al., submitted Verbeke et al., submitted Károly Szuhai 8 MPT, Siófok, 21

9 FFPE-DNA & array-cgh II Rare chondrosarcoma Dediff., clear cell, mesench. Primary angiosarcoma of bone cases >1 EuroBoNet institutions Decalcified samples EDTA formic acid Pearson, Bland-Altman correlation 2 ng DNA loaded per sample (Nanodrop) Agilent 4x44k oligo array FFPE chemical labeling kit from Agilent/Kreatech 5ng DNA FFPE RP kit (Invitrogen) 5 and 15 ng de Jong et al., submitted Verbeke et al., submitted Károly Szuhai 9 MPT, Siófok, 21

10 FFPE-DNA & array-cgh III Detection sensitivity ~7kb amplified region at CMYC locus 6kb homozygous deletion region at CDKN2a/p16 locus DNA obtained after extended digestion in combination with double purification Suitable for novel FFPE labeling kits de Jong et al., submitted Meijer et al., submitted Károly Szuhai 1 MPT, Siófok, 21

11 FFPE Next-generation Sequencing: Helicos 1 ng non-amplified DNA used for direct sequencing Optimal fragment size ~1bps Degraded FFPE DNA is a pro Varian Ewing sarcoma sample tested with cloned breakpoints After QC and blasting 12.2 million fragment reads at unique locations 97. with > 15 read/loci likely to be repeat elements 2.4 million unique loci >3 and <15 read / 5bp bin Szuhai et al., Clin Cancer Res, 29 Károly Szuhai 11 MPT, Siófok, 21

12 Helicos Read: Chromosomes 2 & 22 One channel Helicos run ~2.4 million reads ~~ 2.4 million array Breakpoints localized within 1 bps after a single run within EWSR1 and NFATc2 genes Károly Szuhai 12 MPT, Siófok, 21

13 MPT, Siófok, 21 Summary Sample quality Test complexity Károly Szuhai 13

14 Summary Test com mplexity Sample quality Sample Detailed sample annotations treatment, type of surgery, time Standardized protocols Cut out Fixation (time, fixative, volume, sampling) Storage Isolation process Extensive retriaval and purification DNA, RNA and protein Double QC (size & concentration) Methodolocigal improvements Novel applications/platforms Improved/adapted labeling biospecimens.cancer.org Károly Szuhai 14 MPT, Siófok, 21

15 Summary Test com mplexity Sample quality Sample Detailed sample annotations treatment, type of surgery, time Standardized protocols Cut out Fixation (time, fixative, volume, sampling) Storage Isolation process Extensive retriaval and purification DNA, RNA and protein Double QC (size & concentration) Methodolocigal improvements Novel applications/platforms Improved/adapted labeling biospecimens.cancer.org Károly Szuhai 15 MPT, Siófok, 21

16 Károly Szuhai 16 MPT, Siófok, 21

17 Acknowledgements Molecular Cell Biology LUMC Marja van der Burg Ronald Duim Marjie IJszenga Daniëlle de Jong Anneliene Jonker Willem Sloos Hans Tanke Károly Szuhai 17 Pathology LUMC Judith Bovee Pancras Hogendoorn Daniëlle Meijers Jan Oosting Sofie Verbeke Erasmus MC, Rotterdam Herman van Dekken LGTC LUMC Yavuz Ariyurek Johan den Dunnen Matthew Hestand MPT, Siófok, 21