PURE AND MIXED CLEAR CELL CARCINOMA 81 4 deficiency (MMRd) was defined as total loss of nuclear staining of either PMS2 or MSH6, in the presence of a positive internal control. DNA extraction and library preparation Representative tumor tissue was selected by means of microdissection from 8 x 10 µm thick formalin-fixed, paraffin-embedded (FFPE) sections. In case of mixed histology, both components were microdissected separately. Next, tissue was digested at 56°C for at least 16 hours in the presence of TET-lysis buffer (1M Tris/HCL pH 8.5, 0.5M EDTA pH 8.0, 20% Tween-20) with 5% Chelex- 100 (Bio-Rad, Hercules, CA) and 10% Proteinase K (Qiagen, Valencia, CA), followed by inactivation at 95°C for 10 minutes. Twice, the supernatant was transferred to a clean tube after centrifugation at 14 000 xg for 10 minutes. DNA concentration was determined using the Qubit 1x dsDNA High Sensitivity assay kit (Thermo Fisher Scientific, Waltham, MA, US). The isolated DNA was stored at -20°C. The samples were analyzed with single-molecule Molecular Inversion Probes (smMIPs, Integrated DNA Technologies, Leuven, Belgium) 23. The panel consisted of twelve relevant genes involved in EC oncogenesis as well as a number of genes informative for ProMisE classification (AKT1, ARID1A, CTNNB1, ERBB2, FGFR2, KRAS, MTOR, NRAS, PIK3CA, PTEN, POLE, TP53, Supplementary B), in addition to markers for microsatellite instability (MSI). Targeted sequencing with smMIPs was performed as previously described 23. All smMIPs were designed in a tiling manner for hotspots in oncogenes and all coding as well as splice site consensus sequences of tumor suppressor genes (TSGs), with preferential targeting of both strands by two smMIPs. The smMIP probes were constructed by an extension and ligation probe arm (40bp long) with a 112bp gap and a common backbone sequence for PCR-based library amplification. The ligation probe arm and backbone were connected by means of an 8bp degenerate sequence (8xN) serving as a Unique Molecular Identifier (UMI, ‘single molecule tag’). The smMIP probes were mixed and phosporylated with 1μL of T4 polynucleotide kinase (M0201; New England Biolabs, Ipswich, MA, US) per 25μL of 100 μmol/L smMIPs and ATP-containing G4 DNA ligase buffer (B0202, New England Biolabs). The molecular ratio between gDNA and smMIPs was set at 1:3200 for each individual smMIP and the standard genomic DNA input was 100ng. Next, a capture mix was made (volume: 25μL) with the phosporylated smMIP pool, 1 unit of Ampligase DNA ligase (A0110K; EpiBio, Madison, WI) and Ampligase Buffer (A1905B, DNA ligase buffer), 3.2 units of Hemo Klentaq (M0332; New England Biolabs), 8mmol of dNTPs (28-406520/-12/-22/-32; GE Healthcare, Little Chalfont, UK) and 100ng of genomic DNA in a 20μL volume. This capture mix was denatured at 95ºC for 10 minutes and subsequently incubated for probe hybridization, extension and ligation at 60ºC for 18 hours. After cooling, to perform exonuclease treatment, Exonuclease I (10 units; M0293; New England Biolabs) and III (50 units; M0206; New England Biolabs) and Ampligase Buffer was added to the capture mix (total of 27μL). The mix was incubated at 37ºC for 45 minutes, with subsequent inactivation at
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