Array–comparative genomic hybridization (a-CGH) analysis
DNA copy number analysis was performed using an oligonucleotide array-CGH platform (SurePrint G3 Human CGH Microarray 8x60K; Agilent Technologies Inc., Santa Clara, CA, USA), using a previously established protocol for FFPE samples 23, 24. DNA was isolated using the standard phenol-chloroform method. Reference DNA was prepared from the peripheral blood of a pool of ten healthy donors25. Equal amounts of tumor and reference genomic DNA (1-2 µg) were digested and enzymatically labeled using the SureTag Complete DNA Labeling Kit (Agilent Technologies, Inc., Santa Clara, CA, USA) and hybridized to the arrays. The array data were analyzed with the Feature Extraction (FE) v.10.10 software and Agilent CytoGenomics v.3.0 software (Agilent Technologies Inc., Santa Clara, CA, USA), using the ADM-2 algorithm, threshold 6.0, and an aberration filter with a minimum of > 3 probes 25. Copy number gains and losses were defined as previously described 24. Cyto­bands that showed copy number alterations (CNAs) in the Ch11q-deleted locus in pre- and post-CT samples from three patients were identified using Agilent CytoGenomics v.7.0 interval base reports (Agilent Technologies, Inc., Santa Clara, CA, USA). Genes were extracted from these cytobands using the UCSC genome browser 26and the RefSeq gene model. The online data mining software BioMart of the Ensembl platform (http://m.ensembl.org/; release 101, August 2020) was used to select the protein-coding genes within this region. DIANA-miRPath v.3 and TarBase v.7.0 27, 28 were used to identify the predicted miRNA targets. Only miRNA/mRNA targets that had a TargetScan context score of -0.4 (default) were included.