Array Comparative Genomic Hybridization analysis of resected thymomas on paraffin embedded material

Introduction: Thymomas are rare tumors derived from the thymic epithelial cells. They show heterogeneous biological characteristics and survival outcome. The WHO classification identifies different histotypes with prognostic implications. They range from type A with around 100% 10-year survival to the most aggressive type C with 0% 10 years survival reported. There is very little known of the biology of thymomas and of the genetic alterations present in these tumors. Array comparative genomic hybridization (CGH) is a powerful technique to define compy number abnormalities. Materials and Methods: We selected 60 thymoma samples showing a proportion of cancer cells > 80% from 151 formalin fixed paraffin embedded samples of 134 patients who underwent surgery at Humanitas Hospital, Milan, Italy. The DNA extraction and labeling was performed according to Genomic DNA ULS Labeling Kit (Agilent, Santa Clara, California, USA). We used as reference Human Genomic DNA: Male (Promega, Madison, Wisconsin, USA). Eleven samples were hybridized on Human Genome CGH Array 105A (Agilent) and 29 on SurePrint G3 Human CGH Array 180K (Agilent). Slides were scanned on a laser-based microarray scanner (Agilent). The data were extracted and normalized by Feature Extraction 10.5 (Agilent). We performed data analysis using Nexus 4.0 (Biodiscovery Inc, El Segundo California, USA) by Log Rank segmentation algorithm. We selected for the analysis 40 samples with a Robust Variance Sample QC< 0.2 as recommended. We evaluated aberration of autosomal chromosomes. Common Aberrant Region (CAR) are defined by a imbalance frequencies of 50% in type C and of 35% in the remaining categories. Results: In 40 thymoma samples we identified 12 common aberrant regions: 6 are gains involving 1037 genes and 6 are losses involving 96 genes. These aberrations are present in each histotype. In 9 type A thymoma samples we discovered 11 common aberrant gains (CAG) involving 112 genes and 3 common aberrant losses (CAL) involving 11 genes. For the 11 AB specimens we identified 18 CAG involving 931 genes and 3 CAL involving 56 genes. We evaluated 15 B3 samples presenting 10 CAG involving 1172 genes and 9 CAL involving 389 genes. Five thymic carcinomas showed 20 CAG with 1356 genes and 8 CAL with 540 genes involved. Conclusion: CGH analysis is feasible using FFPE samples commonly stored for diagnosis. The number of genes involved in karyotypic aberration increase from type A to the more aggressive type C. Chromosome imbalance is present in each histotype analyzed.