448 chapter 11 CNA patterns The results of the CNA-LOH analysis using the GWLOH panel were interpreted as described in the flowchart in Figure 2, identifying the different types of CNA patterns and establishing a molecular diagnosis. First, the CNA type is identified as GH type, or reciprocal chromosomal imbalance (RCI) type, or no CNA. GH type CNA are defined by LOH and chromosomal losses, as observed by chromosomal imbalances on the VAF plot (Figure 1B and 1C, bottom panel) and a lower normalized median amplicon read count of the affected chromosomes as compared to the unaffected chromosomes, indicating copy number losses (Figure 1B and 1C, middle panel). This CNA pattern requires further characterization by assessing the number of affected chromosomes, any heterogenicity of the alterations, and the possible presence of endoreduplication (Figure 2). We defined three categories for the number of affected chromosomes: 1-5, 6-10, and 11-23. Heterogenicity is defined as varying VAF amplitudes of the chromosomal imbalances among the affected chromosomes (Figure 1B, bottom panel) and is associated with benign disease. Endoreduplication is also assessed using the VAF plot amplitudes. GH type CNA with (suspected) endoreduplication (Figure 1B) are characterized by an extreme amplitude of the SNP segregation of the LOH-affected chromosomes (Figure 1B, bottom panel). GH type CNA without endoreduplication (Figure 1C) are characterized by other, non-extreme VAF amplitudes of the chromosomal imbalances (Figure 1C, bottom panel). As the former is associated with OCA, primarily higher stage disease, and the latter is observed in both OA and OCA, it is important to aim to distinguish these two clinically relevant subtypes of the GH type CNA pattern [343, 483, 484, 649, 673-676, 678, 679]. RCI type CNA (Figure 1D) are characterized by (imbalanced) chromosomal copy number gains, as observed by an SNP imbalance on the VAF plot (Figure 1D, bottom panel) and a higher normalized median amplicon read count of the affected chromosomes (Figure 1D, middle panel), indicating copy number gains. In contrast to GH type CNA, all chromosomes may be involved. No further characterization of RCI type CNA are required (Figure 2). The RCI type, consistent with genotype AAB, is foremost associated with benign oncocytic cell proliferations [649, 673]. No CNA are observed when the VAF plot indicates heterozygosity (Figure 1A, bottom panel) and the normalized median amplicon read count is similar across all chromosomes (Figure 1A, middle panel). Next, the results of somatic mutation and fusion analysis are considered alongside the results of the CNA-LOH analysis (Figure 2). When additional driver mutations that are associated with OCA (e.g., TERT promoter, TP53) are observed, a malignant molecular diagnosis is considered more likely.
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