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Copy‐number methods dramatically underestimate loss of heterozygosity in cancer

In the first seven months of 2006, Genes, Chromosomes and Cancer published eight studies using comparative genomic hybridization (CGH) to analyze genomic gains and losses in cancer (Aarts et al., 2006; Coe et al., 2006; Diep et al., 2006; Horsley et al., 2006; Hosoya et al., 2006; Mendrzyk et al., 2006; Ribeiro et al., 2006; Rossi et al., 2006). Such studies, and those based upon other copy-number methods (Pinkel et al., 1988; Schrock et al., 1996; Lucito et al., 2003), are most informative when copy-number changes closely mimic changes in allelic status, for otherwise they will under-represent the extent of genomic alterations (Cavenee et al., 1983; Huang et al., 2004). Beroukhim and colleagues, using 10K SNP arrays to study prostate cancer, suggested that the extent of under-representation of loss of heterozygosity (LOH) in copy number methods may be very high (Beroukhim et al., 2006). Copy-neutral (i.e., having two copies) and copy-gain (i.e., having more than two copies) forms of allelic loss accounted for a surprising 80% of all LOH identified using paired normal/tumor tissues. Given the interest in deletion assessments by CGH, we felt a need to provide an independent confirmation of these findings. Therefore, we compared copy-number estimates with genotype (allele-discriminating) data obtained from 24 pancreatic cancer cell lines previously evaluated on 100K SNP arrays (Calhoun et al., 2006). Copy-number estimates were determined by the Affymetrix Copy Number Analysis Tool (version 2.2) and variation was smoothed using a moving-average algorithm. All copy-number estimates were rounded to the nearest integer and cataloged with respect to allelic status (i.e., LOH vs. retention of heterozygosity, ROH). Figure 1 summarizes these data, representing more than 2.7 million SNPs. We found that copy-neutral and copy-gain LOH events accounted for 67.5% of all LOH identified in pancreatic cancer (15.0% represented copy-gain LOH), while LOH associated with copy-reduction occurred at a rate of only 32.2% of LOH identified. Before drawing conclusions from these results, an assessment of likely sources of error is in order. For example, *9.5% of ROH SNPs were estimated to have a reduction in copy-number (i.e., having one copy). Such a counter-intuitive result is inherently erroneous and could be due to the failure to correctly classify candidate regions of LOH. Such errors could derive from a number of sources. For example, a previous estimate suggests that 17.5% of LOH went undetected when analyzed in the absence of matched normal tissue (Calhoun et al., 2006). Beroukhim et al., (2006) provided a similar false-negative rate (19.8%) in their study. Such misclassifications would decrease the frequency of copy-reduction LOH. Conversely, an estimated false-positive rate of LOH of 11.1% (ROH incorrectly classified as LOH) would artificially inflate the level of copy-neutral and copy-gain LOH seen in our study. Assuming these represent maximal false-negative and false-positive rates and using our SNP assessment as ‘‘true LOH’’, we did a virtual reassignment of all copy-reduced ROH SNPs as copy-reduced LOH SNPs and reassigned 11.1% of copy-neutral and copy-gain LOH SNPs as ROH SNPs. From this analysis, we predict that methods depending solely upon copy number analysis would theoretically identify a maximum of only 47.2% of all forms of LOH. The results of our study in pancreatic cancer support those of Beroukhim et al., (2006) in prostate cancer and suggest the levels of LOH to be underestimated markedly in recent studies using CGH. In a recent review by Karhu et al., (2006), the authors summarize the spectrum of genetic alterations reported in pancreatic cancer using copy-number methods and reinforce the idea that pancreatic adenocarcinoma is a genetically highly-complex disease. The apparent poor accuracy in illustrating the true level of LOH, however, appears to undermine the effectiveness of these studies in characterizing genomic losses in cancer tissues.