Genome-Wide Analysis of Copy Number Alterations/LOH/Allelic Imbalances in Non-Hodgkin Lymphoma Using Ultrahigh-Density SNP-Genotyping Microarrays with the Robust CNAG Algorithms.

Non-Hodgkin lymphomas (NHL) are hematopoietic malignancies originated from diversity of peripheral lymphoid organs. During the past two decades, there have been significant advances in the pathogenesis of NHL including identification of a number of genes associated with the disease-specific translocations and other genetic alterations. In view of cytogenetics, however, NHL frequently shows complex chromosomal abnormalities involving copy number alterations as well as other unbalanced translocations, many of which have not been unveiled at the molecular levels.

Affymetrix® 100K/500K mapping arrays were originally developed for large-scale SNP typing required for genome-wide association studies, but the quantitative nature of the whole-genome amplification and hybridization used in these platforms also makes them powerful tools for genome-wide analysis of cancer genomes with use of uniformly distributed 116,204/520,000 SNP-specific probes. Moreover the use of SNP specific probes enables allele-specific copy number analysis that is totally impossible with other platforms. Here we developed the robust algorithms (Copy number analyzer for Affymetrix® GeneChip®; CNAG) for high-quality processing of 100K/500K data and analyzed a total of 72 NHL samples (61 primary samples including 34 diffuse large B-cell lymphoma, 18 follicular lymphoma and 11 cell lines including 3 adult T cell leukemia/ lymphoma) for genome-wide copy number alterations, LOH, and allelic imbalances at the resolutions of 23.6/5.4 kb.

In 100K analysis, 34 homozygous deletions and 42 high-grade amplifications and other numerous copy number alternations and/or LOH, were identified together with possible gene targets as for some regions. 500K analysis disclosed even more subtle changes. Common overlapping alternations included deletions in 1p31.1 and 9p21.3, and 19p13.32 and high-grade amplifications in 3p14.2–p14.1,7q21.13–q21.3, and 20q11.21.

Of particular importance is, however, the finding of otherwise undetected copy number neutral LOHs, which are revealed only by allele-specific copy-number analysis. In fact the copy number neutral LOHs represented a novel type of genetic abnormality in NHL because they were very frequent and found in more than 87% (20/23) of NHL cases examined with allele-specific copy number analysis, making a stark contrast to ALL, in which these abnormalities were rare. They typically involved chromosomal ends, indicating somatic recombinations are the potential mechanism of generating these abnormalities. Notably, there was a clear predisposition of the copy number neutral LOH to specific chromosomal loci including 1p, 1q, 6p, 9p, 17q, and 19p suggesting existence of relevant genes to NHL pathogenesis within these common regions.

In conclusion, Affymetrix® SNP-genotyping microarrays and our CNAG algorithms provide a powerful platform of dissecting NHL genomes and could facilitate identification of the novel molecular mechanisms for lymphomagenesis.