Gene-Specific Delineation of Copy Number Aberrations in Follicular Lymphoma with Molecular Inversion Probes.

Background: Follicular lymphoma, derived from germinal center B cells, is one of the most common types of lymphoma. Follicular lymphoma like many other malignancies demonstrates genomic instability, a phenomenon where tumor cells accumulate various genetic changes including point mutations, genomic deletions and gene amplifications. Such genomic aberrations may contribute to cancer phenotype such as transformation. Previous studies of follicular lymphoma have shown a combination of cytogenetic - aneuploidy type events from gross chromosomal alterations to smaller deletions and amplifications. For example, deletions in chromosome 6q may be associated with follicular lymphoma aggressiveness. Efforts to identify these changes have used traditional karyotyping, comparative genomic hybridization (CGH) or traditional genetic markers (microsatellites) for loss of heterozygosity (LOH) analysis. Characterization of of genomic instability in follicular lymphoma has not been thoroughly investigated on a genomic scale.

Objectives: We have employed a novel genomic technology relying on molecular inversion probes (MIPs) to conduct a large scale analysis of gene copy aberrations in follicular lymphoma. MIPs technology enables querying any gene sequence in the genome at a high resolution for allele-specific gene copy alterations and polymorphisms simultaneously, a significant improvement over other genomic methods such as array CGH. We are using this genomic technology to delineate concordant gene copy aberrations in follicular lymphoma that may influence a variety of clinical outcomes using a probe set with over 7,000 cancer-related genes among 25,000 loci.

Methods: DNA was extracted from a cohort of follicular lymphoma clinical samples obtained at the time of diagnosis and prior to treatment. B-cell lymphoma cells were purified using a Rosette purification scheme, genomic DNA was isolated from the purified tumor cell populations from 37 individual lymphoma samples and the cohort was analyzed with a MIP cancer probe set described above. Each probe contains two unique recognition sequences to a genomic DNA target and a unique barcode sequence. The molecular inversion probe assay was conducted. The final products were hybridized to a generic barcode microarray overnight and interrogated by an Affymetrix scanner. Barcode array data was preprocessed and gene copy number was extrapolated for each molecular inversion probe. Concordant gene copy aberrations were determined across the sample set.

Results: Follicular lymphoma samples from pretreated patients have common patterns of chromosomal losses, gains and multiple gene deletions with large overlap in some areas of deletion. Common large scale chromosomal deletions and amplifications included regions of chromosome 6 and 18. Multiple individual genes were found to be commonly amplified or deleted as well including BTK which is suspected to play a role in follicular lymphoma.

Conclusions: MIP technology has been used to analyze follicular lymphoma for highly sensitive and specific gene copy analysis. We identified concordant gene copy alterations found across the majority of samples in the cohort, suggesting that these aberrations may play a role in follicular lymphoma development. We are pursuing additional studies to confirm these findings.