Deletion in Chromosome 17p12 and Gains in Chromosome 9q33.3 by Array Comparative Hybridization Are Associated with R-CHOP Treatment Failure in Patients with Diffuse Large B Cell Lymphoma

Abstract

Background: The genetic alterations associated with survival in patients with diffuse large B cell lymphomas (DLBCL) treated with combined rituximab-CHOP (R-CHOP) chemo-immunotherapy are not well understood. Methods: 92 patients with DLBCL treated with R-CHOP who had a biopsy available at the time of diagnosis were included in the study. 31 patients were classified as treatment failures defined as progression < 6 months of completing R-CHOP and 61 patients were classified as treatment successes defined as a maintained remission >2 years after diagnosis. We used genome-wide BAC array comparative genomic hybridization (aCGH) to determine the genomic copy number imbalances. The presence of genetic gains and losses were determined using the intersection between visual annotations and a Hidden Markov model algorithm. DLBCL cell of origin (COO) subtype distinctions (GCB vs ABC) were determined using a Bayesian predictor model on gene expression derived from custom Affymetrix arrays (Dave, N Engl J Med, 2006;354:2431). A permutation test was used to identify genetic regions that were significantly different between treatment failures and treatment successes. Functional pathway analysis was performed using Ingenuity software. A novel model based clustering algorithm was applied to the normalized data to determine if any association with outcome correlated with the observed genetic alterations. Results: Lymphoma progressed in 31/92 (34%) patients < 6 months after R-CHOP (median follow-up = 4 y). All 92 patients had successful aCGH and 81 had COO available for this analysis. The International Prognostic Index (IPI) and COO were predictive of outcome (p=0.04, p=0.02, respectively). 451 regions containing 338 genes were associated with treatment failure with a p-value of <10−6. Gains in 9q33.3 were found in 13 patients (14%) and were significantly associated with treatment failure p<10−8. This region contains genes such as HSPA5, a negative regulator of apoptosis and PPP6C, a positive regulator of the cell cycle by targeting IKBe thereby removing inhibition of the NFkB pathway. Deletions in 17p12 were detected in 24 (26%) and were the most statistically significantly associated with treatment failure p<10−9. This region contains tumor necrosis factor (TNF) receptor superfamily member (TNFRSF13B or TACI) which, when deleted or mutated, has been previously shown to lead to activation of the noncanonical NFkB pathway and B cell proliferation. 21 of these 24 patients also had deletion of 17p13 at the TP53 locus (p<10−6). Neither 9p33.3 nor 17p12 deletion was associated with COO distinctions. Using Ingenuity, pathways involving apoptosis and cellular proliferation, specifically those involving P53, MYC and HSPA5 genes, were over-represented in treatment failures (p=2.04 × 10−4). Unsupervised clustering of the aCGH data demonstrated that 60% of cases could be stratified into 4 genetic sub-groups based on the presence of 1q+, 6q−, +7 and the concurrent presence of +3 and +18. Supervised analysis demonstrated that the +3/+18 group and the 6q− group were associated with ABC subtype of DLBCL whereas the +7 and 1q+ groups were associated with the GCB subtype. However, these genetic groups did not correlate with treatment outcome. Conclusions: Some genetic alterations cluster together and can distinguish COO subtypes of DLBCL. Gains on 9q33.3 and deletions of 17p12 are common alterations detected by high resolution aCGH in DLBCL. Most importantly, these alterations involve genes known to be critical in B cell proliferation and apoptosis and alterations at these sites are strongly associated with treatment failure (p values <10−8) in patients treated with R-CHOP.

Model-based clustering of array CGH data

“…In a simulation study, with realistic data, we show how our method is more accurate than other clustering methods, including hierarchical clustering (van Wieringen and van de Wiel, 2008 ) and K-medoids (KM) (an approach not previously applied to data of this kind). More importantly, we show how HMM-Mix reveals clinically relevant subgroups in data derived from a cohort of 106 follicular lymphoma (FL) patients, originally reported in Cheung et al ( 2008 ), and reveals previously unreported patterns of alteration in a cohort of 92 diffuse large B-cell lymphoma (DLBCL) patients (Johnson et al , 2008 ).…”

“…The DLBCL data (Johnson et al , 2008 ), contains aCGH data for 92 patients with de novo DLBCL, all treated uniformly with multi-agent chemotherapy (CHOP) and anti-CD20 monoclonal antibody rituximab.…”