Mantle cell lymphoma (MCL) is an aggressive non-Hodgkin's lymphoma with median patient survival times of approximately 3 years. Although the characteristic t(11;14)(q13;q32) is found in virtually all cases, experimental evidence suggests that this event alone is insufficient to result in lymphoma and secondary genomic alterations are required. Using a newly developed DNA microarray of 32 433 overlapping genomic segments spanning the entire human genome, we can for the first time move beyond marker based analysis and comprehensively search for secondary genomic alterations concomitant with the t(11;14) in eight commonly used cell models of MCL (Granta-519, HBL-2, NCEB-1, Rec-1, SP49, UPN-1, Z138C and JVM-2). Examining these genomes at tiling resolution identified an unexpected average of 35 genetic alterations per cell line, with equal numbers of amplifications and deletions. Recurrent high-level amplifications were identified at 18q21 containing BCL2, and at 13q31 containing GPC5. In addition, a recurrent homozygous deletion was identified at 9p21 containing p15 and p16. Alignment of these profiles revealed 14 recurrent losses and 21 recurrent gains as small as 130 kb. Remarkably, even the intra immunoglobulin gene deletions at 2p11 and 22q11 were detected, demonstrating the power of combining the detection sensitivity of array comparative genomic hybridization (CGH) with the resolution of an overlapping whole genome tiling-set. These alterations not only coincided with previously described aberrations in MCL, but also defined 13 novel regions. Further characterization of such minimally altered genomic regions identified using whole genome array CGH will define novel dominant oncogenes and tumor suppressor genes that play important roles in the pathogenesis of MCL.

BackgroundHodgkin lymphoma (HL) and Anaplastic Large Cell Lymphoma (ALCL), are forms of malignant lymphoma defined by unique morphologic, immunophenotypic, genotypic, and clinical characteristics, but both overexpress CD30. We used sub-megabase resolution tiling (SMRT) array-based comparative genomic hybridization to screen HL-derived cell lines (KMH2 and L428) and ALCL cell lines (DEL and SR-786) in order to identify disease-associated gene copy number gains and losses.ResultsSignificant copy number gains and losses were observed on several chromosomes in all four cell lines. Assessment of copy number alterations with 26,819 DNA segments identified an average of 20 genetic alterations. Of the recurrent minimally altered regions identified, 11 (55%) were within previously published regions of chromosomal alterations in HL and ALCL cell lines while 9 (45%) were novel alterations not previously reported. HL cell lines L428 and KMH2 shared gains in chromosome cytobands 2q23.1-q24.2, 7q32.2-q36.3, 9p21.3-p13.3, 12q13.13-q14.1, and losses in 13q12.13-q12.3, and 18q21.32-q23. ALCL cell lines SR-786 and DEL, showed gains in cytobands 5p15.32-p14.3, 20p12.3-q13.11, and 20q13.2-q13.32. Both pairs of HL and ALCL cell lines showed losses in 18q21.32-18q23.ConclusionThis study is considered to be the first one describing HL and ALCL cell line genomes at sub-megabase resolution. This high-resolution analysis allowed us to propose novel candidate target genes that could potentially contribute to the pathogenesis of HL and ALCL. FISH was used to confirm the amplification of all three isoforms of the trypsin gene (PRSS1/PRSS2/PRSS3) in KMH2 and L428 (HL) and DEL (ALCL) cell lines. These are novel findings that have not been previously reported in the lymphoma literature, and opens up an entirely new area of research that has not been previously associated with lymphoma biology. The findings raise interesting possibilities about the role of signaling pathways triggered by membrane associated serine proteases in HL and aggressive NHL, similar to those described in epithelial tumors.

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.

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