Purpose: Follicular lymphoma (FL) constitutes the second most common non-Hodgkin's lymphoma in the Western world. The clinical course is variable and only in part explained by known tumor-intrinsic or -extrinsic factors. FL carries the hallmark chromosomal translocation t(14;18), deregulating the expression of Bcl-2, but this is not sufficient to explain either FL biology or clinical behavior. Experimental Design: We have employed high-density genomic profiling technology using the Affymetrix 50K-XbaI oligonucleotide single nucleotide polymorphism^chip platform to interrogate the genomes of 58 fluorescence-activated cell^sorted (FACS) FL specimens for chromosomal copy number changes and 46 specimens for loss of heterozygosity (LOH). Results: We report (a) previously unknown high-frequency copy-neutral LOH (uniparental disomy) in FL on chromosomes1p (f50%) and 6p (f30%); (b) that del6q is complex, as reported, with at least two regions of minimal common loss at 6q13-15 and 6q23-24, and that in addition, f8% of FL specimens contain a homozygous deletion at 6q23.3-24.1 that spans the negative NFnB regulator A20 and the p53 apoptosis effector PERP ; (c) that combined analysis of chromosome 17p for LOH, copy number, and p53 mutations shows that most p53 mutations in FL do not involve del17p. Finally, we map high-frequency LOH with and without copy loss on chromosomes 9p, 10q, and 16p and genomic gains on 2p15-16 and 8q24.22-24.3. Conclusions: This comprehensive description of the pathologic anatomy of the FL genome uncovers novel genetic lesions and should aid with identification of genes relevant to FL biology and clinical behavior.
Chronic lymphocytic leukemia (CLL) is the most common leukemia in the Western world and remains incurable with conventional therapies. Patients with relapsed or resistant CLL have a significantly shortened lifespan. MDM2 inhibitors have been developed and may have significant potential in the treatment of CLL. Clinical development of these compounds would be aided through knowledge of molecular predictors of activity. To understand determinants of sensitivity or resistance to MDM2 inhibitor therapy in CLL, we comprehensively analyzed a large cohort of CLL patient-derived samples for response to MDM2 inhibition and correlated these responses with clinically important biomarkers. Furthermore, we employed high-density single nucleotide polymorphism (SNP) arrays to analyze genomewide changes of copy number and allele status, including that of p53. The results of these studies conclusively demonstrate that p53 status is the major determinant of response to MDM2 inhibitors in CLL. Additional defects in the p53 regulatory cascade do not appear operational in this leukemia. Further, we identify a novel subgroup of patients with CLL with early progressive disease that appears particularly sensitive to MDM2 inhibitor treatment. These data provide definitive evidence for target-specific and predictive activity and a rationale to proceed with this potentially important class of compounds in the treatment of CLL. IntroductionChronic lymphocytic leukemia (CLL) is the most common form of adult leukemia in the Western world, and is characterized by a highly variable clinical course. 1,2 Treatment of CLL is reserved for symptomatic patients or patients in advanced clinical stage. Despite improvements in response rates using chemoimmunotherapy combinations, CLL remains incurable, and patients refractory to fludarabine or patients who have suffered multiple disease relapses have a poor outlook. [3][4][5] Therefore, novel therapeutics are needed to advance the outlook for afflicted patients.Inhibitors of murine double minute 2 (Mdm2) represent a novel therapeutic approach. [6][7][8][9][10][11][12][13][14][15][16][17][18][19] In cells with functional p53, the p53 activity is primarily inhibited through direct and tonic interaction with the MDM2 protein. [20][21][22] Treatment of various tumor cells with inhibitors of the MDM2-p53 interaction results in rising p53 levels and subsequent induction of cell-cycle arrest and apoptosis. For poorly understood reasons, nonmalignant cells appear relatively resistant to MDM2 inhibition. 23,24 In CLL, in contrast to many solid tumors, p53 is mutated in only about 10% of patients at presentation and in 10% to 30% of patients with pretreated disease. [25][26][27][28] Thus, small-molecule inhibitors that block the MDM2-p53 interaction could represent a new therapeutic strategy for the treatment of most patients with CLL.Human cancers are biologically heterogeneous and respond nonuniformly to therapeutic intervention. Therefore, understanding the molecular mechanisms underlying cancer susceptibili...
Chronic lymphocytic leukemia (CLL) has a variable clinical course. Presence of specific genomic aberrations has been shown to impact survival outcomes and can help categorize CLL into clinically distinct subtypes. We studied 178 CLL patients enrolled in a prospective study at the University of Michigan, of whom 139 and 39 were previously untreated and previously treated, respectively. We obtained unbiased, high-density, genome-wide measurements of subchromosomal copy number changes in highly purified DNA from sorted CD19 ؉ cells and buccal cells using the Affymetrix 50kXbaI SNP array platform (Santa Clara, CA). Genomic complexity scores were derived and correlated with the surrogate clinical end points time to first therapy (TTFT) and time to subsequent therapy (TTST): measures of disease aggressiveness and/or therapy efficaciousness. In univariate analysis, progressively increasing complexity scores in previously untreated CLL patients identified patients with short TTFT at high significance levels. Similarly, TTST was significantly shorter in pretreated patients with high as opposed to low genomic complexity. In multivariate analysis, genomic complexity emerged as an independent risk factor for short TTFT and TTST. Finally, algorithmic subchromosomal complexity determination was developed, facilitating automation and future routine clinical application of CLL whole-genome analysis. (Blood. 2008
To understand determinants of sensitivity or resistance to MDM2 inhibitor therapy in chronic lymphocytic leukemia (CLL), we comprehensively analyzed highly pure CD19+ cells derived from 106 CLL patients for response to the MDM2 inhibitors MI-63 and Nutlin3 ex vivo and correlated these responses with clinically important biomarkers, including hierarchical FISH, IgVH-mutations and ZAP70 expression. P53 exons 5–9 were sequenced for all cases, and p53 induction pre- and post-inhibitor treatment was determined by immunoblotting for all 106 samples. Furthermore, we employed high-density 50kSNP-arrays to analyze changes of copy number and allele status, including that of p53 on chromosome 17p, in 171 CLL patients and correlated this finding with p53 mutations and p53 expression. Patient-derived samples were incubated for 36 hours with both inhibitors in parallel and apoptotic (annexin-V) and necrotic cell death (PI) was assayed for all incubations using flow cytometry. IC50 values were calculated using XL-fit. Nineteen CLL cases demonstrated either p53 mutations or aberrant expression and all were relatively resistant to MDM2 inhibitor treatment; eighty-seven cases had functional p53 and all but 3 cases demonstrated sensitivity to MDM2 inhibitor treatment. Very few cases with functional p53 demonstrated IC50 values for both inhibitors that were within the range of p53 mutant cases. Del17p patient samples were resistant to MDM2 inhibitor-mediated cell kill, while all other CLL biomarker-based subgroups appeared equally sensitive. Univariate time to first therapy (TTFT) estimates demonstrated a statistically significant (p=0.02 using the log-rank test) shorter TTFT for the group of 19 CLL cases with aberrant p53 function as opposed to the group of 87 cases with functional p53. Importantly, TTFT analysis for the 87 patients with functional p53 found that patients with low IC50 values (high sensitivity to MDM2 inhibitors) had more aggressive CLL (shorter TTFT), identifying for the first time a susceptible target group for these inhibitors that demonstrates aggressive disease and wt p53. In conclusion, the results of these studies conclusively demonstrate that p53 status is the major determinant of response to MDM2 inhibitors in CLL. Additional defects in the p53 regulatory cascade do not appear operational in this leukemia. This comprehensive dataset provides supportive rationale for the design of early phase human trials using these compounds and supports ex vivo measurements of p53 induction pre- and post-MDM2 inhibitor treatment as part of future trial schemas.
Follicular lymphoma (FL) constitutes the second most common non-Hodgkin’s lymphoma in the Western world. The clinical course is variable and only in part explained by known tumor-intrinsic (genetic) or -extrinsic (infiltrating immune cells, infiltrating macrophage content) factors. FL carries the hallmark chromosomal translocation t(14;18), deregulating expression of Bcl-2, but this is not sufficient to explain either FL biology or clinical behavior. We have employed genomic profiling technology using the Affymetrix 50K-Xba1 oligonucleotide single nucleotide polymorphism (SNP) chip platform to interrogate the genomes of 58 FL specimens for loss of heterozygosity (LOH) and chromosomal copy number changes. Where available, these high-density genomic data were correlated with published FL gene expression data. Using this integrated approach, we provide data that suggest that i) Bcl11A is a likely target gene for the amplification on chromosome 2p15–16 in FL, ii) that the amplification of der(18)t(14;18) does not include Bcl-2, iii) high-frequency copy-neutral LOH exists at 1p36 (50%) and 6p (33%), and that 6p LOH possibly deregulates IRF4 and, iv) deletion 6q is non-overlapping, complex and possibly involves four or more gene loci of importance. In addition, high-frequency LOH (with and without chromosomal copy loss) exists on chromosomes 9p, 10q and 16p. These data should facilitate analysis of specific genes in FL and should allow for development of a SNP -and genomics-based assay for FL risk stratification and outcome prognostication.
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