Cross-platform assessment of genomic imbalance confirms the clinical relevance of genomic complexity and reveals loci with potential pathogenic roles in diffuse large B-cell lymphoma

Abstract Despite excellent initial responses of diffuse large B-cell lymphoma (DLBCL) patients to current frontline immunotherapy (RCHOP), only about 40% of patients are ultimately cured, with most relapses occurring within the first 2-3 years. Apart from clinical features, very few biomarkers are available and validated for risk stratification of DLBCL patients, other than cell-of-origin (COO) subtyping and MYC rearrangement. Genomic copy number changes identified using a variety of genomic profiling technologies including massively parallel sequencing, have variously been reported to have prognostic value in DLBCL. Our previous studies used a common analytical approach across three different clinical datasets to identify and define 32 common regions (CR) of overlapping genomic imbalance (17 gain, 15 loss), comprising 36 minimal common regions (MCR) observed in newly-diagnosed DLBCL. We now report the establishment of standardized calling criteria for a total of 50 aberrations within the 32 CR, taking into consideration GISTIC-defined peaks based on copy number data from three publicly available datasets: IS-172 (GSE11318, n=170), IS-51HR (E-MEXP-3463, n=51 with high IPI), and one similarly analyzed dataset IS-180 (GSE34171, n=180). As a form of validation of the scoring criteria, the frequencies of the aberrations detected in IS-172 in the known GCB and ABC COO subtypes were compared with those previously reported (PMID:18765795). The relative imbalance of the aberrations between the subtypes were re-capitulated including six occurring at higher frequency in the GCB subtype and five in the ABC subtype. Integration of the expression profiles of 162 samples of IS-172 by univariate t-test with the 50 aberrations revealed 569 unique positively-correlated Refseqs mapping to 24 of the MCRs with at least 1.2 fold change in expression (Univariate t-test with P ≤ 0.05 after Benjamini-Hochberg false discovery rate [FDR]). Of these, 27 were located within overlapping peaks of gain/loss. Ingenuity Pathway Analysis determined five significant pathways (P ≤ 0.001, FDR<0.10): p53 signaling, PKC theta signaling in T lymphocytes, geranylgeranyl-diphosphate biosynthesis, B-cell receptor signaling, and RANK signaling in osteoclasts. Association of each of the defined 50 aberrations with overall survival (OS) was performed with the Kaplan-Meier method and log-rank statistic in three RCHOP datasets: IS-124 (GSE15127, n=124), a subset of 70 of IS-180 (IS-70), and a dataset of 41 for which array-CGH was performed in-house using a targeted oligonucleotide (Agilent Technologies) with DNA extracted from sections of formalin–fixed paraffin-embedded de novo DLBCL (IH-41). Ten aberrations significantly associated (P ≤0.05) with poor outcome in at least one of the datasets: gain of 12q (12q13 and 12q14), 16q24, 19q13, and loss of 2q24, 2 sites at 6q21, 8p22, 9p21, 15q15, and 17p13. Of these 10 aberrations, loss of 17p significantly associated with gain of 16q, and losses of 6q, 8p, and 15q (P <0.05), while loss of 9p21 correlated with loss of 2q, 6q, and 8p as evaluated using the exact t-test. Gain of the 2 sites on 12q did not significantly correlate with any of the other 8 aberrations. Genomic complexity was assessed by two methods. In the first, the median number of MCR aberrations was determined to be 1, across three datasets (IS-172, IS-180, and IS-124). Specimens in RCHOP-treated datasets IS-124 and IS-70 were then called “complex” if 2 or more MCR aberrations were detected: 31% and 67% respectively. In both datasets, a complex genome was significantly associated with overall unfavorable outcome (P=0.037, 0.006 respectively). Specimens in IS-172 and IS-70 were additionally classified as “complex” based on the presence of at least one of nine aberrations involving the CDKN2A-TP53-RB-E2F axis (PMID:22975378) where 33% and 57% of cases were scored as complex respectively. Using this approach, significant association with outcome was only found for IS-70 (P<0.001). Of note, between “complex” scoring approaches, 11% and 13% discordance was evident. In summary, establishment of robust scoring criteria for copy number changes afforded correlative analyses with clinical features to reveal that genomic complexity is indeed associated with overall inferior survival in DLBCL and that integrated expression analysis identified biological pathways in DLBCL that may represent potential therapeutic targets. Disclosures Dias: Cancer Genetics,Inc: Employment. Thodima:Cancer Genetics,Inc: Employment, Stock option holder Other. Guttapalli:Cancer Genetics, Inc: Employment, Stock option holder Other. Mendiratta:Cancer Genetics,Inc: Employment. Houldsworth:Cancer Genetics, Inc.: Employment, Stock and stock option holder Other.

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Genomic Complexity in Diffuse Large B-Cell Lymphoma Is Associated with p53 Expression and Inferior Survival

Blood ◽

10.1182/blood.v124.21.3023.3023 ◽

2014 ◽

Vol 124 (21) ◽

pp. 3023-3023

Author(s):

Charles Ma ◽

Imran N Siddiqi ◽

Yi Xie ◽

Anil Tulpule ◽

Jane Houldsworth

Keyword(s):

B Cell ◽

Tp53 Mutation ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Stock Option ◽

P53 Expression ◽

Genomic Complexity ◽

Large B Cell Lymphoma ◽

Gain Loss ◽

Large B Cell

Abstract Genomic complexity in diffuse large-B-cell lymphoma has recently been reported to have strong prognostic value in patients with diffuse large B-cell lymphoma (DLBCL) treated with rituximab-based immunochemotherapy (PMID:22975378). In that study, the presence of the respective gain/loss of at least one of nine genomic markers along the CDKN2A-TP53-RB-E2F axis was used to define cases with “complex” genomes. Genomic imbalance at these specific loci were suggested to functionally contribute toward cell cycle deregulation resulting in increased overall genomic instability. Other clinicopathologic studies have clearly demonstrated that inferior survival is also associated with elevated expression of p53, which serves as a surrogate for TP53 mutation. To date, there have been very few studies, if any, that have examined the relationship between genomic imbalance and complexity, and clinicopathologic features in DLBCL, in particular with relevance to TP53. To this end, DNA was extracted from either sections of formalin-fixed paraffin-embedded biopsies with greater than 70% tumor burden or tumor-enriched cores of 85 DLBCL specimens from patients treated at a single institution (with IRB approval). DNAs from 39 specimens have been blindly submitted to array-CGH to date, using a targeted array (Agilent Technologies). The design permitted assessment at 50 loci commonly gained/lost in DLBCL (arising within 36 minimal common regions [MCRs]) using well defined scoring criteria. Specimens were classified as “complex” if any one of the following aberrations were detected: gain of 1q23, 6p21, 7q22, 12q15, or 19p13, or loss of 9p21, 13q14, 16q12, or 17p13. For all cases, expression of p53, MYC, BCL2, Ki-67, and Epstein-Barr virus (EBV) had previously been examined and reported (PMID:24619762) as was the COO subtype. Correlative analyses with overall survival (OS) were tested using the Kaplan Meier method and log rank statistic. Significance of pathogenomic correlations were examined using the Fisher’s exact t-test (P<0.05 was considered significant). Of the 39 cases evaluated to date, IPI was available for 34. For the preliminary analysis comprising 39 cases, genomic complexity (observed for 22 cases) did not significantly correlate with IPI, consistent with the prior report where genomic complexity associated with inferior survival independent of IPI (PMID:22975378). However, genomic complexity did portend shortened survival within the preliminary 39 patients studied to date (P<0.01) and a trend was observed for the 29 who received RCHOP or RCHOP-like therapy (P=0.05) (for the remaining 10 cases, treatment status was unknown for 7 and 3 received palliative care). Genomic complexity based on absolute number of aberrations for the entire 50 assessed (>1), did not exhibit significant association with outcome. Fourteen of 18 cases with p53 expression (>30%) had complex genomes, which was significantly enriched compared with cases with low or no p53 expression (P <0.03). No significant correlation was found between genomic complexity and BCL2 (>70%) and MYC (45%) expression, nor with COO subtype. Specifically for TP53, loss of 17p13 was detected in 7/39 cases, and positively correlated with adverse outcome both for the entire dataset (P=0.05) and for RCHOP-treated patients (P=0.02). Interestingly, of those with loss, 3 were not positive for p53 expression and overall, p53 expression did not correlate with 17p loss. Previous analyses in this dataset had also revealed that co-expression of p53 and MYC, but not BCL2 had an enhanced negative effect on outcome. Only one of 5 cases with co-expression of p53 and MYC also displayed 17p loss. Overall then, p53 expression was associated with underlying genomic complexity but specific loss of the TP53 locus appeared to mark another smaller subset of DLBCL patients with inferior survival, independent of p53 expression. Expansion of the study to include the additional 46 cases is currently ongoing to confirm these emerging correlative patterns, as is determination of the TP53 mutation status of each specimen. Additional correlative pathogenomic studies will also be afforded with the larger dataset, examining the role of the 8 other loci of genomic gain/loss suggested to underlie genomic complexity in DLBCL. Disclosures Ma: Cancer Genetics, Inc.: Employment, Stock option holder Other. Houldsworth:Cancer Genetics, Inc: Employment, Stock and stock option holder Other.

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