Adult high-grade B-cell lymphoma with Burkitt lymphoma signature: genomic features and potential therapeutic targets

Abstract

The adult high-grade B-cell lymphomas sharing molecular features with Burkitt lymphoma (BL) are highly aggressive lymphomas with poor clinical outcome. High-resolution structural and functional genomic analysis of adult Burkitt lymphoma (BL) and high-grade B-cell lymphoma with BL gene signature (adult-molecularly defined BL [mBL]) revealed the MYC-ARF-p53 axis as the primary deregulated pathway. Adult-mBL had either unique or more frequent genomic aberrations (del13q14, del17p, gain8q24, and gain18q21) compared with pediatric-mBL, but shared commonly mutated genes. Mutations in genes promoting the tonic B-cell receptor (BCR)→PI3K pathway (TCF3 and ID3) did not differ by age, whereas effectors of chronic BCR→NF-κB signaling were associated with adult-mBL. A subset of adult-mBL had BCL2 translocation and mutation and elevated BCL2 mRNA and protein expression, but had a mutation profile similar to mBL. These double-hit lymphomas may have arisen from a tumor precursor that acquired both BCL2 and MYC translocations and/or KMT2D (MLL2) mutation. Gain/amplification of MIR17HG and its paralogue loci was observed in 50% of adult-mBL. In vitro studies suggested miR-17∼92's role in constitutive activation of BCR signaling and sensitivity to ibrutinib. Overall integrative analysis identified an interrelated gene network affected by copy number and mutation, leading to disruption of the p53 pathway and the BCR→PI3K or NF-κB activation, which can be further exploited in vivo by small-molecule inhibitors for effective therapy in adult-mBL.

Figure 1.

Adult and pediatric-mBL cases are indistinguishable by the Burkitt classifier but have distinct CNAs. (A) Heat map showing expression of genes in the Dave et al and Hummel et al Burkitt classifiers for a BL and DLBCL training cohort and a validation cohort consisting of adult-BL, pediatric-BL, nonmolecularly classified Burkitt, and t(8;14)-positive DLBCL tumors. MYC and BCL2 translocation status of the 61 cases in the study are noted, and cases in which the translocation status comes solely from NGS data are marked (·). (B) Comparison of the mBL probability score predicted by the Dave et al and Hummel et al BL classifiers. Three cases classified as Burkitt by the Dave classifier would be classified as intermediate by the Hummel classifier. (C) Kaplan-Meier curves comparing overall survival of adult and pediatric molecularly classified Burkitt cases. The adult-BL series shows dismal clinical outcome, which may be attributed to suboptimal treatment, diagnostic challenges (before the GEP era), double-hit cases with mBL features, and advanced age (median, 55 years).

Figure 2.

CNAs in germinal center B-cell-derived lymphomas. (A) Box plot of the proportion of the aberrant genome (change in genome content) for the noted GC-B-cell-derived NHL entities. (B) Bar graph of the frequency or recurrent CNAs in different lymphomas. CNAs with significantly a different (P < .05) frequency of CNAs are denoted (*). (C) Circos plots comparing the frequency of gains and losses found in the noted lymphoma types. Darker shades denote regions of amplifications or homozygous copy loss and axis lines represent 20%.

Figure 3.

Select CNA or genes found to be recurrently mutated in 52 mBL cases. (A) The block color represents the type of mutation. Blocks with 2 colors indicate that more than 1 type of mutation was observed. Genes also affected by copy number abnormalities are noted (G, copy gain; L, copy loss; N, copy neutral loss of heterozygosity). MYC and BCL2 translocation status is shown and MYC, BCL2, and KI67 mRNA expression relative to the median is depicted. (B) Copy number and/or mutation status of RB1, CDKN2A/CDKN2B, and CCND3 for individual mBL tumors.

Figure 4.

MIR17HG and Chr8q are gained in mBL. (A) Frequency of DNA CN gains found in adult (red-orange) or pediatric (blue) mBL for MIR17HG and paralogues MIR106B and MIR106A. (B) Heat maps of expression of the MIR17∼92 cluster members measured by TaqMan Array Human MicoRNA A Card (ABI) in tumors with and without MIR17HG DNA copy number gain. (C) Validation of the frequency of MIR17HG DNA CN gain in adult-BL using a published BL genomic/WES dataset. CN status was assessed using Varscan2 CNV followed by segmentation by the Circular binary segmentation algorithm. For WES data, regions of gain were cut off at a threshold of 0.2 log₂ ratio. (D) Copy number status of MIR17HG and paralogues for individual tumors (gain = red, loss = green). (E) Kaplan-Meier curves comparing overall survival adult-mBL cases by MIR17HG copy number status. Several MIR17HG gain/amplified cases also had double MYC/ BCL2 translocation/mutation, but showed BL mutation and GEP profile. (F) Plot of CNAs along chromosome 8 for mBL cases with abnormalities. (G) Bar graph of the fold increase in expression of genes along chromosome 8 that are significantly upregulated (P < .05, 1-sided Student t test) in cases with CN gain. Genes of interest are noted. (H) Boxplot of MYC mRNA expression by MYC DNA copy number status and by age. Red points represent expression values of individual cases. Expression levels in normal centroblasts (CB), CD77-negative cells, and naive B cells (NBC) are noted for comparison. (I) MYC protein expression in 3 cases with MYC gain and 3 cases with a MYC DNA copy number=2. (J) Signatures upregulated on MYC overexpression were enriched in pediatric-mBLs compared with adult-mBLs.

Figure 5.

18q21 gain or BCL2 translocation is associated with BCL2 protein expression. (A) BCL2 IHC was performed and scored on adult-mBL cases with no evidence of BCL2 gain/translocation based on clinical data, NGS, and SNP array (n = 7) and cases with BCL2 gain/translocation (n = 6). Bars represent the mean expression score and error bars represent 1 standard deviation. (B) Example of BCL2-positive and BCL2-negative staining cases (original magnification, ×20). (C) BL cell lines are sensitive to a pan-BCL2 inhibitor compared with single-agent inhibitors. Bars represent the mean of 3 replicates from 1 representative experiment of at least 3 separate experiments, and error bars represent 1 standard deviation.

Figure 6.

Evaluation of functional significance of MYC and MIR17HG in BL tumors and cell lines. (A) Comparison of average miR-17-92 expression and MYC expression in mBL tumors. miR17∼92 expression levels were inversely correlated with MYC expression in tumors (r = −0.23). (B) Whole-cell lysates were western blotted for PTEN, BIM, MYC, and β-actin expression in the noted BL cell lines expressing empty vector (C) or a vector containing mIR17-92 sponge (S). (C) BIM, PTEN, and MYC expression was normalized to β-actin, and relative expression (sponge/vector control) was quantified from 2 separate western blot experiments. Error bars represent 1 standard deviation. (D) Proliferation in BL cell lines after miR17∼92 sponge expression or MIR17HG knock out (Daudi-CRISPR) and/or ibrutinib (IBT) treatment. Line graph depicts 1 representative experiment of triplicate experiments, and points are the average of 3 biological replicates. Error bars represent 1 standard deviation. BL cell lines were treated with a specific concentration of IBT based on the 50% infective dose calculation for each line. (E) Comparison of mRNA levels of CD22 and FCGR2B in control (vector or parental) or miR17∼92 sponge or knockout (Daudi-CRISPR) cells with or without IgM stimulation. Fold change was calculated relative to the vector or parental controls. Graph depicts 1 representative experiment of 3 separate experiments. Error bars depict 1 standard deviation of 3 technical replicates. (F) Cell proliferation in BL cell lines after MYC KO and/or miR17∼92 knockdown (sponge). Points represent the mean of 3 biological replicates from 1 representative experiment of at least 3 separate experiments. Error bars represent the standard error of the mean. (G) Western blots of whole-cell lysates from control (GFP or parental) or miR17∼92 sponge-expressing or MIR17HG knock-out (Daudi-Cas-9) BL cell lines after induction of BCR signaling with anti-IgM. (H) Relative expression of phosphorylated/total SYK, BTK, BLNK, and ERK was quantified from the western blot, suggesting decreased BCR signaling in miR17∼92 sponge-expressing or KO BL cell lines.