Gene profiling of canine B-cell lymphoma reveals germinal center and postgerminal center subtypes with different survival times, modeling human DLBCL

Abstract

Diffuse large B-cell lymphoma (DLBCL) is the most common lymphoma subtype, and fewer than half of patients are cured with standard first-line therapy. To improve therapeutic options, better animal models that accurately mimic human DLBCL (hDLBCL) are needed. Canine DLBCL, one of the most common cancers in veterinary oncology, is morphologically similar to hDLBCL and is treated using similar chemotherapeutic protocols. With genomic technologies, it is now possible to molecularly evaluate dogs as a potential large-animal model for hDLBCL. We evaluated canine B-cell lymphomas (cBCL) using immunohistochemistry (IHC) and gene expression profiling. cBCL expression profiles were similar in many ways to hDLBCLs. For instance, a subset had increased expression of NF-κB pathway genes, mirroring human activated B-cell (ABC)-type DLBCL. Furthermore, immunoglobulin heavy chain ongoing mutation status, which is correlated with ABC/germinal center B-cell cell of origin in hDLBCL, separated cBCL into two groups with statistically different progression-free and overall survival times. In contrast with hDLBCL, cBCL rarely expressed BCL6 and MUM1/IRF4 by IHC. Collectively, these studies identify molecular similarities to hDLBCL that introduce pet dogs as a representative model of hDLBCL for future studies, including therapeutic clinical trials.

Figure 1

Histologic appearance and immunophenotyping of canine BCL. (A) cDLBCL is characterized by lymph node effacement with diffuse sheets of large B-cells (H&E stain, first panel) that are CD79a-positive (second panel) and CD3-negative (third panel) by immunohistochemistry. (B) Canine MZL is characterized by nodules of large B-cells with “fading germinal centers” (H&E stain, first panel) that are CD79a-positive (second panel) and CD3-negative (third panel) by immunohistochemistry. Insets show the cell size similarity between the two morphologic types.

Figure 2

Principal component analysis does not distinguish cMZL and cDLBCL. The 1079 genes that were most differentially expressed (Supplemental Fig. 2) among cBCL were used for principal component analysis. There is no division between histologic subtypes of cBCL by gene expression data.

Figure 3

cBCL and hDLCBL co-cluster using human ABC/GCB classifier genes. Expression data from 203 hDLBCLs was combined with 58 cBCL expression profiles. Distance Weighted Discrimination (DWD) was used to remove systematic biases between the two groups, and unsupervised hierarchical clustering was performed using the listed genes. Red branches are cBCL samples and black branches are hDLBCL samples.

Figure 4

Clustering of cBCLs using human ABC/GCB classifier genes. (A) Hierarchical clustering of gene expression data reveal two groups of cBCLs that are not robustly separated. Also, genes more highly expressed in ABC lymphomas (teal) do not cluster distinctly from the genes more highly expressed in GCB lymphomas (orange) on the vertical axis. (B) Kaplan-Meier survival curves and Cox regression analysis were performed on the two groups. Overall and progression-free survival by ABC/GCB grouping approaches, but does not reach, statistical significance.

Figure 5

Clustering of cBCLs using “dog specific” ABC/GCB classifier genes. (A) Hierarchical clustering with 1180 “dog specific” ABC/GCB genes. Differentially expressed genes categorized as being in the “B-cell receptor pathway”, are listed on the left. (B) Kaplan-Meier survival curves and Cox regression were performed on the two groups.

Figure 6

Immunoglobulin heavy chain mutation status predicts survival in cBCL. The IGHV gene was subcloned and sequenced in 53 cBCLS, which were then categorized as “ongoing” (many different subclones, indicating ongoing SHM) or “static” (subclones identical or nearly identical, indicating completed SHM) (A) Kaplan-Meier survival curves and Cox regression were performed on the two groups. (B) Overlap between groups defined by canine “ABC/GCB” genes (Fig. 5) and IGHV status is shown. p=0.097 for the X² test.