Duodenal follicular lymphoma: comprehensive gene expression analysis with insights into pathogenesis

Abstract

Follicular lymphoma (FL) of the gastrointestinal tract, particularly duodenal follicular lymphoma (DFL), is a rare variant of FL with indolent clinical behavior, and this disease is included in the 2008 World Health Organization classification system. In contrast to nodal follicular lymphoma (NFL), DFL occurs most frequently in the second part of the duodenum, lacks follicular dendritic cell meshworks and has memory B-cell characteristics. However, its molecular pathogenesis is still unclear. In the present study, we examined 10 DFL, 18 NFL and 10 gastric MALT lymphoma samples using gene expression analysis. Quantitative RT-PCR experiments and immunohistochemical analysis for 72 formalin-fixed, paraffin-embedded tissues from an independent series, including 32 DFL, 19 gastric MALT lymphoma and 27 NFL samples, were performed for validation of microarray data. Gene expression profiles of the three lymphoma types were compared using 2918 differentially expressed genes (DEG) and results suggested that DFL shares characteristics of MALT lymphoma. Among these DEG, CCL20 and MAdCAM-1 were upregulated in DFL and MALT but downregulated in NFL. In contrast, protocadherin gamma subfamily genes were upregulated in DFL and NFL. Quantitative RT-PCR and immunohistochemical studies demonstrated concordant results. Double immunofluorescence studies revealed that CCL20 and CCR6 were co-expressed in both DFL and MALT. We hypothesize that increased expression of CCL20 and MAdCAM-1 and co-expression of CCL20 and CCR6 may play an important role in tumorigenesis.

Keywords: CCL20; MALT lymphoma; MAdCAM-1; duodenal follicular lymphoma; gene expression profile.

Figure 1

Hierarchical sample clustering was conducted using the 2918 differentially expressed genes (see Fig. S2). Although a few exceptions were observed, normal tissues (normal duodenal mucosa [NDU] and normal gastric mucosa [STCH]) were clustered together as one group, and the other group included lymphomas (duodenal follicular lymphoma [DFL], MALT and nodal follicular lymphoma [NFL]) and control disease samples (reactive lymphoid hyperplasia [RLH]). Clustering was conducted using MultiExperiment Viewer (MeV) version 4.8.1. The cluster for NFL is shown in red, RLH is in green, DFL is in blue, and MALT is in yellow. A clustering of the 2918 genes is shown in Supplementary Figure S3.

Figure 2

Hierarchical gene clustering using 116 upregulated genes (a) and 445 downregulated genes (b). Clustering was conducted using MultiExperiment Viewer (MeV) version 4.8.1.

Figure 3

Heatmap of genes of interest, including 84 differentially expressed genes (DEG) and 9 non-DEG: IL2RG, ITGB2, MMP25, IL12RB1, TNFAIP8L2, TNFRSF8, ILDR2, CXCL10 and C3.

Figure 4

Validation of microarray data by quantitative RT-PCR and immunohistochemical studies. (a, b) Quantitative RT-PCR for CCL20 and MAdCAM-1, respectively. duodenal follicular lymphoma (DFL) and MALT showed higher expression levels of CCL20 and MAdCAM-1 than nodal follicular lymphoma (NFL) (P < 0.001). The coefficient of correlation with the microarray data was 0.925 for CCL20 and 0.7934 for MAdCAM-1. (c–k) Immunohistochemical results for CCL20, MAdCAM-1 and CCR6 in DFL, MALT and NFL. DFL and MALT were positive for CCL20 (c) and (d), while NFL was negative for CCL20 (e). Vascular endothelial cells were highly expressed in DFL and MALT (f) and (g), but not in NFL (h). DFL (i), MALT (j) and NFL (k) were positive for CCR6.

Figure 5

Validation of microarray data and double immunohistochemical staining for CCL20 and CCR6. (a–i) Immunohistochemical results for PCDHGA3, PCDHGA8 and PCDHGB4. Duodenal follicular lymphoma (DFL) and nodal follicular lymphoma (NFL) were positive for PCDHGA3 (a) and (c), PCDHGA8 (d) and (f), and PCDHGB4 (g) and (i), while MALT was negative for all three markers (b), (e) and (h). (j) Double immunohistochemical staining for CCL20 and CCR6 in DFL. Tumor cells co-expressed CCL20 and CCR6.