GSEA-assisted gene signatures valid for combinations of prognostic markers in PCNSL

Abstract

Primary central nervous system lymphoma (PCNSL) is a brain malignant non-Hodgkin's B-cell lymphoma. The standard treatments are high-dose methotrexate (MTX)-based chemotherapies and deferred whole brain radiotherapy. However, MTX resistance-dependent global expression and signaling pathway changes and their relationship with prognoses have not yet been elucidated. Here, we conducted a global expression analysis with next-generation sequencing and gene set enrichment analysis (GSEA) in MTX-resistant PCNSL cell lines (HKBML-MTX and TK-MTX) and PCNSL tissues. In rank scores, genes listed in HKBML-MTX and TK-MTX were enriched in PCNSL with poor prognoses. In fold changes, a part of differentially-expressed genes in PCNSL tissues were also detected in HKBML-MTX and TK-MTX cells; FOXD2-AS1 and MMP19 were commonly expressed in both HKBML-MTX and TK-MTX, FABP5 and CD70 were HKBML-MTX-specifically expressed, and CLCN2, HOXB9, INE1, and LRP5L were TK-MTX-specifically expressed, which may provide a combination of prognostic markers on MTX-sensitivities in PCNSL. Additionally, PCNSL subgroups, divided with hierarchical clustering and Kaplan-Meier methods, included twenty commonly expressed genes in both HKBML-MTX and TK-MTX, ten HKBML-MTX-specifically expressed genes, and two TK-MTX-specifically expressed genes. These results suggest that the GSEA-assisted gene signatures can provide a combination for prognostic markers in recurrent PCNSL with MTX resistances.

Figure 1

Gene set enrichment analysis (GSEA) in MTX-resistant PCNSL cells. (a,b) GSEA for upregulated genes of MTX-resistant PCNSL cells in PCNSL with poor and good prognoses. The upregulated genes in (a) HKBML-MTX and (b) TK-MTX, compared with HKBML and TK, respectively. (c,d) GSEA for downregulated genes of MTX-resistant PCNSL cells in poor and good prognoses. The downregulated genes in (c) HKBML-MTX and (d) TK-MTX, compared with HKBML and TK, respectively. ES; enrichment score. (e,f) Venn diagram of genes in MTX-resistant PCNSL cells, compared with control cells. Genes associated with high enrichment score in GSEA were selected. Numbers of genes were shown in Venn diagram. (e) The numbers of upregulated genes in HKBML-MTX and TK-MTX, compared with control cells. (f) The numbers of downregulated genes in HKBML-MTX and TK-MTX, compared with control cells. (g,h) Lists of cell-type-specifically and commonly expressed genes in HKBML-MTX and TK-MTX, compared with control cells, associated with ESs in GSEA. (g) Upregulated genes. (h) Downregulated genes.

Figure 2

Identification of cell-type-dependent marker candidates for PCNSL with poor survivals and MTX resistances. (a-c) Differential expression in (a) PCNSL with poor survivals, (b) HKBML-MTX, and (c) TK-MTX. (d) Clustering of differential expression marker candidates. Green-black-red as low-median-high expression. (e–l) Expression in PCNSL samples divided by prognoses in box-whisker plots. (e) FOXD2-AS1 and (f) MMP19 as gene marker candidates for PCNSL with poor survivals. (g) FABP5 and (h) CD70 as gene marker candidates for HKBML-MTX. (i) CLCN2, (j) HOXB9, (k) INE1, and (l) LRP5L as gene marker candidates for TK- MTX. FPKM; fragments per kilobase of exon model per million reads mapped.

Figure 3

Differential expression of MTX-resistant PCNSL cells-agitated genes in PCNSL with poor prognoses. (a–d) Clustering of gene expression of MTX-resistant PCNSL cells in PCNSL specimens. GSEA-assisted upregulated genes in (a) HKBML-MTX and (c) TK-MTX. GSEA-assisted downregulated genes in (b) HKBML-MTX and (d) TK-MTX. Green-black-red as low-median-high expression. (e–h) Survival distribution of PCNSL patients divided into clusters. Clusters 1, 2, and 3 in each panel of (e–h), correspond to those in (a–d). Kaplan-Meier survival curves were evaluated with log-rank tests. OS; overall survival.

Figure 4

Protein-protein interaction (PPI) networks in MTX-resistant PCNSL cells. Networks were estimated by MCODE and STRING. (a,b) PPI networks on upregulated genes in (a) HKBML-MTX and (b) TK-MTX, compared with control cells. (c,d) PPI networks on cell-type-dependent upregulated genes in (c) HKBML and (d) TK.