Dysregulated CXCR4 expression promotes lymphoma cell survival and independently predicts disease progression in germinal center B-cell-like diffuse large B-cell lymphoma

Abstract

Abnormal expression of the chemokine receptor CXCR4 plays an essential role in tumor cell dissemination and disease progression. However, the significance of CXCR4 overexpression in de novo diffuse large B cell lymphoma (DLBCL) is unknown. In 743 patients with de novo diffuse large B cell lymphoma (DLBCL) who received standard Rituximab-CHOP immunochemotherapy, we assessed the expression of CXCR4 and dissected its prognostic significance in various DLBCL subsets. Our results showed that CXCR4+ patients was associated with male, bulky tumor, high Ki-67 index, activated B-cell-like (ABC) subtype, and Myc, Bcl-2 or p53 overexpression. Moreover, CXCR4+ was an independent factor predicting poorer progression-free survival in germinal-center B-cell-like (GCB)-DLBCL, but not in ABC-DLBCL; and in patients with an IPI of ≤2, but not in those with an IPI>2. The lack of prognostic significance of CXCR4 in ABC-DLBCL was likely due to the activation of p53 tumor suppressor attenuating CXCR4 signaling. Furthermore, concurrent CXCR4+ and BCL2 translocation showed dismal outcomes resembling but independent of MYC/BCL2 double-hit DLBCL. Gene expression profiling suggested that alterations in the tumor microenvironment and immune responses, increased tumor proliferation and survival, and the dissemination of CXCR4+ tumor cells to distant organs or tissues were underlying molecular mechanisms responsible for the CXCR4+ associated poor prognosis.

Keywords: BCL2; CXCR4; DLBCL; Myc; TP53 mutation.

Figure 1. Expression and prognostic significance of CXCR4 in DLBCL

(A-C) Representative CXCR4 immunohistochemistry staining (showing 100%, 60%, 0% CXCR4 cell surface expression in DLBCL cells). (D) Histogram of CXCR4 expression frequency distribution in the DLBCL study cohort. X-axis, percentage of immunopositive cells in tumors; Y-axis, numbers of DLBCL patients. (E-F) ABC-DLBCL compared to GCB-DLBCL had increased CXCR4 cell surface expression, but did not differ in CXCL12 mRNA significantly. (G) CXCR4 cell surface expression correlated with decreased CXCL12 mRNA levels, both in GCB- and ABC-DLBCL. (H-I) CXCR4 expression correlated with significantly poorer OS and PFS in the overall DLBCL cohort. (J-K) CXCR4 expression correlated with significantly poorer PFS (but not OS) in GCB-DLBCL. (L-M) CXCR4 expression correlated with significantly poorer OS (but not PFS) in ABC-DLBCL. (N-O) CXCR4 expression correlated with significantly poorer survival in DLBCL patients with a low IPI, but not in DLBCL patients with a high IPI.

Figure 2. Expression and prognostic significance of CXCR4 in nodal and extranodal DLBCL

(A-B) CXCR4 cell surface and CXCL12 mRNA expression levels in nodal and extranodal DLBCL. (C) CXCR4 cell surface expression correlated with decreased CXCL12 mRNA levels, both in nodal and extranodal DLBCL. (D) CXCR4 cell surface expression correlated with decreased CXCL12 mRNA levels in DLBCL patients without bone marrow (BM) involvement. (E-F) CXCR4 expression correlated with significantly poorer OS and PFS in the nodal DLBCL. (G-H) CXCR4 expression in extranodal sites did not correlate with survival significantly in DLBCL. (I-L) the prognostic significance of CXCR4 expression was independent of BM involvement.

Figure 3. Association of CXCR4 expression with Myc/Bcl-2 expression and the synergism of prognostic significance in DLBCL

(A-D) Association between CXCR4 and Myc expression levels. (E-H) Association between CXCR4 and Bcl-2 expression levels. (I-J) CXCR4 expression synergized with Bcl-2 expression in GCB-DLBCL. (K-L) CXCR4 expression synergized with BCL2 translocation in GCB-DLBCL. (M) The synergism between BCL2 translocation and CXCR4 expression was independent of double-hit MYC/BCL2 translocations. (N) CXCR4 expression synergized with Myc expression in DLBCL. (O-P) the prognostic significance of CXCR4 in GCB-DLBCL patients with concurrent Myc/Bcl-2 expression.

Figure 4. Regulation of and signaling pathways related to CXCR4 expression

(A-C) p53 mutations were associated increased CXCR4 and decreased CXCL12 mRNA expression, especially in GCB-DLBCL. (D-E) CXCR4 expression was associated with decreased PRDM1 mRNA and BLIMP-1 expression in GCB-DLBCL. (F) CXCR4 expression was associated with increased AICDA mRNA expression both in GCB- and ABC-DLBCL. (G-H) Heatmaps and differentially expressed genes between CXCR4⁺ and CXCR4⁻ patients in the overall DLBCL, GCB-DLBCL and ABC-DLBCL cohorts. (J-K) CXCR4 expression was associated with increased PI3K mRNA expression in GCB-DLBCL, and increased protein expression in ABC-DLBCL.

Figure 5. Effects of BTK140 on proliferation and the growth patterns of DLBCL cells

(A) Expression levels of CXCR4 mRNA in DLBCL cell lines. (B) Ten DLBCL cell lines were treated with BTK140 in a dose-dependent manner. Cell proliferation was measured using ³H-thymiding incorporation assay after 72 hours incubation. Dates shown are the means and ranges of triplicate samples relative to control samples of three independent experiments. (C) The proliferation pattern of McA cells incubated without BKT140 (i), with 6.25uM BKT140 (ii), and with 50uM BKT140 (iii). (C) The proliferation pattern of LY19 incubated without BKT140 (I), with 6.25uM BKT140 (II), and with 50uM BKT140 (III).