Biomarker potential of nuclear Nrf2 activation in the ABC subtype of diffuse large B‑cell lymphoma

Abstract

Diffuse large B-cell lymphoma (DLBCL) is an aggressive B-cell lymphoma characterized by distinct subtypes and heterogeneous treatment outcomes. Oxidative stress and the dysregulation of related regulatory genes are prevalent in DLBCL, prompting an investigation into the nuclear factor erythroid 2-related factor 2 (Nrf2)-kelch-like ECH-associated protein 1 (Keap1) signaling pathway and associated genes. The present study assessed pathological specimens and clinical data from 43 newly diagnosed patients with DLBCL, comparing the associations and correlations between the expression of Nrf2, Keap1, microtubule-associated protein 1 light chain 3β (LC3B) and nitrotyrosine and the activated B-cell (ABC) and germinal center B-cell (GCB) subtypes of DLBCL using immunohistochemistry and digital image analysis software. Nuclear Nrf2 activation was observed in 33.3% of patients with DLBCL ABC, demonstrating a higher prevalence of hepatitis B surface antigen positivity, calcium ions and significant body weight loss (P<0.05). Total Nrf2 expression was associated with the DLBCL GCB subtype and inversely correlated with Keap1 expression in the DLBCL ABC subtype. Furthermore, a positive correlation was demonstrated between Nrf2 and LC3, indicating that total Nrf2 is inhibited by Keap1 and regulates LC3 expression. The ABC subtype was also associated with lower white blood cell counts and more frequent chemotherapy courses than the GCB subtype. These findings suggest that nuclear Nrf2 could be a biomarker for DLBCL clinical diagnosis.

Keywords: activated B-cell; diffuse large B-cell lymphoma; germinal center B-cell; kelch-like ECH-associated protein 1; microtubule-associated protein 1 light chain 3β; nitrotyrosine; nuclear factor erythroid 2-related factor 2.

Figure 1.

Nrf2 is differentially distributed in different cancer stages and subtypes of DLBCL. (A) Elevated NFE2L2 (Nrf2) mRNA levels in DLBCL tissues compared with in normal tissues. (B) Increased % Nrf2-positive cancer cells in advanced stages of DLBCL. (C) Higher frequency of Nrf2-positive cancer cells in the GCB subtype of DLBCL compared with that in the ABC subtype. (D) Higher Nrf2-positive cancer cell numbers in the GCB subtype across all stages of DLBCL. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001. All IHC sample images are ×20 magnification. ns, no significance; DLBCL, diffuse large B-cell lymphoma; Nrf2, nuclear factor erythroid 2-related factor 2; ABC, activated B-cell; GCB, germinal center B-cell; TPM, transcripts per million; IHC, immunohistochemistry.

Figure 2.

Immunohistochemical staining of Nrf2 localization in DLBCL. (A) Active Nrf2 is distributed in both the nucleus and cytoplasm, covering the entire cell (black arrows). (B) Nrf2 localized in the cytoplasm surrounds the nucleus (red arrows indicate cytoplasmic Nrf2; nucleus stained with hematoxylin). (C) Nuclear Nrf2 is predominantly observed in the ABC subtype of DLBCL and is absent in the GCB subtype. All IHC sample images are ×40 magnification. DLBCL, diffuse large B-cell lymphoma; Nrf2, nuclear factor erythroid 2-related factor 2; ABC, activated B-cell; GCB, germinal center B-cell.

Figure 3.

Keap1 expression in DLBCL, especially in the advanced staged and ABC subtype. (A) KEAP1 mRNA is significantly upregulated in DLBCL compared with that in the controls in The Cancer Genome Atlas and Genotype-Tissue Expression databases. (B) Significantly higher Keap1 expression in the advanced stages of DLBCL than in the early stages. (C) Significantly higher Keap1 expression in patients with DLBCL of the ABC subtype than the GCB subtype. (D) KEAP1 mRNA expression in DLBCL cell lines (HT and U2932). *P<0.05; **P<0.01. All IHC sample images are ×20 magnification. DLBCL, diffuse large B-cell lymphoma; Keap1, kelch-like ECH-associated protein 1; ABC, activated B-cell; GCB, germinal center B-cell; TPM, transcripts per million; IHC, immunohistochemistry.

Figure 4.

Increased LC3B (encoded by MAP1LC3B) expression in DLBCL. (A) Elevated MAP1LC3B mRNA expression in DLBCL compared with controls in The Cancer Genome Atlas and Genotype-Tissue Expression databases. (B) LC3B expression across the early and advanced stages of DLBCL. (C) LC3B expression in the DLBCL GCB and ABC subtypes. All IHC sample images are ×20 magnification. ns, no significance; DLBCL, diffuse large B-cell lymphoma; TPM, transcripts per million; IHC, immunohistochemistry; MAP1LC3B, microtubule-associated protein 1 light chain 3β.

Figure 5.

Nitrotyrosine expression in different DLBCL stages and subgroups. (A) Nitrotyrosine-positive cells were more prevalent in the advanced stages of DLBCL. (B) Higher frequency of nitrotyrosine-positive cells was observed in the ABC subtype of DLBCL. All IHC sample images are ×20 magnification. ns, no significance; DLBCL, diffuse large B-cell lymphoma; IHC, immunohistochemistry.