Lipoprotein-Associated Phospholipase A2: A Novel Contributor in Sjögren's Syndrome-Related Lymphoma?

Abstract

Background: B-cell non-Hodgkin's lymphoma (B-NHL) is one of the major complications of primary Sjögren's syndrome (SS). Chronic inflammation and macrophages in SS minor salivary glands have been previously suggested as significant predictors for lymphoma development among SS patients. Lipoprotein-associated phospholipase A2 (Lp-PLA2)-a product mainly of tissue macrophages-is found in the circulation associated with lipoproteins and has been previously involved in cardiovascular, autoimmune, and malignant diseases, including lymphoma.

Objective: The purpose of the current study was to investigate the contributory role of Lp-PLA2 in B-NHL development in the setting of primary SS.

Methods: Lp-PLA2 activity in serum samples collected from 50 primary SS patients with no lymphoma (SS-nL), 9 primary SS patients with lymphoma (SS-L), and 42 healthy controls (HC) was determined by detection of [³H]PAF degradation products by liquid scintillation counter. Moreover, additional sera from 50 SS-nL, 28 SS-L, and 32 HC were tested for Lp-PLA2 activity using a commercially available ELISA kit. Lp-PLA2 mRNA, and protein expression in minor salivary gland (MSG) tissue samples derived from SS-nL, SS-L patients, and sicca controls (SC) were analyzed by real-time PCR, Western blot, and immunohistochemistry.

Results: Serum Lp-PLA2 activity was significantly increased in SS-L compared to both SS-nL and HC by two independent methods implemented [mean ± SD (nmol/min/ml): 62.0 ± 13.4 vs 47.6 ± 14.4 vs 50.7 ± 16.6, p-values: 0.003 and 0.04, respectively, and 19.4 ± 4.5 vs 15.2 ± 3.3 vs 14.5 ± 3.0, p-values: <0.0001, in both comparisons]. ROC analysis revealed that the serum Lp-PLA2 activity measured either by radioimmunoassay or ELISA has the potential to distinguish between SS-L and SS-nL patients (area under the curve [AUC]: 0.8022, CI [95%]: 0.64-0.96, p-value: 0.004 for radioimmunoassay, and AUC: 0.7696, CI [95%]: 0.66-0.88, p-value: <0.0001, for ELISA). Lp-PLA2 expression in MSG tissues was also increased in SS-L compared to SS-nL and SC at both mRNA and protein level. ROC analysis revealed that both MSG mRNA and protein Lp-PLA2 have the potential to distinguish between SS-nL and SS-L patients (area under the curve [AUC] values of 0.8490, CI [95%]: 0.71-0.99, p-value: 0.0019 and 0.9444, CI [95%]: 0.79-1.00, p- value: 0.0389 respectively). No significant difference in either serum Lp-PLA2 activity or MSG tissue expression was observed between SS-nL and HC.

Conclusions: Lp-PLA2 serum activity and MSG tissue mRNA/protein expression could be a new biomarker and possibly a novel therapeutic target for B-cell lymphoproliferation in the setting of SS.

Keywords: Sjögren’s syndrome; lipoprotein-associated phospholipase A2 (Lp-PLA2); lymphomagenesis; novel therapeutic target; serum biomarker.

Figure 1

Serum Lp-PLA2 activity in the two SS sets of samples and receiver-operating characteristic analysis (ROC) curve analysis for both radioimmunoassay and ELISA protocols. (A) Increased Lp-PLA2 activity measured by liquid scintillation in SS-L patients compared to SS-nL (p-value: 0.003) and HC (p=0.04). (B) ROC analysis revealed that serum Lp-PLA2 activity measured by radioimmunoassay has the potential to distinguish between SS-L and SS-nL patients [area under the curve (AUC) = 0.8022, CI (95%): 0.64–0.96, p-value: 0.004]. (C) When ROC curves for the predictive models were fitted, AUC were 0.7196, CI (95%): 0.56–0.88, p-value: 0.04 for SS-L vs HC. (D) Increased Lp-PLA2 activity measured by ELISA in patients with SS-L compared to SS-nL patients (p-value: <0.0001) and HC (p-value: <0.0001). (E) ROC analysis showed that serum Lp-PLA2 activity measured by ELISA has the potential to distinguish between SS-L and SS-nL patients with AUC 0.7696, CI (95%): 0.66–0.88, p-value <0.0001 and (F) For SS-L patients vs HC, ROC curves showed an AUC 0.8114, CI (95%): 0.70–0.92, p-value < 0.0001. HC, healthy controls; SS-nL, Sjogren’s syndrome without lymphoma; SS-L, Sjogren’s syndrome complicated by lymphoma; ROC, receiver operating characteristic; AUC, area under the curve.

Figure 2

Lp-PLA2 (encoded by the PLA2G7 gene) mRNA expression (measured by real-time PCR) and protein density (tested by Western blot) in MSG tissues of our study participants and ROC curve analysis for both methods. (A) PLA2G7 gene expression was significantly increased in SS-L patients compared to both SS-nL patients and SC (p-values: 0.0012 and 0.0013, respectively). (B) ROC analysis could distinguish between SS-L and SS-nL patients with an AUC 0.8490, CI (95%): 0.71–0.99, p-value: 0.0019. (C) For SS-nL vs SC patients ROC curves showed an AUC 0.9444, CI (95%): 0.84–1.00, p- value: 0.0027. (D) Increased Lp-PLA2 protein expression was found in MSG total protein extracts derived from SS-L patients compared to both SS-nL patients and SC as tested by Western Blot (p-values: 0.0476 and 0.0357, respectively). (E) ROC analysis for Lp-PLA2 protein expression depicted an AUC of 0.94, CI (95%): 0.79–1.00, p-value: 0.0389 for SS-L vs SS-nL patients. (F) ROC analysis revealed an AUC of 1.00, CI (95%): 1.00–1.00, p-value: 0.0253. (G) Representative image of Lp-PLA2 and GAPDH MSG protein expression by Western Blot. SC, controls with sicca complaints; SS-n, Sjogren’s syndrome; SS-L, Sjogren’s syndrome complicated by lymphoma; ROC, receiver operating characteristic; AUC, area under the curve.

Figure 3

Lp-PLA-2 immunohistochemical expression in minor salivary gland (MSG) tissue sections from patients with sicca features (SC), Sjögren’s syndrome with no lymphoma (SS-nL), Sjogren’s syndrome with lymphoma (SS-L) (A–F), as well as in a lymph node derived from a patient with marginal zone lymphoma (G, H). (A) Isotype Control staining (grade 0), (B) Lp-PLA2 staining in SC (grade 1), (C) Lp-PLA2 staining in SS-nL (grade 2), (D) Lp-PLA2 staining in SS-L (grade 3), (E) Lp-PLA2 in SS-L, (F) CD68 staining (denoting the presence of tissue macrophages) in SS-L (same patient as E), (G) Hematoxylin & Eosin (H&E staining), marginal zone lymphoma (MZL), (H) Lp-PLA2 in a lymph node from a non-SS patient with MZL (same patient as G).