Elevated IgG4 in patient circulation is associated with the risk of disease progression in melanoma

Abstract

Emerging evidence suggests pathological and immunoregulatory functions for IgG4 antibodies and IgG4⁺ B cells in inflammatory diseases and malignancies. We previously reported that IgG4 antibodies restrict activation of immune effector cell functions and impair humoral responses in melanoma. Here, we investigate IgG4 as a predictor of risk for disease progression in a study of human sera (n = 271: 167 melanoma patients; 104 healthy volunteers) and peripheral blood B cells (n = 71: 47 melanoma patients; 24 healthy volunteers). IgG4 (IgG4/IgG_total) serum levels were elevated in melanoma. High relative IgG4 levels negatively correlated with progression-free survival (PFS) and overall survival. In early stage (I-II) disease, serum IgG4 was independently negatively prognostic for progression-free survival, as was elevation of IgG4⁺ circulating B cells (CD45⁺CD22⁺CD19⁺CD3^-CD14^-). In human tissues (n = 256; 108 cutaneous melanomas; 56 involved lymph nodes; 60 distant metastases; 32 normal skin samples) IgG4⁺ cell infiltrates were found in 42.6% of melanomas, 21.4% of involved lymph nodes and 30% of metastases, suggesting inflammatory conditions that favor IgG4 at the peripheral and local levels. Consistent with emerging evidence for an immunosuppressive role for IgG4, these findings indicate association of elevated IgG4 with disease progression and less favorable clinical outcomes. Characterizing immunoglobulin and other humoral immune profiles in melanoma might identify valuable prognostic tools for patient stratification and in the future lead to more effective treatments less prone to tumor-induced blockade mechanisms.

Keywords: B cells; IgG4; biomarker; cancer inflammation; humoral response; immunomodulation; immunomonitoring; immunosuppression; melanoma; prognosis.

Figure 1.

Experimental study design, collection and processing of clinical samples. Cohorts of 171 melanoma patients and 104 healthy volunteers were identified for evaluations of IgG4 and lactate dehydrogenase (LDH). IgG subclass levels were measured using a standardized Luminex bead array assay (n = 171) and in the same samples, LDH levels were analyzed through a diagnostic laboratory protocol (n = 158). *Researchers working on this study were blinded to prevent bias; **Two independent medical professionals, not involved in the quantification of IgG4 or LDH, evaluated patient information; ***Patients with co-morbidities that may influence IgG4 levels were excluded from the analysis.

Figure 2.

For figure legend, see next page.

Figure 3.

For figure legend, see next page.Figure 3 (See previous page). Increased frequencies of peripheral blood IgG4+ B cells from melanoma patients compared to healthy volunteers. (A) Representative cytofluorimetric dot plots and flow cytometry gating strategy for evaluation of the circulating IgG4⁺ B cell compartment. Lymphoid cells were gated according to their FSC-A and SSC-A properties and viable CD45⁺ were selected and cell doublets excluded using FSC-A and FSC-H dot plots. CD3⁻CD14⁻ cells were selected and B cells identified as CD19⁺CD22⁺ cells (top panel). Although the overall number of circulating B cells did not differ significantly between melanoma patients and healthy volunteer samples (lower panel, left for % of total PBMCs and numbers of B cell events), the IgG4⁺ cells were selected from the CD45⁺CD22⁺CD19⁺CD3⁻CD14⁻ cell compartment. Representative dot plots depicting IgG4⁺ (CD45⁺CD22⁺CD19⁺CD3⁻CD14⁻) peripheral B cells from PBMCs of a healthy volunteer (middle) and of a melanoma patient (right) (lower panel). (B) Left and Middle: The number (left) and frequency (middle) of the IgG4⁺ peripheral B cell compartment (based on counted CD45⁺CD22⁺CD19⁺CD3⁻CD14⁻ cells) of 24 healthy volunteer and 47 melanoma patient samples showed statistically significantly higher levels of IgG4⁺ B cells in the patient group (Mann-Whitney-U-test; P = 0.01; P = 0.003); Right: IgG4⁺ B cell frequencies differed significantly between Stages I–II (n = 24; *** P <0.001) or Stage III–IV (n = 23; * P<0.05) vs. healthy volunteers (n = 24). Statistical analysis was performed by Kruskal-Wallis one-way analysis of variance with post-hoc Dunn's test; lines represent medians and error bars indicate interquartile range. (C) Frequency of circulating IgG4⁺ B cells from patients with local (Stages I–II) or metastatic (Stages III–IV) melanoma and correlation with risk of disease progression. Statistical analysis was performed by Mann-Whitney-U-test; lines represent medians and error bars indicate interquartile range. Corresponding ROC analyses are depicted directly underneath each column graph. In the Stage I–II patient cohort, patients with stable disease had a significantly lower frequency of IgG4⁺ B cells vs. patients with progressive disease (*P = 0.014). For this cohort, a median AUC of 0.81 was calculated by ROC analysis.

Figure 4.

IgG4+ cell infiltrates are detected in melanoma skin tumors. A tissue microarray (TMA; BioMax, n = 256) consisting of melanoma skin tumors (n = 108), melanoma lymph nodes metastases (n = 56), distant organ metastases (n = 60) and healthy skin (n = 32) specimens (top panel) was examined for the presence of IgG4 by immunohistochemistry. IgG4 positive infiltrates were detected by alkaline phosphatase (in red, selected areas shown by black arrows) and sections were counterstained in hematoxylin (in blue). Representative images of IgG4 immunohistochemical staining for IgG4⁺ infiltration revealed positive staining in skin lesions (top and second panels), lymph node and distant metastases (third panel), while staining was less frequent in healthy skin (bottom panel). Black bar represents 800 µm (third bottom right panel).

Figure 5.

IgG4+ cell infiltration is higher in melanoma skin tumors, melanoma lymph nodes metastases, and distant organ metastases, compared to healthy skin. Top panel: Evaluation of tissue microarray (TMA) sections for frequency of IgG4⁺ cell infiltration in melanoma tissues in skin, lymph node metastases and distant metastases vs. healthy skin (Pie charts represent % of total numbers). Middle panel: In skin lesions (n = 108), IgG4⁺ infiltration is found in early (Stage I–II) and metastatic (Stage III–IV) disease. Lower panel: In melanoma skin lesions, IgG4⁺ cell infiltration is demonstrated across different melanoma skin tumor thicknesses (T1-T4). Analyses were performed by 2 independent researchers to identify the density of infiltration per high power field using the following criteria: “negative” = 0% infiltration; “low” < 25% infiltration; “high” > 25% infiltration.