Role of Tumor-Associated Macrophages in the Clinical Course of Pancreatic Neuroendocrine Tumors (PanNETs)

Abstract

Purpose: This study evaluated the potential role of immune cells and molecules in the pathogenesis and clinical course of pancreatic neuroendocrine tumors (PanNET).

Experimental design: Surgically resected PanNETs (N = 104) were immunohistochemically analyzed for Ki67 index, mitotic rate, macrophage, CD4⁺ cells, and CD8⁺ T-cell infiltration, as well as HLA class I, PD-L1, and B7-H3 expression. Results were correlated with clinicopathologic characteristics as well as with disease-free (DFS) and disease-specific (DSS) survival.

Results: The median age of the 57 WHO grade 1 and 47 WHO grade 2 patients was 55 years. High intratumoral CD8⁺ T-cell infiltration correlated with prolonged DFS (P = 0.05), especially when the number of tumor-associated macrophages (TAM) was low. In contrast, high peritumoral CD4⁺ cell and TAM infiltration was associated with a worse DFS and DSS. PD-L1 and B7-H3 were expressed in 53% and 78% PanNETs, respectively. HLA class I expression was defective in about 70% PanNETs. HLA-A expression correlated with favorable DSS in PD-L1-negative tumors (P = 0.02). TAM infiltration (P = 0.02), WHO grade (P = 0.04), T stage (P = 0.01), and lymph node positivity (P = 0.04) were independent predictors of DFS. TAM infiltration (P = 0.026) and T stage (P = 0.012) continued to be predictors of DFS in WHO grade 1 PanNET patients. TAM infiltration was the sole independent predictor of DSS for WHO grade 1 and 2 patients (P = 0.02). Therefore, this biomarker may contribute to identifying WHO grade 1 patients with poor prognosis.

Conclusions: TAM infiltration appears to be the most informative prognostic biomarker in PanNET. It may represent a useful immunotherapeutic target in patients with PanNET.

Figure 1.

Immune cell infiltration and HLA class I expression in PanNET tumors. A, PanNET tissue sections were IHC stained with rabbit anti-CD4, mouse anti-CD8, mouse anti-CD68, and rabbit anti-CD163 mAbs. Different levels of CD4⁺ cell, CD8⁺ T cell, and TAM (CD68⁺CD163⁺) infiltration (X200 original magnification, insets X800 original magnification) are shown. B, PanNET tissue sections were IHC stained with mouse anti-HLA-A mAb HCA2, anti-HLA-B/C mAb HC-10, and anti-β2m mAb NAMB-1. Representative patterns of positive, heterogeneous, and negative HLA-A, HLA-B/C and β2m staining (X200 original magnification) are shown. C, HLA-A expression is significantly associated with HLA-B/C expression (P<0.001). D, HLA-A expression is significantly associateted with β2m expression (P<0.001). P values derived from Fisher exact test.

Figure 2.

Association of PD-L1, B7-H3, phospho-STAT1, and RIG-I expression with the extent of peritumoral CD8⁺T-cell infiltration and with HLA class I expression in PanNETs. A, PanNET tissue sections were IHC stained with rabbit anti-PD-L1 mAb and goat anti-B7-H3 pAb. Representative patterns of highly positive embrane/cytoplasmic, scattered positive membrane/cytoplasmic and negative PD-L1 staining (X200 original magnification) are shown. Representative patterns of highly positive membrane/cytoplasmic, low membrane/cytoplasmic and negative B7-H3 staining (X200 original magnification) are shown. B, Peritumoral CD8⁺ T-cell number is significantly associated with PD-L1 expression (P=0.02), but not with cytoplasmic and/or membranous B7-H3 expression (P=0.06). C, PanNET tissue sections were IHC stained with mouse anti-HLA-A mAb HCA2, mouse anti-HLA-B/C mAb HC-10, rabbit anti-phosphoStat1 (pSTAT1) mAb, and rabbit anti-RIG-I pAb. The positive HLA-A and HLA-B/C expression in PanNETs with positive pSTAT1 expression and the low/negative HLA-A and HLA-B/C expression in PanNETs with negative phosphoStat1 expression (X200 original magnification, insets X400 original magnification) are shown. D, Positive HLA-A and HLA-B/C expression is significantly associated with positive pSTAT1 expression (P<0.001). E, Peritumoral and intratumoral CD8⁺ T-cell number is significantly associated with pSTAT1 expression (P=0.001 and P=0.003). F, HLA-A and HLA-B/C expression is significantly associated with RIG-I expression (P<0.001). G, Peritumoral and intratumoral CD8⁺ T-cell number is significantly associated with positive RIG-I expression (P=0.009 and P=0.028). Bars, median values. Error bars, interquartile range. P values derived from Mann–Whitney U tests.

Figure 3.

Association of the extent of tumor infiltration by immune cells with DFS and DSS in patients with PanNET. DFS based on peritumoral CD4⁺ cell, TAM, and intratumoral CD8⁺ T-cell number is shown. Prognosis is depicted with Kaplan–Meier curves: (A) for peritumoral CD4⁺ cell number: high versus medium and low (mean survival: high: 113 vs. medium and low: 123 months, P=0.02), (B) for TAM number: high versus medium and low (mean survival: high: 113 versus medium and low: 121 months, P<0.05); and (C) for intratumoral CD8⁺ T-cell number: high versus medium and low (mean survival: high: 170 vs. medium and low: 104 months, P=0.05). Disease-specific survival based on peritumoral CD4⁺ cells and TAM number is shown. Prognosis is depicted with Kaplan–Meier curves: (D) for peritumoral CD4⁺ cell number: high versus medium and low (mean survival: high: 150 vs. medium and low: 173 months, P=0.04), and (E) for TAM number: high versus medium and low (mean survival: high: 149 vs. medium and low: 175 months, P =0.04). P values derived from log-rank tests.

Figure 4.

Impact of PD-L1 expression on the association of HLA-A and β2m expression by PanNET cells and of TAM number with DSS in patients with PanNET. A and B, Prognosis is depicted with Kaplan-Meier curves for HLA-A expression (positive and heterogeneous vs. negative) in PD-L1-negative (P=0.02) and positive (P=0.057) group. C and D, Prognosis is depicted with Kaplan-Meier curves for β2m expression (positive and heterogeneous vs. negative) in PD-L1–negative (P=0.03) and positive (P=0.085) group. E and F, Prognosis is depicted with Kaplan-Meier curves for TAM number (high vs. medium and low) in PD-L1-negative (P=0.02) and positive (P=0.057) group. P values derived from logrank tests.