Tertiary Lymphoid Structures Are Associated with Progression-Free Survival of Peripheral Neuroblastic Tumor Patients

Abstract

Background/Objectives: Peripheral neuroblastic tumors (pNT) are a biologically heterogeneous group of embryonal tumors that derive from the neural crest and affect the sympathetic nervous system. So far, little is known about the complex immune landscape in these rare childhood cancers. Methods: We focused on the immune cell infiltrate of treatment-naïve pNT from 24 patients, including high-risk neuroblastoma (HR-NBL), non-high-risk neuroblastoma (NHR-NBL), ganglioneuroblastoma (GNBL), and rare ganglioneuroma (GN). To gain novel insights into the immune architecture of these pNT subtypes, we used multiplex immunohistochemistry, multispectral imaging, and algorithm-based data evaluation to detect and characterize T cells, B cells, neutrophils, macrophages, and tertiary lymphoid structures (TLS). Results: The majority of the investigated tumor-infiltrating immune cells were macrophages and T cells. Their detailed phenotypic characterization revealed high proportions of M2-like macrophages as well as activated GrzB⁺ CD8⁺ and PD-1⁺ T lymphocytes. Proportions of these T cell phenotypes were significantly increased in GN compared to HR-NBL, NHR-NBL, or GNBL. In addition, TLS occurred in 11 of 24 patients, independent of immune cell frequencies in the whole tissues. Interestingly, all GN, most GNBL, but only a few NBL contained TLS. We distinguished between three TLS maturation stages that were present irrespective of the pNT subtype. The majority belonged to mature TLS of the primary follicle state. Mature LAMP3⁺ dendritic cells were also found, predominantly in T cell zones of TLS. Furthermore, TLS presence identified pNT patients with significantly prolonged progression-free survival in contrast to all other analyzed immunological features. Conclusions: We propose TLS to be a potential prognostic marker for pNT to predict patient outcomes.

Keywords: T cells; macrophages; multiplex immunohistochemistry; peripheral neuroblastic tumors; tertiary lymphoid structures; tumor immune microenvironment.

Figure 1

Overview of the general immune cell infiltrates in human pNT. (A) Representative multiplex IHC images visualize tumor cells (tumor marker [TM]: synaptophysin/CD56, white), T cells (CD3, green), B cells (CD20, yellow), neutrophils (CD66b, cyan), and macrophages (CD68, red). Nuclei were stained with DAPI (blue). Scale bars indicate 100 µm (merge image) and 25 µm (magnified image). (B) Bar plot shows the proportion of CD3⁺, CD20⁺, CD66b⁺, and CD68⁺ immune cells for the individual HR-NBL, NHR-NBL, GNBL, and GN patients. (C) Quantified densities of CD3⁺, CD20⁺, CD66b⁺, and CD68⁺ cells are depicted for all 24 pNT tissues investigated and (D) pNT subtypes HR-NBL, NHR-NBL, GNBL, and GN. (E) Representative multiplex IHC images illustrate CD4⁺ CD3⁺ (T helper cells, red/yellow), CD8⁺ CD3⁺ (cytotoxic T cells, magenta/yellow), and FoxP3⁺ CD3⁺ (Treg, cyan/yellow). Scale bars indicate 5 µm. (F) Calculated proportions of T cells in all pNT tissues and (G) classified in pNT subtypes are shown in dot plots. Median with 95% confidence interval (CI); Mann–Whitney test; * p ≤ 0.05. (H) The heatmap created by unsupervised clustering presents the stratification of HR-NBL, NHR-NBL, GNBL, and GN patients in a low and a high immune cell infiltrate cluster. (I) Kaplan–Meier curves show progression-free survival (PFS) for pNT patients stratified based on their high/low immune cell infiltrate. p-values calculated by Log-rank test.

Figure 2

Detailed phenotypic characterization of T cell populations infiltrating pNT tissue samples. (A) Representative image of a pNT section demonstrates tissue infiltration by CD3⁺ (yellow), CD4⁺ (red), or CD8⁺ (cyan) T cells expressing inhibitory receptors PD-1 (green), LAG-3 (magenta), and/or cytotoxic marker GrzB (orange). Nuclei were stained with DAPI (blue). PD-1⁺, LAG-3⁺, and GrzB⁺ cells are highlighted by white arrows. Scale bars indicate 100 µm (merge image) and 25 µm (magnified area). (B) Calculated proportions of T helper (CD4⁺ CD3⁺) and cytotoxic T cell (CD8⁺ CD3⁺) populations defined by the (co-)expression (+) or absence (−) of investigated markers as presented in the graph. (C) CD4⁺ CD3⁺ (left) and CD8⁺ CD3⁺ T cell proportions (right) distributed to HR-NBL, NHR-NBL, GNBL, and GN are shown in dot plots. Median with 95% confidence interval (CI); Mann–Whitney test (B) and Kruskal–Wallis test with Dunn‘s multiple comparisons post hoc test (C); * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001. (D) The heatmap created by unsupervised clustering presents the clustering of patients with high and low active T cell phenotype infiltration. (E) Kaplan–Meier curves demonstrate PFS for pNT patients stratified according to their high/low infiltration of active T cell phenotypes. p-values calculated by Log-rank test.

Figure 3

Investigation of pNT TIME regarding macrophage infiltration and polarization. (A) Representative images of a pNT section visualize stained marker molecules IRF8 (M1, magenta), CD68 (pan-macrophage marker, yellow), CD163 (M2, red), and CD206 (M2, green). Nuclei were stained with DAPI (blue). Scale bars indicate 100 µm (merge image) and 25 µm (magnified area). (B) Calculated M1- and M2-like macrophage proportions of all CD68⁺ cells are given as dot plots for all pNT samples and (C) HR-NBL, NHR-NBL, GNBL, and GN patients. (D) Calculated proportions of dissected M1-like (IRF8⁻ CD68⁺ CD163⁻ CD206⁻ and IRF8⁺ CD68⁺ CD163⁻ CD206⁻) and M2-like (IRF8⁻ CD68⁺ CD163⁺ CD206⁻, IRF8⁻ CD68⁺ CD206⁺ CD163⁻, and IRF8⁻ CD68⁺ CD163⁺ CD206⁺) macrophage subpopulations are displayed for all pNT patients and (E) pNT subtypes. Positive marker expression (+) or absent marker expression (−) was defined for each investigated marker as displayed below the axes. Median with 95% confidence interval (CI); Mann–Whitney test (B) and Kruskal–Wallis test with Dunn‘s multiple comparisons post hoc test (D); ** p ≤ 0.01, *** p ≤ 0.001. (F) The heatmap created by unsupervised clustering shows the stratification of patients based on single CD68⁺ (M1-like) and triple CD68⁺CD163⁺CD206⁺ (M2-like) macrophages. (G) Kaplan–Meier curves demonstrate PFS for pNT patients stratified based on the unsupervised clustering of the two above-mentioned macrophage populations. p-values calculated by Log-rank test.

Figure 4

Frequency of TLS in NBL, GNBL, and GN samples. (A) Representative H&E images of pNT tissues show a lymphocyte aggregates in the tumor area (left) and a clearly defined, mature TLS (framed by a black dashed line) in the Schwannian stroma containing two HEVs marked by arrows (right). Scale bars indicate 50 µm. (B) Dot plots demonstrate the median enrichment scores of 83 TLS-related gene signatures calculated by gene set variation analysis (GSVA) using publically available gene expression datasets. Median with 95% confidence interval (CI); Kruskal–Wallis test with Dunn‘s multiple comparisons post hoc test; * p ≤ 0.05, ** p ≤ 0.01. (C) Representative multiplex IHC images of TLS in tissue sections of patients diagnosed with HR-NBL, NHR-NBL, GNBL, and GN. Scale bars indicate 200 µm. (D) Pie charts visualize the distribution of TLS-containing tissues among pNT subtypes.

Figure 5

Multiplex immunohistochemical characterization of TLS maturation and LAMP3⁺ DC infiltration. Representative images of an immature TLS (A), a primary follicle TLS (B), and a secondary follicle TLS (C) stained for tumor marker (TM: synaptophysin/chromogranin A/CD56; yellow), CD3 (T cells, green), CD20 (B cells, red), Ki67 (proliferating cells, white), and PNAd (high endothelial venules (HEVs), orange). Nuclei were detected with DAPI (blue). The germinal center (GC) of secondary follicle TLS is highlighted by a white dashed line. Scale bars indicate 100 µm (A), 200 µm (B), and 300 µm (C). (D) Bar plot shows areas of immature TLS, primary follicle TLS, or secondary follicle TLS normalized to the whole tissue areas of the corresponding individual pNT patients. (E) Representative images highlight LAMP3⁺ cells (mature DC, yellow) infiltration in a primary follicle TLS (TM: synaptophysin/chromogranin A/CD56; cyan), CD3 (T cells, green), CD20 (B cells, red), Ki67 (proliferating cells, white), and PNAd (high endothelial venules (HEVs), orange). Scale bars indicate 200 µm in the overview images and 50 µm in the magnified areas. Densities of LAMP3⁺ DC in individual TLS are shown for (F) HR-NBL, NHR-NBL, GNBL, GN, and (G) TLS maturation stages. Median with 95% confidence interval (CI).

Figure 6

Association of relevant immune cell populations and TLS to patient characteristics and tumor attributes. (A) Correlation matrix comprising TLS area, densities of tumor-infiltrated CD3⁺, CD20⁺, and CD68⁺ cells, as well as proportions of FoxP3⁺ CD3⁺ T cells and cytotoxic CD8⁺ T cells in conjunction with Schwannian stroma content and patient age at diagnosis was build showing Spearman r correlation coefficient (left) and corresponding p-values (right). (B) Calculated proportions of CD4⁺, CD8⁺ as well as FoxP3⁺ CD3⁺ T cells are demonstrated for TLS⁺ and TLS⁻ groups. Median with 95% confidence interval (CI); * p ≤ 0.05 (C) Kaplan–Meier curves show PFS for pNT patients stratified based on the presence/absence of TLS. p-values calculated by Log-rank test.