Spatial biology analysis reveals B cell follicles in secondary lymphoid structures may regulate anti-tumor responses at initial melanoma diagnosis

Abstract

Introduction: B cells are key regulators of immune responses in melanoma. We aimed to explore differences in the histologic location and activation status of B cell follicles in sentinel lymph nodes (SLN) of melanoma patients.

Methods: Flow cytometry was performed on fresh tumor draining lymph nodes (LN). Paraffin slides from a separate cohort underwent NanoString Digital Spatial Profiling (DSP)®. After staining with fluorescent markers for CD20 (B cells), CD3 (T cells), CD11c (antigen presenting cells) and a nuclear marker (tumor) was performed, regions of interest (ROI) were selected based on the location of B cell regions (B cell follicles). A panel of 68 proteins was then analyzed from the ROIs.

Results: B cell percentage trended higher in patients with tumor in LN (n=3) compared to patients with nSLN (n=10) by flow cytometry. B cell regions from a separate cohort of patients with tumor in the (pSLN) (n=8) vs. no tumor (nSLN) (n=16) were examined with DSP. Within B cell regions of the SLN, patients with pSLN had significantly higher expression of multiple activation markers including Ki-67 compared to nSLN patients. Among 4 patients with pSLN, we noted variability in arrangement of B cell follicles which were either surrounding the tumor deposit or appeared to be infiltrating the tumor. The B cell follicle infiltrative pattern was associated with prolonged recurrence free survival.

Conclusion: These data suggest a role for B cell follicles in coordinating effective adaptive immune responses in melanoma when low volume metastatic disease is present in tumor draining LN.

Keywords: B cells; digital spatial profiling; melanoma; sentinel lymph nodes (SLN); tumor immune microenvironment.

Figure 1

Flow cytometry analysis of fresh lymph node tissue. Data are presented as percent (%) of live cells in the lymph node (Y-axis). X axis shows cell subsets. Blue bars are patients with tumor in the lymph nodes and gray bars are patients with no tumor in the lymph nodes. P values for CD3+ T cell and CD19+ B cells are also shown.

Figure 2

Example of DSP analysis. We used 2 patients for DSP analysis: patient P22 (positive sentinel node), left and patient N97 (negative sentinel lymph node), right. (A) Patient P22 SLN low power fluorescent staining for CD11c (green), CD20 (gray), CD3 (red), DNA (blue). Tumor region is labeled “tumor.” (B) H&E of sentinel lymph node for patient P22. (C) High power image of white rectangle from (A), white circles indicate B cell regions selected for protein analysis (not all B cell regions selected are shown). Orange circle indicates an example of a tumor region chosen for protein analysis. (D) Patient N97, low power fluorescent staining for CD11c (green), CD20 (gray), CD3 (red), DNA (blue). (E) H&E of sentinel lymph node for patient N97. (F) High power image of white rectangle from (D), white circles indicate B cell regions selected for protein analysis (not all B cell regions selected are shown).

Figure 3

Sentinel lymph node tissue analysis. Comparison of sentinel lymph node tissue (SLN) from patients P22, P23, P7, (top) and P47 (bottom). Fluorescent antibodies were used to examine CD11c (green), CD20 (gray), CD3 (red), and DNA (blue.) (A1) Patient P7 SLN H&E. (A2) Patient P7 stained SLN. (B1) Patient P22 SLN H&E (B2) Patient P22 (an additional cut separate from Figure 2 ) stained SLN. (C1) Patient P23 SLN H&E. (C2) Patient P23 SLN stained. (D1) Patient P47 SLN H&E. (D2) Patient P47 SLN stained low power. (D3) Patient P47 SLN high power of white rectangle from (D2).

Figure 4

Proteins that were significantly differentially expressed between B cell regions among patients with tumor in the sentinel lymph nodes. The signal to noise ratio (y axis) of protein levels from patients P22, P23, and P7 with shorter recurrence free survival in blue bars compared to protein expression from B cell regions from patient P47 in red bars. Protein target names are listed on the x axis. *= q value less than 0.05, **=q value < 0.01; q value adjusted for false discovery rate set at 5%.

Figure 5

Cluster analysis of protein expression in B cell regions. Cluster analysis of protein expression from 3 B cell regions from patient P23 (purple) and 2 B cells regions from patient P47 (tan).