High Numbers and Densities of PD1+ T-Follicular Helper Cells in Triple-Negative Breast Cancer Draining Lymph Nodes Are Associated with Lower Survival

Abstract

Breast cancer tumor draining lymph nodes (TDLNs) display distinct morphologic changes depending on the breast cancer subtype. For triple-negative breast cancers (TNBC), draining LNs display a higher amount of secondary lymphoid follicles, which can be regarded as a surrogate marker for an activated humoral immune response. In the present study, we focus on PD1⁺ T-follicular helper cells (Tfh) in TDLNs of TNBC, since PD1⁺ Tfh are drivers of the germinal center (GC) reaction. We quantified PD1⁺ Tfh in 22 sentinel LNs with 853 GCs and interfollicular areas from 19 patients with TNBC by morphometry from digitalized immunostained tissue sections. Overall survival was significantly worse for patients with a higher number and area density of PD1⁺ Tfh within GCs of TDLNs. Further, we performed T-cell receptor gamma chain (TRG) analysis from microdissected tissue in the primary tumor and TDLNs. Eleven patients showed the same TRG clones in the tumor and the LN. Five patients shared the same TRG clones in the tumor and the GCs. In two patients, those clones were highly enriched inside the GCs. Enrichment of identical TRG clones at the tumor site vs. the TDLN was associated with improved overall survival. TDLNs are important relays of cancer immunity and enable surrogate approaches to predict the outcome of TNBC itself.

Keywords: PD1+ T-follicular helper cells; breast cancer; germinal center; tumor draining lymph node.

Figure 1

(A,B) Exemplary immunohistochemical triple stainings showing BCL6⁺ germinal centers (GCs, brown), PD1⁺ T-follicular helper cells (Tfh, blue), and CD8⁺ cytotoxic T-cells (red); 100µm indicated by white bar in the upper left, respectively. (A) Exemplary GC with polarized PD1⁺ Tfh distribution, lower PD1⁺ Tfh number and lower area density. (B) Exemplary GC with non-polarized PD1⁺ Tfh distribution, higher PD1⁺ Tfh number and higher area density. (C–E) Separation of groups by mean split. (C) Mean PD1⁺ Tfh number ≥ 61.5 cells per GC is associated with lower survival (p = 0.03, logrank test, 15 deaths for 72 months follow-up observation time, 1 patient still alive in the PD1 high group, 3 patients still alive in the PD1 low group after 72 months follow-up). (D) Mean density ≥ 0.002475 PD1⁺ Tfh per mm² GC area is associated with lower survival (p = 0.03, logrank test, 15 deaths for 72 months follow-up observation time, 1 patient still alive in the PD1 high group, 3 patients still alive in the PD1 low group after 72 months follow-up). (E–I) Association between mean numbers of CD8⁺ GC cells, morphometrical, and clinical findings. (E) Higher mean numbers of CD8⁺ GC cells are associated with higher GC numbers, higher mean GC areas (F), higher mean and median CD8⁺ GC cell density (G,H), and higher tumor grade (I).

Figure 2

(A) Tumor/lymph node ratio of identical T-cell clones > 2.476 is associated with improved survival (p = 0.04, logrank test, 11 deaths for 72 months follow-up observation time, 1 patient still alive in the high ratio group after 72 months follow-up; ratio was calculated as (sum of % TRG reads tumor)/(sum of % TRG reads lymph node) of identical TRG sequences from tumors and matched lymph nodes). (B) Displays the percentage (%) of reads (TRG sequencing) of identical clones in different compartments to see whether clones are expanded: (I) identical clones between draining lymph node (LN) and tumor: % reads in the tumor sample; (II) identical clones between tumor and draining LN: % reads in the LN sample; (III) identical clones between tumor and germinal center (GC): % reads in the tumor sample; (IV) identical clones between tumor and germinal GC: % reads in the GC sample, showing two patients with strongly enriched clones inside the GCs; (V) identical clones between lymph node and GC: % reads in the LN sample; (VI) identical clones between lymph node and GC: % reads in the GC sample.