Tertiary lymphoid structures critical for prognosis in endometrial cancer patients

Abstract

B-cells play a key role in cancer suppression, particularly when aggregated in tertiary lymphoid structures (TLS). Here, we investigate the role of B-cells and TLS in endometrial cancer (EC). Single cell RNA-sequencing of B-cells shows presence of naïve B-cells, cycling/germinal center B-cells and antibody-secreting cells. Differential gene expression analysis shows association of TLS with L1CAM overexpression. Immunohistochemistry and co-immunofluorescence show L1CAM expression in mature TLS, independent of L1CAM expression in the tumor. Using L1CAM as a marker, 378 of the 411 molecularly classified ECs from the PORTEC-3 biobank are evaluated, TLS are found in 19%. L1CAM expressing TLS are most common in mismatch-repair deficient (29/127, 23%) and polymerase-epsilon mutant EC (24/47, 51%). Multivariable Cox regression analysis shows strong favorable prognostic impact of TLS, independent of clinicopathological and molecular factors. Our data suggests a pivotal role of TLS in outcome of EC patients, and establishes L1CAM as a simple biomarker.

Fig. 1. Single cell RNA sequencing of tumor-infiltrating B-cells.

a UMAP projection of endometrial cancer B-cell scRNA-seq data (1501 cells; 6 donors) annotated by cluster. b Dotplot of canonical B cell subtype marker genes per cluster. c Predicted cell identity for Cluster 1, Cluster 2 and Cluster 3 cells assigned using reference scRNA-seq data of a human lymph node. Cell identities are projected onto the endometrial cancer B-cell UMAP from a. d Quantification of predicted cell identities per cluster. e UMAP projection of Cluster 3 plasmablasts with Feature Plots depicting IGHG1, IGHA1, IGLC3 and IGKC expression in single cells. UMAP Uniform Manifold Approximation and Projection; MBC Memory B cell. Source data are provided as a Source Data file.

Fig. 2. L1CAM expression in mature germinal centers of tertiary lymphoid structures.

a Representative H&E image of a TLS in EC, which were observed in 29 of the 273 cases of the Uterine Corpus Endometrial Carcinoma (UCEC) cohort of The Cancer Genome Atlas (TCGA) research consortium. b Frequency of molecular subgroups by TLS status in endometrial cancer patients included in the UCEC TCGA. Source data are available at: https://portal.gdc.cancer.gov. c Differential gene expression of TLS-positive versus TLS-negative TCGA UCEC cases. d Representative L1CAM-positive TLS case. Arrows indicate TLS. e Co-immunofluorescent analysis of L1CAM-positive TLS with L1CAM and CD21. The experiment was repeated four times with similar results. n Number of cases, POLEmut Pathogenic polymerase epsilon mutation, MMRd Mismatch repair-deficient, p53abn p53 abnormal, NSMP No specific molecular profile, TLS Tertiary lymphoid structure, L1CAM Ligant-1 cell adhesion molecule, DAPI 4′,6-diamidino-2-phenylindole. Source data are provided as a Source Data File.

Fig. 3. Hallmarks of mature TLS in L1CAM positive TLS.

Representative example of a single endometrial cancer case showing hallmark features of TLS maturation in L1CAM-positive TLS as determined by Bcl6, CD20, CD4 and CD8 immunohistochemistry.

Fig. 4. Relation between molecular group, TLS and CD8 densities and prognosis in high-risk endometrial cancer.

a Heat map of included PORTEC-3 patients (N = 378) with available data on molecular classification, TLS, CD8⁺ and CD20⁺ densities (N = 252). Each patient is represented by a row in the graph. Clustering of CD8⁺ and CD20⁺ densities stratified by molecular group was done by hierarchical clustering using Ward’s minimum variance method. b Endometrial cancer recurrence-free survival calculated according to Kaplan-Meier’s methodology using the log rank test (two-sided alpha of 0.05) for all included PORTEC-3 patients (N = 378). c Endometrial cancer-specific survival calculated according to Kaplan-Meier’s methodology using the log-rank test (two-sided alpha of 0.05) for all included PORTEC-3 patients (N = 378).

Fig. 5. Characteristics of prognostic models in high-risk endometrial cancer.

a Boxplots showing concordance (C index) of the pathologic model, molecular model and the molecular-immune model. Box and whisker (Tukey) plots use results of 1000 bootstrap resamples from study population; lower and upper limits of box indicate 25th and 75th percentiles; and whiskers extend to 1.5x interquartile range below and above these values, respectively. The thick black bars indicate the C index from original population. b Pie charts showing relative importance of variables within these three multivariable models based on the proportion of the χ² statistic. LVSI Lymphovascular space invasion; TLS Tertiary lymphoid structure.

Fig. 6. Endometrial cancer recurrence by TLS presence across the endometrial cancer molecular classes.

Endometrial cancer recurrence-free survival calculated according to Kaplan-Meier’s methodology and tested between patients with and without TLS using a two-sided log rank test (alpha 0.05). a Patients with POLEmut endometrial cancer (N = 47) and MMRd endometrial cancer (N = 127). b Patients with p53abn endometrial cancer (N = 83) and NSMP endometrial cancer (N = 121). TLS Tertiary lymphoid structure; POLEmut Pathogenic polymerase epsilon mutation; MMRd Mismatch repair deficient; p53abn p53 abnormal; NSMP No specific molecular profile.