Dados do Trabalho

Título

Mature tertiary lymphoid structures predict responses to chemoimmunotherapy in pancreatic ductal adenocarcinomas

Introdução

Given its success in many cancer types, immunotherapies hold promise for the standard of care of pancreatic ductal adenocarcinoma (PDAC), though initial studies underscore the need of reliable biomarkers of clinical outcome. Immune aggregates called tertiary lymphoid structures (TLSs), composed of B, T and dendritic cells, were shown to predict responses to immune checkpoint blockade in melanoma, sarcoma, renal cell carcinoma and urothelial carcinoma. These findings may be applicable to other cancer types, as TLSs could act as sheltered niches for optimal lymphocyte activity.

Objetivo

To provide a framework for understanding the biological and clinical relevance of TLSs in PDACs, we characterized the functional states and spatial organization of tumor-infiltrating T and B cells, and investigated the impact of TLS maturation on tumor-specific immunity and response to immunotherapy.

Métodos

We analyzed single-cell RNAseq (scRNA-seq) data from 24 treatment-naïve PDACs (Peng et al. Cell Res. 2019). We also performed flow cytometry, multicolor immunofluorescence, immunohistochemistry, gene expression profiling of laser-capture microdissected TLS and in vitro validation assays using samples from a cohort of treatment-naïve PDACs that underwent surgical resection at the A.C.Camargo Cancer Center or Santa Casa de Misericordia de Sao Paulo (n = 70, CEP 2712/19). To evaluate the clinical relevance of our findings, we used pathology slides and RNA-seq data from 147 treatment-naïve PDACs in The Cancer Genome Atlas (TCGA), and RNA-seq data from 105 pre-treatment biopsies of metastatic PDAC patients that received first-line chemoimmunotherapy (PRINCE trial).

Resultados

Analysis of scRNA-seq and flow cytometry data revealed that ~10% of PDACs harbor fully developed TLSs where B cells undergo antibody-affinity maturation and differentiate towards plasma cells. Using immunohistochemistry and laser-capture microdissection, we found that these mature TLSs are key hubs of lymphocyte communication and support the activation and clonal expansion of tumor-reactive T cells. Importantly, immunofluorescence staining and in vitro assays showed that chronically activated, tumor-reactive T cells exposed to fibroblast-derived TGF-β may act as TLS organizers by producing the B cell chemoattractant CXCL13. Finally, by integrated RNA-seq data and paired H&E-stained sections from TCGA patients, we defined a refined gene signature that specifically reflected tumors harboring mature TLSs. Analysis of RNA-seq data from the PRINCE trial revealed that patients with a high signature score had longer overall survival after chemoimmunotherapy, independent of de novo/recurrent staging at initial diagnosis, prior chemotherapy usage and biopsy site.

Conclusões

We provided an in-depth view of PDAC-infiltrating lymphocytes, from cell spatiality to their network of interactions, revealing the benefit of lymphocyte compartmentalization within TLSs for the generation of adaptive immunity in situ. We also developed a method to infer the presence of mature TLSs using gene expression data from small tissue samples, bypassing the need of high-quality pathology slides from surgical pieces. This strategy can have a direct clinical impact, guiding the selection of PDAC patients for future biomarker-based immunotherapy trials.

Palavras-chave

Immunotherapy, tertiary lymphoid structure, tumor-reactive T cells

Financiador do resumo

FAPESP 19/25129-2 (fellowship to G.S.K.), 18/14034-8 (grant to T.S.M).