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Germinal center reactions in tertiary lymphoid structures associate with neoantigen burden, humoral immunity and long-term survivorship in pancreatic cancer.

ABSTRACT: Pancreatic ductal adenocarcinoma (PDAC) has traditionally been thought of as an immunologically quiescent tumor type presumably because of a relatively low tumor mutational burden (TMB) and poor responses to checkpoint blockade therapy. However, many PDAC tumors exhibit T cell inflamed phenotypes. The presence of tertiary lymphoid structures (TLS) has recently been shown to be predictive of checkpoint blockade response in melanomas and sarcomas, and are prognostic for survival in PDAC. In order to more comprehensively understand tumor immunity in PDAC patients with TLS, we performed RNA-seq, single and multiplex IHC, flow cytometry and predictive genomic analysis on treatment naïve, PDAC surgical specimens. Forty-six percent of tumors contained distinct T and B cell aggregates reflective of "early-stage TLS" (ES-TLS), which correlated with longer overall and progression-free survival. These tumors had greater CD8⁺ T cell infiltration but were not defined by previously published TLS gene-expression signatures. ES-TLS⁺ tumors were enriched for IgG1 class-switched memory B cells and memory CD4⁺ T cells, suggesting durable immunological memory persisted in these patients. We also observed the presence of active germinal centers (mature-TLS) in 31% of tumors with lymphocyte clusters, whose patients had long-term survival (median 56 months). M-TLS-positive tumors had equivalent overall T cell infiltration to ES-TLS, but were enriched for activated CD4⁺ memory cells, naive B cells and NK cells. Finally, using a TCGA-PDAC dataset, ES-TLS⁺ tumors harbored a decreased TMB, but M-TLS with germinal centers expressed significantly more MHCI-restricted neoantigens as determined by an in silico neoantigen prediction method. Interestingly, M-TLS⁺ tumors also had evidence of increased rates of B cell somatic hypermutation, suggesting that germinal centers form in the presence of high-quality tumor neoantigens leading to increased humoral immunity that confers improved survival for PDAC patients. AbbreviationsTLS: tertiary lymphoid structures; GC: germinal center(s); PDAC: pancreatic ductal adenocarcinoma; RNA-seq: RNA sequencing; BCRseq: B cell receptor sequencing; HEV: high endothelial venule; PNAd: peripheral node addressin; TMB: tumor mutational burden; TCGA: the cancer genome atlas; PAAD: pancreatic adenocarcinoma; FFPE: formalin fixed paraffin embedded; TIME: tumor immune microenvironment.

SUBMITTER: J Gunderson A

PROVIDER: S-EPMC7993148 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:There is increasing evidence that tertiary lymphoid structures (TLS) control not only local adaptive B cell responses at melanoma tumor sites but also the cellular composition and function of other immune cells. In human melanoma, however, a comprehensive analysis of TLS phenotypes, density and spatial distribution at different disease stages is lacking. Here we used 7-color multiplex immunostaining of whole tissue sections from 103 human melanoma samples to characterize TLS phenotypes along the expression of established TLS-defining molecular and cellular components. TLS density and spatial distribution were determined by referring TLS counts to the tissue area within defined intra- and extratumoral perimeters around the invasive tumor front. We show that only a subgroup of primary human melanomas contains TLS. These TLS rarely formed germinal centers and mostly located intratumorally within 1 mm distance to the invasive tumor front. In contrast, melanoma metastases had a significantly increased density of secondary follicular TLS. They appeared preferentially in stromal areas within an extratumoral 1 mm distance to the invasive tumor front and their density varied over time and site of metastasis. Interestingly, secondary follicular TLS in melanoma often lacked BCL6+ lymphatic cells and canonical germinal center polarity with the formation of dark and light zone areas. Our work provides an integrated qualitative, quantitative and spatial analysis of TLS in human melanoma and shows disease progression- and site-associated changes in TLS phenotypes, density and spatial distribution. The frequent absence of canonical germinal center polarity in melanoma TLS highlights the induction of TLS maturation as a potential additive to future immunotherapy studies. Given the variable evaluation strategies used in previous TLS studies of human tumors, an important asset of this study is the standardized quantitative evaluation approach that provides a high degree of reproducibility.

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Germinal center reactions in tertiary lymphoid structures associate with neoantigen burden, humoral immunity and long-term survivorship in pancreatic cancer.

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