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.

KEYWORDS:

• TLS
• b cells
• pancreatic cancer
• immunotherapy
• t cells
• neoantigens

Acknowledgments

We would like to thank members of the Providence HPB surgical team and the Integrated therapies laboratory not listed in the author list that contributed critical data and sample curation toward the completion of this study. We would also like to thank Dr. Walter Urba for critical reading of this manuscript. The results published here are in part based upon data generated by the TCGA Research Network: https://www.cancer.gov/tcga.

Competing Interests

A.J.G. declares no competing interests. K.H.Y. is the principal investigator of clinical trial NCT02688712 sponsored by Providence Health and Services in collaboration with Eli Lilly and Company. M.J.G. and M.R.C. have research support from Mavupharma, Bristol Myers Squibb, Nanobiotix, and Jounce that is not directly related to the subject of this manuscript. The funders had no part in the subject or content of this manuscript.

Authors’ Contributions

Conceptualization - A.J.G. and K.H.Y.; Methodology - A.J.G., K.H.Y., C.D., B.P., V.R., B.C., M.J.G, T.M., M.S., E.T., J.P.; Board Pathologist – C.B.; Software - C.D., B.P., M.S., V.R., M.J.G., J.P, E.T., A.J.G.; Validation – A.J.G., V.R.; Formal Analysis – A.J.G., M.S., V.R., B.P., C.D., J.P., C.B., K.H.Y.; Investigation – A.J.G, K.H.Y., V.R., B.P., M.S., B.C., J.P., T.M., K.M., M.P., C.D., E.T., M.J.G.; Resources – C.B., P.N., P.H., E.T., B.C., M.R.C., M.J.G., K.H.Y.; Data Curation – M.J.G., K.H.Y., C.B., E.T., A.J.G., V.R.; Writing – A.J.G, K.H.Y.; Visualization – A.J.G., K.H.Y., B.P., M.S.; Supervision – A.J.G., K.H.Y., M.J.G, M.R.C.; Project Administration – A.J.G., K.H.Y., M.J.G., M.R.C.; Funding acquisition – A.J.G., K.H.Y., M.J.G., M.R.C.

Ethics approval and consent to participate

Sample collection from this study was approved by the institutional review board at Providence Portland Medical Center, Portland, OR with the study ID numbers PHS: 06-108 and PHS 10-037. All patients provided informed consent for participation in this study.

Availability of data and materials

The datasets analyzed during the current study are available from the corresponding and last author on reasonable request.

Supplementary material

Supplemental data for this article can be accessed on the publisher’s website.

Additional information

Funding

A.J.G. was supported in part by the Collins Medical Trust of Oregon. K.H.Y. is funded by the 2016 Sydney Kimmel Foundation Translational Research Scholar Grant. M.R.C. and M.J.G. supported this study through grants: NIH R01 CA182311 (M.J.G.) and NIH R01 CA208644 (M.R.C.);Collins Medical Trust;National Institutes of Health [NIH RO1 CA182311];National Institutes of Health [NIH RO1 CA208644];Adaptive Biotechnologies;Susan G. Komen;