Pancreatic ductal adenocarcinoma (PDAC) is considered a “non-Immunogenic” neoplasm. Single agent immunotherapies have failed to demonstrate significant clinical activity in PDAC and other “non-immunogenic” tumors, in part due to a complex tumor microenvironment (TME) that provides a formidable barrier to immune infiltration and function. We designed a neo-adjuvant and adjuvant clinical trial comparing an irradiated, granulocyte-macrophage colony-stimulating factor (GM-CSF)-secreting, allogeneic PDAC vaccine (GVAX) given as single agent, or in combination with low dose cyclophosphamide (Cy) to deplete regulatory T cells (Tregs), to study how the TME is altered by immunotherapy. Examination of resected PDACs revealed the formation of vaccine-induced intratumoral tertiary lymphoid aggregates in 33/39 patients two weeks following vaccine treatment. Immunohistochemical analysis showed these aggregates to be regulatory structures of adaptive immunity. Microarray analysis of microdissected aggregates identified gene-expression signatures in five signaling pathways involved in regulating immune cell activation and trafficking that were associated with improved post-vaccination responses. A suppressed Treg pathway and an enhanced Th17 pathway within these aggregates were associated with improved survival, enhanced post-vaccination mesothelin-specific T-cell responses and increased intratumoral Teffector/Treg ratios. This study provides the first example of immune-based therapy converting a “non-immunogenic” neoplasm into an “immunogenic” neoplasm by inducing infiltration of T cells and development of tertiary lymphoid structures in the TME. Post-GVAX T-cell infiltration and aggregate formation resulted in the upregulation of immunosuppressive regulatory mechanisms including the PD-1/PD-L1 pathway, suggesting that vaccine-primed PDAC patients may be better candidates than vaccine–naïve patients for immune checkpoint and other immunomodulatory therapies.

Pancreatic ductal adenocarcinoma (PDA) has a poor prognosis due to late detection and resistance to conventional therapies. Published studies show that the PDA tumor microenvironment (TME) is predominantly infiltrated with immune suppressive cells and signals that if altered, would allow effective immunotherapy. However, single-agent checkpoint inhibitors including agents that alter immune suppressive signals in other human cancers such as cytotoxic T lymphocyte antigen-4 (CTLA-4), programmed death 1 (PD-1) and its ligand PD-L1, have failed to demonstrate objective responses when given as single agents to PDA patients. We recently reported that inhibition of the CTLA-4 pathway when given together with a T cell inducing vaccine gives objective responses in metastatic PDA patients. In this study, we evaluated blockade of the PD-1/PD-L1 pathway. We found that PD-L1 is weakly expressed at a low frequency in untreated human and murine PDAs but treatment with a GM-CSF secreting PDA vaccine (GVAX) significantly upregulates PD-L1 membranous expression after treatment of tumor bearing mice. In addition, combination therapy with vaccine and PD-1 antibody blockade improved murine survival compared to PD-1 antibody monotherapy or GVAX therapy alone. Furthermore, PD-1 blockade increased effector CD8+ T lymphocytes and tumor-specific interferon-γ production of CD8+ T cells in the TME. Immunosuppressive pathways, including regulatory T cells (Tregs) and CTLA-4 expression on T cells were overcome by the addition of vaccine and low dose cyclophosphamide to PD-1 blockade. Collectively, our study supports combining PD-1 or PD-L1 antibody therapy with a T cell inducing agent for PDA treatment.

These results illustrate the complexity of the role of stroma in PDAs. Further exploration of the prognostic ability of the characteristics of stroma is warranted.

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