Serum immune checkpoint profiling identifies soluble CD40 as a biomarker for pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) responds poorly to systemic treatment, including new immunotherapeutic approaches. Biomarkers are urgently needed for early disease detection, patient stratification for treatment, and response prediction. The role of soluble CD40 (sCD40) is unknown in PDAC. In this study, we performed a quantitative multiplex analysis of 17 immune checkpoint proteins in serum samples from patients with various stages of PDAC in a discovery study (n = 107) and analyzed sCD40 by ELISA in a validation study (n = 317). Youden's J statistic was used for diagnostic cut-off optimization. A Cox proportional hazards regression model was applied in an empiric approach for prognostic threshold optimization. Kaplan-Meier estimator and multivariable Cox regression analyses were used for survival analysis. sCD40 was significantly increased in the serum of patients with PDAC compared to healthy cohorts and patients with IPMN. In the validation cohort, the area under the receiver operating characteristic (ROC) c-statistic was 0.8, and combining sCD40 with CA19-9 yielded a c-statistic of 0.95. sCD40 levels were independent of the tumor stage. However, patients who received neoadjuvant chemotherapy had significantly lower sCD40 levels than those who underwent upfront surgery. Patients with a sCD40 level above the empirical threshold of 0.83 ng/ml had a significantly reduced overall survival with a hazard ratio of 1.4. This observation was pronounced in patients after neoadjuvant chemotherapy. Collectively, soluble CD40 may be considered as both a diagnostic and prognostic non-invasive biomarker in PDAC.

VISTA Ligation Reduces Antitumor T-Cell Activity in Pancreatic Cancer.

Immunotherapy has shown promising results in multiple solid tumors and hematological malignancies. However, pancreatic ductal adenocarcinoma (PDAC) has been largely refractory to current clinical immunotherapies. The V-domain Ig suppressor of T-cell activation (VISTA) inhibits T-cell effector function and maintains peripheral tolerance. Here, we determine VISTA expression in nontumorous pancreatic (n = 5) and PDAC tissue using immunohistochemistry (n = 76) and multiplex immunofluorescence staining (n = 67). Additionally, VISTA expression on tumor-infiltrating immune cells and matched blood samples (n = 13) was measured with multicolor flow cytometry. Further, the effect of recombinant VISTA on T-cell activation was investigated in vitro, and VISTA blockade was tested in an orthotopic PDAC mouse model in vivo. PDAC showed significantly higher VISTA expression compared to that of a nontumorous pancreas. Patients with a high density of VISTA-expressing tumor cells had reduced overall survival. The VISTA expression of CD4+ and CD8+ T cells was increased after stimulation and particularly after a coculture with tumor cells. We detected a higher level of proinflammatory cytokine (TNFα and IFNγ) expression by CD4+ and CD8+ T cells, which was reversed with the addition of recombinant VISTA. A VISTA blockade reduced tumor weights in vivo. The VISTA expression of tumor cells has clinical relevance, and its blockade may be a promising immunotherapeutic strategy for PDAC.

TIGIT Expression Delineates T-cell Populations with Distinct Functional and Prognostic Impact in Pancreatic Cancer.

PURPOSE: Immunotherapy has led to a fundamental shift in the treatment of several cancers. However, its efficacy in pancreatic ductal adenocarcinoma (PDAC) is limited. Understanding the expression of inhibitory immune checkpoint receptors (ICR) by intratumoral T cells may help to unravel their involvement in insufficient T-cell-mediated antitumor immunity. EXPERIMENTAL DESIGN: Using multicolor flow cytometry, we analyzed circulating and intratumoral T cells from blood (n = 144) and matched tumor samples (n = 107) of patients with PDAC. We determined the expression of programmed cell death protein 1 (PD-1) and T-cell immunoreceptor with Ig and immunoreceptor tyrosine-based inhibition motif (ITIM) domains (TIGIT) by CD8+ T-cells, conventional CD4+ T-cells (Tconv) and regulatory T cells (Treg) and their association with T-cell differentiation, tumor reactivity, and cytokine expression. A comprehensive follow-up was used to determine their prognostic value. RESULTS: Intratumoral T cells were characterized by increased PD-1 and TIGIT expression. Both markers delineated distinct T-cell subpopulations. PD-1+TIGIT- T cells highly expressed proinflammatory cytokines and markers of tumor reactivity (CD39, CD103), whereas TIGIT expression was linked to antiinflammatory and exhausted phenotypes. In addition, the enhanced presence of intratumoral PD-1+TIGIT- Tconv was associated with improved clinical outcomes, while high ICR expression on blood T cells was a significant hazard for overall survival (OS). CONCLUSIONS: Our results uncover the association between ICR expression and T-cell functionality. PD-1 and TIGIT characterized intratumoral T cells with highly divergent phenotypes linked to clinical outcomes, further underscoring the relevance of TIGIT for immunotherapeutic approaches in PDAC. The prognostic value of ICR expression in patient blood may be a valuable tool for patient stratification.

Neoadjuvant chemotherapy drives intratumoral T cells toward a proinflammatory profile in pancreatic cancer.

BACKGROUNDPancreatic ductal adenocarcinoma (PDAC) has a dismal prognosis. At diagnosis, only 20% of patients with PDAC are eligible for primary resection. Neoadjuvant chemotherapy can enable surgical resection in 30%-40% of patients with locally advanced and borderline resectable PDAC. The effects of neoadjuvant chemotherapy on the cytokine production of tumor-infiltrating T cells are unknown in PDAC.METHODSWe performed multiplex immunofluorescence to investigate T cell infiltration in 91 patients with PDAC. Using flow cytometry, we analyzed tumor and matched blood samples from 71 patients with PDAC and determined the frequencies of T cell subsets and their cytokine profiles. Both cohorts included patients who underwent primary resection and patients who received neoadjuvant chemotherapy followed by surgical resection.RESULTSIn human PDAC, T cells were particularly enriched within the tumor stroma. Neoadjuvant chemotherapy markedly enhanced T cell density within the ductal area of the tumor. Whereas infiltration of cytotoxic CD8+ T cells was unaffected by neoadjuvant chemotherapy, the frequency of conventional CD4+ T cells was increased, and the proportion of Tregs was reduced in the pancreatic tumor microenvironment after neoadjuvant treatment. Moreover, neoadjuvant chemotherapy increased the production of proinflammatory cytokines by tumor-infiltrating T cells, with enhanced TNF-α and IL-2 and reduced IL-4 and IL-10 expression.CONCLUSIONNeoadjuvant chemotherapy drives intratumoral T cells toward a proinflammatory profile. Combinational treatment strategies incorporating immunotherapy in neoadjuvant regimens may unleash more effective antitumor responses and improve prognosis of pancreatic cancer.FUNDINGThis work was supported by the Jung Foundation for Science and Research, the Monika Kutzner Foundation, the German Research Foundation (SE2980/5-1), the German Cancer Consortium, and the Faculty of Medicine Carl Gustav Carus, Technische Universität Dresden.

Clinical Significance of Tumor-Infiltrating Conventional and Plasmacytoid Dendritic Cells in Pancreatic Ductal Adenocarcinoma.

Dendritic cells (DCs) play a key role in the orchestration of antitumor immunity. Activated DCs efficiently enhance antitumor effects mediated by natural killer cells and T lymphocytes. Conversely, tolerogenic DCs essentially contribute to an immunosuppressive tumor microenvironment. Thus, DCs can profoundly influence tumor progression and clinical outcome of tumor patients. To gain novel insights into the role of human DCs in pancreatic ductal adenocarcinoma (PDAC), we explored the frequency, spatial organization, and clinical significance of conventional DCs type 1 (cDC1s) and type 2 (cDC2s) and plasmacytoid DCs (pDCs) in primary PDAC tissues. A higher density of whole tumor area (WTA)- and tumor stroma (TS)-infiltrating cDC1s was significantly associated with better disease-free survival (DFS). In addition, an increased frequency of intraepithelial tumor-infiltrating cDC2s was linked to better DFS and overall survival (OS). Furthermore, an increased density of WTA- and TS-infiltrating pDCs tended to improve DFS. Moreover, a higher frequency of WTA- and TS-infiltrating cDC1s and pDCs emerged as an independent prognostic factor for better DFS and OS. These findings indicate that tumor-infiltrating DCs can significantly influence the clinical outcome of PDAC patients and may contribute to the design of novel treatment options that target PDAC-infiltrating DCs.

LAG-3-Expressing Tumor-Infiltrating T Cells Are Associated with Reduced Disease-Free Survival in Pancreatic Cancer.

T cells are the predominant immune cell population in the pancreatic tumor microenvironment. High CD8+ and Th1-polarized CD4+ T cell infiltration is associated with prolonged survival in human pancreatic ductal adenocarcinoma (PDAC). However, the expression pattern of co-stimulatory and inhibitory receptors by PDAC-infiltrating T cells and their prognostic significance are not well defined. In this study, we employed multiplex immunofluorescence to investigate the intratumoral expression of the co-stimulatory receptor inducible T-cell co-stimulator (ICOS), the inhibitory receptors lymphocyte-activation gene 3 (LAG-3), programmed death 1 (PD-1), and V-domain immunoglobulin suppressor of T cell activation (VISTA) by tumor-infiltrating T cells (CD3) in a cohort of 69 patients with resected PDAC. T cells were enriched particularly within the stromal area and were highly heterogeneous across tumors. Further, T cells were associated with prolonged disease-free survival (DFS). However, LAG-3 expression by PDAC-infiltrating T cells was correlated with reduced DFS. Our study highlights the biological importance of LAG-3 expression by tumor-infiltrating T cells. LAG-3+ T cells may represent a novel prognostic marker and a particularly attractive target for immunotherapeutic strategies in PDAC.

PD-1 Expression by Lymph Node and Intratumoral Regulatory T Cells Is Associated with Lymph Node Metastasis in Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by a mostly immunosuppressive microenvironment. Tumor-draining lymph nodes (TDLN) are a major site for priming of tumor-reactive T cells and also tumor metastasis. However, the phenotype and function of T cells in TDLNs from PDAC patients is unknown. In this study, lymph nodes from the pancreatic head (PH), the hepatoduodenal ligament (HDL) and the interaortocaval (IAC) region were obtained from 25 patients with adenocarcinoma of the pancreatic head. Additionally, tumors and matched blood were analyzed from 16 PDAC patients. Using multicolor flow cytometry, we performed a comprehensive analysis of T cells. CD4+ T cells were the predominant T cell subset in PDAC-draining lymph nodes. Overall, lymph node CD4+ and CD8+ T cells had a similar degree of activation, as measured by CD69, inducible T cell co-stimulator (ICOS) and CD137 (4-1BB) expression and interferon-γ (IFNγ) secretion. Expression of the inhibitory receptor programmed death 1 (PD-1) by lymph node and tumor-infiltrating regulatory T cells (Tregs) correlated with lymph node metastasis. Collectively, Treg cells and PD-1 are two relevant components of the immunosuppressive network in PDAC-draining lymph nodes and may be particularly attractive targets for combinatorial immunotherapeutic strategies in selected patients with node-positive PDAC.

Gamma-delta T cells stimulate IL-6 production by pancreatic stellate cells in pancreatic ductal adenocarcinoma.

INTRODUCTION: The immunosuppressive tumor microenvironment promotes progression of pancreatic ductal adenocarcinoma (PDAC). γδ T cells infiltrate the pancreatic tumor stroma and support tumorigenesis through αß T cell inhibition. Pancreatic stellate cell (PSC) activation contributes to pancreatic fibrosis in PDAC, limiting the delivery and efficacy of therapeutic agents. Whether γδ T cells have direct effects on PSC activation is unknown. METHODS: In this study, we analyzed tumor tissue from 68 patients with PDAC and determined the frequency and location of γδ T cells using immunohistochemistry and immunofluorescence. PDAC samples from the TCGA database with low and high TRGC2 expression were correlated with the expression of extracellular matrix genes. Further, PSCs were isolated from pancreatic tumor tissue and co-cultured with γδ T cells for 48 hours and cytokine production was measured using a cytometric bead array. RESULTS: γδ T cells infiltrated the pancreatic tumor stroma and were located in proximity to PSCs. A high infiltration of γδ T cells was associated with increased expression of several extracellular matrix genes in human PDAC. In vitro, γδ T cells stimulated IL-6 production by PDAC-derived PSCs. CONCLUSION: γδ T cells activated PSCs and modulation of this interaction may enhance the efficacy of combinational therapies in human PDAC.

Detection of pancreatic ductal adenocarcinoma with galectin-9 serum levels.

Pancreatic ductal adenocarcinoma (PDAC) responds poorly to checkpoint blockade, such as anti-CTLA-4 and anti-PD-1. Galectin-9, a ß-galactoside-binding lectin, promotes immune suppression through T-cell inhibition, and programming of tolerogenic macrophages. Of all cancers tested, PDAC showed the highest expression of LGALS9 (galectin-9) mRNA. We analyzed formalin-fixed and paraffin-embedded specimens from 83 patients with PDAC stained for galectin-9. Using flow cytometry, we determined galectin-9 expression on immune cells from tumor and matched blood samples from 12 patients with resectable PDAC. Furthermore, we analyzed galectin-9 serum levels by enzyme-linked immunosorbent assay using serum samples from 70 patients with PDAC, from 36 individuals with benign pancreatic disease, and from 28 healthy controls. Galectin-9 was highly expressed in human PDAC compared with normal pancreas and present on both tumor and immune cells. Tumor-infiltrating immune cells, especially CD3+ T cells, showed upregulation of galectin-9 compared with immune cells from matched blood. Blood γδ T cells from PDAC patients had higher galectin-9 expression than γδ T cells from healthy individuals. Galectin-9 polarized macrophages toward a protumoral M2 phenotype leading to suppressed T-cell cytokine secretion. Furthermore, serum concentration of galectin-9 was able to discriminate PDAC from benign pancreatic disease and healthy individuals, and was prognostic for stage IV patients. Galectin-9 is a new biomarker for the detection of PDAC.

Colorectal Carcinogenesis: Connecting K-RAS-Induced Transformation and CREB Activity In Vitro and In Vivo.

UNLABELLED: Oncogenic transformation is often associated with an increased expression of the cAMP response element binding (CREB) transcription factor controlling the expression of genes involved in cell proliferation, cell cycle, apoptosis, and tumor development, but a link between K-RAS(V12)-induced transformation and CREB has not yet been determined. Therefore, the constitutive and/or inhibitor-regulated mRNA and protein expression of CREB and signal transduction components and growth properties of parental fibroblasts, K-RAS(V12)-transformed counterparts, shCREB K-RAS(V12) transfectants and human colon carcinoma cells were determined. Increased CREB transcript and protein levels accompanied by an enhanced CREB activity was detected in K-RAS(V12)-transformed murine fibroblasts and K-RAS(V12)-mutated human tumor cells, which is dependent on the MAPK/MEK, PI3K, and/or PKC signal transduction. Immunohistochemical (IHC) staining of colorectal carcinoma lesions and murine tumors, with known KRAS gene mutation status, using antibodies specific for CREB and phospho-CREB, revealed a mechanistic link between CREB expression and K-RAS(V12)-mutated colorectal carcinoma lesions when compared with control tissues. Silencing of CREB by shRNA and/or treatment with a CREB inhibitor (KG-501) reverted the neoplastic phenotype of K-RAS(V12) transformants as demonstrated by a more fibroblast-like morphology, enhanced apoptosis sensitivity, increased doubling time, decreased migration, invasion and anchorage-independent growth, reduced tumorigenesis, and enhanced immunogenicity in vivo. The impaired shCREB-mediated invasion of K-RAS(V12) transformants was accompanied by a transcriptional downregulation of different matrix metalloproteinases (MMP) coupled with their reduced enzymatic activity. IMPLICATIONS: CREB plays a key role in the K-RAS(V12)-mediated neoplastic phenotype and represents a suitable therapeutic target for murine and human K-RAS(V12)-induced tumors.