Search

Current treatments for chronic pancreatitis focus on symptom management and therapeutics targeting disease reversal are lacking. Given the role of the cyclooxygenase-2 (COX-2) enzyme in producing prostaglandin EThis pilot two-center randomized controlled trial seeks to examine 32 subjects with chronic pancreatitis who have no contraindications to indomethacin. Subjects will be randomized to either oral indomethacin 50 mg twice a day or placebo twice a day for a total of 28 days. Baseline (pre-treatment) assessment of pain and quality of life will be performed using the Brief Pain Inventory and the PROMIS-10 questionnaires, respectively. Biological specimens including blood, urine, and saliva will be collected at pre-treatment and post-treatment(day 28). Endoscopic pancreatic function testing with concomitant pancreatic fluid collection will also be performed pre- and post-treatment to assess the change in pancreatic fluid PGEThis study will elucidate the effect of oral indomethacin on PGE

Purpose: Biliary tract cancers (BTC) are aggressive malignancies refractory to chemotherapy and immunotherapy. MEK inhibition (MEKi)-based regimens may have utility in this disease when combined with PD-L1 blockade. We hypothesize that dual MEK/PD-L1 inhibition alters circulating soluble and cellular immune mediators to improve clinical outcomes in patients with advanced BTC. Experimental Design: We examined immune features in peripheral blood from 77 patients with advanced BTC enrolled in a phase II clinical trial investigating atezolizumab with or without cobimetinib. Plasma and peripheral blood mononuclear cells (PBMC) were isolated from whole blood to evaluate soluble factors and immune cell populations. Baseline blood samples were additionally compared with healthy donors to identify immune signatures unique to BTC. Results: At baseline, the soluble factors platelet-derived growth factor B (PDGF)-BB, placental growth factor (PlGF)-1, IL5, and IL17A were elevated in patients with BTC compared with healthy adult donors, and higher baseline frequencies of CD8+BTLA+ T cells correlated with better overall survival (OS) in this trial. There were also significant treatment-related alterations in several factors, including decreased PDGF-BB following combination treatment, that correlated with improved OS and progression-free survival (PFS). Higher baseline levels of IL23 and RANTES corresponded to improved clinical outcomes following combination treatment. Dual MEK/PD-L1 inhibition increased populations of CD4+TIM3+ and decreased CD8+VISTA+ T cells, correlating with worse OS and better PFS, respectively. Conclusions: This work represents a comprehensive analysis of peripheral immune features in patients with BTC and systemic responses to dual MEK/PD-L1 inhibition. These data support further investigation to understand how MEKi combines with immunotherapeutic approaches to improve clinical outcomes for patients with advanced BTC.

MEK inhibition (MEKi) is proposed to enhance antitumor immunity but has demonstrated mixed results as an immunomodulatory strategy in human clinical trials. MEKi exerts direct immunomodulatory effects on tumor cells and tumor-infiltrating lymphocytes (TIL), but these effects have not been independently investigated. Here we modeled tumor-specific MEKi through CRISPR/Cas-mediated genome editing of tumor cells [MEK1 knockout (KO)] and pharmacologic MEKi with cobimetinib in a RAS-driven model of colorectal cancer. This approach allowed us to distinguish tumor-mediated and tumor-independent mechanisms of MEKi immunomodulation. MEK1 KO tumors demonstrated upregulation of JAK/STAT signaling, enhanced MHCI expression, CD8+ T-cell infiltration and T-cell activation, and impaired tumor growth that is immune dependent. Pharmacologic MEKi recapitulated tumor-intrinsic effects but simultaneously impaired T-cell activation in the tumor microenvironment. We confirmed a reduction in human peripheral-lymphocyte activation from a clinical trial of anti–PD-L1 (atezolizumab) with or without cobimetinib in biliary tract cancers. Impaired activation of TILs treated with pharmacologic MEKi was reversible and was rescued with the addition of a 4-1BB agonist. Collectively, these data underscore the ability of MEKi to induce context-dependent immunomodulatory effects and suggest that T cell–agonist therapy maximizes the beneficial effects of MEKi on the antitumor immune response.

Supplementary Figures 1-5 and Supplementary Table 1

Supplemental Table S3. Demographic, clinical, and pathological characteristics of 25 men with biochemical recurrent prostate cancer

We hypothesized that soy phytochemicals may have immunomodulatory properties that may affect prostate carcinogenesis and progression. A randomized, phase II trial was conducted in 32 patients with prostate cancer with asymptomatic biochemical recurrence but no measurable disease on standard staging studies. Patients were randomized to two slices of soy bread (34 mg isoflavones/slice) or soy bread containing almond powder daily as a source of β-glucosidase. Flow cytometry and bioplex assays were used to measure cytokines or immune cell phenotype in blood at baseline (day 0) and following intervention (day 56). Adequate blood samples were available at enrollment and day 56 and evaluated. Multiple plasma cytokines and chemokines were significantly decreased on day 56 versus baseline. Subgroup analysis indicated reduced TH1 (P = 0.028) and myeloid-derived suppressor cell (MDSC)-associated cytokines (P = 0.035). TH2 and TH17 cytokines were not significantly altered. Phenotypic analysis revealed no change in CD8+ or CD4+ T cells but showed increased CD56+ natural killer (NK) cells (P = 0.038). The percentage of cells with a T regulatory cell phenotype (CD4+CD25+FoxP3+) was significantly decreased after 56 days of soy bread (P = 0.0136). Significantly decreased monocytic (CD33+HLADRnegCD14+) MDSC were observed in patients consuming soy bread (P = 0.0056). These data suggest that soy bread modulates systemic soluble and cellular biomarkers consistent with limiting inflammation and suppression of MDSCs. Additional studies to elucidate impact on the carcinogenic process or as a complement to immune-based therapy are required. Cancer Prev Res; 8(11); 1036–44. ©2015 AACR.

Supplemental Data Table 2A and 2B to describe changes in cytokine and cellular biomarkers between soy and soy-almond bread.

Supplemental Figures S1-S7

Selinexor, a selective inhibitor of nuclear export (SINE) compound targeting exportin-1, has previously been shown to inhibit melanoma cell growth in vivo. We hypothesized that combining selinexor with antibodies that block or disrupt T-cell checkpoint molecule signaling would exert superior antimelanoma activity. In vitro, selinexor increased PDCD1 and CTLA4 gene expression in leukocytes and induced CD274 gene expression in human melanoma cell lines. Mice bearing syngeneic B16F10 melanoma tumors demonstrated a significant reduction in tumor growth rate in response to the combination of selinexor and anti-PD-1 or anti-PD-L1 antibodies (P < 0.05). Similar results were obtained in B16F10-bearing mice treated with selinexor combined with anti-CTLA4 antibody. Immunophenotypic analysis of splenocytes by flow cytometry revealed that selinexor alone or in combination with anti-PD-L1 antibody significantly increased the frequency of both natural killer cells (P ≤ 0.050) and CD4+ T cells with a Th1 phenotype (P ≤ 0.050). Further experiments indicated that the antitumor effect of selinexor in combination with anti-PD-1 therapy persisted under an alternative dosing schedule but was lost when selinexor was administered daily. These data indicate that the efficacy of selinexor against melanoma may be enhanced by disrupting immune checkpoint activity. Mol Cancer Ther; 16(3); 417–27. ©2017 AACR.See related article by Tyler et al., p. 428.

Supplemental Figure S1: Effects of alternative dosing schedules for selinexor (15 mg/kg) and anti-PD-1 on systemic immune populations; Supplemental Figure S2: Effects of alternative dosing schedules for selinexor (10 mg/kg) and anti-PD-1 on systemic immune populations.

Supplementary Material contains Supplementary Figures 1-5 and Supplementary Table 1. Supplementary Figure 1. MEK162 treatment prevents cancer-induced weight loss and muscle wasting. Supplementary Figure 2. Development of a MEK162 resistant C-26 tumor cell line. Supplementary Figure 3. MEK162 prevents cancer-induced weight loss and muscle wasting in part via a tumor extrinsic mechanism. Supplementary Figure 4. MEK162 prevents cancer-induced weight loss and muscle wasting when used in combination with buparlisib. Supplementary Figure 5. MEK162 and buparlisib modulate the immune system. Supplementary Table 1. Primer sequences used for real-time RT-PCR.

Supplemental Methods details the methods for preparation and standardization of study soy breads and the quantification of isoflavonoids in blood samples

Supplemental Figure S5. Supplemental Figure S5. Prostate specific antigen levels pre-study, during study, and post-study. Mean {plus minus} SEM. Inset reports the slope of average prostate specific antigen values.

Mean Differences in Cytokines.

Pancreatic ductal adenocarcinoma (PDAC) has a prominent fibrotic stroma, which is a result of interactions between tumor, immune and pancreatic stellate cells (PSC), or cancer-associated fibroblasts (CAF). Targeting inflammatory pathways present within the stroma may improve access of effector immune cells to PDAC and response to immunotherapy. Heat shock protein-90 (Hsp90) is a chaperone protein and a versatile target in pancreatic cancer. Hsp90 regulates a diverse array of cellular processes of relevance to both the tumor and the immune system. However, to date the role of Hsp90 in PSC/CAF has not been explored in detail. We hypothesized that Hsp90 inhibition would limit inflammatory signals, thereby reprogramming the PDAC tumor microenvironment to enhance sensitivity to PD-1 blockade. Treatment of immortalized and primary patient PSC/CAF with the Hsp90 inhibitor XL888 decreased IL6, a key cytokine that orchestrates immune changes in PDAC at the transcript and protein level in vitro. XL888 directly limited PSC/CAF growth and reduced Jak/STAT and MAPK signaling intermediates and alpha-SMA expression as determined via immunoblot. Combined therapy with XL888 and anti–PD-1 was efficacious in C57BL/6 mice bearing syngeneic subcutaneous (Panc02) or orthotopic (KPC-Luc) tumors. Tumors from mice treated with both XL888 and anti–PD-1 had a significantly increased CD8+ and CD4+ T-cell infiltrate and a unique transcriptional profile characterized by upregulation of genes associated with immune response and chemotaxis. These data demonstrate that Hsp90 inhibition directly affects PSC/CAF in vitro and enhances the efficacy of anti–PD-1 blockade in vivo.

Supplementary Table from Combined MEK/PD-L1 Inhibition Alters Peripheral Cytokines and Lymphocyte Populations Correlating with Improved Clinical Outcomes in Advanced Biliary Tract Cancer

Supplementary Figure from Combined MEK/PD-L1 Inhibition Alters Peripheral Cytokines and Lymphocyte Populations Correlating with Improved Clinical Outcomes in Advanced Biliary Tract Cancer

Purpose:Biliary tract cancers (BTC) are aggressive malignancies refractory to chemotherapy and immunotherapy. MEK inhibition (MEKi)-based regimens may have utility in this disease when combined with PD-L1 blockade. We hypothesize that dual MEK/PD-L1 inhibition alters circulating soluble and cellular immune mediators to improve clinical outcomes in patients with advanced BTC.Experimental Design:We examined immune features in peripheral blood from 77 patients with advanced BTC enrolled in a phase II clinical trial investigating atezolizumab with or without cobimetinib. Plasma and peripheral blood mononuclear cells (PBMC) were isolated from whole blood to evaluate soluble factors and immune cell populations. Baseline blood samples were additionally compared with healthy donors to identify immune signatures unique to BTC.Results:At baseline, the soluble factors platelet-derived growth factor B (PDGF)-BB, placental growth factor (PlGF)-1, IL5, and IL17A were elevated in patients with BTC compared with healthy adult donors, and higher baseline frequencies of CD8+BTLA+ T cells correlated with better overall survival (OS) in this trial. There were also significant treatment-related alterations in several factors, including decreased PDGF-BB following combination treatment, that correlated with improved OS and progression-free survival (PFS). Higher baseline levels of IL23 and RANTES corresponded to improved clinical outcomes following combination treatment. Dual MEK/PD-L1 inhibition increased populations of CD4+TIM3+ and decreased CD8+VISTA+ T cells, correlating with worse OS and better PFS, respectively.Conclusions:This work represents a comprehensive analysis of peripheral immune features in patients with BTC and systemic responses to dual MEK/PD-L1 inhibition. These data support further investigation to understand how MEKi combines with immunotherapeutic approaches to improve clinical outcomes for patients with advanced BTC.

supplemental legend

Purpose: Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer with a 5-year survival rate Experimental Design: Peripheral blood was collected from 73 patients presenting with previously untreated metastatic PDAC. Extensive immunologic profiling was conducted to assess relationships between OS and the level of soluble plasma biomarkers or detailed immune cell phenotypes as measured by flow cytometry.Results: Higher baseline levels of the immunosuppressive cytokines IL6 and IL10 were strongly associated with poorer OS (P = 0.008 and 0.026, respectively; HR = 1.16 and 1.28, respectively), whereas higher levels of the monocyte chemoattractant MCP-1 were associated with significantly longer OS (P = 0.045; HR = 0.69). Patients with a greater proportion of antigen-experienced T cells (CD45RO+) had longer OS (CD4 P = 0.032; CD8 P = 0.036; HR = 0.36 and 0.61, respectively). Although greater expression of the T-cell checkpoint molecule CTLA-4 on CD8+ T cells was associated with significantly shorter OS (P = 0.020; HR = 1.53), the TIM3 molecule had a positive association with survival when expressed on CD4+ T cells (P = 0.046; HR = 0.62).Conclusions: These data support the hypothesis that baseline immune status predicts PDAC disease course and overall patient survival. To our knowledge, this work represents the largest cohort and most comprehensive immune profiling of treatment-naïve metastatic PDAC patients to date. Clin Cancer Res; 22(10); 2565–74. ©2015 AACR.

Supplementary table 1. Selected early phase clinical trials evaluating Reolysin single-agent and combination treatment regimens. Abbreviations: SD = stable disease, PR = partial response, CR = complete response; ER = endoplasmic reticulum.

Supplementary Table 1. List of antibodies used for immunohistochemical staining of mouse pancreas.

The accessibility of adoptive T-cell transfer therapies (ACT) is hindered by the cost and time required for product development. Here we describe a streamlined ACT protocol using Th17 cells expanded only 4 days ex vivo. While shortening expansion compromised cell yield, this method licensed Th17 cells to eradicate large tumors to a greater extent than cells expanded longer term. Day 4 Th17 cells engrafted, induced release of multiple cytokines including IL6, IL17, MCP-1, and GM-CSF in the tumor-bearing host, and persisted as memory cells. IL6 was a critical component for efficacy of these therapies via its promotion of long-term immunity and resistance to tumor relapse. Mechanistically, IL6 diminished engraftment of FoxP3+ donor T cells, corresponding with robust tumor infiltration by donor effector over regulatory cells for the Day 4 Th17 cell product relative to cell products expanded longer durations ex vivo. Collectively, this work describes a method to rapidly generate therapeutic T-cell products for ACT and implicates IL6 in promoting durable immunity of Th17 cells against large, established solid tumors.Significance:An abbreviated, 4-day ex vivo expansion method licenses Th17 cells to confer long-lived immunity against solid malignancies via induction of systemic IL6 in the host.See related commentary by Fiering and Ho, p. 3795

Table S1. Antibodies used for flow cytometry Table S2. Monte Carlo cross-validation of continuous measure markers. Table S3. Monte Carlo cross-validation of dichotomous measure markers. Table S4. Pancreatic cancer patient circulating immune cell frequencies Figure S1. Overall survival does not predict plasma MCP-1 levels

supplemental legend

Lipocalin-2 (LCN2) promotes malignant development in many cancer types. LCN2 is upregulated in patients with pancreatic ductal adenocarcinoma (PDAC) and in obese individuals, but whether it contributes to PDAC development is unclear. In this study, we investigated the effects of Lcn2 depletion on diet-induced obesity, inflammation, and PDAC development. Mice with acinar cell–specific expression of KrasG12D were crossed with Lcn2-depleted animals and fed isocaloric diets with varying amounts of fat content. Pancreas were collected and analyzed for inflammation, pancreatic intraepithelial neoplasia (PanIN), and PDAC. We also used a syngeneic orthotopic PDAC mouse model to study tumor growth in the presence or absence of Lcn2 expression. In addition, to understand the mechanistic role of how LCN2 could be mediating PDAC, we studied LCN2 and its specific receptor solute carrier family 22 member 17 (SLC22A17) in human pancreatic cancer stellate cells (PSC), key mediators of the PDAC stroma. Depletion of Lcn2 diminished extracellular matrix deposition, immune cell infiltration, PanIN formation, and tumor growth. Notably, it also increased survival in both obesity-driven and syngeneic orthotopic PDAC mouse models. LCN2 modulated the secretion of proinflammatory cytokines in PSC of the PDAC tumor microenvironment, whereas downregulation of LCN2-specific receptor SLC22A17 blocked these effects. Our results reveal how LCN2 acts in the tumor microenvironment links obesity, inflammation, and PDAC development. Cancer Res; 77(10); 2647–60. ©2017 AACR

Purpose: IFN-α is administered to melanoma patients and its endogenous production is essential for immune-mediated tumor recognition. We hypothesized that a reduced capacity for signal transducer and activator of transcription (STAT) 1 activation allows melanoma cells to evade the direct actions of IFN-α.Experimental Design: Tyr701-phosphorylated STAT1 (P-STAT1) was measured by flow cytometry in IFN-α–stimulated human melanoma cell lines, melanoma cells derived from patient tumors, and peripheral blood mononuclear cells (PBMC). Expression of other Janus-activated kinase (Jak)-STAT intermediates (STAT1, STAT2, Jak1, tyrosine kinase 2, IFN-α receptor, STAT3, and STAT5) was evaluated by flow cytometry, immunoblot, or immunohistochemistry.Results: Significant variability in P-STAT1 was observed in human melanoma cell lines following IFN-α treatment (P < 0.05) and IFN-α–induced P-STAT1 correlated with the antiproliferative effects of IFN-α (P = 0.042). Reduced formation of P-STAT1 was not explained by loss of Jak-STAT proteins or enhanced STAT5 signaling as reported previously. Basal levels of P-STAT3 were inversely correlated with IFN-α–induced P-STAT1 in cell lines (P = 0.013). IFN-α–induced formation of P-STAT1 was also variable in melanoma cells derived from patient tumors; however, no relationship between P-STAT3 and IFN-α–induced P-STAT1 was evident. Because IFN-α acts on both tumor and immune cells, we examined the ability of IFN-α to induce P-STAT1 in patient-derived melanoma cells and PBMCs. IFN-α induced significantly lower levels of P-STAT1 in melanoma cells compared with matched PBMCs (P = 0.046). Melanoma cells and human melanocytes required 10-fold higher IFN-α doses to exert P-STAT1 levels comparable with PBMCs.Conclusions: Melanoma cells are variable in their IFN-α responsiveness, and cells of the melanocytic lineage exhibit a lower capacity for IFN-α–induced Jak-STAT signaling compared with immune cells.

Supplementary Table S1 from Gene Expression Profiling Reveals Similarities between the In vitro and In vivo Responses of Immune Effector Cells to IFN-α

Purpose: Reolysin, a proprietary isolate of reovirus type III dearing, enters and preferentially induces apoptosis of malignant cells. RAS pathway activation has been associated with more efficient reoviral infectivity and enhanced oncolysis. Reovirus is currently in advanced solid tumor phase I-II trials; no clinical trials have been conducted in patients with hematologic malignancies.Experimental Design: A phase I trial treated 12 relapsed myeloma patients at two dose levels. Reolysin was infused daily for 5 days every 28 days. Bone marrow specimens were examined by in situ-based hybridization (ISH) for CD138, p38, caspase-3, reoviral RNA, and capsid protein at screening and cycle 1 day 8. Junctional adhesion molecule 1 (JAM-1) and cancer upregulated gene 2 (CUG2) were evaluated in patient samples and multiple myeloma cell lines. Neutralizing anti-reovirus antibody assay was performed weekly during cycle 1.Results: There were no dose-limiting toxicities, patients reached the 3 × 1010 TCID50 daily on days 1 to 5 dose level, and grade 3 laboratory toxicities included neutropenia, thrombocytopenia, and hypophosphatemia. ISH demonstrated reoviral genome confined in multiple myeloma cells. Reoviral capsid protein and caspase-3 were rarely identified within reoviral RNA-positive cells. The longest durations of stable disease were 4, 5, and 8 months.Conclusions: Treatment with single-agent Reolysin was well tolerated and associated with avid reoviral RNA myeloma cell entry but only minimal intracellular reoviral protein production within multiple myeloma cells. Our data support that in multiple myeloma cells, Reolysin-induced oncolysis requires combination therapy, similar to other cancers. Clin Cancer Res; 20(23); 5946–55. ©2014 AACR.

Supplementary Table 2. List of human (h) and mouse (m) primer sequences used in study.

Additional Experimental Methods

Supplementary Data from Melanoma Cells Exhibit Variable Signal Transducer and Activator of Transcription 1 Phosphorylation and a Reduced Response to IFN-α Compared with Immune Effector Cells

Purpose: The precise molecular targets of IFN-α therapy in the context of malignant melanoma are unknown but seem to involve signal transducers and activators of transcription 1 signal transduction within host immune effector cells. We hypothesized that the in vitro transcriptional response of patient peripheral blood mononuclear cells (PBMC) to IFN-α would be similar to the in vivo response to treatment with high-dose IFN-α.Experimental Design: The gene expression profiles of PBMCs and immune cell subsets treated in vitro with IFN-α were evaluated, as were PBMCs obtained from melanoma patients receiving adjuvant IFN-α.Results: Twenty-seven genes were up-regulated in PBMCs from normal donors after treatment with IFN-α in vitro for 18 hours (>2-fold, P < 0.001). A subset of these genes (in addition to others) was significantly expressed in IFN-α–treated T cells, natural killer cells, and monocytes. Analysis of gene expression within PBMCs from melanoma patients (n = 13) receiving high-dose IFN-α-2b (20 MU/m2 i.v.) revealed significant up-regulation (>2-fold) of 21 genes (P < 0.001). Also, the gene expression profile of in vitro IFN-α–stimulated patient PBMCs was similar to that of PBMCs obtained from the same patient after IFN-α therapy.Conclusions: This report is the first to describe the transcriptional response of T cells, natural killer cells, and monocytes to IFN-α and characterize the transcriptional profiles of PBMCs from melanoma patients undergoing IFN-α immunotherapy. In addition, it was determined that microarray analysis of patient PBMCs after in vitro stimulation with IFN-α may be a useful predictor of the in vivo response of immune cells to IFN-α immunotherapy.

Supplementary table 2. Five patients experienced at least grade 2 adverse events. Two of the 5 patients received at least 4 cycles of treatment, and the associated adverse events were noted after cycle 1. All cytopenias resolved prior to the completion of the cycle that adverse event occurred. Abbreviations: DL = dose level, m-protein = monoclonal protein, CXDX = cycle X, day X, NA = not available.

Supplementary figure 1. The top ten worst adverse events include grade 1 - 3 events. Grade 1 events included decreased neutrophil, white blood cell, lymphocyte, and platelet counts, increased aspartate aminotransferase and alkaline phosphatase, and nausea. Grade 2 events included decreased neutrophil, white blood cell, and platelet counts, anemia, and myalgia. Grade 3 events included decreased neutrophil, white blood cell, and platelet counts, and hypophosphatemia.

Supplementary Figure 2 from Myeloid-Derived Suppressor Cell Inhibition of the IFN Response in Tumor-Bearing Mice