CD200 is overexpressed in the pancreatic tumor microenvironment and predictive of overall survival.

Pancreatic cancer is an aggressive disease with a 5 year survival rate of 13%. This poor survival is attributed, in part, to limited and ineffective treatments for patients with metastatic disease, highlighting a need to identify molecular drivers of pancreatic cancer to target for more effective treatment. CD200 is a glycoprotein that interacts with the receptor CD200R and elicits an immunosuppressive response. Overexpression of CD200 has been associated with differential outcomes, depending on the tumor type. In the context of pancreatic cancer, we have previously reported that CD200 is expressed in the pancreatic tumor microenvironment (TME), and that targeting CD200 in murine tumor models reduces tumor burden. We hypothesized that CD200 is overexpressed on tumor and stromal populations in the pancreatic TME and that circulating levels of soluble CD200 (sCD200) have prognostic value for overall survival. We discovered that CD200 was overexpressed on immune, stromal, and tumor populations in the pancreatic TME. Particularly, single-cell RNA-sequencing indicated that CD200 was upregulated on inflammatory cancer-associated fibroblasts. Cytometry by time of flight analysis of PBMCs indicated that CD200 was overexpressed on innate immune populations, including monocytes, dendritic cells, and monocytic myeloid-derived suppressor cells. High sCD200 levels in plasma correlated with significantly worse overall and progression-free survival. Additionally, sCD200 correlated with the ratio of circulating matrix metalloproteinase (MMP) 3: tissue inhibitor of metalloproteinase (TIMP) 3 and MMP11/TIMP3. This study highlights the importance of CD200 expression in pancreatic cancer and provides the rationale for designing novel therapeutic strategies that target this protein.

Decoupling FcRn and tumor contributions to elevated immune checkpoint inhibitor clearance in cancer cachexia.

High baseline clearance of immune checkpoint inhibitors (ICIs), independent of dose or systemic exposure, is associated with cachexia and poor outcomes in cancer patients. Mechanisms linking ICI clearance, cachexia and ICI therapy failure are unknown. Here, we evaluate in four murine models and across multiple antibodies whether altered baseline catabolic clearance of administered antibody requires a tumor and/or cachexia and whether medical reversal of cachexia phenotype can alleviate altered clearance. Key findings include mild cachexia phenotype and lack of elevated pembrolizumab clearance in the MC38 tumor-bearing model. We also observed severe cachexia and decreased, instead of increased, baseline pembrolizumab clearance in the tumor-free cisplatin-induced cachexia model. Liver Fcgrt expression correlated with altered baseline catabolic clearance, though elevated clearance was still observed with antibodies having no (human IgA) or reduced (human H310Q IgG1) FcRn binding. We conclude cachexia phenotype coincides with altered antibody clearance, though tumor presence is neither sufficient nor necessary for altered clearance in immunocompetent mice. Magnitude and direction of clearance alteration correlated with hepatic Fcgrt, suggesting changes in FcRn expression and/or recycling function may be partially responsible, though factors beyond FcRn also contribute to altered clearance in cachexia.

BTK inhibition potentiates anti-PD-L1 treatment in murine melanoma: potential role for MDSC modulation in immunotherapy.

Myeloid-derived suppressor cells (MDSC) have been linked to loss of immune effector cell function through a variety of mechanisms such as the generation of reactive oxygen and nitrogen species and the production of inhibitory cytokines. Our group has shown that signaling through Bruton's tyrosine kinase (BTK) is important for MDSC function. Ibrutinib is an orally administered targeted agent that inhibits BTK activation and is currently used for the treatment of B cell malignancies. Using a syngeneic murine model of melanoma, the effect of BTK inhibition with ibrutinib on the therapeutic response to systemic PD-L1 blockade was studied. BTK was expressed by murine MDSC and their activation was inhibited by ibrutinib. Ibrutinib was not directly cytotoxic to cancer cells in vitro, but it inhibited BTK activation in MDSC and reduced expression of inducible nitric oxide synthase (NOS2) and production of nitric oxide. Ibrutinib treatments decreased the levels of circulating MDSC in vivo and increased the therapeutic efficacy of anti-PD-L1 antibody treatment. Gene expression profiling showed that ibrutinib decreased Cybb (NOX2) signaling, and increased IL-17 signaling (upregulating downstream targets Mmp9, Ptgs2, and S100a8). These results suggest that further exploration of MDSC inhibition could enhance the immunotherapy of advanced melanoma.PrécisInhibition of Bruton's tyrosine kinase, a key enzyme in myeloid cellular function, improves therapeutic response to an anti-PD-L1 antibody in an otherwise fairly resistant murine melanoma model.

The phase 1/2 trial of indomethacin in chronic pancreatitis (The PAIR trial): Protocol for a parallel multi-center randomized controlled trial.

BACKGROUND/OBJECTIVES: 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 E2 (PGE2), a key component in the inflammatory pathway of chronic pancreatitis, this study evaluates the physiologic effect of oral indomethacin, a COX-2 inhibitor, on PGE2 levels in pancreatic fluid. METHODS: This 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 PGE2 levels. The relationship between pancreatic fluid PGE2 levels with blood and saliva PGE2 levels will be examined. CONCLUSIONS: This study will elucidate the effect of oral indomethacin on PGE2 levels in the pancreas to assess its role in the inflammatory pathway of chronic pancreatitis. Should indomethacin significantly reduce PGE2 levels, this may represent a potential disease-altering treatment for chronic pancreatitis.

The Neonatal Fc Receptor Is Elevated in Monocyte-Derived Immune Cells in Pancreatic Cancer.

The neonatal Fc receptor (FcRn) is responsible for recycling of IgG antibodies and albumin throughout the body. This mechanism has been exploited for pharmaceutic delivery across an array of diseases to either enhance or diminish this function. Monoclonal antibodies and albumin-bound nanoparticles are examples of FcRn-dependent anti-cancer therapeutics. Despite its importance in drug delivery, little is known about FcRn expression in circulating immune cells. Through time-of-flight mass cytometry (CyTOF) we were able to characterize FcRn expression in peripheral blood mononuclear cell (PBMC) populations of pancreatic ductal adenocarcinoma (PDAC) patients and non-cancer donors. Furthermore, we were able to replicate these findings in an orthotopic murine model of PDAC. Altogether, we found that in both patients and mice with PDAC, FcRn was elevated in migratory and resident classical dendritic cell type 2 (cDC2) as well as monocytic and granulocytic myeloid-derived suppressor cell (MDSC) populations compared to tumor-free controls. Furthermore, PBMCs from PDAC patients had elevated monocyte, dendritic cells and MDSCs relative to non-cancer donor PBMCs. Future investigations into FcRn activity may further elucidate possible mechanisms of poor efficacy of antibody immunotherapies in patients with PDAC.

A multi-center, single-arm, phase Ib study of pembrolizumab (MK-3475) in combination with chemotherapy for patients with advanced colorectal cancer: HCRN GI14-186.

Modified FOLFOX6 is an established therapy for patients with metastatic colorectal cancer (mCRC). We conducted a single-arm phase Ib study to address the hypothesis that addition of pembrolizumab to this regimen could safely and effectively improve patient outcomes (NCT02375672). The relationship between immune biomarkers and clinical response were assessed in an exploratory manner. Patients with mCRC received concurrent pembrolizumab and modified FOLFOX6. The study included safety run-in for the first six patients. The primary objective was median progression-free survival (mPFS), with secondary objectives including median overall survival, safety, and exploratory assessment of immune changes. To assess immunological impact, peripheral blood was collected at baseline and during treatment. The levels of soluble factors were measured via bioplex, while a panel of checkpoint molecules and phenotypically defined cell populations were assessed with flow cytometry and correlated with RECIST and mPFS. Due to incidences of grade 3 and grade 4 neutropenia in the safety lead-in, the dose of mFOLFOX6 was reduced in the expansion cohort. Median PFS was 8.8 months and median OS was not reached at data cutoff. Best responses of stable disease, partial response, and complete response were observed in 43.3%, 50.0%, and 6.7% of patients, respectively. Several soluble and cellular immune biomarkers were associated with improved RECIST and mPFS. Immunosuppressive myeloid and T cell subsets that were analyzed were not associated with response. Primary endpoint was not superior to historic control. Biomarkers that were associated with improved response may be informative for future regimens combining chemotherapy with immune checkpoint inhibitors.

Suppressive myeloid cells are expanded by biliary tract cancer-derived cytokines in vitro and associate with aggressive disease.

BACKGROUND: BTC is an aggressive disease exacerbated by inflammation and immune suppression. Expansion of immunosuppressive cells occurs in biliary tract cancer (BTC), yet the role of BTC-derived cytokines in this process is unclear. METHODS: Activated signalling pathways and cytokine production were evaluated in a panel of human BTC cell lines. Human peripheral blood mononuclear cells (PBMCs) were cultured with BTC supernatants, with and without cytokine neutralising antibodies, and analysed by flow cytometry or immunoblot. A human BTC tissue microarray (TMA, n = 69) was stained for IL-6, GM-CSF, and CD33+S100a9+ cells and correlated with clinical outcomes. RESULTS: Immunomodulatory factors (IL-6, GM-CSF, MCP-1) were present in BTC supernatants. BTC supernatants expanded CD33dimCD11b+HLA-DRlow/- myeloid-derived suppressor cells (MDSCs) from human PBMCs. Neutralisation of IL-6 and GM-CSF in BTC supernatants inhibited activation of STAT3/5, respectively, in PBMCs, with heterogeneous effects on MDSC expansion in vitro. Staining of a BTC TMA revealed a positive correlation between IL-6 and GM-CSF, with each cytokine and more CD33+S100a9+ cells. Increased CD33+S100a9+ staining positively correlated with higher tumour grade, differentiation and the presence of satellite lesions. CONCLUSION: BTC-derived factors promote suppressive myeloid cell expansion, and higher numbers of CD33+S100a9+ cells in resectable BTC tumours correlates with more aggressive disease.

Exploratory analysis of immune checkpoint receptor expression by circulating T cells and tumor specimens in patients receiving neo-adjuvant chemotherapy for operable breast cancer.

BACKGROUND: While combinations of immune checkpoint (ICP) inhibitors and neo-adjuvant chemotherapy (NAC) have begun testing in patients with breast cancer (BC), the effects of chemotherapy on ICP expression in circulating T cells and within the tumor microenvironment are still unclear. This information could help with the design of future clinical trials by permitting the selection of the most appropriate ICP inhibitors for incorporation into NAC. METHODS: Peripheral blood samples and/or tumor specimens before and after NAC were obtained from 24 women with operable BC. The expression of CTLA4, PD-1, Lag3, OX40, and Tim3 on circulating T lymphocytes before and at the end of NAC were measured using flow cytometry. Furthermore, using multi-color immunohistochemistry (IHC), the expression of immune checkpoint molecules by stromal tumor-infiltrating lymphocytes (TILs), CD8+ T cells, and tumor cells was determined before and after NAC. Differences in the percentage of CD4+ and CD8+ T cells expressing various checkpoint receptors were determined by a paired Student's t-test. RESULTS: This analysis showed decreased ICP expression by circulating CD4+ T cells after NAC, including significant decreases in CTLA4, Lag3, OX40, and PD-1 (all p values < 0.01). In comparison, circulating CD8+ T cells showed a significant increase in CTLA4, Lag3, and OX40 (all p values < 0.01). Within tumor samples, TILs, CD8+ T cells, and PD-L1/PD-1 expression decreased after NAC. Additionally, fewer tumor specimens were considered to be PD-L1/PD-1 positive post-NAC as compared to pre-NAC biopsy samples using a cutoff of 1% expression. CONCLUSIONS: This work revealed that NAC treatment can substantially downregulate CD4+ and upregulate CD8+ T cell ICP expression as well as deplete the amount of TILs and CD8+ T cells found in breast tumor samples. These findings provide a starting point to study the biological significance of these changes in BC patients. TRIAL REGISTRATION: NCT04022616.

Lipocalin-2 expression and function in pancreatic diseases.

Lipocalin-2 (LCN2) is a secreted molecule, expressed in various cell types, that is involved in the progression of numerous diseases and disorders. The biological functions and expression levels of LCN2 in diseases including pancreatic cancer, pancreatitis (acute and chronic), and diabetes mellitus, suggest the potential role of LCN2 as a biomarker and/or therapeutic target. However, findings on the role of LCN2 in pancreatic diseases have been contradictory. In pancreatic cancer and pancreatitis, LCN2 has been identified as a potential biomarker; increased expression levels in various biological specimens correlate with the presence of the disease and may be able to differentiate cancer and chronic pancreatitis from healthy subjects. LCN2 is also known to be an adipokine; it is upregulated in obesity and is a common co-factor in the development of pancreatic diseases. Emerging research suggests LCN2 is elevated in type 2 diabetes mellitus, but the exact role of LCN2 in this disease is not clear. In this review, we summarize research on LCN2 as it relates to pancreatic diseases, highlighting the discrepancies in the literature. By explaining and clarifying the role of LCN2 in these disorders, we aim to promote research in developing novel diagnostic and treatment strategies to reduce the burden of pancreatic diseases.

Reduction of inflammation in chronic pancreatitis using a soy bread intervention: A feasibility study.

INTRODUCTION: Chronic pancreatitis is a chronic inflammatory disease, which progresses to fibrosis. Currently there are no interventions to delay or stop the progression to irreversible organ damage. In this study, we assessed the tolerability and feasibility of administering soy bread to reduce circulating inflammatory mediators. METHODS: Subjects with chronic pancreatitis diagnosed using the American Pancreatic Association diagnostic guidelines were enrolled. During the dose escalation (DE) phase, subjects received one week of soy bread based using a 3 + 3 dose-escalation design, which was then followed by a maximally tolerated dose (MTD) phase with four weeks of intervention. Dose-limiting toxicities (DLTs) were monitored. Plasma cytokine levels were measured using a Meso Scale Discovery multiplex assay kit. Isoflavonoid excretion in 24-h urine collection was used to measure soy bread compliance. RESULTS: Nine subjects completed the DE phase, and one subject completed the MTD phase without any DLTs at a maximum dosage of three slices (99 mg of isoflavones) per day. Reported compliance to soy bread intervention was 98%, and this was confirmed with urinary isoflavones and their metabolites detected in all subjects. There was a significant decline in the TNF-α level during the DE phase (2.667 vs 2.382 pg/mL, p = 0.039); other levels were similar. CONCLUSIONS: In this feasibility study, there was excellent compliance with a short-term intervention using soy bread in chronic pancreatitis. Reduction was seen in at least one pro-inflammatory cytokine with short-term intervention. Larger cohorts and longer interventions with soy are warranted to assess the efficacy of reducing pro-inflammatory mediators of disease.

An IL-15-based superagonist ALT-803 enhances the NK cell response to cetuximab-treated squamous cell carcinoma of the head and neck.

Squamous cell carcinoma of the head and neck (SCCHN) is the sixth most common cancer worldwide and epidermal growth factor receptor (EGFR) is overexpressed in greater than 90% of patient tumors. Cetuximab is a monoclonal antibody that binds to EGFR and can activate immune cells, such as natural killer (NK) cells, that express receptors for the Fc (constant region) of immunoglobulin G. IL-15 (interleukin-15) is a critical factor for the development, proliferation and activation of effector NK cells. A novel IL-15 compound known as ALT-803 that consists of genetically modified IL-15 plus the IL-15 receptor alpha protein (IL15Rα) fused to the Fc portion of IgG1 has recently been developed. We hypothesized that treatment with ALT-803 would increase NK cell-mediated cytotoxicity of cetuximab-coated head and neck squamous cells. CD56+ NK cells from normal healthy donors were treated overnight with ALT-803 and tested for their ability to lyse cetuximab-coated tumor cells. Cytotoxicity was greater following NK cell ALT-803 activation, as compared to controls. ALT-803-treated NK cells secreted significantly higher levels of IFN-γ than control conditions. Additionally, NK cells showed increased levels of phospho-ERK and phospho-STAT5 when co-cultured with cetuximab-coated tumors and ALT-803. Administration of both cetuximab and ALT-803 to mice harboring Cal27 SCCHN tumors resulted in significantly decreased tumor volume when compared to controls and compared to single-agent treatment alone. Overall, the present data suggest that cetuximab treatment in combination with ALT-803 in patients with EGFR-positive SCCHN may result in significant NK cell activation and have important anti-tumor activity.

Circulating interleukin-6 is associated with disease progression, but not cachexia in pancreatic cancer.

BACKGROUND: Cachexia is a wasting syndrome characterized by involuntary loss of >5% body weight due to depletion of adipose and skeletal muscle mass. In cancer, the pro-inflammatory cytokine interleukin-6 (IL-6) is considered a mediator of cachexia and a potential biomarker, but the relationship between IL-6, weight loss, and cancer stage is unknown. In this study we sought to evaluate IL-6 as a biomarker of cancer cachexia while accounting for disease progression. METHODS: We retrospectively studied 136 subjects with biopsy-proven pancreatic ductal adenocarcinoma (PDAC), considering the high prevalence of cachexia is this population. Clinical data were abstracted from subjects in all cancer stages, and plasma IL-6 levels were measured using a multiplex array and a more sensitive ELISA. Data were evaluated with univariate comparisons, including Kaplan-Meier survival curves, and multivariate Cox survival models. RESULTS: On multiplex, a total of 43 (31.4%) subjects had detectable levels of plasma IL-6, while by ELISA all subjects had detectable IL-6 levels. We found that increased plasma IL-6 levels, defined as detectable for multiplex and greater than median for ELISA, were not associated with weight loss at diagnosis, but rather with the presence of metastasis (p < 0.001 for multiplex and p = 0.007 for ELISA). Further, while >5% weight loss was not associated with worse survival, increased plasma IL-6 by either methodology was. CONCLUSION: Circulating IL-6 levels do not correlate with cachexia (when defined by weight loss), but rather with advanced cancer stage. This suggests that IL-6 may mediate wasting, but should not be considered a diagnostic biomarker for PDAC-induced cachexia.

IL-6 and PD-L1 antibody blockade combination therapy reduces tumour progression in murine models of pancreatic cancer.

OBJECTIVE: Limited efficacy of immune checkpoint inhibitors in pancreatic ductal adenocarcinoma (PDAC) has prompted investigation into combination therapy. We hypothesised that interleukin 6 (IL-6) blockade would modulate immunological features of PDAC and enhance the efficacy of anti-programmed death-1-ligand 1 (PD-L1) checkpoint inhibitor therapy. DESIGN: Transcription profiles and IL-6 secretion from primary patient-derived pancreatic stellate cells (PSCs) were analyzed via Nanostring and immunohistochemistry, respectively. In vivo efficacy and mechanistic studies were conducted with antibodies (Abs) targeting IL-6, PD-L1, CD4 or CD8 in subcutaneous or orthotopic models using Panc02, MT5 or KPC-luc cell lines; and the aggressive, genetically engineered PDAC model (KrasLSL-G12D, Trp53LSL-R270H, Pdx1-cre, Brca2F/F (KPC-Brca2 mice)). Systemic and local changes in immunophenotype were measured by flow cytometry or immunohistochemical analysis. RESULTS: PSCs (n=12) demonstrated prominent IL-6 expression, which was localised to stroma of tumours. Combined IL-6 and PD-L1 blockade elicited efficacy in mice bearing subcutaneous MT5 (p<0.02) and Panc02 tumours (p=0.046), which was accompanied by increased intratumoural effector T lymphocytes (CD62L-CD44-). CD8-depleting but not CD4-depleting Abs abrogated the efficacy of combined IL-6 and PD-L1 blockade in mice bearing Panc02 tumours (p=0.0016). This treatment combination also elicited significant antitumour activity in mice bearing orthotopic KPC-luc tumours and limited tumour progression in KPC-Brca2 mice (p<0.001). Histological analysis revealed increased T-cell infiltration and reduced α-smooth muscle actin cells in tumours from multiple models. Finally, IL-6 and PD-L1 blockade increased overall survival in KPC-Brca2 mice compared with isotype controls (p=0.0012). CONCLUSIONS: These preclinical results indicate that targeted inhibition of IL-6 may enhance the efficacy of anti-PD-L1 in PDAC.

Circulating myeloid-derived suppressor cells increase in patients undergoing neo-adjuvant chemotherapy for breast cancer.

This study sought to evaluate whether myeloid-derived suppressor cells (MDSC) could be affected by chemotherapy and correlate with pathologic complete response (pCR) in breast cancer patients receiving neo-adjuvant chemotherapy. Peripheral blood levels of granulocytic (G-MDSC) and monocytic (M-MDSC) MDSC were measured by flow cytometry prior to cycle 1 and 2 of doxorubicin and cyclophosphamide and 1st and last administration of paclitaxel or paclitaxel/anti-HER2 therapy. Of 24 patients, 11, 6 and 7 patients were triple negative, HER2+ and hormone receptor+, respectively. 45.8% had pCR. Mean M-MDSC% were <1. Mean G-MDSC% and 95% confidence intervals were 0.88 (0.23-1.54), 5.07 (2.45-7.69), 9.32 (4.02-14.61) and 1.97 (0.53-3.41) at draws 1-4. The increase in G-MDSC by draw 3 was significant (p < 0.0001) in all breast cancer types. G-MDSC levels at the last draw were numerically lower in patients with pCR (1.15; 95% CI 0.14-2.16) versus patients with no pCR (2.71; 95% CI 0-5.47). There was no significant rise in G-MDSC from draw 1 to 3 in African American patients, and at draw 3 G-MDSC levels were significantly lower in African Americans versus Caucasians (p < 0.05). It was concluded that G-MDSC% increased during doxorubicin and cyclophosphamide therapy, but did not significantly differ between patients based on pathologic complete response.

The Raf Kinase Inhibitor Sorafenib Inhibits JAK-STAT Signal Transduction in Human Immune Cells.

Sorafenib is an oral multikinase inhibitor that was originally developed as a Raf kinase inhibitor. We hypothesized that sorafenib would also have inhibitory effects on cytokine signaling pathways in immune cells. PBMCs from normal donors were treated with varying concentrations of sorafenib and stimulated with IFN-α or IL-2. Phosphorylation of STAT1 and STAT5 was measured by flow cytometry and confirmed by immunoblot analysis. Changes in IFN-α- and IL-2-stimulated gene expression were measured by quantitative PCR, and changes in cytokine production were evaluated by ELISA. Cryopreserved PBMCs were obtained from cancer patients before and after receiving 400 mg sorafenib twice daily. Patient PBMCs were thawed, stimulated with IL-2 or IFN-α, and evaluated for phosphorylation of STAT1 and STAT5. Pretreatment of PBMCs with 10 µM sorafenib decreased STAT1 and STAT5 phosphorylation after treatment with IFN-α or IL-2. This inhibitory effect was observed in PBMCs from healthy donors over a range of concentrations of sorafenib (5-20 µM), IL-2 (2-24 nM), and IFN-α (10(1)-10(6) U/ml). This effect was observed in immune cell subsets, including T cells, B cells, NK cells, regulatory T cells, and myeloid-derived suppressor cells. Pretreatment with sorafenib also inhibited PBMC expression of IFN-α- and IL-2-regulated genes and inhibited NK cell production of IFN-γ, RANTES, MIP1-α, and MIG in response to IFN-α stimulation. PBMCs from patients receiving sorafenib therapy showed decreased responsiveness to IL-2 and IFN-α treatment. Sorafenib is a Raf kinase inhibitor that could have off-target effects on cytokine-induced signal transduction in immune effector cells.

Randomized Phase 2 Trial of the Oncolytic Virus Pelareorep (Reolysin) in Upfront Treatment of Metastatic Pancreatic Adenocarcinoma.

Pelareorep causes oncolysis in tumor cells with activated Ras. We hypothesized that pelareorep would have efficacy and immunomodulatory activity in metastatic pancreatic adenocarcinoma (MPA) when combined with carboplatin and paclitaxel. A randomized phase 2 study (NCT01280058) was conducted in treatment-naive patients with MPA randomized to two treatment arms: paclitaxel/carboplatin + pelareorep (Arm A, n = 36 evaluable patients) versus paclitaxel/carboplatin (Arm B, n = 37 evaluable patients). There was no difference in progression-free survival (PFS) between the arms (Arm A PFS = 4.9 months, Arm B PFS = 5.2 months, P = 0.6), and Kirsten rat sarcoma viral oncogene (KRAS) status did not impact outcome. Quality-adjusted Time without Symptoms or Toxicity analysis revealed that the majority of PFS time was without toxicity or progression (4.3 months). Patient immunophenotype appeared important, as soluble immune biomarkers were associated with treatment outcome (fractalkine, interleukin (IL)-6, IL-8, regulated on activation, normal T cell expressed and secreted (RANTES), and vascular endothelial growth factor (VEGF)). Increased circulating T and natural killer (NK)-cell subsets were also significantly associated with treatment outcome. Addition of pelareorep was associated with higher levels of 14 proinflammatory plasma cytokines/chemokines and cells with an immunosuppressive phenotype (Tregs, cytotoxic T lymphocyte associated protein 4 (CTLA4)(+) T cells). Overall, pelareorep was safe but does not improve PFS when administered with carboplatin/paclitaxel, regardless of KRAS mutational status. Immunologic studies suggest that chemotherapy backbone improves immune reconstitution and that targeting remaining immunosuppressive mediators may improve oncolytic virotherapy.

Bioactive compounds or metabolites from black raspberries modulate T lymphocyte proliferation, myeloid cell differentiation and Jak/STAT signaling.

Bioactive phytochemicals from natural products, such as black raspberries (BRB; Rubus occidentalis), have direct anticancer properties on malignant cells in culture and in xenograft models. BRB components inhibit cancer progression in more complex rodent carcinogenesis models. Although mechanistic targets for BRB phytochemicals in cancer cells are beginning to emerge, the potential role in modulating host immune processes impacting cancer have not been systematically examined. We hypothesized that BRB contain compounds capable of eliciting potent immunomodulatory properties that impact cellular mediators relevant to chronic inflammation and tumor progression. We studied both an ethanol extract from black raspberries (BRB-E) containing a diverse mixture of phytochemicals and two abundant phytochemical metabolites of BRB produced upon ingestion (Cyanidin-3-Rutinoside, C3R; Quercitin-3-Rutinoside, Q3R). BRB-E inhibited proliferation, and viability of CD3/CD28 activated human CD4(+) and CD8(+) T lymphocytes. BRB-E also limited in vitro expansion of myeloid-derived suppressor cells (MDSC) and their suppressive capacity. Pre-treatment of immune cells with BRB-E attenuated IL-6-mediated phosphorylation of signal transducer and activator of transcription-3 (STAT3) and IL-2-induced STAT5 phosphorylation. In contrast, pre-treatment of immune cells with the C3R and Q3R metabolites inhibited MDSC expansion, IL-6-mediated STAT3 signaling, but not IL-2-induced STAT5 phosphorylation and were less potent inhibitors of T cell viability. Together these data indicate that BRB extracts and their physiologically relevant metabolites contain phytochemicals that affect immune processes relevant to carcinogenesis and immunotherapy. Furthermore, specific BRB components and their metabolites may be a source of lead compounds for drug development that exhibits targeted immunological outcomes or inhibition of specific STAT-regulated signaling pathways.

Elevating body temperature enhances hematopoiesis and neutrophil recovery after total body irradiation in an IL-1-, IL-17-, and G-CSF-dependent manner.

Neutropenia is a common side effect of cytotoxic chemotherapy and radiation, increasing the risk of infection in these patients. Here we examined the impact of body temperature on neutrophil recovery in the blood and bone marrow after total body irradiation (TBI). Mice were exposed to either 3 or 6 Gy TBI followed by a mild heat treatment that temporarily raised core body temperature to approximately 39.5°C. Neutrophil recovery was then compared with control mice that received either TBI alone heat treatment alone. Mice that received both TBI and heat treatment exhibited a significant increase in the rate of neutrophil recovery in the blood and an increase in the number of marrow hematopoietic stem cells and neutrophil progenitors compared with that seen in mice that received either TBI or heat alone. The combination treatment also increased G-CSF concentrations in the serum, bone marrow, and intestinal tissue and IL-17, IL-1ß, and IL-1α concentrations in the intestinal tissue after TBI. Neutralizing G-CSF or inhibiting IL-17 or IL-1 signaling significantly blocked the thermally mediated increase in neutrophil numbers. These findings suggest that a physiologically relevant increase in body temperature can accelerate recovery from neutropenia after TBI through a G-CSF-, IL-17-, and IL-1-dependent mechanism.

Antibody Drug Clearance: An Underexplored Marker of Outcomes with Checkpoint Inhibitors.

Immune-checkpoint inhibitor (ICI) therapy has dramatically changed the clinical landscape for several cancers, and ICI use continues to expand across many cancer types. Low baseline clearance (CL) and/or a large reduction of CL during treatment correlates with better clinical response and longer survival. Similar phenomena have also been reported with other monoclonal antibodies (mAb) in cancer and other diseases, highlighting a characteristic of mAb clinical pharmacology that is potentially shared among various mAbs and diseases. Though tempting to attribute poor outcomes to low drug exposure and arguably low target engagement due to high CL, such speculation is not supported by the relatively flat exposure-response relationship of most ICIs, where a higher dose or exposure is not likely to provide additional benefit. Instead, an elevated and/or increasing CL could be a surrogate marker of the inherent resistant phenotype that cannot be reversed by maximizing drug exposure. The mechanisms connecting ICI clearance, therapeutic efficacy, and resistance are unclear and likely to be multifactorial. Therefore, to explore the potential of ICI CL as an early marker for efficacy, this review highlights the similarities and differences of CL characteristics and CL-response relationships for all FDA-approved ICIs, and we compare and contrast these to selected non-ICI mAbs. We also discuss underlying mechanisms that potentially link mAb CL with efficacy and highlight existing knowledge gaps and future directions where more clinical and preclinical investigations are warranted to clearly understand the value of baseline and/or time-varying CL in predicting response to ICI-based therapeutics.

Physical Activity Decreases Inflammation and Delays the Development of Obesity-Associated Pancreatic Ductal Adenocarcinoma.

Obesity is a risk factor for pancreatic ductal adenocarcinoma (PDAC), a deadly disease with limited preventive strategies. Lifestyle interventions to decrease obesity represent a potential approach to prevent obesity-associated PDAC. In this study, we examined whether decreasing obesity through physical activity (PA) and/or dietary changes could decrease inflammation in humans and prevent obesity-associated PDAC in mice. Comparison of circulating inflammatory-associated cytokines in subjects (overweight and obese) before and after a PA intervention revealed PA lowered systemic inflammatory cytokines. Mice with pancreatic-specific inducible KrasG12D expression were exposed to PA and/or dietary interventions during and after obesity-associated cancer initiation. In mice with concurrent diet-induced obesity and KrasG12D expression, the PA intervention led to lower weight gain, suppressed systemic inflammation, delayed tumor progression, and decreased proinflammatory signals in the adipose tissue. However, these benefits were not as evident when obesity preceded pancreatic KrasG12D expression. Combining PA with diet-induced weight loss (DI-WL) delayed obesity-associated PDAC progression in the genetically engineered mouse model, but neither PA alone nor combined with DI-WL or chemotherapy prevented PDAC tumor growth in orthotopic PDAC models regardless of obesity status. PA led to the upregulation of Il15ra in adipose tissue. Adipose-specific overexpression of Il15 slowed PDAC growth but only in nonobese mice. Overall, our study suggests that PA alone or combined with DI-WL can reduce inflammation and delay obesity-associated PDAC development or progression. Lifestyle interventions that prevent or manage obesity or therapies that target weight loss-related molecular pathways could prevent progression of PDAC. Significance: Physical activity reduces inflammation and induces changes to adipose-related signaling to suppress pancreatic cancer, supporting the potential of obesity management strategies to reduce the risk of developing pancreatic cancer. See related commentary by Sogunro and Muzumdar, p. 2935.