Safety and Efficacy of Avelumab in Small Bowel Adenocarcinoma.

INTRODUCTION: Small bowel adenocarcinomas (SBAs) are rare and frequently treated like large intestinal adenocarcinomas. However, SBAs have a very different microenvironment and could respond differently to the same therapies. Our previous data suggested that SBAs might benefit from targeting the PD-1/PD-L1 axis based on PD-L1 staining in almost 50% of SBA tissue samples tested. Thus, we designed a phase 2 study to explore safety and efficacy of avelumab in SBA. PATIENTS AND METHODS: Patients with advanced or metastatic disease were enrolled; ampullary tumors were considered part of the duodenum and allowed. Prior PD-1/PD-L1 inhibition was not allowed. Avelumab (10 mg/kg) was given every 2 weeks, and imaging was performed every 8 weeks. Primary endpoint was response rate. RESULTS: Eight patients (n = 5, small intestine; n = 3, ampullary) were enrolled, with a majority (88%) being male and a median age of 61 years. Of 7 efficacy-evaluable patients, 2 (29%) experienced partial responses; stable disease occurred in 3 additional patients (71%). Median progression-free survival was 3.35 months. Most frequent, related toxicities were anemia, fatigue, and infusion-related reaction (25% each), mostly grade ≤2; grade 3 hypokalemia and hyponatremia occurred in one patient, and another reported grade 4 diabetic ketoacidosis. CONCLUSIONS: Despite the observed benefit, accrual was slower than expected and the study was closed early due to feasibility. A general clinic observation was that patients were receiving immunotherapy off-label as the availability of these agents increased. Off-label availability and disease rarity were likely drivers of insufficient accrual.

High Sensitivity Troponin T and NT-proBNP in Patients Receiving Chimeric Antigen Receptor (CAR) T-Cell Therapy.

Retrospective studies suggest that chimeric antigen receptor T-cell (CAR T) therapy may lead to cardiac injury, but this has not been assessed systematically or prospectively. In this prospective study of 40 patients who received CAR T, we systematically measured high-sensitivity troponin T (hsTropT) and N-terminal pro-B natriuretic peptide (NTproBNP) at baseline and on day 1, days 7, and 21 after CAR T. Biomarker elevations with respect to timepoint and cytokine release syndrome (CRS) status were examined using repeated measure analysis of variance. hsTropT did not differ with time or with the presence of grade 2 CRS. Median hsTropT was 12.1 ng/L [interquartile range (IQR): 9.2, 20.1] at baseline, 13.1 ng/L (IQR: 9.6, 24.2) at day 1, 11.9 ng/L (IQR: 9.6, 18.0) at day 7, and 15.3 ng/L (10.8, 20.2) at day 21. In contrast, NTproBNP rose on day 1 (P Wilcox = 0.0002) and day 7 (P Wilcox = 2.7 × 10-5), and the degree of elevation differed by the presence of grade 2 CRS (P interaction = 0.002). Median NTproBNP was 179 pg/mL (IQR: 116, 325) at baseline, 357 pg/mL (IQR: 98, 813) at day 1, 420 pg/mL (IQR: 239, 1242) at day 7, and 177 pg/mL (IQR: 80, 278) at day 21. In conclusion, hsTropT l did not differ across timepoints after CAR T therapy, but NTproBNP rose at day 7, the prognostic implications of which should be the target of future research, as the indications for this therapy expand.

Cyclooxygenase inhibition during allergic sensitization increases STAT6-independent primary and memory Th2 responses.

Immune sensitization and memory generation are required for the development of allergic inflammation. Our previous studies demonstrate that the cyclooxygenase (COX) metabolic pathway is actively involved in allergic responses and COX inhibition increases allergic airway inflammation in a STAT6-independent fashion. To test the hypothesis that COX inhibition augments allergic inflammation by enhancing immune sensitization and memory, we sensitized STAT6 knockout mice with an i.p. injection of OVA with aluminum hydroxide as an adjuvant and treated the mice with the COX inhibitor indomethacin or vehicle for analyses of the primary and memory immune responses. We found that COX inhibition during immune sensitization, but not the allergic challenge phase, was necessary and sufficient to increase allergic inflammation. COX inhibition during sensitization increased the numbers of mature dendritic cells and activated CD4 T cells in the spleen and augmented OVA-specific IL-5 and IL-13 responses of the splenic CD4 T cells at day 5 after sensitization. COX inhibition during sensitization also augmented allergic Th2 response to OVA challenge 90 days after the sensitization. Therefore, COX inhibition during allergic sensitization augments allergic responses by enhancing Th2 cell activation and memory generation and the proallergic effect is STAT6-independent. These findings provide a mechanistic explanation for the increased allergic inflammation previously shown in the mice treated with COX inhibitors and in COX-deficient mice and suggest that use of COX-inhibiting drugs during initial allergen exposure may increase the risk of developing allergic responses.

Metastatic Melanoma Patient-Derived Xenografts Respond to MDM2 Inhibition as a Single Agent or in Combination with BRAF/MEK Inhibition.

PURPOSE: Over 60% of patients with melanoma respond to immune checkpoint inhibitor (ICI) therapy, but many subsequently progress on these therapies. Second-line targeted therapy is based on BRAF mutation status, but no available agents are available for NRAS, NF1, CDKN2A, PTEN, and TP53 mutations. Over 70% of melanoma tumors have activation of the MAPK pathway due to BRAF or NRAS mutations, while loss or mutation of CDKN2A occurs in approximately 40% of melanomas, resulting in unregulated MDM2-mediated ubiquitination and degradation of p53. Here, we investigated the therapeutic efficacy of over-riding MDM2-mediated degradation of p53 in melanoma with an MDM2 inhibitor that interrupts MDM2 ubiquitination of p53, treating tumor-bearing mice with the MDM2 inhibitor alone or combined with MAPK-targeted therapy. EXPERIMENTAL DESIGN: To characterize the ability of the MDM2 antagonist, KRT-232, to inhibit tumor growth, we established patient-derived xenografts (PDX) from 15 patients with melanoma. Mice were treated with KRT-232 or a combination with BRAF and/or MEK inhibitors. Tumor growth, gene mutation status, as well as protein and protein-phosphoprotein changes, were analyzed. RESULTS: One-hundred percent of the 15 PDX tumors exhibited significant growth inhibition either in response to KRT-232 alone or in combination with BRAF and/or MEK inhibitors. Only BRAFV600WT tumors responded to KRT-232 treatment alone while BRAFV600E/M PDXs exhibited a synergistic response to the combination of KRT-232 and BRAF/MEK inhibitors. CONCLUSIONS: KRT-232 is an effective therapy for the treatment of either BRAFWT or PAN WT (BRAFWT, NRASWT) TP53WT melanomas. In combination with BRAF and/or MEK inhibitors, KRT-232 may be an effective treatment strategy for BRAFV600-mutant tumors.

Prostaglandin I2 analogs inhibit Th1 and Th2 effector cytokine production by CD4 T cells.

An anti-inflammatory effect of PGI(2) has been suggested by increased inflammation in mice that are deficient in the PGI(2) receptor (IP) or in respiratory syncytial viral- or OVA-induced CD4 T cell-associated responses. To determine the mechanism of the anti-inflammatory effect, we hypothesized that PGI(2) analogs inhibit CD4 T cell effector cytokine production. To test this hypothesis, we activated purified CD4 T cells with anti-CD3 and anti-CD28 antibodies under Th1 and Th2 polarizing conditions for 4 days and restimulated the T cells with anti-CD3 in the presence of PGI(2) analogs for 2 days. We found that PGI(2) analogs (cicaprost and iloprost) inhibited the production of Th1 cytokines (IFN-gamma) and Th2 cytokines (IL-4, IL-10, and IL-13) in a dose-dependent pattern. The inhibitory effect was partially dependent on the IP receptor signaling and was correlated with elevated intracellular cAMP and down-regulated NF-kappaB activity. Pretreatment of the CD4 T cells with 8-bromoadenosine-3',5'-cyclic monophosphorothioate, Rp-isomer, to inhibit a key signaling molecule in the cAMP pathway, protein kinase A (PKA), attenuated the suppressive effect of PGI(2) analogs significantly, suggesting that PKA, in part, mediates the inhibition of the cytokine production. These data indicate that PGI(2) analogs have an immune-suppressive effect on previously activated and differentiated CD4 T cells in vitro and suggest that PGI(2) may have a similar function in vivo.

IL-13 is associated with reduced illness and replication in primary respiratory syncytial virus infection in the mouse.

The role of IL-13 in respiratory syncytial virus (RSV) immunopathogenesis is incompletely described. To assess the effect of IL-13 on primary RSV infection, transgenic mice which either overexpress IL-13 in the lung (IL-13 OE) or non-transgenic littermates (IL-13 NT) were challenged intranasally with RSV. IL-13 OE mice had significantly decreased peak viral titers four days after infection compared to non-transgenic littermates. In addition, IL-13 OE mice had significantly lower RSV-induced weight loss and reduced lung IFN-gamma protein expression compared with IL-13 NT mice. In contrast, primary RSV challenge of IL-13 deficient mice resulted in a small, but statistically significant increase in viral titers on day four after infection, no difference in RSV-induced weight loss compared to wild type mice, and augmented IFN-gamma production on day 6 after infection. In STAT1-deficient (STAT1 KO) mice, where primary RSV challenge produced high levels of IL-13 production in the lungs, treatment with an IL-13 neutralizing protein resulted in greater peak viral titers both four and six days after RSV and greater RSV-induced weight loss compared to mice treated with a control protein. These results suggest that IL-13 modulates illness from RSV-infection.

Jnk2 effects on tumor development, genetic instability and replicative stress in an oncogene-driven mouse mammary tumor model.

Oncogenes induce cell proliferation leading to replicative stress, DNA damage and genomic instability. A wide variety of cellular stresses activate c-Jun N-terminal kinase (JNK) proteins, but few studies have directly addressed the roles of JNK isoforms in tumor development. Herein, we show that jnk2 knockout mice expressing the Polyoma Middle T Antigen transgene developed mammary tumors earlier and experienced higher tumor multiplicity compared to jnk2 wildtype mice. Lack of jnk2 expression was associated with higher tumor aneuploidy and reduced DNA damage response, as marked by fewer pH2AX and 53BP1 nuclear foci. Comparative genomic hybridization further confirmed increased genomic instability in PyV MT/jnk2-/- tumors. In vitro, PyV MT/jnk2-/- cells underwent replicative stress and cell death as evidenced by lower BrdU incorporation, and sustained chromatin licensing and DNA replication factor 1 (CDT1) and p21(Waf1) protein expression, and phosphorylation of Chk1 after serum stimulation, but this response was not associated with phosphorylation of p53 Ser15. Adenoviral overexpression of CDT1 led to similar differences between jnk2 wildtype and knockout cells. In normal mammary cells undergoing UV induced single stranded DNA breaks, JNK2 localized to RPA (Replication Protein A) coated strands indicating that JNK2 responds early to single stranded DNA damage and is critical for subsequent recruitment of DNA repair proteins. Together, these data support that JNK2 prevents replicative stress by coordinating cell cycle progression and DNA damage repair mechanisms.

Cutting Edge: Oseltamivir decreases T cell GM1 expression and inhibits clearance of respiratory syncytial virus: potential role of endogenous sialidase in antiviral immunity.

The sialoglycosphingolipid GM1 is important for lipid rafts and immune cell signaling. T cell activation in vitro increases GM1 expression and increases endogenous sialidase activity. GM1 expression has been hypothesized to be regulated by endogenous sialidase. We tested this hypothesis in vivo using a mouse model of respiratory syncytial virus (RSV) infection. RSV infection increased endogenous sialidase activity in lung mononuclear cells. RSV infection increased lung CD8+ T cell surface GM1 expression. Activated CD8+ T cells in the lungs of RSV-infected mice were GM1(high). Treatment of RSV-infected mice with the sialidase/neuraminidase inhibitor oseltamivir decreased T cell surface GM1 levels. Oseltamivir treatment decreased RSV-induced weight loss and inhibited RSV clearance. Our data indicate a novel role for an endogenous sialidase in regulating T cell GM1 expression and antiviral immunity. Also, oseltamivir, an important anti-influenza drug, inhibits the clearance of a respiratory virus that lacks a neuraminidase gene, RSV.

Prostaglandin I2 analogs inhibit proinflammatory cytokine production and T cell stimulatory function of dendritic cells.

Signaling through the PGI(2) receptor (IP) has been shown to inhibit inflammatory responses in mouse models of respiratory syncytial viral infection and OVA-induced allergic responses. However, little is known about the cell types that mediate the anti-inflammatory function of PGI(2.) In this study, we determined that PGI(2) analogs modulate dendritic cell (DC) cytokine production, maturation, and function. We report that PGI(2) analogs (iloprost, cicaprost, treprostinil) differentially modulate the response of murine bone marrow-derived DC (BMDC) to LPS in an IP-dependent manner. The PGI(2) analogs decreased BMDC production of proinflammatory cytokines (IL-12, TNF-alpha, IL-1alpha, IL-6) and chemokines (MIP-1alpha, MCP-1) and increased the production of the anti-inflammatory cytokine IL-10 by BMDCs. The modulatory effect was associated with IP-dependent up-regulation of intracellular cAMP and down-regulation of NF-kappaB activity. Iloprost and cicaprost also suppressed LPS-induced expression of CD86, CD40, and MHC class II molecules by BMDCs and inhibited the ability of BMDCs to stimulate Ag-specific CD4 T cell proliferation and production of IL-5 and IL-13. These findings suggest that PGI(2) signaling through the IP may exert anti-inflammatory effects by acting on DC.

Respiratory syncytial virus infection in the absence of STAT 1 results in airway dysfunction, airway mucus, and augmented IL-17 levels.

BACKGROUND: Respiratory syncytial virus (RSV) is the leading infectious cause of respiratory failure and wheezing in infants and young children. Prematurity is the greatest risk factor for severe RSV-induced disease, and recent studies suggest that premature children have lower levels of the type I IFNs (alpha/beta), for which signal transducer and activator of transcription (STAT) 1 is a critical intracellular signaling molecule. OBJECTIVE: We hypothesized that RSV infection in STAT 1 knockout (STAT 1 KO) mice would result in both increased airway resistance and airway hyperresponsiveness. METHODS: Wild-type (WT) and STAT 1 KO mice on a BALB/c background were either RSV or mock infected. Phenotypic response to infection was assessed by means of plethysmography, immunohistochemistry, and lung cytokine measurement. RESULTS: We found that STAT 1 KO mice infected with RSV (STAT 1 KO-RSV) had greater baseline lung resistance (P=.05) and airway responsiveness (P<.001) than mock-infected STAT 1 KO (STAT 1 KO-MOCK), RSV-infected wild type (WT-RSV), and mock-infected wild type (WT-MOCK) mice. In addition, the STAT 1 KO-RSV mice showed induction of mucus production and expression of gob-5 and Muc5ac, conditions not present in any of the other 3 groups. IL-17, a cytokine that regulates Muc5ac expression, was expressed in the lungs of the STAT 1 KO-RSV mice, whereas lung levels of IL-17 were undetectable in the remaining groups. Expression of the IL-23-specific p19 subunit was also increased in the STAT 1 KO-RSV mice but not in the WT-RSV mice. CONCLUSION: These results show that STAT 1 has an important regulatory role in RSV-induced alteration of airway function.

Respiratory syncytial virus in allergic lung inflammation increases Muc5ac and gob-5.

Respiratory syncytial virus (RSV) is associated with wheezing and childhood asthma. We previously reported that RSV infection prolongs methacholine-induced airway hyperresponsiveness in ovalbumin (OVA)-sensitized mice. In addition, allergically sensitized RSV-infected (OVA/RSV) mice had more abundant airway epithelial mucus production compared with OVA mice 14 days after infection, whereas there was almost no mucus in mice that were only RSV infected. We hypothesized that this increased mucus was associated with mucosal expression of Muc5ac, a mucus gene expression in airways, and gob-5, a member of the Ca(2)(+)-activated chloride channel family. By histochemical analysis, we found that there was significantly increased staining for gob-5 and Muc5ac in the airways of OVA/RSV mice compared with either OVA mice or allergically sensitized mice that were challenged with inactivated RSV, and virtually no detectable staining in the RSV group. These findings were confirmed by Western blot analysis. The increased mucus expression in the OVA/RSV group was associated with increased lung levels of interleukin-17, a factor known to stimulate airway mucin gene expression. The impact of virus infection combined with allergic inflammation on mucus production may partially explain the more severe disease and airway hyperresponsiveness associated with RSV in the setting of atopy.

Signaling through the prostaglandin I2 receptor IP protects against respiratory syncytial virus-induced illness.

The role of prostanoids in modulating respiratory syncytial virus (RSV) infection is unknown. We found that RSV infection in mice increases production of prostaglandin I(2) (PGI(2)). Mice that overexpress PGI(2) synthase selectively in bronchial epithelium are protected against RSV-induced weight loss and have decreased peak viral replication and gamma interferon levels in the lung compared to nontransgenic littermates. In contrast, mice deficient in the PGI(2) receptor IP have exacerbated RSV-induced weight loss with delayed viral clearance and increased levels of gamma interferon in the lung compared to wild-type mice. These results suggest that signaling through IP has antiviral effects while protecting against RSV-induced illness and that PGI(2) is a potential therapeutic target in the treatment of RSV.