Induction and Therapeutic Targeting of Human NPM1c+ Myeloid Leukemia in the Presence of Autologous Immune System in Mice.

Development of targeted cancer therapy requires a thorough understanding of mechanisms of tumorigenesis as well as mechanisms of action of therapeutics. This is challenging because by the time patients are diagnosed with cancer, early events of tumorigenesis have already taken place. Similarly, development of cancer immunotherapies is hampered by a lack of appropriate small animal models with autologous human tumor and immune system. In this article, we report the development of a mouse model of human acute myeloid leukemia (AML) with autologous immune system for studying early events of human leukemogenesis and testing the efficacy of immunotherapeutics. To develop such a model, human hematopoietic stem/progenitor cells (HSPC) are transduced with lentiviruses expressing a mutated form of nucleophosmin (NPM1), referred to as NPM1c. Following engraftment into immunodeficient mice, transduced HSPCs give rise to human myeloid leukemia, whereas untransduced HSPCs give rise to human immune cells in the same mice. The de novo AML, with CD123+ leukemic stem or initiating cells (LSC), resembles NPM1c+ AML from patients. Transcriptional analysis of LSC and leukemic cells confirms similarity of the de novo leukemia generated in mice with patient leukemia and suggests Myc as a co-operating factor in NPM1c-driven leukemogenesis. We show that a bispecific conjugate that binds both CD3 and CD123 eliminates CD123+ LSCs in a T cell-dependent manner both in vivo and in vitro. These results demonstrate the utility of the NPM1c+ AML model with an autologous immune system for studying early events of human leukemogenesis and for evaluating efficacy and mechanism of immunotherapeutics.

Effect of Standardized Infliximab Dose Rounding on an Outpatient Infusion Center.

BACKGROUND: Infliximab dose rounding is a commonly accepted practice at many institutions to contain costs. Currently, there is limited data on the clinical and financial implications of infliximab dose rounding standardization. OBJECTIVE: To determine whether standardized infliximab dose rounding is comparable with nonstandardized dosing in patients with Crohn's disease or ulcerative colitis in terms of cost and efficiency, using a cost comparison between the 2 dosing methods at an outpatient infusion center attached to a community teaching hospital. METHODS: A retrospective electronic chart review was conducted to identify patients who received infliximab for ulcerative colitis or Crohn's disease over a 6-month period. The primary endpoint was cost comparison between the 2 dosing methods. The secondary outcomes were estimated time taken for order verification, number of order clarifications, increase in dose or frequency of infliximab, number of patients who switched to alternative therapy, and use of medications for adverse drug effects. Descriptive statistics and Fisher's exact test were used for data analysis. RESULTS: 72 patients met the inclusion criteria. Because of patient overlap during the study period, 45 patients (62.5%) were in the standardized rounding arm, and 69 patients (95.8%) were in the nonstandardized rounding arm. One patient in each arm required an increased dose or frequency of infusion (2.2% vs. 1.5%, P = 1.000). Standardized infliximab dose rounding had a theoretical cost savings of at least $104,640 per year (based on our rough annual census of 480 patients) compared with the nonstandardized method that had been used previously. The cost savings can also be translated as $218 per patient per month on average. The mean times to order verification were 10 vs. 12 minutes in the nonstandardized and standardized groups, respectively. Two patients in the nonstandardized group switched to alternative therapy. There was no difference in usage of rescue medications for adverse drug effects. CONCLUSIONS: Standardization of infliximab dose rounding resulted in increased efficiency in the pharmacy workflow by reducing time for order verification. Furthermore, standardized dose rounding resulted in a significant reduction in expenditure for infliximab for the institution. DISCLOSURES: No outside funding supported this research. The authors have nothing to disclose. This research was presented as a poster at the ASHP Midyear Clinical Meeting & Exhibition 2017; December 3-7, 2017; Orlando, FL.

Fear of hypoglycemia in adults with type 1 diabetes: impact of therapeutic advances and strategies for prevention - a review.

PURPOSE: This review summarizes the current state of the science related to fear of hypoglycemia (FOH) in adults with type 1 diabetes. Fear of hypoglycemia is a critical deterrent to diabetes self-management, psychological well-being, and quality of life. We examine the influence of contemporary treatment regimens, technology, and interventions to identify gaps in knowledge and opportunities for research and practice. BASIC PROCEDURES: A literature search was conducted of MEDLINE, PsycINFO, and EMBASE. Fifty-three studies that examined fear of hypoglycemia were included. MAIN FINDINGS: Fear of hypoglycemia influences diabetes management and quality of life. Gender and age differences exist in experiences and responses. Responses vary from increased vigilance to potentially immobilizing distress. Fear of hypoglycemia is greater at night and may contribute to poor sleep quality. Strategies to reduce fear of hypoglycemia have had varying success. Newer technologies hold promise but require further examination. CONCLUSIONS: Fear of hypoglycemia remains a problem, despite advances in technology, insulin analogs, and evidence-based diabetes management. Clinical care should consistently include assessment for its influence on diabetes self-management and psychological health. Further research is needed regarding the influence of newer technologies and individualized strategies to reduce fear of hypoglycemia while maintaining optimal glucose control.

Tumor-associated macrophages promote invasion while retaining Fc-dependent anti-tumor function.

Tumor-associated macrophages (TAMs) have been shown to promote tumor progression, and increased TAM infiltration often correlates with poor prognosis. However, questions remain regarding the phenotype of macrophages within the tumor and their role in mAb-dependent cytotoxicity. This study demonstrates that whereas TAMs have protumor properties, they maintain Fc-dependent anti-tumor function. CD11b(+)CD14(+) TAMs isolated from primary human breast tumors expressed activating FcγRs. To model breast cancer TAMs in vitro, conditioned medium from breast cancer cells was used to drive human peripheral monocyte differentiation into macrophages. Tumor-conditioned macrophages were compared with in vitro derived M1 and M2a macrophages and were found to promote tumor cell invasion and express M2a markers, confirming their protumor potential. However, unlike M2a macrophages, tumor-conditioned macrophages expressed FcγRs and phagocytosed tumor cells in the presence of a tumor Ag-targeting mAb, unmasking an underappreciated tumoricidal capacity of TAMs. In vivo macrophage depletion reduced the efficacy of anti-CD142 against MDA-MB-231 xenograft growth and metastasis in SCID/beige mice, implicating a critical role for macrophages in Fc-dependent cell killing. M-CSF was identified in tumor-conditioned media and shown to be capable of differentiating macrophages with both pro- and anti-tumor properties. These results highlight the plasticity of TAMs, which are capable of promoting tumor progression and invasion while still retaining tumoricidal function in the presence of tumor-targeting mAbs.

Cyclophosphamide creates a receptive microenvironment for prostate cancer skeletal metastasis.

A number of cancers predominantly metastasize to bone, due to its complex microenvironment and multiple types of constitutive cells. Prostate cancer especially has been shown to localize preferentially to bones with higher marrow cellularity. Using an experimental prostate cancer metastasis model, we investigated the effects of cyclophosphamide, a bone marrow-suppressive chemotherapeutic drug, on the development and growth of metastatic tumors in bone. Priming the murine host with cyclophosphamide before intracardiac tumor cell inoculation was found to significantly promote tumor localization and subsequent growth in bone. Shortly after cyclophosphamide treatment, there was an abrupt expansion of myeloid lineage cells in the bone marrow and the peripheral blood, associated with increases in cytokines with myelogenic potential such as C-C chemokine ligand (CCL)2, interleukin (IL)-6, and VEGF-A. More importantly, neutralizing host-derived murine CCL2, but not IL-6, in the premetastatic murine host significantly reduced the prometastatic effects of cyclophosphamide. Together, our findings suggest that bone marrow perturbation by cytotoxic chemotherapy can contribute to bone metastasis via a transient increase in bone marrow myeloid cells and myelogenic cytokines. These changes can be reversed by inhibition of CCL2.

The opioid component of delayed gastrointestinal recovery after bowel resection.

INTRODUCTION: Patients undergoing bowel resection or other major abdominal surgery experience a period of delayed gastrointestinal recovery associated with increased postoperative morbidity and longer hospital length of stay. Symptoms include nausea, vomiting, abdominal distension, bloating, pain, intolerance to solid or liquid food, and inability to pass stool or gas. The exact cause of delayed gastrointestinal recovery is not known, but several factors appear to play a central role, namely the neurogenic, hormonal, and inflammatory responses to surgery and the response to exogenous opioid analgesics and endogenous opioids. DISCUSSION: Stimulation of opioid receptors localized to neurons of the enteric nervous system inhibits coordinated gastrointestinal motility and fluid absorption, thereby contributing to delayed gastrointestinal recovery and its associated symptoms. Given the central role of opioid analgesics in delayed gastrointestinal recovery, a range of opioid-sparing techniques and pharmacologic agents, including opioid receptor antagonists, have been developed to facilitate faster restoration of gastrointestinal function after bowel resection when used as part of a multimodal accelerated care pathway. This review discusses the etiology of opioid-induced gastrointestinal dysfunction as well as clinical approaches that have been evaluated in controlled clinical trials to reduce the opioid component of delayed gastrointestinal recovery.

Blockade of interleukin-6 signalling with siltuximab enhances melphalan cytotoxicity in preclinical models of multiple myeloma.

Signalling through the interleukin (IL)-6 pathway induces proliferation and drug resistance of multiple myeloma cells. We therefore sought to determine whether the IL-6-neutralizing monoclonal antibody siltuximab, formerly CNTO 328, could enhance the activity of melphalan, and to examine some of the mechanisms underlying this interaction. Siltuximab increased the cytotoxicity of melphalan in KAS-6/1, INA-6, ANBL-6, and RPMI 8226 human myeloma cell lines (HMCLs) in an additive-to-synergistic manner, and sensitized resistant RPMI 8226.LR5 cells to melphalan. These anti-proliferative effects were accompanied by enhanced activation of drug-specific apoptosis in HMCLs grown in suspension, and in HMCLs co-cultured with a human-derived stromal cell line. Siltuximab with melphalan enhanced activation of caspase-8, caspase-9, and the downstream effector caspase-3 compared with either of the single agents. This increased induction of cell death occurred in association with enhanced Bak activation. Neutralization of IL-6 also suppressed signalling through the phosphoinositide 3-kinase/Akt pathway, as evidenced by decreased phosphorylation of Akt, p70 S6 kinase and 4E-BP1. Importantly, the siltuximab/melphalan regimen demonstrated enhanced anti-proliferative effects against primary plasma cells derived from patients with myeloma, monoclonal gammopathy of undetermined significance, and amyloidosis. These studies provide a rationale for translation of siltuximab into the clinic in combination with melphalan-based therapies.

JAK1 activates STAT3 activity in non-small-cell lung cancer cells and IL-6 neutralizing antibodies can suppress JAK1-STAT3 signaling.

Members of the signal transducer and activator of transcription (STAT) family of transcription factors are potential targets for the treatment and prevention of cancers including non-small-cell lung cancer. STAT proteins can be phosphorylated and activated by diverse upstream kinases including cytokine receptors and tyrosine kinases. We examined STAT protein activation in lung cancer cell lines including those with activating mutations in the EGFR and examined upstream kinases responsible for STAT3 phosphorylation and activation using small molecules, antibodies, and RNA interference. We found more pronounced STAT3 activation in cells with activating EGFR mutations, yet inhibition of EGFR activity had no effect on STAT3 activation. Inhibition of JAK1 with small molecules or RNA interference resulted in loss of STAT3 tyrosine phosphorylation and inhibition of cell growth. An interleukin-6 neutralizing antibody, siltuximab (CNTO 328) could inhibit STAT3 tyrosine phosphorylation in a cell-dependent manner. Siltuximab could completely inhibit STAT3 tyrosine phosphorylation in H1650 cells, and this resulted in inhibition of lung cancer cell growth in vivo. Combined EGFR inhibition with erlotinib and siltuximab resulted in dual inhibition of both tyrosine and serine STAT3 phosphorylation, more pronounced inhibition of STAT3 transcriptional activity, and translated into combined effects on lung cancer growth in a mouse model. Our results suggest that JAK1 is responsible for STAT3 activation in lung cancer cells and that indirect attacks on JAK1-STAT3 using an IL-6 neutralizing antibody with or without EGFR inhibition can inhibit lung cancer growth in lung cancer subsets.

Effects of siltuximab on the IL-6-induced signaling pathway in ovarian cancer.

PURPOSE: To explore potential therapeutic strategies for interrupting the interleukin-6 (IL-6) signaling pathway, we measured IL-6 expression in ovarian cancer tissues, and evaluated the effects of a monoclonal anti-IL-6 antibody; siltuximab (CNTO 328), on levels of IL-6-induced Stat3 phosphorylation, Stat3 nuclear translocation, and Stat3 downstream antiapoptotic genes. We then looked for enhancing paclitaxel sensitivity in multidrug-resistant ovarian cancer cell lines. EXPERIMENTAL DESIGN: Expressions of IL-6 in ovarian cancer patient specimens were assessed by immunohistochemistry. Effects of siltuximab on IL-6-induced activation of Stat3 in an ovarian cancer cell line were determined by Western blot and real-time analysis of Stat3 nucleocytoplasmic translocation. Influence of combination of siltuximab and paclitaxel on tumor growth was evaluated in a xenograft mouse mode in vivo. RESULTS: Metastatic and drug-resistant recurrent tumors have significantly higher IL-6 expression when compared with the matched primary tumors. Siltuximab specifically suppressed IL-6-induced Stat3 phosphorylation and Stat3 nuclear translocation. Treatment with siltuximab significantly decreased the levels of Stat3 downstream proteins such as MCL-1, Bcl-X(L), and survivin. Treatment with siltuximab reduced expression of multiple IL-6-induced genes in these cell lines. Furthermore, siltuximab increased the cytotoxic effects of paclitaxel in a paclitaxel resistant ovarian cancer cell line in vitro, but combination therapy with siltuximab did not have a significant effect on paclitaxel resistant tumor growth in vivo. CONCLUSIONS: These results show that siltuximab effectively block the IL-6 signaling pathways and IL-6-induced gene expression. Blockage of IL-6 signaling may provide benefits for the treatment of ovarian cancer.

Down-regulation of suppressor of cytokine signaling-3 causes prostate cancer cell death through activation of the extrinsic and intrinsic apoptosis pathways.

Suppressor of cytokine signaling-3 (SOCS-3) acts as a negative feedback regulator of the Janus-activated kinase/signal transducers and activators of transcription factors signaling pathway and plays an important role in the development and progression of various cancers. To better understand the role of SOCS-3 in prostate cancer, SOCS-3 expression was down-regulated in DU-145, LNCaP-IL-6+, and PC3 cells by consecutive SOCS-3 small interfering RNA transfections. SOCS-3 mRNA and protein expression as measured by quantitative reverse transcription-PCR and Western blot, respectively, were decreased by approximately 70% to 80% compared with controls. We observed a significant decrease in cell proliferation and viability in all SOCS-3-positive cell lines but not in the parental LNCaP cell line, which is SOCS-3 negative. In this study, we show that down-regulation of SOCS-3 leads to an increased cell death in prostate cancer cell lines. We found a considerable increase in the activation of the proapoptotic caspase-3/caspase-7, caspase-8, and caspase-9. A significant up-regulation of cleaved poly(ADP-ribose) polymerase and inhibition of Bcl-2 expression was observed in all SOCS-3-positive cell lines. Overexpression of Bcl-2 could rescue cells with decreased SOCS-3 levels from going into apoptosis. Tissue microarray data prove that SOCS-3 is highly expressed in castration-refractory tumor samples. In conclusion, we show that SOCS-3 is an important protein in the survival machinery in prostate cancer and is overexpressed in castration-resistant tumors. SOCS-3 knockdown results in an increase of cell death via activation of the extrinsic and intrinsic apoptosis pathways.

Targeted inhibition of interleukin-6 with CNTO 328 sensitizes pre-clinical models of multiple myeloma to dexamethasone-mediated cell death.

Interleukin (IL)-6-mediated signalling attenuates the anti-myeloma activity of glucocorticoids (GCs). We therefore sought to evaluate whether CNTO 328, an anti-IL-6 monoclonal antibody in clinical development, could enhance the apoptotic activity of dexamethasone (dex) in pre-clinical models of myeloma. CNTO 328 potently increased the cytotoxicity of dex in IL-6-dependent and -independent human myeloma cell lines (HMCLs), including a bortezomib-resistant HMCL. Isobologram analysis revealed that the CNTO 328/dex combination was highly synergistic. Addition of bortezomib to CNTO 328/dex further enhanced the cytotoxicity of the combination. Experiments with pharmacologic inhibitors revealed a role for the p44/42 mitogen-activated protein kinase pathway in IL-6-mediated GC resistance. Although CNTO 328 alone induced minimal cell death, it potentiated dex-mediated apoptosis, as evidenced by increased activation of caspases-8, -9 and -3, Annexin-V staining and DNA fragmentation. The ability of CNTO 328 to sensitize HMCLs to dex-mediated apoptosis was preserved in the presence of human bone marrow stromal cells. Importantly, the increased activity of the combination was also seen in plasma cells from patients with GC-resistant myeloma. Taken together, our data provide a strong rationale for the clinical development of the CNTO 328/dex regimen for patients with myeloma.

Circulating human interleukin-8 as an indicator of cancer progression in a nude rat orthotopic human non-small cell lung carcinoma model.

Clinically relevant animal models of human cancer are necessary for the evaluation of putative therapeutics. We hypothesized that circulating human lung cancer-associated proteins would correlate with physiologic measurements from an orthotopic H460 human non-small cell lung carcinoma model that we developed in immunodeficient rats. Physiologic measurements and serum samples were collected over time. Serum interleukin-8 (IL-8), p53, vascular endothelial growth factor, and matrix metalloproteinase-9 were quantitated for correlation with physiologic measurements. Matrix metalloproteinase-9 and p53 were not significantly detectable. Circulating vascular endothelial growth factor was detected at high levels in some tumor-bearing animals. Human IL-8 was detectable in all tumor-bearing animals and correlated positively with markers of respiratory acidosis (pH, P = 0.012; TCO(2), P = 0.024; pCO(2), P = 0.007; and HCO(3)(-), P = 0.029) and with surface body temperature (P = 0.001) beginning on day 16 after implantation. IL-8 levels negatively correlated with survival (P < 0.001), indicating an association with tumor burden. Circulating human IL-8 might be a useful, clinically relevant circulating tumor protein marker due to its positive correlation with multiple physiologic variables associated with lung cancer progression.

Anti-integrin monoclonal antibody CNTO 95 enhances the therapeutic efficacy of fractionated radiation therapy in vivo.

Selective targeting of up-regulated integrins on tumor cells is a novel antiangiogenesis strategy for treating solid tumors. CNTO 95 is a fully human anti-alpha(v) integrin monoclonal antibody and has shown antitumor activity when used as a single agent in preclinical studies. We previously showed that radiation combined with an integrin alpha(v)beta(3) antagonist cRGD peptide increased the therapeutic efficacy of radiation in preclinical tumor models. We hypothesized that the combination of radiation and CNTO 95 would synergistically enhance the efficacy of radiation therapy. The in vitro studies showed that CNTO 95 radiosensitized and induced apoptosis in M21 cells in vitronectin-coated dishes. In mice bearing established human cancer xenograft tumors, CNTO 95 alone had only a moderate effect on tumor growth. The combined therapy of CNTO 95 and fractionated radiation significantly inhibited tumor growth and produced the longer tumor growth delay time in multiple tumor models. Maintenance dosing of CNTO 95 following irradiation contributed to efficacy and was important for continued inhibition of tumor regrowth. Immunohistochemistry studies showed that the combined use of CNTO 95 and radiation reduced the alpha(v) integrin and vascular endothelial growth factor receptor expression and the microvessel density and increased apoptosis in tumor cells and the tumor microenvironment. CNTO 95 alone and in combination with radiation did not produce any obvious signs of systemic toxicity. These results show that CNTO 95 can potentiate the efficacy of fractionated radiation therapy in a variety of human cancer xenograft tumor types in nude mice. These findings are very promising and may have high translational relevance for the treatment of patients with solid tumors.

Mcl-1 is regulated by IL-6 and mediates the survival activity of the cytokine in a model of late stage prostate carcinoma.

The proinflammatory cytokine interleukin-6 (IL-6) has been considered a positive growth factor in late stage prostate cancer (PC) cells and a potential target for therapeutic interference. We studied the effects of inhibition of IL-6 in LNCaP-IL6+ cells, a model system for advanced PC, which produce IL-6. By using the chimeric anti-IL-6 antibody, CNTO 328, we showed that the autocrine IL-6 loop is responsible for decreased sensitivity of LNCaP-IL-6+ cells to die by apoptosis. Dysregulation of Bcl-2 family members could be implicated in the acquisition of resistance to apoptosis in malignant cell lines. Myeloid cell leukemia 1 (Mcl-1) is an antiapoptotic member of this family that is overexpressed in the IL-6 selected cells compared with control. Specific knock-down of Mcl-1 gene expression by siRNA yielded an increase in apoptosis of LNCaP-IL-6+ cells. Interestingly, inactivation of IL-6 autocrine loop was not able to increase apoptosis levels in the absence of Mcl-1, thus suggesting this molecule as a mediator of the survival action of IL-6. Finally, using selective kinase inhibitors we provide evidence for the involvement of p38 and ERK1/2 mitogen-activated protein kinases pathways in the IL-6-mediated regulation of Mcl-1. In conclusion, these data suggest that endogenous IL-6 acts as an antiapoptotic factor in LNCaP-IL-6+ cells and that Mcl-1 is critical for its survival activity. CNTO 328, in our experimental conditions, is able to render LNCaP-IL-6+ cells more sensitive to apoptosis. These data support the concept of anti-IL-6 therapy in human PC.

Postoperative blood loss and transfusion associated with use of Hextend in cardiac surgery patients at a blood conservation center.

BACKGROUND: Hydroxyethyl starch (HES) solutions are readily available colloids, but their widespread use is shadowed by controversies surrounding their effects on bleeding. This retrospective study was conducted to evaluate the relationship between Hextend (HEX; Hospira, Inc.) doses of 1 to 20 mL/kg and allogeneic transfusion and 24-hour chest tube drainage (CTD) in cardiac surgeries at a blood conservation center. STUDY DESIGN AND METHODS: After institutional review board approval, data on 748 patients undergoing coronary artery bypass grafting (CABG), valve, or combined CABG and valve surgeries were collected. Cases not receiving HEX (due to contraindications, e.g., renal failure, bleeding diathesis) or receiving more than 20 mL per kg HEX, not accepting transfusions, or requiring more extensive surgery were excluded, and the remaining 621 cases were analyzed. RESULTS: Overall transfusion rate and mean CTD were 12.7 percent and 460.4 mL, respectively. Patients who received transfusions received more HEX (10.8 mL/kg vs. 9.8 mL/kg; p = 0.043) but HEX per kg was not associated with higher transfusion rates in multivariate analysis (p = 0.077). HEX per kg was associated with CTD in both uni- and multivariate analyzes (p < 0.001) with 1.66 percent increase in CTD for every 1 mL per kg increase in HEX. CONCLUSIONS: Although HEX was associated with transfusion in univariate analysis and with CTD in uni- and multivariate analysis, the former was no longer significant when adjusted for other predictors of transfusion in our selected patient population at a blood conservation center. The clinical significance of the observed increase in CTD remains undetermined. To minimize transfusion and bleeding in these patients, it is recommended that HEX be used in amounts of not more than 20 mL per kg together with point-of-care coagulation tests and other blood conservation strategies.

Alpha-v integrins as therapeutic targets in oncology.

Integrins are heterodimeric cell adhesion receptors that mediate intercellular communication through cell-extracellular matrix interactions and cell-cell interactions. Integrins have been demonstrated to play a direct role in cancer progression, specifically in tumor cell survival, tumor angiogenesis, and metastasis. Therefore, agents targeted against integrin function have potential as effective anticancer therapies. Numerous anti-integrin agents, including monoclonal antibodies and small-molecule inhibitors, are in clinical development for the treatment of solid and hematologic tumors. This review focuses on the role of alpha(v) integrins in cancer progression, the current status of integrin-targeted agents in development, and strategies for the clinical development of anti-integrin therapies.