Tissue factor expression, angiogenesis, and thrombosis in pancreatic cancer. Cancer Gene Ther. Khorana AA, Ahrendt SA, Ryan CK, Francis CW, Hruban RH, Hu YC, et al. The slides were incubated for 1 h at room temperature (RT) with primary antibodies for rabbit anti neutrophil elastase (ab68672, Abcam) at 1:200, sheep anti fibrinogen (ab61352, Abcam) 1:1000, and mouse anti tissue factor (ab17375, Abcam) 1:200, in 0.5% BSA solution. Treatment of mice with CQ led to a decrease in aggregation in tumor bearing animals with no change in sham (b, AUC 52.6 ± 5.3 vs. 68.1 ± 8.8, n = 4, p < 0.05). Tohme S, Yazdani HO, Al-Khafaji AB, Chidi AP, Loughran P, Mowen K, et al. Circ Cardiovasc Genet. Venous thromboembolism predicts poor prognosis in irresectable pancreatic cancer patients. PAD4 and RAGE knockout mice, deficient in NET formation, were used to study the role of NETs in platelet aggregation, release of tissue factor and hypercoagulability. The Dana-Farber trial of hydroxychloroquine, led by Kimmelman and oncologist Brian Wolpin, MD, is designed to enroll 36 pancreatic cancer patients in whom first- … Binimetinib may stop the growth of tumor cells by blocking some of the enzymes needed for cell growth. NETs promote hypercoagulability in PDA by releasing circulating tissue factor. Whole blood platelet aggregation was measured using impedance aggregometry (ChronoLog aggregometer, Model 700, Havertown, PA, USA). Both in vitro treatment of whole blood (Fig. 4a) and in vivo treatment of mice (Fig. 4b) with chloroquine resulted in decreased platelet aggregation and activation (Additional file 2: Figure S2C). Because autophagy is critical to the process of NET formation, we studied the novel use of the autophagy inhibitor chloroquine to target NET mediated hypercoagulability. Platelet aggregometry was performed on RAGE knockout (RAGE KO) animals, which have global genetic depletion of RAGE. Activated platelets present high mobility group box 1 to neutrophils, inducing autophagy and promoting the extrusion of neutrophil extracellular traps. There was no difference in pretreatment patient demographics between the two randomized groups (Additional file 4: Table S1). Mice were housed in ventilated caging units in the Hillman Cancer Center Specific Pathogen Free (SPF) facility with standard housing and husbandry and free access to food and water. Isolation of mouse neutrophils. Serum was collected after blood was allowed to clot for 30 min and then spun at 1000 g for 10 min. There were no significant differences in pretreatment patient demographics or characteristics. Patients received 600 mg hydroxychloroquine orally twice per day. 2009;27(Suppl 1):63–74. Tumor cell-induced platelet aggregation in vitro by human pancreatic cancer cell lines. Patients received 400 mg hydroxychloroquine orally twice per day. Activated platelets are capable of inducing NETs [32] and NETs in turn promote platelet aggregation as observed in sepsis and deep vein thrombosis [33, 34]. Tissue factor ELISA was performed on serum from orthotopic mice, demonstrating that tumor burdened mice had elevated levels of circulating tissue factor compared to sham (a, 255 ± 49 vs. 159 ± 26 pg/mL, p < 0.05). For target lesions, complete response (CR) is disappearance of all target lesions and partial response (PR) is at least a 30% decrease in the sum of longest diameter (LD) of target lesions, taking as reference baseline sum LD. In patients treated as part of a phase I/II dose escalation trial of preoperative hydroxychloroquine with gemcitabine, the 90 day VTE rate was 3% (n = 1 of 33) [18]. HCQ led to a statistically greater reduction in tissue factor in those patients who had elevated tissue factor prior to treatment, defined by preoperative level greater than the median (40 ng/mL), (− 240 ± 120 vs. -8.74 ± 26.1 pg/mL, p < 0.05, Fig. 4e). Experimental: Hydroxychloroquine 600 mg b.i.d. Neutrophil extracellular trap-derived enzymes oxidize high-density lipoprotein: an additional proatherogenic mechanism in systemic lupus erythematosus. Patients without an event were censored at date of last disease evaluation. The human F3/CD142/Tissue factor ELISA kit was used to measure tissue factor in patient blood samples (LS Bio, LS-F433). Chloroquine reverses hypercoagulability in pancreatic cancer. Study record managers: refer to the Data Element Definitions if submitting registration or results information. RAGE knockout tumor bearing mice, who we have previously shown have decreased NET formation, also had lower levels of tissue factor compared to WT controls (b, 331 ± 39 vs. 390 ± 34 pg/mL, p < 0.05). Traditional coagulation tests such as prothrombin time (PT), partial thromboplastin time (PTT), and international normalized ratio (INR) are frequently normal in hypercoagulability and provide limited information regarding the mechanisms driving a prothrombotic state. Boone BA, Bahary N, Zureikat AH, Moser AJ, Normolle DP, Wu WC, et al. Trials to test hydroxychloroquine, an inhibitor of autophagy, for pancreatic cancer are in progress. Brian A. Boone. Objective: To investigate whether hydroxychloroquine treatment is associated with major adverse cardiovascular events (MACE) (myocardial infarction, ischemic stroke, or cardiovascular-associated death) in patients with cutaneous LE (CLE) or systemic LE (SLE). Furthermore, treatment of whole blood from RAGE KO mice with NET supernatant led to diminished platelet aggregation compared with WT mice (Fig. 2d). Reprod Sci. Proceedings: AACR Annual Meeting 2018; April 14-18, 2018; Chicago, IL Autophagy, a regulated metabolic process that enables energy conservation in adverse environmental circumstances, has been shown in animal models to confer resistance to chemotherapy, which can be reversed by hydroxychloroquine (HCQ). Inhibiting NET formation by genetic depletion of PAD4 resulted in a decrease in serum tissue factor (Fig. 3a). Genetic and Rare Diseases Information Center. During the formation of NETs, DNA is the principle factor released, however many other intracellular components including tissue factor, myeloperoxidase, and histones are also released. Results are reported from at least two independent experiments performed with at least duplicate samples. Correlative patient samples and data were included from two clinical trial protocols that were approved by the Institutional Review Board for the University of Pittsburgh (Protocol #10010028 and #13080444). The first trial was a dose escalation Phase I/II trial of preoperative gemcitabine with hydroxychloroquine for patients with high risk pancreatic adenocarcinoma (UPCI 09–122, IRB Protocol #10010028) [18]. PubMed Central  To elucidate the potential mechanism of decreased platelet aggregation after CQ treatment, we treated PAD4KO mice with CQ and found that it had minimal effect in these mice, suggesting that CQ mediates decreased platelet aggregation through inhibition of NETs (Fig. 4c). Conditions: Metastatic Pancreatic Carcinoma; Stage II Pancreatic Cancer; Stage IIA Pancreatic Cancer; Stage IIB Pancreatic Cancer; Stage III Pancreatic Cancer; Stage IV Pancreatic Cancer; Unresectable Pancreatic Carcinoma Interventions: Drug: Hydroxychloroquine; Drug: Trametinib Sponsors: University of Utah; Novartis Pharmaceuticals Recruiting Hydroxychloroquine is an autophagy inhibitor. 2016;114(5):581–6. NETs promote hypercoagulability in murine PDA through stimulation of platelets and release of tissue factor. Chloroquine treatment led to a significant reduction in serum tissue factor levels in tumor bearing mice with no significant change in sham mice (Fig. 4d). PAD4 KO mice are unable to form NETs as a result of genetic deficiency in protein arginine deiminase 4, an enzyme critical for NET formation that citrullinates histones to allow for DNA unwinding and expulsion from the cell [22]. Data is reported as the area under the curve (AUC), which incorporates both the slope and amplitude of the aggregation curve. 2016;115(3):332–8. ], Histologically confirmed unresectable pancreatic adenocarcinoma that is metastatic to distant sites, Measurable disease, defined as at least one lesion that can accurately be measured in at least one dimension, Patients must have been treated with one or two previous lines of chemotherapy for metastatic disease with documented tumor progression or intolerance due to toxicity, Minimum of two weeks since any major surgery, completion of radiation, or completion of all prior systemic anticancer therapy, Normal organ and marrow function as outlined in the protocol. Binimetinib may stop the growth of tumor cells … This phase I trial studies the best dose of hydroxychloroquine when given together with binimetinib in treating patients with KRAS gene mutated pancreatic cancer that has spread to other places in the body (metastatic). Removing DNA from NET supernatant using DNase I treatment prior to exposure to whole blood reversed the treatment effects of NET supernatant on platelet aggregation in human blood (a, 25.9 ± 2.2 vs. 11.35 ± 0.31, n = 4, p < 0.05). Google ScholarÂ. However, because CQ also has direct antiplatelet effects, it is difficult to completely attribute all its effects to inhibition of NETosis. Risk of site-specific cancer in incident venous thromboembolism: a population-based study. Median PFS follow-up in this study cohort was 46.5 days (95% CI 33-61). TEG was performed on 340 μl murine whole blood drawn via submandibular bleed mixed with 1:9 dilution of 3.4% sodium citrate and 10 units/mL heparin using a Haemoscope 5000 analyzer (Haemonetics, Braintree, MA, USA) as previously described [19]. Proc Natl Acad Sci U S A. Curve analysis was performed using Haemonetics TEG software (version 4.2.3) and the R, K, angle, and MA were measured. (DOCX 109 kb), Figure S3. DNA is released from neutrophils into the circulation during NET formation, therefore this data suggests that NETs may play a role in VTE in patients with pancreatic cancer. Slides were washed three times with BSA solution and incubated for 1 h at RT with Alexa 488 donkey anti mouse secondary antibody (A21202, Invitrogen) diluted 1:500, combined with donkey anti rabbit CY3 (711–165-152, Jackson Immuno) 1:1000, and donkey anti sheep Cy5 (713–175-147, Jackson) in BSA solution. PubMed Central  Among all patients, those with VTE had a mean increase of 6 ng/mL with treatment compared with decrease of 70 ng/mL in those that did not have VTE (p < 0.05). Mice were treated with DNase I (Sigma Aldrich, St Louis, MO, USA) for 5 consecutive daily intraperitoneal injections (5 mg/kg) prior to sacrifice. Annals of oncology: official journal of the European society for. Biochemical response rate was defined as the percentage of patients achieving on treatment a decrease in serum CA 19-9 by > 30% from baseline. Article  BMC Cancer 18, 678 (2018). The findings presented today focused on advanced pancreatic cancer patients who enrolled in XCELSIOR and were treated with a MEK inhibitor, trametinib, in combination with an autophagy inhibitor, hydroxychloroquine, a generic medication approved by the U.S. Food and Drug Administration (FDA) for treatment of malaria, as part of their clinical cancer care. Int J Mol Sci. Animals were sacrificed 4 weeks following injection at which time they had palpable left upper quadrant abdominal tumors. 2017;129(10):1357–67. Venous thromboembolism (VTE) in patients with cancer: epidemiology and risk factors. Information provided by (Responsible Party): Brian Wolpin, MD, MPH, Dana-Farber Cancer Institute. Waterfall plot demonstrating individual treatment response to gemcitabine/nab-paclitaxel with and without hydroxychloroquine in patients with elevated preoperative levels (e). A more recent trial randomized patients to two cycles of preoperative gemcitabine/nab-paclitaxel with or without 1200 mg/day oral hydroxychloroquine (UPCI 13–074, IRB Protocol #13080444). Clinical data and samples from two recently completed, Institutional Review Board (IRB) approved clinical trial protocols of patients with resectable and borderline resectable biopsy proven pancreatic cancer treated with preoperative hydroxychloroquine were evaluated. There were 2 arms in this study because the study was amended to evaluate a second cohort of patients treated at a higher dose using the same two-stage statistical design. LY3214996 is an extracellular signal-regulated kinase (ERK) inhibitor. Platelet aggregation was assessed using collagen-activated impedance aggregometry. 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