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    Bone Pain as a Clinical Presentation of Acute Lymphoblastic Leukemia
    (2019-03-05) Bowman, Paul MD; Hamby, Tyler PhD; Vivatson, Sara
    Introduction: Acute lymphoblastic leukemia (ALL) is the most common malignancy in children, accounting for approximately 1/3 of pediatric cancers. Treatment of ALL has progressed extensively over the years, and approximately 85% of pediatric ALL cases achieve remission. Many factors are used in determining the treatment for individuals, including central nervous system infiltration, chromosomal abnormalities, and various mutations. Most patients present with systemic signs and symptoms that lead the pediatrician towards the diagnosis of ALL, but some present with the chief complaint of bone pain. Case Information: Case 1: A 5-year-old female presented to an urgent care clinic complaining of low back pain for the past 2 weeks. Initially, an X-ray was performed which showed anterolisthesis and diminished vertebral height. Following the X-ray, she was evaluated and found to have additional symptoms including a low-grade fever, headache, nasal discharge, and cough. Labs drawn revealed pancytopenia and an MRI scan revealed an infiltrative process of the L3 vertebral body. Bone marrow biopsy confirmed the diagnosis of B-cell ALL. The patient was started on standard risk therapy, but had positive minimal residual disease (MRD) at day 29 of treatment. Patient was then placed on the high risk treatment protocol. Patient is now in maintenance therapy. Case 2: A 4-year-old female presented to an emergency department with left upper arm pain and a recent episode of strep throat. Exam showed some lymphadenopathy and painful range of motion testing in the left arm. X-ray was unremarkable. Labs showed the patient to be anemic. White blood cell numbers were within normal range, however the differential count identified abnormal lymphocytes to be present. Bone marrow biopsy confirmed diagnosis of B-cell ALL. Patient was started on standard risk therapy, achieved MRD negative remission, and is currently in maintenance therapy. Conclusions: This case study reviews 2 cases out of many that presented with similar complaints of bone pain. Often children experience minor trauma, such as falling out of a chair, which may cause pain. Pediatricians may see many such cases, but sometimes the bone pain is a manifestation of something more serious. Lack of awareness can lead to a delay in diagnosis of ALL. Therefore, ALL should always be considered when evaluating a child for bone pain.
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    Reconstituted high-density lipoprotein as a potential delivery vehicle for TAMs re-polarization agents
    (2019-03-05) Sabnis, Nirupama; Raut, Sangram; Lacko, Andras; Dossou, Akpedje
    Purpose: As part of the tumor microenvironment, tumor-associated macrophages (TAMs) form a functionally heterogeneous population where the pro-inflammatory M1 type macrophages exert anti-tumoral function by enabling the activation of cytotoxic immune cells while immunosuppressive M2 type macrophages support tumor progression, angiogenesis, immune system evasion and metastasis. However, TAMs display a high ratio of M2 to M1 macrophages, and this polarization is promoted by tumor secretions. Thus, their presence in tumor microenvironment is associated with poor prognosis. Because the reversal from M2 to M1 constitutes an attractive cancer immunotherapy strategy, there is a need for targeted selective delivery carriers for reversal agents to avoid off-target effects. Reconstituted high density lipoprotein (rHDL) nanoparticles (NPs) are biocompatible with various administration routes, and they have been confirmed to deliver their cargo to targeted cells via a scavenger receptor class B type 1 (SR-B1)-mediated uptake. In addition, Apolipoprotein A1 (ApoA1), one of the components of the rHDL NP, has been shown to promote a M2 to M1 reversal of TAMs. Hence, we propose that rHDL NPs are particularly appropriate to deliver specific re-polarizing agents to TAMS to achieve a predominately M1 type TAM population, and thus enhance the effectiveness of tumor immunotherapy. Methods: The rHDL NPs were prepared using egg yolk phosphatidylcholine, free cholesterol, cholesterol oleate and the ApoA1 protein. The size, polydispersity index and zeta potential of the NPs were assessed by dynamic light scattering. Transmission electron microscopy confirmed size and uniformity of the rHDL NP preparation. Raw 264.7 macrophages were polarized to M1, M2a and M2c by using respectively lipopolysaccharide + interferon-gamma, interleukin-4, and interleukin-10. Protein expression was confirmed via immunoblot. Results: The rHDL NPs show a sub-50 nm size and form a fairly homogeneous preparation. M2 type macrophages display a higher SR-B1 expression than the M1 type (Raw 264.7 macrophages). Conclusions: Findings of these and earlier studies show that the rHDL NPs may be particularly suited to deliver reversal (re-polarizing) agents to M2 macrophages.
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    Understanding the Mechanism of Action of Copper-Tolfenamic Acid’s Anti-cancer Activity in Pancreatic Cancer Cells
    (2019-03-05) Prokai, Laszlo PhD; Umesh, Sankpal; Basha, Riyaz; Hurtado, Myrna; Levesque, Blair
    Purpose: Non-steroidal anti-inflammatory (NSAID) agents have been proven to have anti-cancer activity. Our group is investigating the use of NSAIDs as sensitizing agents to use alongside standard chemotherapy. This combination increases the efficacy of chemotherapy to accomplish higher anti-cancer activity at relatively low doses, thereby reducing the side effects. It is desirable to find anti-cancer NSAIDs with low Inhibitory concentration (IC50) values. The NSAID, tolfenamic acid (TA), has been tested in preclinical studies for anti-cancer activity against pancreatic cancer (PaCa). Recently, it is shown that metal complexes of NSAIDs enhances the efficacy. Methods: We investigated the anti-proliferative activity of copper-tolfenamic acid (Cu-TA) using 12 cancer cells and reported that the Cu-TA complex had a stronger therapeutic response and lower IC50 values by 30-80% compared to TA. The goal of this investigation is to determine the mechanism by which Cu-TA induces anti-cancer activity in PaCa cell lines. MIAPaCa2 cells were treated with vehicle or Cu-TA (IC50 value) and processed by Next Generation Sequencing (NGS). Ingenuity Pathway Analysis was used to determine the functional significance of the altered gene expression. The top upstream regulators were confirmed by Western blot analysis. Results: Several networks, regulators, and molecular and cellular functions were found to be affected by the Cu-TA treatment. qPCR and Western blot analysis were used to assess and confirm the alterations in the expression of the candidate markers in PaCa cells. Previously, confirmatory studies were performed using MIAPaCa2 cells. Due to the heterogeneity of PaCa, in this study we used a second cell line PANC1 for similar experiments. Tumor protein p53, human epidermal receptor growth factor 2, Specificity protein 1 and signal transducer and activator of transcription 3 were the top upstream regulators confirmed by Western blot analysis. It was demonstrated by qPCR of selected genes, Centromere protein F, DNA damage inducible transcript 3 and S-phase kinase associated protein 2 that Cu-TA is efficacious at a lower dose than TA. Conclusion: NGS and Ingenuity Pathway analysis identified important pathways and genes effected by Cu-TA. In this investigation, PANC1 showed similar results as MIAPaCa2 cells. The genes and pathways that were altered by treatment with Cu-TA involved cell survival or apoptosis demonstrating that Cu-TA is modulating genes associated with cancer. This identifies the potential of Cu-TA as an effective anti-cancer agent.
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    Exploring Less Toxic Combination Treatment Options for Inducing Anti-Cancer Activity in Medulloblastoma Cells
    (2019-03-05) Smith, Kolton OMS-II; Grebennikov, Sarah OMS-II; Sankpal, Umesh PhD; Bowman, W. MD; Basha, Riyaz PhD; Schullek, Melissa OMS-II
    Purpose.Medulloblastoma (MB) is the most common type of malignant pediatric brain cancer and is typically located in the posterior fossa. There are four subgroups within MB: Wnt, Sonic-Hedgehog, Group 3, and Group 4. Due to differences in pathology, signaling pathways, and gene expression, each subgroup is approached differently with respect to treatment, based upon differences in prognosis. Currently, the standard treatment approaches include surgical resection, radiotherapy, and chemotherapeutic agents such as etoposide, vincristine, and cisplatin. Survivors often suffer from severe long-term side effects including neurocognitive deficits and the potential for a future second neoplasm due to the tumorigenic potential of aggressive combination therapies. Because of these side-effects, there is an urgent need for effective and less toxic therapeutic strategies for the treatment of MB. Through prior research we have demonstrated the combination of etoposide alongside less toxic anti-cancer agents potentially increases anti-cancer activity in Ewing Sarcoma. We hypothesize that using a combination of etoposide with other sensitizing agents can also enhance the anti-cancer activity in MB cell lines. Methods. DAOY cells were cultured with increasing concentrations of Etoposide (ETO), Mithramycin-A (MIT), BNS-22 and Tolfenamic acid (TA) and the cell growth was monitored at 48 hours using CellTiter-Glo kit (luminescence cell viability assay). Dose-curves were then generated using sigma-plot software. After calculating the IC50 values for each agent, low dose of ETO (half of IC50 value) and IC50 value of other agents were tested for the combination treatment. Results. Overall, we observed decreased cell viability in a dose and time dependent manner for all tested agents. The IC50 values derived from the dose curves were 1 µg/ml for ETO, 33.3 nM for MIT, 15 µg/ml for TA, and 14.5 µM for BNS. The combination treatment using 0.5 µg/ml ETO and other agents (IC50 values) showed cell growth inhibition greater than any single agent in DAOY cells. The analysis revealed that the combination of ETO (0.5 µg/ml) plus BNS-22 was very effective. Conclusions: These preliminary data demonstrate promise in creating combination therapy of ETO with BNS-22 to treat DAOY cell lines. For better applications, similar experiments should be done with more cell lines representing various sub-groups of MB and to be confirmed by in vivoassays.
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    Diffuse Cutaneous Mastocytosis and its potential comorbidities in pediatric patients – a case study.
    (2019-03-05) Basha, Riyaz; Bowman MD, Paul; Hamby, Tyler; smith, john
    Background: Mastocytosis is the pathologic proliferation and accumulation of mast cells in various tissues of the body. There are different forms of mastocytosis that can present in pediatric patients including systemic (SM), cutaneous (CM) and diffuse cutaneous mastocytosis (DCM). Both the CM and DCM forms have the potential to progress into SM as the patient reaches adulthood. Mastocytosis has been shown to be comorbid with joint pathologies including Ehlers-Danlos syndrome and inflammatory gastrointestinal conditions such as eosinophilic esophagitis. The greatest risk among patients with mastocytosis is anaphylaxis. Case information:A13-week-old male presented to his primary care physician with erythematous spots on his torso and arms, and was diagnosed with eczema. The spots grew and transformed morphologically over the next month and a referral to dermatology was made. Upon biopsy of the original lesion (on the torso), the diagnosis of DCM was made. Over the coming months, symptoms progressed and comorbidities—including joint hypermobility (diagnosed with Ehlers-Danlos syndrome), dysphagia and diarrhea—arose. The patient broke his distal radius while crawling, due to his mast cell disorder and severe vitamin D deficiency. Conclusions: In most children with DCM, symptoms will partially or fully resolve by adolescence. But for some patients, the disease can progress to SM. Numerous comorbidities can occur, as did in this case. Current treatment strategies are wide ranging, from topical glucocorticoids to specialized UV radiating therapy. The specific approaches to this disease are still being understood, with recent investigations into immunological treatment modalities. The individuality of each case is crucial for health care professionals to recognize.
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    Optimization of Antisense Oligonucleotides (ASO) Entrapped HDL Mimetic Lipo-peptide Nanoparticles
    (2019-03-05) Raut, Sangram; Sabnis, Nirupama; Lacko, Andras; Conjeevaram Nagarajan, Bhavani Saranya
    Purpose: Antisense Oligonucleotides (ASO) are short single stranded DNA oligomers that represent a novel class of Nucleic acid therapeutics with high potential to treat a broad range of disorders including cancer by modulating gene expression. Phosphorodiamidate Morpholinos (PMOs) are third generation ASOs which are chemically modified to possess neutral charge, high stability and resisitivity to nucleases and low toxicity/immunogenicity. Despite the enhanced pharmacokinetics, the cellular internalization of PMO is very poor and necessitates the need for a delivery vehicle. Recently, we have developed a simple formulation using an Apo-A1 mimetic peptide conjugated to myristic acid (Myr5A) that self-assembles into nanoparticles (NP) and has same functional properties as endogenous high density lipoproteins (HDL). Hence, we hypothesize that Myr 5A, an HDL mimetic lipo-peptide can potentially be used as a delivery vehicle to entrap ASO. Methods: Myr5A PMO NPs were prepared using microfluidics based Nanoassemblr platform with various concentrations of Myr5A peptide (2.5 5,10 mg/ml) and carboxyfluorescein PMO 80µg/ml. Entrapment efficiency of PMO was determined by fluorescence spectrometry. Dynamic Light Scattering (DLS) was used to analyze the size and the zeta potential. Time resolved (TR) anisotropy was measured to confirm the binding of PMO. Additionally, size exclusion chromatography by FPLC was also carried out to determine the entrapment of PMO and the heterogeneity of the population (if any). Results: Based on our preliminary findings, formulations prepared with 10mg/ml Myr 5A and 80ug/ml PMO resulted in more homogenous particles with entrapment efficiency of 40%. This data was further supported by the TR anisotropy as values for Free PMO and Myr5A PMO NP were 0.09±0.01 and 0.12±0.01 respectively. Furthermore, DLS measurements revealed the presence of homogenous particles with a diameter of 4.98+0.9nm and zeta potential of 0.9mV. Additionally, FPLC has shown distinctive difference in the elution of Free Myr 5A and Myr 5A PMO nanoparticles as they eluted at 12.95 and 17.8 minutes respectively indicating the difference in molecular size. Conclusion: The studies show that HDL mimetic Myr 5A peptide can be a promising candidate for the entrapment of PMOs. However, our future experiments designed to determine the knockdown efficiency of these nanoparticles in-Vitro and in-vivo would hold definitive answers for determining the efficiency of Myr5A PMO NP.
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    A Liposomal Platform Using a Microfluidic Mixing Method for Drug Delivery and Targeting of Metastatic Prostate Cancer
    (2019-03-05) Ranjan, Amalendu Dr.; Joshi, Rohan; Vishwanatha, Jamboor Dr.; Lampe, Jana
    Purpose: The success rate for the treatment of localized prostate cancer (PCa) is very high. However, the overall survival rate for patients with metastatic PCa drops to 28%. Bone is the primary metastatic site in 90% of PCa patients, which not only shortens survival, but also causes a significant decrease in the quality of life. The objective of the project is to develop a dual-targeted nanotherapeutic for bone metastatic PCa. We will engineer liposomes composed of two lipids, DOPE and DOTAP. A bone-targeting moiety with a high affinity to the Ca2+ in bone will be conjugated to the outside of the liposome. This liposome will be loaded with a cabazitaxel-ligand conjugate that has a high affinity for the receptors that are upregulated in 95% of PCa cells. Methods: In this formulation we have used a lipid ratio between 40% and 60%. Then the size and polydispersity were optimized by selecting the best Flow Ratio (FR) and Total Flow Rate (TFR) settings in the NanoAssemblr. We also measured the zeta potential (ZP). Cellular uptake studies were performed using the PC3 cell line and DID dye-loaded, DOPE-DOTAP liposomes, then imaged with a Zeiss LSM 510 confocal microscope. Additionally, liposomes were loaded with curcumin to determine their % drug loading (DL) and encapsulation efficiency (EE). Results: The liposomes were optimized with a 50:50 mol% ratio, a 1:1 FR, and a 6 ml/min TFR. Size (~150 nm) and polydispersity index (0.2), were measured and found to be consistent over a 7-day period to show stability. The ZP (~+40 mV) was also measured. The cellular uptake studies showed that the liposomes were increasingly taken up by the cells over time. The EE was ~93% and DL was ~5%. Conclusion: By finding a desirable ratio of lipids, FR and TFR, we have optimized our liposomal formulation based on size, PDI, and ZP. Also, we have demonstrated that our liposome can easily be taken up by cancer cells and have shown excellent DL and EE. As a result, we are prepared to continue with the next steps of the project. In the next step we will attach the bone-targeting moiety to the liposome, conjugate cabazitaxel with the targeting ligand, and load the liposomes with the cabazitaxel-ligand. Fully functional liposomes will be tested for in vitro and in vivo functionality.
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    Metformin inhibits medulloblastoma cell growth and increases sensitivity to chemotherapy drugs
    (2019-03-05) Basha, Riyaz PhD; Bowman, Paul MD; Sankpal, Umesh PhD; Hong, Julie
    Purpose: Medulloblastoma (MB) is the most common malignant pediatric brain tumor. Standard treatment is chemotherapy and radiation, both of which can be associated with long-term toxicity for pediatric patients. This project is focused on the use of metformin in the treatment of medulloblastoma. Metformin (MET) is an anti-diabetic drug with low toxicity that has been shown to have anti-cancer properties. We hypothesize that MET will inhibit MB cell growth and enhance the effect of chemotherapy and anti-cancer agents such as vincristine (VCR) and valproic acid (VPA) when used in combination, possibly by inhibiting the expression of survivin protein. Methods: MB cells (DAOY) were treated with increasing doses of MET (1-30 mM), VCR (1-16 nM), and VPA (1-30 mM). For combination treatment, DAOY cells were treated with selected doses of VCR (1, 2, 4 nM) and VPA (0.9, 1.8, 3.5 mM) alone or in the presence of MET (10 and 20 mM). Cell viability was assessed at 48 h post-treatment using the CellTiter-Glo cell viability assay kit. For western blot analysis, DAOY cells were treated with increasing doses of MET (0, 5, 10, 20 mM) for 24 and 48 h. Cells were harvested and protein extracts were prepared and used for determining survivin expression. Results: Treatment with MET, VCR, and VPA alone resulted in a decrease in cell viability in a dose and time dependent manner. The combination of MET+VCR resulted in greater inhibition of cell proliferation with 78.99% inhibition in comparison to MET alone (51.5%) or VCR alone (46.02%). The combination of MET+VPA resulted in greater inhibition of cell proliferation with 84.88% inhibition in comparison to MET alone (52.6%) or VPA alone (47.81%). Western blot analysis of MET treated cells showed a dose and time dependent decrease in survivin expression. Conclusion: Our experiments demonstrate the potential of MET as a novel therapeutic agent for the treatment of MB based on its ability to inhibit proliferation and enhance the activity of anti-cancer agents. These results also suggest that MET’s effect could be partially mediated by the down-regulation of survivin, a protein known to be involved in the inhibition of apoptosis and resistance of cells to chemotherapy. The low toxicity of metformin and its ability to sensitize medulloblastoma cells could potentially result in lowering chemotherapy associated toxicities, leading to improved quality of life for long-term survivors.
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    Phosphorylation of Annexin A2 is Essential for its Association with Exosomes in Triple Negative Breast Cancer
    (2019-03-05) Chaudhary, Pankaj; Sun, Xiangle; Vishwanatha, Jamboor; Desai, Priyanka
    Introduction: Studying triple negative breast cancer (TNBC) is the utmost importance as treatment lacks targeted-based therapies. High expression of exosomal Annexin A2 (AnxA2), a Ca+2-dependent phospholipid binding protein, plays an important role in pre-metastatic niche formation and promoting cancer metastasis in TNBC. Also, N-terminal phosphorylation of AnxA2 at Tyrosine (Tyr) 23 has been implicated in several cancer progression. However, the mechanism through which AnxA2 enters into the exosomes has not been elucidated in TNBC. Methods: Plasmids expressing constitutive phosphomimetic AnxA2 (AnxA2-Y23E) and non-phosphomimetic AnxA2 (AnxA2-Y23F) mutant gene were transfected in MDA-MB-231 cells. Transfected cells were functionally validated for AnxA2 specific functions like migration, invasion and proliferation. Exosomes isolated from AnxA2-Y23E (exo-AnxA2-Y23E) and AnxA2-Y23F (exo-AnxA2-Y23F) mutant cells were analyzed for surface expression of AnxA2. Effect of exosomes containing AnxA2-Y23E and AnxA2-Y23F mutant proteins was analyzed on invasiveness and proliferation in cancer cells. Results: Our results showed that MDA-MB-231 TNBC cells expressing phosphomimetic AnxA2 showed increased migratory, invasive and proliferative capacity compared to cells expressing non-phosphomimetic AnxA2. Exosomes derived from phosphomimetic cells had increased AnxA2 expression at surface compared to exosomes derived from non-phosphomimetic cells. In addition, high surface expression of AnxA2 in exosomes derived from phosphomimetic cells induced invasive and proliferative characteristics in CAL-148 breast adenocarcinoma cells compared to exosomes derived from non-phosphomimetic cells (exo-AnxA2-Y23F). Conclusion: Phosphorylation of AnxA2 at Tyr23 plays an important role in imparting metastatic phenotype to the TNBC cells. In addition, the phosphorylation of AnxA2 at Tyr23 is an important event for its entry into the exosomes that promotes invasion and proliferation in cancer cells.
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    Long Term Survival in Pediatric Renal Cell Carcinoma despite multiple relapses: A Case Study
    (2019-03-05) Bowman, William; Dzurik, Yvette; Patel, Nidhi
    Background Although common in adults, renal cell carcinoma (RCC) is extremely rare in pediatric populations, comprising of less than 5% of malignant renal tumors. Literature on the topic is scarce, and there currently is no protocol for the treatment of pediatric RCC. This study focuses on a pediatric RCC patient who experienced multiple relapses of the disease, and aims to highlight the multifocal approach taken to care for the patient. Case History A 6 year old male presented in 2006 with a chief complaint of 1 week of painless hematuria, and was found to have a renal mass by abdominal examination. He was diagnosed with type II papillary RCC based on pathological examination of tissue following radical nephroureterectomy and regional lymph node excision. Over the next 7 years the patient had several regional and distal metastases of the disease, which required different approaches including a right pneumonectomy, stereotactic spinal radiosurgery, external beam radiotherapy, and chemotherapy with Sunitinib. In addition, patient developed posterolateral right sided rotation of his heart (iatrogenic retrodextrocardia) after the pneumonectomy. Despite several relapses, the patient has done well with treatment, and has not shown any evidence of metastasis or active disease in the last five years, with the most recent follow up being in 2018. Conclusion Renal cell carcinoma is an extremely rare malignancy in pediatric populations, and research on it is sparse. Studies for the development of appropriate treatment protocols are being explored currently, however, nothing concrete exists currently. The patient in our case study portrays the success of an individualized approach, and calls for attention to the need for further research in treatment options, and the development of a standardized protocol. We hope that this case report serves as a source of optimism, and increases clinicians’ knowledge of treatment approaches that have shown positive results for this rare pediatric neoplasm.
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    Survivin as a New Target for Neuroblastoma
    (2019-03-05) Basha, Riyaz; Umesh, Sankpal; Hernandez, Yazmin; Zakhary, Emily
    Background: Neuroblastoma is the most common solid peripheral nervous system pediatric tumor found in pediatrics. Neuroblastoma (NB) commonly metastasizes through the lymphatic system and bone marrow, and has a particularly poor prognosis in children older than 18 months, with five-year survival rates typically around 40-50%. Our group is investigating for less toxic agents against NB cells. We found that Tolfenamic acid (TA), a small molecule had anti-cancer effects in high risk neuroblastoma (HRNB) cell lines. Survivin has been associated with poor prognosis in several types of cancers. Survivin is a protein that specifically inhibits the caspase apoptotic proteins thus negatively regulating apoptosis. We conducted a search of various databases in order to investigate the association of Survivin with the survival of NB patients. The other objective was to test an agent that could target Survivin an inhibit NB cell growth. Materials: We utilized R2 genomics and visualization platform to generate survival curves, looking specifically at overall and relapse-free survival probabilities in NB patients. The graphs were made using the data from 88 patients. We also used NB cell line for in vitro testing. SH-SY 5Y cells were cultured it in the presence of copper TA and cell viability was assessed at 24 and 48 hours. Protein extracts were prepared and analyzed for the expression of Survivin using Western blot analysis. Results: We found that the overall survival probability for NB patients with high expression of Survivin had a significantly poorer prognosis (p: 8.5e-9), than those with lower expression of Survivin. Similarly, the relapse-free survival probability curve also demonstrated that high expression of Survivin was associated with a poorer prognosis (p: 1.9e-6) than patients who had lower expression levels. Thus demonstrating that there is a strong association with high Survivin expression and poor prognosis. We also found that Cu-TA acted as an effective inhibitor to Survivin in our laboratory research with the SHSY-5Y cell line. Conclusion: The survival curves showed a strong association of Survivin with poor prognosis. The complexed agent, Cu-TA acts as an efficacious inhibitor of the HRNB cell line SHSY-5Y potentially inhibiting Survivin. Going forward, further research should be done to identify novel less toxic therapeutic agents to target Survivin, in hopes of better treating HRNB cell lines.
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    Lipoprotein Based Targeted Therapy for AML
    (2019-03-05) Raut, Sangram; Sabnis, Nirupama; Lacko, Andras; Mai, Tu
    Background: Acute myeloid leukemia (AML) is the most common acute leukemia in adults; it is only cured in less than 50% of the patients that are diagnosed with the disease. A new approach to the AML treatment is proposed using a nanoparticle system (rHDL) that mimics the structure and function of endogenous high density lipoproteins (HDL) specifically targeting the scavenger receptor type B1 [SR-B1] that is overexpressed in cancer cells. The drug-rHDL complex would be more efficient in specifically delivering the anti-cancer drugs to its target. Objective: Create a new drug delivery system for AML chemotherapy by formulating rHDL nanoparticles with current AML pharmaceutical drugs (e.g. etoposide and azacitadine) for preclinical studies. Method: Revising the existing protocol for rHDL nanoparticle synthesis from Lipoprotein Drug Delivery Research Laboratory at UNTHSC, etoposide was encapsulated in rHDL nanoparticles and left to be characterized. Characterization involved using the high performance liquid chromatography (HPLC) instrument to analyze the compound and the drug content within the nanoparticles. Nanoparticle size, zeta potential and composition will be determined using established methods in the lab. Revisions to the synthesis protocol will be made accordingly in order to maximize the amount of drug encapsulated. Conclusions: A standard curve has been established to characterize the nanoparticle-drug loading created by utilizing high performance liquid chromatography (HPLC). Current work involves characterizing the rHDL nanoparticles for drug loading and physical attributes such as size, zeta potential and chemical composition. After characterization and revising the protocol to maximize the drug content within the nanoparticles, the next step in the process would involve evaluating cytotoxicity in SR-B1 positive AML cells before moving to animal models. The long term goal of the project is to create a new delivery method for AML in order to minimize the side effects and enhance the therapeutic index of multiple AML drugs used in management of AML.
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    Bioengineered Mesenchymal Stem Cell Exosome-Coated Polymeric Nanoparticles for the Treatment of Triple Negative Breast Cancer
    (2019-03-08) Lampe, Jana; Ranjan, Amalendu; Vishwanatha, Jamboor; Joshi, Rohan
    Background: Metastasis is the leading cause of death in breast cancer worldwide. Although there have been many new agents approved for metastatic breast cancer, they are poorly efficacious. Mesenchymal stem cells (MSC) and their exosomes play a role in the tumor microenvironment and they may have tumor homing properties. Our goal is to bioengineer MSC exosome-coated polymeric nanoparticles (BioExoNP) to deliver a chemotherapeutic drug for targeted therapy of TNBC. Methods: To isolate the MSC exosomes, we grew MSC cells in exosome free media and used ultracentrifugation at 100,000 x g for exosome isolation. We used Dynamic Light Scattering (DLS) for size analysis, polydispersity index (PDI) and zeta potential (ZP). Western blotting was used for exosomal protein identification. PLGA polymeric nanoparticles were formulated using the microfluidic based Nanoassembler. Their size, PDI and ZP were obtained using DLS. To make the coated NP we used an extrusion method. High performance liquid chromatography was used for drug loading and encapsulation efficiency. Results: The MSC exosomes had a size of 77 nm, ZP of -14 mV, and PDI of 0.24. The NP also have similar results with a size 76 nm, and PDI of 0.2, however the ZP was -38. Our exosome sample was positive for known exosomal proteins and negative for all other extracellular vesicle markers. After extrusion, the ZP of our sample was closer to -14 mV which, tells us that our sample was coated in exosomal membrane. However, after extrusion we did obtain three populations of NPs: bare NPs, coated NPs and just exosomes. Our sample was further purified using centrifugation. Conclusion: In this study, we have demonstrated that exosome-coated polymeric nanoparticles can be successfully formulated with optimal characteristics. These hybrid nanoparticles were stable and uniform. In future applications, we will use this platform to formulate BioEXoNP and evaluate its therapeutic potential using in vitro and in vivo studies. Funding: Supported by a grant award number RP170301 from the Cancer Prevention and Research Institute of Texas (CPRIT).
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    Anti-Proliferative Effect of Copper Tolfenamic Acid in Neuroblastoma Cell Lines
    (2019-03-05) Basha, Riyaz; Sankpal, Umesh; Maram, Rajasekhar; Grebennikov, Sarah
    Anti-Proliferative Effect of Copper Tolfenamic Acid in Neuroblastoma Cell Lines Sarah Grebennikov, Yazmin Hernandez, Rajasekhar Maram, Umesh T. Sankpal, and Riyaz Basha Background:Neuroblastoma (NB) is a neuroendocrine tumor of the sympathetic nervous system most commonly found in the adrenal medulla. It is the most common extracranial tumor in infants with an average age of onset of 1 year. While presentation in children over the age of 5 is rare, the prognosis is markedly worse due to the higher likelihood of an aggressive malignancy with metastasis to lymph nodes and bone marrow. Current treatment modalities include surgical resection, chemotherapy, radiotherapy, and autologous stem cell transplant. These treatments are highly efficacious, however there are several associated side effects. Specifically, chemotherapeutic side effects are dose dependent and can range from mild stomach pain to more severe and serious complications including hearing loss, myelosuppression, and neurotoxicity. To limit these side effects, we are investigating anti-cancer agents with limited side-effects. Copper Tolfenamic Acid (Cu-TA) is metallic complex of an anti-cancer Non-Steroidal Anti-inflammatory Drug, Tolfenamic acid which is known to inhibit anti-apoptotic protein, Survivin and inhibits cancer cell growth. Hypothesis: We hypothesize that Cu-TA down-regulates survivin and inhibits neuroblastoma cell growth more effectively than TA. Methods: NB cell lines, SMS-KCNR and LA155n cell lines (from ATCC) were treated with increasing concentrations of Cu-TA or TA (0, 5, 10, 20, 40 and 80 µM). CellTiter-Glo reagent was added to the 96-well plate, and readings were taken at 24 and 48 hours. Using this data, IC50 values were calculated using SigmaPlot software. The effect of Cu-TA on Survivin protein expression was measured using western blot analysis. Results: Cell viability data showed a dose dependent decrease due to Cu-TA treatment in both cell lines. Analysis of the Western Blot confirms that there was a decrease in the survivin protein in the cells treated with Cu-TA. Conclusion:These results demonstrate that Cu-TA is an inhibitor of survivin and more effective at inhibiting NB cancer cells than TA alone. Since survivin is associated with resistance to chemotherapy, if Cu-TA sensitizes NB cells to chemotherapy, it will help reduce the side effects of chemotherapy while maintaining the efficacy of treatment.
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    Delayed Diagnosis of Ewing Sarcoma: A Case Series
    (2019-03-05) Bowman, Paul MD; Hamby, Tyler PhD; Richie-Gillespie, Mayme MD; St. Louis, Blake
    Background: Ewing Sarcoma, the second most common bone cancer in adolescents, affects young adults between the ages of 15 to 24. Ewing Sarcoma presents with a variety of non-specific symptoms, including pain and possible mass; the average delay in diagnosis is 6 months. Physician-related delays, defined as the time when a patient first presents to a physician to the time of diagnosis, account for 63.6% of delays. Adolescent (15-19 years old) patients have a notably longer patient-related delay, defined as the time when a patient first notices a symptom to the time they present to a physician, contributing to an overall longer delay in diagnosis. Patients diagnosed without metastasis have a 70% survival rate, whereas patients with metastasis present have only a 20% survival rate. By reviewing three cases of delayed diagnosis in Ewing Sarcoma, we aim to increase awareness of providers to improve earlier diagnosis of Ewing Sarcoma. Methods: Electronic medical records of three patients were reviewed at Cook Children’s Medical Center. Case Information: Three cases of adolescent Ewing Sarcoma diagnosed between 2010-2018 were evaluated. One patient was diagnosed with sciatica followed by a herniated disc after a car accident and had a six-month delay. The patient did not respond to treatment and was referred to a pain management specialist. At diagnosis, the patient had metastasis to multiple areas and is currently undergoing treatment. The other two patients had delays of approximately 12 months and were being treated by either a chiropractor or physical therapist. Both patients associated their pain to the musculoskeletal system and delayed going to a physician. One patient had metastasis to multiple sites at diagnosis, while the other patient had no metastasis. Both patients are currently in remission. Conclusions: Ewing Sarcoma presents with nonspecific symptoms and can have lengthy delays in diagnosis. Timely diagnosis is important because longer delays can result in metastasis, a need for more intensive therapy, and a worse prognosis. As demonstrated by these cases, pain onset, proper imaging guidelines, age-specific statistics, avoiding specialty bias, and enhanced provider awareness are important considerations in developing an appropriate differential diagnosis and earlier recognition of Ewing Sarcoma. In presenting these cases we seek to improve awareness and suspicion of Ewing Sarcoma among practitioners evaluating patients such as we have described.
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    Identifying Top Gene Contributors to Triple Negative Breast Cancer Health Disparities Among African American Women: A Machine Learning Approach
    (2019-03-05) Liu, Jin Ph.D.; Hayatshahi, Hamed Ph.D.; Morid, Mohammad Amin Ph.D; Green, Amyia; Fluker, Kenneth Jr.; Ahuactzin, Emilio; Radler, Charlene
    Purpose: Triple Negative Breast Cancer (TNBC) is a breast cancer subtype which multiple studies have shown to be disproportionately prevalent among premenopausal African American women. The factors contributing to the TNBC health disparities remain unclear. Methods: Here, we developed a highly accurate, reproducible machine learning classification model that used patient gene expression values as predictor attributes to classify 100 TNBC patients as either African American or non-African American. Results: By using weighting methods and comparison of classification performance at varying levels of attributes, our study identified a subset of genes able to accurately classify TNBC patients by race. Intriguingly, the top genes of this subset are linked to diabetes, indicating that diabetes may associate with the TNBC health disparities. Conclusions: Our study demonstrated the factors contributing the TNBC health disparities and provided a subset of genes that may be targetable for precision medicine development to address disparity of TNBC among the African American female population.
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    Trial of Sirolimus in Gorham Disease
    (2019-03-05) Heym, Kenneth MD; Akers, Lauren DO; Ray, Anish MD; Merchant, Zahra
    Background: Gorham Disease, also known as Gorham-Stout Disease, massive osteomyelitis, or vanishing bone disease, is a rare disorder of poorly understood etiology. Patients undergo bone resorption without proliferation and have significant soft-tissue swelling. No standard treatment is currently available. Novel use of the MTOR inhibitor sirolimus has shown some promise in slowing the progression of this devastating disorder. Immunosuppressant agents such as sirolimus have been used effectively in transplant patients for prophylaxis of organ rejection. We hypothesize that this agent can also successfully slow the progression of bone resporption in patients with Gorham Disease. This case series describes the diagnosis, progression, and therapy of three patients in the Cook Children’s Medical Center hematology-oncology department with varying levels of severity of Gorham Disease. Case Information: Three patients were diagnosed with Gorham Disease between 2011 and 2014 and are now being treated within the Cook Children’s Medical Center Hematology-Oncology practice. One patient has involvement of her skull base and ear canals, diagnosed after ear canal abnormalities were detected on CT following meningitis. The second patient has involvement of her posterior ribs and T7-T12 vertebral bodies, with thoracic instability and necessity of either remaining in the supine position or wearing a back brace. The third patient has involvement of his left lower extremity and left hemipelvis, necessitating a left above knee amputation and subsequent disease progression. The first two patients have had radiographic improvement after the addition of twice daily sirolimus, while the third patient has had steadying of his disease. Conclusions: Gorham Disease is a rare condition with possibly devastating effects. The introduction of sirolimus, in select cases, has appeared to either steady or slowly reverse the progression of the disease. While more studies need to be performed to understand the full effects of sirolimus on this disease, it has the potential to have a significant role in the treatment of Gorham.
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    The role of Specificity Protein 1 and downstream target Survivin in Ovarian Adenocarcinoma Survival and Prognosis
    (2019-03-05) Nash, Madeline; Chandra, Ashwin; Nair, Maya; Vishwantha, Jamboor; Basha, Riyaz; Lin, Christine
    Purpose: Ovarian adenocarcinoma is a solid proliferation of ovarian glandular epithelium. These are the most threatening types of gynecologic malignancies. Although many risk factors are involved in determining prognosis, the study of germline mutations related to tumorigenesis is the focus of this study. It is well established that Specificity protein 1 (Sp1), a transcription factor regulates key genes associated with cancer including survivin (baculoviral inhibitor of apoptosis repeat-containing 5 or BIRC5). Survivin is an anti-apoptotic protein which is known to be associated with poor prognosis in multiple cancers and displays therapy-resistant tumor characteristics. Sp1 has also been associated with poor prognosis in several different types of cancers. The role of these two proteins in the development of ovarian adenocarcinoma and their impact on prognosis remains unclear. Methods: The R2 genomics visualization platform was used to generate Kaplan-Meier curves for survival probabilities in patients with ovarian adenocarcinoma who express high and low levels of Sp1 and Survivin. Both curves were generated using data from 107 patients. There were 38 patients in the high Sp1 expression group and 69 patients in the low Sp1 expression group. In the Survivin (BIRC5) group there were 70 patients with high expression and 37 patients in the low expression group. Results: The survival curves show that patients with ovarian adenocarcinoma who express higher levels of Sp1 had a significantly poorer prognosis compared to patients with lower levels of Sp1 expression (p = 0.023). Similarly, patients with high levels of survivin protein expression were found to have a significant decrease in overall survival probability compared to patients who had low levels of survivin expression (p = 0.049). Conclusion: There is a strong association with high expression of either Sp1 or survivin with poor prognosis in patients with ovarian adenocarcinoma. Moving forward, studies are in development to elucidate the roles that Sp1 and survivin play, individually and together, in the evolution of ovarian adenocarcinoma. We are also interested in exploring the relationship of differential expressivity of Sp1 and survivin in association with age, race and ethnicity among patients with ovarian cancer.
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    (2019-03-05) Chaudhary, Pankaj; Vishwanatha, Jamboor; Nsiah, Nana Yaa
    PURPOSE: Breast cancer is the second leading cause of cancer-related deaths in women. Metastasis accounts for majority of breast cancer deaths. It remains a major barrier to cancer treatment due to limitations in diagnosis and lack of effective therapy. Understanding the role of underlying molecular mechanisms involved in metastasis could lead to effective therapy to prevent and treat breast cancer. Migration and Invasion Enhancer 1 (MIEN1), is a novel gene abundantly expressed in different tumors compared to normal cells. Our previous studies have shown it plays a critical role in regulating cell migration and invasion to promote metastases. It is located on chromosome 17q12 near the Her 2/neu oncogene. MIEN1 protein is a membrane-anchored signal protein, with important structural motifs such as the Immunoreceptor tyrosine-based activation motif (ITAM), a redox active motif and a prenylation sequence at the carboxyl terminal. Here, we evaluated the expression of MIEN1 in breast cancer patients with clinical outcome. METHODS: We analyzed The Cancer Genome Atlas (TCGA) Breast Invasive Carcinoma (BRCA) database to observe MIEN1 mRNA expression in breast cancer subtypes and its correlation with survival. Also, we assessed MIEN1 expression in a panel of normal and breast cancer cell lines using Western blot. RESULTS: MIEN1 gene expression was significantly increased in different subtypes of breast carcinomas (Invasive ductal carcinoma, Invasive lobular carcinoma, Mixed Ductal and Lobular, and Mucinous) compared to normal tissues. Moreover, MIEN1 is predominantly overexpressed in HER2+ breast cancer patients compared to other subtypes. However, MIEN1 expression in luminal A, luminal B and basal-like subtypes were also high in comparison to normal breast tissues. High expression levels of MIEN1 was associated with reduced overall survival (HR = 1.61; 95% CI = 1.34-1.94, P = 0.0001). Screening of MIEN1 expression in various breast cancer cell lines suggest that expression of MIEN1 is high in majority of them compared to immortalized normal mammary epithelial cell line. CONCLUSION: Our findings confirm that MIEN1 is an important oncogene, and its increased expression in breast cancer contributes towards an aggressive disease with poor survival.
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    Trial of Anakinra in Secondary Hemophagocytic Lymphohystiocytosis
    (2019-03-05) Guirola, Ricardo MD; Perez, Maria MD; Ray, Anish MD; Merchant, Zahra
    Background: Hemophagocytic lymphohystiocytosis (HLH) is is an uncommon yet potentially devastating systemic disease, arising from uncontrolled activation of the immune system. Secondary HLH can be triggered by malignancy, metabolic disease and infection. Use of the IL-1 receptor antagonist, anakinra, has shown some promise in the treatment of this disorder. Interleukin-1 receptor antagonists such as anakinra have been used effectively in the treatment of pediatric rheumatoid arthritis. We hypothesize that this immunomodulator can also successfully treat patients with secondary HLH. This case series describes the diagnosis, progression, and therapy of three patients at Cook Children’s Medical Center with secondary HLH. Case Information: Three patients within the Cook Children’s network were diagnosed with HLH between 2014 and 2016 and treated per HLH-2004 protocol. The 1st patient, diagnosed with HLH at age 10, sustained relapse after five weeks of therapy with etoposide and dexamethasone, requiring reintensification. Thereafter, she developed fever, body aches and cytopenias with eventual diagnosis of juvenile rheumatoid arthritis, treated with prednisone and Anakinra, leading to remission. Thereafter, she was treated with tociluzumab, progressively weaned and now discontinued for several months, given continued remission. The 2nd patient, 16-year-old male, completed treatment for HLH followed by rising levels of ferritin generalized erythematous rash, swollen right elbow with erythema and pain. At that time, he was diagnosed with systemic rheumatoid arthritis and treated with Anakinra daily. He is currently in remission while continuing Anakinra for 10 months. The 3rd patient, 12-year-old female with past medical history of type 1 diabetes and idiopathic juvenile arthritis, was diagnosed with EBV driven HLH, treated with dexamethasone, etoposide, and rituximab. As part of workup, she was found to have depressed NK cell activity. She presented a year later with symptoms concerning for recurrence of HLH. Given her history of juvenile idiopathic arthritis, she was treated with Anakinra with resolution of her symptoms. She remains in remission for over two years. Conclusions: The diagnosis of HLH can be elusive and is often accompanied with multiple systemic manifestations. For those patients with a concurrent diagnosis of Juvenile Rheumatoid Arthritis, the introduction of anakinra has shown significant improvement in both arthritic symptoms and HLH markers.