Fresh Patient Samples Research Articles

EXTH-73. HETEROGENEITY IN CELL SURFACE ANTIGEN AND HLA CLASS I EXPRESSION IN PEDIATRIC BRAIN TUMORS AND ITS IMPACT ON T-CELL IMMUNOTHERAPY

Abstract Immunotherapy with tumor antigen-specific chimeric antigen receptor (CAR) and/or ab (T-cell receptor) TCR T-cells has the potential to improve clinical outcomes of patients with pediatric brain tumors. As a prerequisite for successful T-cell therapies, we must determine which cell surface antigens are expressed and targetable in these tumors, and if HLA Class I is present, which is necessary for ab TCR T-cell recognition. Therefore, in this study we systematically analyzed pediatric patient-derived orthotopic xenograft (PDOX) brain tumor samples for cell surface expression of five known CAR targets: IL13Ra2, HER2, EphA2, B7-H3, and GD2, as well as HLA Class I. We established and validated a flow cytometry-based method of profiling tumor-associated antigens. Fifty-three PDOX samples have been profiled to date, including medulloblastoma, high grade glioma (HGG), diffuse intrinsic pontine glioma (DIPG), atypical teratoid rhabdoid tumor (ATRT), and ependymoma, among others. Our results showed high variability within and between individual samples. B7-H3 was the most consistently expressed, seen in 98% of the samples tested. We validated these results by conventional immunohistochemistry staining for B7-H3 and found comparable RESULTS: HLA Class I was highly expressed on all HGG samples but was undetectable on 47.8% of other brain tumor samples. This suggests that down-regulation of HLA class I is one mechanism by which brain tumors evade conventional T-cells, and that HLA-independent CAR T-cells would be useful therapies. We also compared expression of antigens in fresh patient samples and corresponding PDOX tumors and saw that they were indeed similar. To our knowledge, this is the largest group of pediatric brain tumor PDOX samples methodically analyzed for potential CAR target antigens and HLA Class I. Taken together, our data demonstrate that elimination of tumors by CAR T-cell immunotherapies will require targeting multiple antigens, and our profiling method could inform how to circumvent antigen-negative relapse.

Abstract B31: Detection of viable tumor cells from cryopreserved buffy coat using the VTX-1 Liquid Biopsy Platform

Abstract Introduction: Several circulating tumor cell (CTC) technologies have been developed amidst growing interests in the “liquid biopsy” field. The fully automated VTX-1 platform is developed to gently isolate intact CTCs from whole blood with high recovery and viability, compatible with many applications (1-3). However, many CTC technologies require processing of fresh patient samples at off-site facilities. Here we present encouraging preliminary results showing viable cell isolation by VTX-1 from cryopreserved buffy coat, using a telomerase-selective replicating adenovirus (TelomeScan). This widens potential applications of viable CTC detection for retrospective clinical studies and drug development. Methods: Buffy coat preparation: Buffy coat was prepared by centrifugation of healthy donor blood collected in EDTA, heparin, or heparin CPT vacutainers. Cell spiking and processing: 1) Cancer cells were spiked into freshly prepared buffy coat and processed with the Vortex chip at different dilutions. 2) Cancer cells were labeled and spiked into healthy donor blood, and buffy coat was prepared and processed by VTX-1 either immediately or following cryopreservation up to 1 week. Imaging was evaluated for cell recovery. Viable, telomerase-activated tumor cell detection: VTX-1 enriched cells were directly collected in chamber slides and infected with the TelomeScan adenovirus (OBP-401) for 24 h (4). The percentage of GFP (+), viable, telomerase-expressing cells was evaluated alongside cell recovery. Results: MCF7 cells were spiked into freshly prepared buffy coat, resuspended in 8mL, and processed at 5X, 10X, and 20X dilutions. The best compromise between recovery, WBC count, and processing time was obtained for the 10X dilution with 46% recovery. The majority of spiked cells (MCF7 and H1299) collected from buffy coat were shown to be viable, as demonstrated by infection with the telomerase-selective replicating adenovirus (TelomeScan, OBP-401). Cryopreservation of the buffy coat samples showed a slight reduction following cryopreservation. Discussion: The optimal performance of the Vortex chip on buffy coat samples has previously been shown at 10X dilution. This capability of the VTX-1 was further demonstrated through infection with a telomerase-selective replicating adenovirus. This combined workflow has shown seamless integration from whole blood. Noteworthy recovery and sensitivity were also observed with cryopreserved buffy coat. Patient biobanked samples are currently being pursued. The overall advantages are: a) viable cell detection, b) direct collection of cells into culture media ready for in vitro testing, c) no requirements for fixation, labeling, backflushing, d) a very low WBC background eliminating false positives, and e) the potential to retrospectively analyze clinical biobanked samples at a centralized laboratory.

Abstract P3-06-04: Tumor specific cargo release in ex vivo patient samples and murine models of triple negative breast cancer by a pH-targeted nanoparticle: V3-RUBY

Abstract Background: Triple negative breast cancer (TNBC), unlike other breast cancer subtypes, lacks a specific targetable receptor. As such tumor specific delivery, which can limit off-target effects of anti-neoplastic therapies, has been an unmet clinical need in treating this aggressive breast cancer subtype. To address this need, we exploited the tumor hallmark of an acidic microenvironment and developed a pH targeted nanoparticle by conjugation of the V3 pH specific peptide on a wormhole pore mesoporous silica nanoparticles (V3-RUBY) and assessed the ability of V3-RUBY to specifically release cargo in ex vivo patient samples and in orthotopically implanted TNBC tumors as detect by multispectral optoacoustic imaging technology (MSOT). Methods: The silica nanoparticles with wormhole pore architecture were synthesized by sol-gel chemistry and characterized by transmission electron microscopy (TEM), zeta potential, and dynamic light scattering (DLS). The surface base particle was crosslinked with a gatekeeper molecule, chitosan. Further targeting of the particle using V3 pHLIP (low insertion peptide), resulted in the V3-RUBY nanoparticle which allows for pH-sensitive cargo release. The particle was loaded with imaging dye to assess tumor specificity. Fresh ex vivo TNBC patient tumor tissues were resected and rapidly treated with V3-RUBY containing propodium iodide (PI) to evaluate tumor uptake of V3-RUBY and cargo release within the tumor cells, as measured by the red fluorescence of PI when bound to nucleic acids. (PI independently cannot cross cell membranes.) In vivo, female athymic mice were implanted with MDA-MB-468 breast cancer cells by the mammary fat pad injection. Once the tumor reached 3mm in size, athymic mice were intravenously injected with V3-RUBY nanoparticles carrying IR780 infrared imaging dye and were imaged with MSOT inVision 512TF. Results: The RUBY nanoparticle with wormhole pores was 27 nm diameter. The dual targeting approach of a nanoparticle with V3 targeting peptide and chitosan demonstrated pH specificity around tumor pH. In ex vivo patient TNBC samples, V3-RUBY demonstrated active targeting and dye release at pH 6.8, which approximated the pH measured at surgical tumor removal, in 10 fresh patient samples compared to pH 7.4 controls (p<0.0001, N=10) (Table). There was a non-significant trend towards lower uptake in histological grade 2 tumors, relative to grade 3 tumors. In the murine models, IR780 uptake in the MDA-MB-468 tumors measured 23.2 a.u (arbitrary units) and was 3.1, a.u. in the liver and 0.9 a.u. in the kidneys (p <0.0001, N=5). Tumor accumulation in the MDA-MB-468 model was significantly increased compared to both liver and kidneys (p=0.0002 and p=0.0003). Conclusion: The tumor-specific release of payload by V3-RUBY suggests the potential of a pH specific target in TNBC, with such a nanoparticle holding promise to deliver both diagnostic and therapeutic cargo directly to the tumor and limit off-target toxicity. Future translation of these technologies could have promise in TNBC, as well as other high-grade breast cancer subtypes and expand treatment options in this challenging area of oncology. TablePatientBreast Cancer SubtypeTumor GradePathologic StagePI Uptake (a.u.) pH 6.8PI Uptake (a.u.) 7.41TNBC3ypT3N3a33.20.82TNBC3ypT2N038.41.53TNBC3ypT4bN1a36.81.74TNBC3T1bN022.40.85TNBC2T1cN021.80.86TNBC2ypT0N021.50.67TNBC3ypT1aN2a22.00.48TNBC3ypT4dN239.52.19TNBC3T2N2a31.52.210TNBC3T2N1a32.21.8 Citation Format: Alexandra Thomas, Akiko Chiba, Abhilash Samykutty, Molly W. McNally, Lacey R. McNally. Tumor specific cargo release in ex vivo patient samples and murine models of triple negative breast cancer by a pH-targeted nanoparticle: V3-RUBY [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-06-04.

LGALS3 is connected to CD74 in a previously unknown protein network that is associated with poor survival in patients with AML

BackgroundGalectin 3 (LGALS3) gene expression is associated with poor survival in acute myeloid leukemia (AML) but the prognostic impact of LGALS3 protein expression in AML is unknown. LGALS3 supports diverse survival pathways including RAS mediated cascades, protein expression and stability of anti-apoptotic BCL2 family members, and activation of proliferative pathways including those mediated by beta Catenin. CD74 is a positive regulator of CD44 and CXCR4 signaling and this molecule may be critical for AML stem cell function. At present, the role of LGALS3 and CD74 in AML is unclear. In this study, we examine protein expression of LGALS3 and CD74 by reverse phase protein analysis (RPPA) and identify new protein networks associated with these molecules. In addition, we determine prognostic potential of LGALS3, CD74, and their protein networks for clinical correlates in AML patients. MethodsRPPA was used to determine relative expression of LGALS3, CD74, and 229 other proteins in 231 fresh AML patient samples and 205 samples were from patients who were treated and evaluable for outcome. Pearson correlation analysis was performed to identify proteins associated with LGALS3 and CD74. Progeny clustering was performed to generate protein networks. String analysis was performed to determine protein:protein interactions in networks and to perform gene ontology analysis. Kaplan-Meir method was used to generate survival curves. FindingsLGALS3 is highest in monocytic AML patients and those with elevated LGALS3 had significantly shorter remission duration compared to patients with lower LGALS3 levels (median 21.9 vs 51.3 weeks, p = 0.016). Pearson correlation of LGALS3 with 230 other proteins identifies a distinct set of 37 proteins positively correlated with LGALS3 expression levels with a high representation of proteins involved in AKT and ERK signaling pathways. Thirty-one proteins were negatively correlated with LGALS3 including an AKT phosphatase. Pearson correlation of proteins associated with CD74 identified 12 proteins negatively correlated with CD74 and 16 proteins that are positively correlated with CD74. CD74 network revealed strong association with CD44 signaling and a high representation of apoptosis regulators. Progeny clustering was used to build protein networks based on LGALS3 and CD74 associated proteins. A strong relationship of the LGALS3 network with the CD74 network was identified. For AML patients with both the LGALS3 and CD74 protein cluster active, median overall survival was only 24.3 weeks, median remission duration was 17.8 weeks, and no patient survived beyond one year. InterpretationThe findings from this study identify for the first time protein networks associated with LGALS3 and CD74 in AML. Each network features unique pathway characteristics. The data also suggest that the LGALS3 network and the CD74 network each support AML cell survival and the two networks may cooperate in a novel high risk AML population. FundLeukemia Lymphoma Society provided funds to SMK for RPPA study of AML patient population. Leukemia Texas provided funds to PPR and SMK to study CD74 and LGALS3 expression in AML patients using RPPA. No payment was involved in the production of this manuscript.

Proteolysis targeting chimeric molecules as therapy for multiple myeloma: efficacy, biomarker and drug combinations.

Proteolysis targeting chimeric molecule ARV 825 causes ubiquitination of bromodomains resulting in their efficient degradation by proteasome activity. Bromodomain degradation down-regulates MYC transcription contributing to growth inhibition of various human cancers. We examined the therapeutic potential of ARV 825 against multiple myeloma (MM) cells both in vitro and in vivo. In a dose-dependent manner, ARV 825 inhibited proliferation of 13 human MM cell lines and three fresh patient samples, and was associated with cell cycle arrest and apoptosis. ARV 825 rapidly and efficiently degraded BRD 2 and BRD 4. Sensitivity of MM cells to ARV 825 was positively correlated with cereblon levels. RNA sequencing analysis showed important genes such as CCR1, RGS, MYB and MYC were down-regulated by ARV 825. A total of 170 small molecule inhibitors were screened for synergy with ARV 825. Combination of ARV 825 with inhibitor of either dual PI3K/mTOR, CRM1, VEGFR, PDGFRα/b, FLT3, IGF-1R, protein kinase C, CBP-EP300 or JAK1/2 showed synergistic activity. Importantly, ARV 825 significantly inhibited the growth of MM xenografts and improved mice survival. Taken together, our results, in conjunction with recently published findings, provide a rationale for investigating the efficacy of ARV 825 for MM, use of cereblon as a biomarker for therapy of MM patients, and the combination of ARV 825 with small molecule inhibitors to improve the outcome of MM patients.

Isolation and Culture of Glioblastoma Brain Tumor Stem Cells.

Cancer stem cells (CSCs) have been identified in glioblastoma (GBM) and are proposed to be the main actors of post-treatment recurrence contributing to the very dismal prognosis of this devastating disease. Consequently, this important population of cells needs to be further studied to uncover potential vulnerabilities, identify novel therapeutic targets, and develop drugs that can be translated to the clinic. One obstacle preventing progress in understanding the biology of GBM and the development of novel therapies has arguably been the absence of biologically relevant in vitro models representative of the CSC population in GBM. Adherent and non-adherent serum-free culture methods, initially developed for culturing neural stem cells, have been adapted to identify, isolate, maintain, and expand brain tumor stem cells (BTSCs) from GBM. In this chapter, we describe a method to isolate and culture these BTSCs from fresh GBM patient samples.

Immunohistochemistry analysis of Pygo2 expression in central nervous system tumors.

Pygo2 as a Wnt signaling pathway component has been detected in multiple cancer types. In this study, we identified Pygo2 expression features by immunohistochemistry in 73 central nervous system tumor specimens, in comparison with 14 normal brain tissues and surrounding non-tumorous tissues of tumor. Our study indicated that 59% of the patient tumor specimens exhibited positive Pygo2-staining and increases intensity with the grade of malignancy, especially for WHO grade III and IV gliomas, was observed high level expression, compared with normal brain tissues. Five out of nine WHO grade III anaplastic astrocytomas and seven out of nine WHO grade IV glioblastomas showed Pygo2-positive staining. The analysis of Pygo2 gene expression by quantitative real-time PCR of additional ten fresh patient samples yielded similar results. Further studies performed with stable cell lines in vitro demonstrated that Pygo2 render cells higher proliferation rate, migration and anchorage-independent colony-forming ability in soft agar. Taken together, our studies suggest an important role of Pygo2 in brain tumor progression.

Electrical Characterization of Human Biological Tissue for Irreversible Electroporation Treatments.

Irreversible electroporation (IRE) is a cancer therapy that uses short, high-voltage electrical pulses to treat tumors. Due to its predominantly non-thermal mechanism and ability to ablate unresectable tumors, IRE has gained popularity in clinical treatments of both liver and pancreatic cancers. Existing computational models use electrical properties of animal tissue that are quantified a priori to predict the area of treatment in three dimensions. However, the changes in the electrical properties of human tissue during IRE treatment are so far unexplored. This work aims to improve models by characterizing the dynamic electrical behavior of human liver and pancreatic tissue. Fresh patient samples of each tissue type, both normal and tumor, were collected and IRE pulses were applied between two parallel metal plates at various voltages. The electrical conductivity was determined from the resistance using simple relations applicable to cylindrical samples. The results indicate that the percent change in conductivity during IRE treatments varies significantly with increasing electric field magnitudes. This percent change versus applied electric field behavior can be fit to a sigmoidal curve, as proposed in prior studies. The generic conductivity data from human patients from this work can be input to computational software using patient-specific geometry, giving clinicians a more accurate and personalized prediction of a given IRE treatment.

305 Speckle-Free and Large Gold Nanorod Enhanced Optical Coherence Tomography for Brain Tumor Margin Detection

Abstract INTRODUCTION Optical coherence tomography (OCT) is an emerging technology with the potential to allow for rapid intraoperative detection of brain tumor margins by detecting differences in structure, intensity, spectral signal, and attenuation. OCT systems are capable of rapid imaging of large three-dimensional volumes with cellular level resolution. However, OCT imaging has previously been limited by speckle artifact and the lack of suitable contrast agents, limitations that are surmounted in this study. METHODS We prepared nude mice with orthotopic U87 glioblastoma xenografts and glass cranial windows. We also created large gold nanorods (LGNR) with plasmonic peaks tuned to the spectral range of the OCT scanner. LGNRs were injected intravenously into tumor-bearing mice and OCT imaging was performed in vivo utilizing a novel method for the removal of speckle artifact called Speckle-Free OCT (SFOCT). Fresh ex-vivo patient samples were also imaged. RESULTS >OCT and SFOCT readily distinguished tumor from normal brain with cellular level spatial resolution and to a depth of 1.5 mm. Additionally, SFOCT allowed for the highest resolution ever seen in vivo of mouse white matter architecture. Cortical layers were also readily visible in SFOCT in both live mice and in the ex-vivo human samples, representing a novel ability to interrogate cortical cytoarchitecture across a large field of view. Systemically administered LGNRs were tumor specific and provided excellent spectral contrast using OCT. Ex-vivo hyperspectral and IHC imaging confirmed the localization of LGNRs within the tumor and found that the LGNRs were largely localized within tumor associated macrophages. CONCLUSION SFOCT and LGNR enhanced OCT imaging are promising state of the art technologies for intraoperative tumor margin detection.

Abstract 5783: In vitro modeling of patient derived bladder cancer cell lines in 3D culture systems

Abstract Background: Drug screening is a key component for drug development and optimizing anti-tumor therapies. Traditionally, in vitro drug testing has been conducted in monolayer systems that are not capable of recapitulating the tumor complexity. Recently, the field has witnessed the rise of interest in 3D culture systems which are capable of reproducing tumor complexity while circumventing the cost associated with in vivo drug testing. Our access to fresh patient samples has enabled us to establish a novel 3D culture system consisting of bladder cancer patient derived cell lines. Using a wide range of matrices and co-culture conditions with tumor associated stromal cells we were able to establish a unique high throughput drug testing tool. Methods: Matrigel and collagen based matrices were used to establish 3D culture systems of bladder cancer patient derived cells. Tumor cells were cultured in 3D conditions either alone or in coculture with tumor associated stromal cells. Response to Cisplatin and PI3K pathway targeted agents (i.e. LY LY3023414) was tested in both conditions. High throughput imaging via Thermo ArrayScan XTI was used to assess the biological behavior of spheroids as well as their response to therapies overtime. Confocal microscopy was used to validate the biological mimicry of tumor derived spheroids to the original patient tumors. Integration of RNA-seq data from the patient-derived tumor cells with the biological behavior and therapeutic response in 3D culture is ongoing for the purpose of characterizing the 3D model Results: In 3D culture conditions; bladder cancer derived cells were able to re-express E-cadherin that was suppressed upon propagation in monolayer. The re-expression of the epithelial marker (E-cadherin) observed in 3D accurately mirrors the original tumors; which are of epithelial origin. Phenotypic differences were observed across different matrix conditions and also among different tumor derived cells. Bladder 3D organoids of luminal origin were more sensitive to both cisplatin and PI3K pathway inhibitors as compared to those of basal origin. This drug response profile was reminiscent of what we observed in vivo using patient derived xenograft (PDX) models derived from the same tumors. The phenotypic as well as the drug response variations observed in our 3D culture correlated with variable gene expression profiles (luminal vs basal) that were detected in our RNA-seq data. Conclusion: As compared to monolayer, 3D culture is more capable of recapitulating tumor complexity and accurately reflects the drug resistance / sensitivity profiles that are observed in PDX models in vivo. Therefore, a 3D culture system provides an invaluable tool for high throughput screening of drugs in bladder cancer and providing a better understanding of tumor biology in the search of more effective treatments for bladder cancer patients. Citation Format: May Elbanna, Sreenivasulu Chintala, Eric Ciamporcero, Remi Adelayie, Ashley Orillion, Sreevani Arisa, Nur Damayanti, Michelle Grimard, TJ Puls, Sherry Harbin, Melissa Fishel, Roberto Pili. In vitro modeling of patient derived bladder cancer cell lines in 3D culture systems [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5783. doi:10.1158/1538-7445.AM2017-5783

Abstract 4562: Exploring the immune stimulatory properties of oncolytic Newcastle Disease Virus

Abstract Newcastle Disease Virus (NDV) is an avian paramyxovirus, which has significant oncolytic activity against mammalian cancers. It is multi-modal in its anti-tumour activity and offers a tumour selective, self-propagating therapeutic with oncolytic activity and immunostimulatory properties. Within the tumour itself, NDV infection is able to modulate the immune suppressive microenvironment and induce positive anti-tumour inflammatory responses. Additionally, we have enhanced the immune modulatory properties of NDV by engineering the virus to express granulocyte/macrophage colony-stimulating factor (GM-CSF). To better understand the immune modulatory mechanisms by which NDV infection is able to alter the tumour microenvironment we investigated the responses of normal human PBMCs and isolated immune cell populations following NDV exposure. Using flow cytometry, cytokine and gene expression analysis we demonstrated activation of the innate immune cells and a robust type I IFN and pro-inflammatory response. 24 hours post infection, innate immune cells (macrophages, natural killer cells and dendritic cells) had upregulated cell surface activation markers and secreted high levels of cytokines in a dose-dependent fashion. Furthermore, by using NDV constructs encoding fluorescent proteins we showed, in a specific subset of myeloid cells, the preferential uptake of virus and subsequent self-limiting viral replication. In a co-culture system, these infected myeloid cells were able to function as ‘virus cellular carriers’ and were able to mediate the efficient transfer of infectious NDV to tumour cells resulting in their oncolytic death. To further investigate the ability of NDV to infect, spread and kill tumour cells in vivo, we utilised tissue slice cultures of fresh patient samples. These studies demonstrated virus replication and transgene expression in tumour slices, as well as the uptake of virus in a small fraction of specific cells in slices of normal liver. Moreover, evidence that NDV was able to alter the tumour microenvironment could be demonstrated in such cultures by sampling the culture supernatants over time. By investigating the immune modulatory properties of NDV in vitro and in vivo we will gain greater insight and understanding of the anti-cancer properties of NDV. This work will also help guide the selection of transgenes for next generation approaches to augment the inherent immunostimulatory properties of NDV, and help inform clinical dosing options such as intravenous infusion or cell-based delivery. Citation Format: Shannon Burke, Ruth Franks, James Harper, Jon Travers, Christel Navarro, Xing Cheng, Robert Wilkinson, Hong Jin, Danielle Carroll. Exploring the immune stimulatory properties of oncolytic Newcastle Disease Virus [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4562. doi:10.1158/1538-7445.AM2017-4562

The Roche Immunoturbidimetric Albumin Method on Cobas c 501 Gives Higher Values Than the Abbott and Roche BCP Methods When Analyzing Patient Plasma Samples.

Serum/plasma albumin is an important and widely used laboratory marker and it is important that we measure albumin correctly without bias. We had indications that the immunoturbidimetric method on Cobas c 501 and the bromocresol purple (BCP) method on Architect 16000 differed, so we decided to study these methods more closely. A total of 1,951 patient requests with albumin measured with both the Architect BCP and Cobas immunoturbidimetric methods were extracted from the laboratory system. A comparison with fresh plasma samples was also performed that included immunoturbidimetric and BCP methods on Cobas c 501 and analysis of the international protein calibrator ERM-DA470k/IFCC. The median difference between the Abbott BCP and Roche immunoturbidimetric methods was 3.3 g/l and the Roche method overestimated ERM-DA470k/IFCC by 2.2 g/l. The Roche immunoturbidimetric method gave higher values than the Roche BCP method: y = 1.111x - 0.739, R² = 0.971. The Roche immunoturbidimetric albumin method gives clearly higher values than the Abbott and Roche BCP methods when analyzing fresh patient samples. The differences between the two methods were similar at normal and low albumin levels.

Comparable Immune Reconstitution Between Ex Vivo Amplification and Un-Manipulated Umbilical Cord Blood Transplantation

Umbilical cord blood transplantation (UCBT) is an alternative in the absence of related or unrelated HLA-matched donor. Defects in the mechanisms of immuno-surveillance, due to the absence of antigen-experienced lymphoid subsets transferred in the cord blood unit (CBU), render recipients more prone to viral infections. Our center conducted a clinical trial testing the benefit of ex vivo amplification of the CD34+ fraction (using SCF, FLT3L, TPO and GCSF) enriched from one CBU on hematopoietic reconstitution, while the CD34- fraction was infused at the same time as the expanded fraction (NCT01034449). We intended to simultaneously evaluate the potential impact of such a procedure on the immune reconstitution following freeze-thaw cycle for the CD34- fraction and on the expansion potential of lymphoid progenitors from CD34+ fraction.We prospectively analyzed fresh patient samples from the clinical trial (called the EVEX), at different time points (Day 42, D100, D180, D360), and, in parallel, from patients receiving one or two un-manipulated CBU during the same period (Control group - CTRL). Immune monitoring included flow cytometry analysis of T-cells (CD3+, CD4+, CD8+) and sub-compartments, B-cells (CD19+), NK cells (CD3-CD16+CD56+) and Dendritic cells (DC).Sixteen patients were included in EVEX, of which 12 were analyzed (4 excluded: 1 not grafted, 1 graft infection, 2 primary rejections before Day 42) and 12 patients in CTRL. In the EVEX group, all patients received reduced intensity conditioning, compared to 6 out of 12 in the CTRL group. GVHD prophylaxis was similar. EVEX showed a shorter duration of neutropenia compared to the CTRL (8.5 versus 17 days, p=0.02). NK and B-cell subsets recovered earlier than T-cells, reaching median normal values at 3 and 6 months, respectively. NK cell count tended to be lower in EVEX, starting at 3 months until 1 year. B-cell counts were lower for EVEX at D360. Both groups recovered T-cells within 12 months, but, interestingly, EVEX recovered earlier, reaching Healthy Donor (HD) median values as soon as D180. The CD4+ T-cells (CD4+T) recovered within 12 months for both groups, with EVEX values tending to plateau at low normal values (Fig 1). CD8+ T-cells (CD8+T) reached HD values within 12 months with an earlier recovery for EVEX (Fig 1). We characterized CD4+T and CD8+T compartments with CD45RA and CD27 delineating: naïve (TN: CD45RA+CD27+), central memory (TCM: CD45RA-CD27+), effector memory (TEM: CD45RA-CD27-) and late effector (TEMRA: CD45RA+CD27-) T-cells. Among the CD4+T, TN and TCM gradually increased until D360 for CTRL, while EVEX reached a plateau at D180. EVEX showed a higher TEMRA CD4+T count at D360. Thus, the plateau observed in the whole CD4+T at D180 in EVEX suggested an altered capacity to induce new CD4+T (thymic output or peripheral expansion), or an exhaustion phenomenon. To this end, we evaluated thymic function via CD31 expression among TN CD4+T and TREC analysis by qPCR. Both methods showed a decreased thymic function in EVEX after D180, probably related to the age difference observed (median: 52 vs. 26 years old for EVEX and CTRL, respectively). TEM and TEMRA CD8+T subsets were higher starting at D180 for the EVEX group. There was no difference between groups for myeloid or plasmacytoid DC.Group outcomes differed in terms of chronic GVHD (cGVHD) (1 vs. 6 patients for EVEX and CTRL respectively, p=0.024). Among the subsets playing a role in cGVHD pathogeny, no difference was found in Treg/Tcon ratio (Treg = CD25hiCD127lo CD4+T, Tcon = non-Treg CD4+T), but memory B-cell subset tended to be greater in CTRL from D42 to D180 (median time of cGVHD onset = 186.5 days).CMV reactivation rate was similar in both groups, occurring around 42 days post transplant. Vdelta-2 negative γd T-cells were greater in EVEX early after infection (D42), suggesting a comparable anti-CMV innate function. However, when looking at adaptive anti-CMV immunity with ELISpot, we found reduced IFNg-secreting cells in EVEX, although group size was small.In conclusion, patients receiving ex vivo expanded CBU showed faster hematopoietic reconstitution than un-manipulated CBU and comparable recovery of the main immune subsets. Baseline clinical differences observed between groups may have impacted on some of the immune findings. A prospective and randomized trial would help to better analyze if the expansion procedure has an impact on immune reconstitution. [Display omitted] DisclosuresMilpied:Celgene: Honoraria, Research Funding.

Phosphoflow-Based Evaluation of Mek Inhibitors as Small-Molecule Therapeutics for B-Cell Precursor Acute Lymphoblastic Leukemia

Upstream mutations that lead to constitutive activation of Erk in B-cell precursor acute lymphoblastic leukemia (BCP-ALL) are relatively common. In the era of personalized medicine, flow cytometry could be used as a rapid method for selection of optimal therapies, which may include drugs that target the Erk pathway. Here, we evaluated the utility of phospho-flow, compared to Western blotting, to monitor Erk pathway activation and its inhibition by targeted Mek kinase inhibitors in human BCP ALL. Because the Erk pathway is not only activated endogenously, by mutations, but also by normal extracellular stimulation through stromal contact and serum growth factors, we compared Erk activation ex vivo in ALL cells in the presence and absence of stroma and serum. Phospho-flow was able to readily detect changes in the pool of pErk1/2 that had been generated by normal microenvironmental stimuli in patient-derived BCP-ALL cells passaged in NSG mice, in viably frozen primary patient samples, and in fresh patient samples. Treatment with the Mek1/2 inhibitor selumetinib resulted in a rapid, complete and persistent reduction of microenvironment-generated pErk1/2. Imaging flow cytometry confirmed reduction of nuclear pErk1/2 upon selumetinib treatment. An ALL relapsing with an activating KRasG12V mutation contained higher endogenous as well as serum/stromal-stimulated levels of pErk1/2 than the matched diagnosis sample which lacked the mutation, but selumetinib treatment reduced pErk1/2 to the same level in both samples. Selumetinib and trametinib as Mek inhibitors were mainly cytostatic, but combined treatment with the PI3K∂ inhibitor CAL101 increased cytotoxicity. Thus phospho-flow cytometry could be used as a platform for rapid, individualized in vitro drug sensitivity assessment for leukemia patients at the time of diagnosis.

Abstract 3317: Radiation induced multinucleated giant cells: A novel therapeutic target to prevent survival and relapse of glioblastoma

Abstract In this study, we address the fundamental issue of radiation resistance in Glioblastoma Multiforme (GBM) a highly malignant form of brain tumor. However, because of the unavailability of multiple biopsies of GBM patients after treatment, we are limited in our understanding of resistance mechanisms. To circumvent this problem we recapitulated clinical scenario of GBM resistance in a cellular model developed from fresh naive primary GBM patient samples and cell lines. Using our model we show that upon lethal dose of radiation, small percentage of GBM cells survive, are non-apoptotic and transiently become non- proliferative. They are arrested in G2/M phase of cell cycle mediated by inhibitory phosphorylation of Cdk1(Y15) and p21. Surprisingly, we find although non -proliferative, these cells are highly motile and undergo homotypic cell-cell fusions at high frequency to form multinucleated and giant cells (MNGCs). Cell fusion leads to the induction of senescence as seen by β-galactosidase staining and accordingly high expression of SASPs (senescence associated secretory proteins like GM-CSF, SCF, IL-6 and IL-8) is also observed. SASPs provide survival and proliferation signals to the cells in an autocrine manner. Accordingly, we find enhanced expression of anti-apoptotic genes BIRC3 and Bcl-xL and unaltered levels of Bax (pro-apoptotic protein) mRNA in the resistant cells. MNGCs also activate AKT, which is a survival signal and a central convergence node of signals downstream to SASPs. AKT activation along with Bcl-xL is reported to regulate apoptosis synergistically. Additionally, resistant cells also undergo active DNA damage repair mediated by sensory kinases ATM and ATR. Interestingly, irrespective of the phase of cell cycle, they adopt Non Homologous End Joining (NHEJ) as their pathway of choice to repair their double strand breaks in DNA. Since we find increased presence of H3K36me2 marks known to modulate NHEJ, a possible link between NHEJ and this modification in GBM is worth exploring. Finally, unlike previous reports we demonstrate that MNGCs do not die of mitotic catastrophe instead escape senescence, undergo normal cell division and proliferation giving rise to mono-nucleated relapse cells with unaltered ploidy compared to parent cells. Importantly, we also show the potential ability to prevent the relapse in Glioma by disrupting the non-proliferative state of radiation resistant cells using mitotic inducer (MK1775) and selectively ablating growth of MNGCs by cytokinesis inhibitor. In summary, our data provides a mechanistic explanation for clinical observations and novel insights into an unexplored multi-step process of radiation survival and relapse in Glioblastoma We identified homotypic cell fusions of resistant Glioma cells as a novel non-genetic mechanism to sustain survival and relapse and generate rationale for novel combinatorial targeted therapies. Citation Format: Ekjot Kaur, Jacinth Rajendra, Sanket Shah, Jyothi Nair, Ankit Khushwaha, Aliasgar Moiyadi, Shilpee Dutt. Radiation induced multinucleated giant cells: A novel therapeutic target to prevent survival and relapse of glioblastoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3317. doi:10.1158/1538-7445.AM2015-3317

Method for the improving of point-of-care HBA1c results harmonization

Objective To establish a new traceability pathway of point-of-care testing (POCT) of HBA1c by using commutable quality controls, in order to improve the accuracy of POCT HBA1c and the harmonization of testing results with those of central laboratories. Methods The study was about measurement traceability. Human frozen whole blood samples with IFCC assigned values were used to calibrate the G8 HBA1c Variant in June, 2013. According to the CLSI EP9-A2-IR guideline, 50 patient samples and 2-level commercial QC samples were then analyzed by G8 system and DCA Ventage system. The best fitting curves for fresh patient samples and the commercial QC materials were established separately. The patient results tested on the DCA Ventage were modified and verified. Paired t-test and Passing Bablok linear regression were used. Results The linear equation of DCA/G8 before calibration was Y=0.899 5X+ 0.389 1(R2=0.991 0). Calibration by fresh patient samples reduced the mean bias of DCA/G8 from -0.40%±0.34% to 0.00%±0.29%. Calibration by QCs reduced the mean bias to 0.15%±0.29%. The linear correlation established by quality controls was stable, which made the bias was lower between DCA and G8 in the consequent six runs. Conclusions The accuracy and the traceability of POC testing could be realized by using commutable QC materials traceable to IFCC assigned values. Through this method, POC testing can become more comparable to the results of clinical laboratory HBA1c instruments.(Chin J Lab Med, 2015, 38: 310-312) Key words: Hemoglobin A, glycosylated; Point-of-care systems; Reproducibility of results

Analytical performance and accuracy verification of four domestic and two imported enzymatic creatinine assay kits

Objective To assessed the analytical performance of six commercial enzymatic creatinine (Cr) reagents and compared the accuracy among them.Four reagents came from domestic manufactures (Maker Biotechnology,Beijing Strong Biotechnologies,Biosino Bio-technology & Science,BaiDing Biological Engineering,labeled as A,B,C,D respectively) and the other two reagents came from oversea manufactures (Sekisui Medical Technology,Japan and Wako Pure Chemical Industries,Japan,labeled as E,F respectively). Methods Performance verification test.70 serum samples were obtained from chronic kidney disease outpatients and medical center healthy volunteers in Peking Union Medical College Hospital from May 2010.Among 30 patients ureh CKD (15 moles,15 females) aged 18 to 80 years old;40 healthy volunteers (20 males,20 females) aged 20 to 60 years old.To evaluate the within-run imprecision and total imprecision according to the CLSI EP15-A protocol,two level control materials were measured with six reagents in five days,with a frequency of four times a day.To verify the linear ranges according to the EP6-A protocol,a series of samples that mixed by high and low level serum or urine pools with specific ratio were tested.To evaluate the anti-interference ability,we tested the sera added specific concentration of substances (bilirubin,hemoglobin,chyle).To verify the reasonable dilution ratio,diluted sera in different ratio were tested.Correlations and deviation among the detection results of six reagents were evaluated by testing 50 fresh patient samples.Accuracy was evaluated by measuring the NIST SRM 967a and NIST SRM 909b standard substances. Results There were no significant differences between the domestic and imported Cr reagents,with the within-run CV ranging from 0.5% to 1.2% and the total CV ranging from 0.5% to 1.9%,all of which were lower than the allowed biological variation for imprecision (3.0%).Linear ranges of the six Cr reagents were all relatively wide.No significant interference (bias%≤±10%) was found in the test of six reagents when conjugated bilirubin≤332μmol/L,unconjugated bilirubin≤327μmol/L,or chyle turbidity≤1530 FTU; no significant interference was found for the test of C,D,F reagents when Hb≤4.83 g/L; and for A,B,E when Hb≤3.381,3.864,4.347g/L,respectively.Percent deviation of the measured results of dulited serum samples diluted by 2 to 16 times compared to the results of the original samples ranged from 95.96% to 108.62%.Fresh patient samples comparison tests exhibited good correlation among the six reagents (r=1.000),although there were some differences (-4.91% to -4.75%).For NIST 967a,the percent bias ranged from -1.85% to 2.81% (level 1,66.5μmol/L),and from -2.28% to 2.05% (level 2,346.2μmol/L),none of the six reagents exceeded the allowed Cr bias derived from biological variation (4.0%).For NIST 909b,the percent bias ranged from -13.4% to 1.01% for level 1 (56.18μmol/L),while its value ranged from -6.49% to -1.79% for level 2 (467.4μmol/L),and these biases all exceeded the allowable range of target value. Conclusions Assessment of four domestic enzymatic Cr reagents and two imported reagents show high precision,wide linear range,and their dilution accuracy can meet the clinical needs.Moreover,the six regents show good anti-interference capability (bilirubin,hemoglobin,chyle) and high accuracy.No significant differences have been found in the above assessed properties among these reagents.(Chin J Lab Med,2013,36:806-811) Key words: Creatinine; Indicators and reagents; Reagent kits; diagnostic

Comparative Study of Blood Collection Tubes and Thromboplastin Reagents for Correction of INR Discrepancies

International normalized ratio (INR) discrepancies were noted between clinical laboratories using various prothrombin time (PT) systems. We studied the influence of different commercial blood collection tubes and different PT systems on INR measurements. INRs of fresh patient samples were determined by 3 laboratories, each using different PT systems. In the first part of the study, samples were drawn with Vacutainer tubes and in the second part with Monovette tubes. In the first part of the study, the maximum bias for all patients amounted to 0.46 INR (14%), and in the second part, to 0.14 INR (4.9%). The maximum bias for all patients could be reduced further by local system calibration using frozen pooled plasma specimens. The sodium citrate solutions in the blood collection tubes were contaminated with magnesium ions (approximately 2.7 mmol/L and 0.3 mmol/L in the Vacutainer and Monovette, respectively). INR discrepancies could be explained largely by this influence of blood collection tubes. The maximum allowable magnesium contamination in sodium citrate anticoagulant solutions should be less than 1 mmol/L.

Functional P2X7 Receptors in Myeloma Cell Lines and Myeloma Patient Bone Marrow Samples Can Be Blocked by Specific P2X7 Receptor Antagonists: Implications for Myeloma Therapy and Chemotherapy Induced Inflammation.

Abstract 1845Inflammation is a driving factor in the pathogenesis of many cancers including multiple myeloma (MM). The active role that cytokines play, IL-1β in particular, during the early stages of MM has been extensively characterized. Myeloma is a plasma cell tumor whose early stages are characterized by cytokine dependent growth, predominantly driven by IL-6. We have demonstrated that IL-1β is a key inducer of IL-6 expressed by bone marrow stromal cells in a paracrine fashion during the progression of multiple myeloma from smoldering myeloma (SMM) to active disease (Mayo Clinic Proc 84:114 (Feb. 2009)). Elevated ATP levels have been documented in vivo in tumor micro-environments and is a potential contributing factor to IL-1β induced inflammation. The P2X7 receptor (P2X7R) is an ATP-gated ion channel expressed by cells of the hematopoetic lineage that has been shown to play a critical role in IL-1b processing and secretion. We investigated the role this receptor may have in the release of IL-1β in the bone marrow microenvironment using bone marrow samples from both chemotherapy treated and untreated patients.Myeloma cell lines were evaluated by RT-PCR for the expression of P2X7R mRNA. Expression was detected and confirmed by sequencing in ANBL-6, MM1.S, U266, RPMI-8226 and KAS-6/1 and was comparable to U937, a previously characterized P2X7R positive cell line. A newly derived plasmacytoma cell line, PCYT3, showed very low levels of message expression. The ability of ATP, the P2X7R agonist, to trigger the processing and release of IL-1β by activation of the P2X7 channel was studied using KAS-Pro, a myeloma cell line stably transfected with a pro- IL-1β gene. IL-1β, measured by ELISA, was released in a dose dependent fashion in response to increasing amounts of ATP ranging from.6mM to 5mM. Four P2X7R specific antagonists were tested for their effect on ATP stimulated IL- 1β release from KAS-Pro; all 4 compounds demonstrated inhibition in a dose dependent manner with varying degrees of potency correlating with P2X7R inhibition.In addition fresh patient samples (8 MGUS/7SMM/9 newly diagnosed MM as well as 12 Treated MM) were evaluated for receptor expression and function. All stages of disease expressed the receptor by RT-PCR. Analysis of samples from all stages of MM, as measured directly by IL-1β ELISA as well as indirectly by measurement of IL-1β induced IL-6 production from normal bone marrow stromal cells, showed functional receptor activation in response to ATP stimulation. IL-6 production detected from MGUS patient samples had a mean result of 2,253 pg/ml, the mean for SMM was 5,495 pg/ml, MM were 6,875 pg/ml while treated MM samples showed a mean result of 15,219 pg/m. Greater than 90% inhibition of IL-1β release by the P2X7R antagonists was seen for all patients who demonstrated a positive ATP induced IL-1β release (see figure). We observed a greater than 2 fold increase in IL-1β release between the treated MM samples and the newly diagnosed MM samples, suggesting a chemotherapy induced inflammation process may be ongoing in these patients. [Display omitted] These results confirm the activity of functional P2X7 receptors on myeloma cell lines and in fresh patient samples and suggest a role for the use of P2X7 receptor antagonists in the therapy of myeloma, particularly in patients undergoing chemotherapy. Disclosures:Hromockyj:Pfizer Corporation: Employment. Meyer:Pfizer Corporation: Employment.

External quality assessment of point-of-care International Normalized Ratio (INR) testing in Europe

Point-of-care testing (POCT) of prothrombin time, expressed as International Normalized Ratio (INR), is widely used to monitor patients in oral anticoagulation treatment. Guidelines recommend that POCT users should participate in an external quality assessment (EQA) scheme whenever available. The aim of this study was to investigate which European countries provide EQA for POCT INR and to compare how these schemes are organized. Thirty European countries were invited to participate in this study. Those who reported that they provide EQA for POCT INR filled in a questionnaire dealing with different aspects of their schemes. Nineteen countries reported that they do not provide EQA for POCT INR, while 12 organizations from nine countries reported that they provide this service. Most of these countries circulate lyophilized samples with for the participants unknown target values. Samples with certified INR values and procedures using split samples with fresh patient samples are also used. The acceptability limits vary from 15% to 30%, and the total number of samples circulated per year varies from 1 to 12. Most of the countries organize educational activities together with their schemes. This study demonstrates that there is a wide variation in the way EQA for POCT INR is performed in Europe and that there are many European countries that do not provide this service. Even though our findings indicate that EQA for POCT INR draws some challenges, especially in providing suitable control materials, participation in such schemes is considered useful.