The aim of the present study was to characterize the activation profiles of 15 opioid ligands in transfected human embryonic kidney cells expressing only delta opioid receptors. Activation profiles ...of most of these ligands at delta opioid receptors had not been previously characterized in vitro. Receptor activation was assessed by measuring the inhibition of forskolin-stimulated cAMP production.
Naltrexone and nalorphine were classified as antagonists at delta opioid receptor. The other ligands studied were agonists at delta opioid receptors and demonstrated IC50 values of 0.1 nM to 2 microM, maximal inhibition of 39-77% and receptor binding affinities of 0.5 to 243 nM. The rank order of efficacy of the ligands tested was metazocine = xorphanol > or = fentanyl = SKF 10047 = etorphine = hydromorphone = butorphanol = lofentanil > WIN 44,441 = Nalbuphine = cyclazocine > or = met-enkephalin >> morphine = dezocine. For the first time these data describe and compare the function and relative efficacy of several ligands at delta opioid receptors.
The data produced from this study can lead to elucidation of the complete activation profiles of several opioid ligands, leading to clarification of the mechanisms involved in physiological effects of these ligands at delta opioid receptors. Furthermore, these data can be used as a basis for novel use of existing opioid ligands based on their pharmacology at delta opioid receptors.
Introduction
Acute myeloid leukemia (AML) is a heterogeneous disease with multiple factors influencing long-term outcome. FLT3 (FMS-like tyrosine kinase 3) mutations (predominantly internal tandem ...duplication ITD) are reported in ~25% of patients with AML and have been associated with poorer outcomes compared with FLT3 non-mutated AML (Kottaridis et al. Blood. 2001). Targeting these mutations with tyrosine kinase inhibitors has become an active area of research. However, patients must undergo molecular testing to identify the presence of this mutation. The specimen of choice for such testing is bone marrow (BM; aspirate or biopsy), as the site of origin of the disease. However, BM testing is invasive and sometimes cannot be performed repeatedly in a timely manner. A simple technique using easily obtainable biological specimens from a source such as peripheral blood (PB) may be desirable if the results accurately reflect status in the active disease site (i.e., BM). We investigated whether a PB specimen could be utilized for FLT3 -ITD determination and if the characteristics of the ITD mutations identified in PB were consistent with those in BM samples from the same patient.
Methods
QuANTUM-R is a global, randomized, phase 3 study to assess the efficacy of quizartinib, a selective FLT3 inhibitor, in patients with FLT3 -ITD-mutated relapsed or refractory (R/R) AML. Paired samples of BM and PB were collected from individual patients as part of the screening process for QuANTUM-R. One to 3 mL each of BM and PB were collected on the same day (window Day -14 to Day 0) from each patient prior to receipt of any study treatment. A statistically powered number of patients (minimum 95) with paired BM and PB samples were selected during a defined period of screening in the phase 3 study. Samples were subjected to DNA isolation followed by PCR and fragment size analysis. Each sample was assessed for the presence of ITD mutation; moreover, the length and allelic frequency of the ITD mutation were measured for each sample. The assay was validated down to a limit of detection (LoD) of 1%. Correlation between the data obtained from BM and PB was assessed using Deming regression analysis and Bland-Altman plot constructed to assess concordance between the 2 sample types.
Results
Paired samples from 107 patients (median age 58y; 32% were ≥65y; 57% were female) were included. Sixteen samples had no ITD mutations in either BM or PB; 3 had ITD only in the BM sample with no corresponding ITD in the paired PB sample (despite having circulating blasts), and 88 had ITD in both BM and PB. In all 88 cases, the length of the ITD was identical in both specimen types. The sizes of ITD observed in this patient population were between 9 and 198 bases; 21/88 samples with ITD demonstrated the presence of multiple ITDs, with samples from 2 patients exhibiting 3 distinct ITDs, each of whose sizes were the same in BM and PB. Across all samples with ITD, the measured ITD allelic ratio ranged from 1% to 96%, with a similar or slightly lower ratio measured in the PB compared to the BM. In 23 (26%) cases, the allelic ratio measured in the PB was lower than that of the BM by ≥10%. Blast counts for these samples were also measured and there was no correlation between blast count and allelic ratio in either specimen type. High correlation between PB and BM samples for the presence of FLT3 -ITD was observed when regressing PB Ratio on BM Ratio via Deming regression analysis, and the following values were obtained: R2 = 0.866; 95% CI for the slope, 0.98 to 1.12; and Bland-Altman difference plot for allelic ratio against difference between PB and BM showed good random scatter around average bias of -5.2%, with 95% CI including 0.0% (95% CI, -27.6 to 17.2).
Conclusions
FLT3 -ITD mutation testing is performed in all AML patients at diagnosis for prognostic purposes and to guide therapeutic decisions. As FLT3 inhibitors are developed for clinical use, regular monitoring of patients' residual disease burden/response to therapy through assessment of FLT3 status may become an important element of monitoring benefit from effective therapy. Mutation assessment is currently performed on BM aspirate or biopsy based on the common belief that PB is not adequate for this assessment. Our results of FLT3 -ITD mutation analysis in paired BM and PB samples collected from >100 patients with R/R AML show that PB specimens have a high degree of concordance with BM specimens for assessment of FLT3 mutation while being less invasive.
Khaled:Daiichi Sankyo, Inc: Other: Travel Support; City of Hope: Research Funding. Ganguly:Amgen: Other: Advisory Board; Seattle Genetics: Speakers Bureau. Perl:Seattle Genetics: Other: Advisory board; Daiichi Sankyo: Consultancy; Astellas: Consultancy; Novartis: Other: Advisory Board; Pfizer: Other: Advisory Board; Actinium Pharmaceuticals: Other: Scientific Advisory Board; Arog Pharmaceuticals: Consultancy; Asana Biosciences: Other: Scientific advisory board. Kobayashi:Daiichi Sankyo, Inc.: Employment. Berisha:Daiichi Sankyo, Inc.: Employment. Lameh:Daiichi Sankyo, Inc.: Research Funding; Navigate BioPharma Service Inc.,: Employment. Martinelli:Celgene: Consultancy; Pfizer: Consultancy; Amgen: Consultancy; Ariad: Consultancy; Incyte-Teva: Speakers Bureau.
Abstract
The renaissance of cancer immunotherapies and the positive clinical responses observed with chimeric antigen receptor modified T cells in the hematomalignancy setting has stimulated ...substantial interest in monitoring immune cell activation and suppression to determine efficacy, prognosis and safety in new agent investigational trials. However, immunophenotyping capacity has been limited and required multi-test tube panels. To address the growing needs of clinical trials, we have developed three fit-for-purpose high complexity (10 or more markers) T-cell phenotyping flow cytometry panels on a qualified LSR FortessaTM platform that enables detection of up to 15 markers in a single test tube. The first panel allows identification of multiple phenotypes along the T-cell differentiation pathway, namely, T-naive (TN), T-stem cell memory (TSCM), T-effector memory (TEM) and T-central memory (TCM) and T-effector memory RA+ (TEMRA). The second panel identifies the most common helper T-cell phenotypes such as Th1, Th2, Treg, and Th17. A third panel determines the functional status of T-cells (e.g., activation vs. suppression) but also enables quantitation of important checkpoint inhibitor expression (e.g., PD-1) on T-cells of interest. These high complexity flow cytometry panels can serve as powerful tools for comprehensive examination of T-cells in a small volume of patient specimen. We believe these new flow cytometry panels will have a substantial impact on the determination of efficacy and safety correlates of immunomodulating agents administered alone or in combination to patients with leukemia.
Citation Format: Ghanashyam Sarikonda, Devika Ashok, Anil Pahuja, Jelveh Lameh, Shabnam Tangri, Naveen Dakappagari. High complexity flow cytometry panels to monitor target expression, T-cell activation and suppresssion by novel immunotherapies in hematomalignancy clinical trials. 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 1302. doi:10.1158/1538-7445.AM2015-1302
The aim of the present study was to describe the activity of a set of opioid drugs, including partial agonists, in a cell system expressing only mu opioid receptors. Receptor activation was assessed ...by measuring the inhibition of forskolin-stimulated cyclic adenosine mono phosphate (cAMP) production. Efficacies and potencies of these ligands were determined relative to the endogenous ligand beta-endorphin and the common mu agonist, morphine.
Among the ligands studied naltrexone, WIN 44,441 and SKF 10047, were classified as antagonists, while the remaining ligands were agonists. Agonist efficacy was assessed by determining the extent of inhibition of forskolin-stimulated cAMP production. The rank order of efficacy of the agonists was fentanyl = hydromorphone = beta-endorphin > etorphine = lofentanil = butorphanol = morphine = nalbuphine = nalorphine > cyclazocine = dezocine = metazocine >or= xorphanol. The rank order of potency of these ligands was different from that of their efficacies; etorphine > hydromorphone > dezocine > xorphanol = nalorphine = butorphanol = lofentanil > metazocine > nalbuphine > cyclazocine > fentanyl > morphine >>> beta-endorphin.
These results elucidate the relative activities of a set of opioid ligands at mu opioid receptor and can serve as the initial step in a systematic study leading to understanding of the mode of action of opioid ligands at this receptor. Furthermore, these results can assist in understanding the physiological effect of many opioid ligands acting through mu opioid receptors.
The N- and C-terminal junctions of the third intracellular loop (i3) of G protein-coupled receptors play a role in the coupling process. We had previously constructed two triple point alanine mutants ...of the i3 junction of the muscarinic Hm1 receptor, W209A/I211A/Y212A and E360A/K362A/T366A, which are defective in mediating carbachol stimulation of phosphatidylinositol (PI) turnover (Moro, O., Lameh, J., Högger, P., and Sadée, W. (1993) J. Biol. Chem. 268, 22273-22276). Each of the corresponding six single point mutations were constructed to determine residues crucial to receptor coupling. Mutants W209A and T366A were similar to or only slightly less effective than wild type Hm1 in stimulating PI turnover. In the N-terminal junction, I211A and Y212A were defective in coupling, and I211A was even more defective than the corresponding triple mutant. Therefore, the triple mutation compensated at least partially for the effect of these two single point mutations. In the C-terminal i3 loop junction, mutant K362A was again more strongly defective than the corresponding triple mutant. In contrast, mutation E360A was found to be activating, leading to elevated PI turnover in the absence of agonist and sensitization toward carbachol activation. Activating mutations in the C-terminal i3 loop junction have been reported previously for the adrenergic receptors, but E360A represents the first muscarinic receptor with substantial basal activity. The effects of the single point mutations observed in this study were not readily predictable from similar mutations from closely related G protein-coupled receptors despite sequence conservation in the i3 loop junctions. Our results caution against defining precise coupling domains in these regions by mutagenesis results.
The presence of MRD in patients with acute myeloid leukemia (AML) who are in morphologic remission has been shown to be a powerful predictor of eventual relapse. FMS-like tyrosine kinase 3 internal ...tandem duplications (FLT3-ITD) confer a negative prognostic impact by increasing the risk of relapse. However, the ability to detect these mutations in remission bone marrow specimens is hampered by the limited sensitivity of conventional polymerase chain reaction (PCR)-based assays, which detect approximately only 1 of every 100 (1%) mutant cells. To address this problem, we have developed a novel NGS-based MRD assay for the detection of FLT3-ITD mutations.
Using isolated genomic DNA from bone marrow aspirates or whole-blood samples, PCR primers flanking exons 14 and 15 of the FLT3 gene were designed and highly diverse NGS libraries were generated. These libraries were then sequenced by Illumina's sequencing-by-synthesis method. The bioinformatics approach we used identifies unique FLT3-ITD mutations of varying length along with wild type sequences and calculates a mutant allelic frequency. The assay was validated using clinical samples to assess accuracy and reproducibility. DNA samples from selected mutant cell lines representing different FLT3-ITD lengths were spiked into normal DNA to evaluate assay sensitivity and linearity. The assay was linear (R2 = 0.958) down to FLT3-ITD allele frequency levels of 0.035% but was capable of detecting FLT3-ITD mutations at a level as low as 0.003%.
We next validated the assay using clinical samples from patients with FLT3-ITD AML. The negative prognostic impact of FLT3-ITD mutations can be mitigated in part when an FLT3 inhibitor is administered in combination with induction chemotherapy, as demonstrated in CALGB10603/RATIFY (N Engl J Med. 2017;377:454). It was reported in this study that patients treated with an FLT3 inhibitor combined with chemotherapy followed by allogeneic transplant in first remission had better overall survival than their counterparts in the control arm. One hypothesis for this outcome is that the FLT3-inhibitor-treated patients had a lower leukemic burden prior to transplant. As a pilot test of this concept, we used our MRD assay on a series of bone marrow aspirate samples collected from 10 patients with newly diagnosed FLT3-ITD AML. The patients were selected to be as uniform as possible. All patients had intermediate-risk karyotype, a detectable FLT3-ITD mutation by conventional PCR, and mutated NPM1. All patients received cytarabine-based intensive induction and achieved morphologic first remission with a single course of chemotherapy. Finally, all patients underwent allogeneic transplant in first remission. The sample analyzed for MRD was the first collected after remission induction, 5-8 weeks after the start of therapy. The investigators performing the MRD assay were blinded to the clinical data. Four patients received chemotherapy alone, while 6 were treated with chemotherapy (7+3) plus an FLT3 inhibitor. In all patients' remission samples, the MRD assay identified the FLT3-ITD mutation that precisely matched the one observed in the original diagnostic specimen. This demonstrates the sensitivity of the assay (all samples had a detectable mutation), and the unique length of each patient's mutation confers a degree of specificity not achievable with MRD detection methods that focus on other AML-associated mutations. Supporting our hypothesis was the observation that patients treated with FLT3 inhibitors had MRD levels lower than those in patients treated with chemotherapy alone (Figure).
Our results help establish the role of NGS-based MRD assays for the clinical management of FLT3-ITD AML. This assay could be used to define the depth of remission, identify persistent disease, and help guide decision making in the use of FLT3 inhibitors as continuation therapy. This study provides validation of the clinical utility of our MRD assay, which will be used to analyze the remission samples from patients in the ongoing phase 3, randomized, double-blind, placebo-controlled QuANTUM-First clinical trial, in which patients with newly diagnosed FLT3-ITD AML are randomized to receive either the highly potent and selective FLT3 inhibitor quizartinib or placebo in combination with chemotherapy, followed by single-agent quizartinib as continuation therapy.
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Shi:Novartis: Employment, Equity Ownership; Daiichi Sankyo: Other: Provide clinical trial testing services. Chang:Daiichi Sankyo: Employment. Laing:Novartis: Employment; Daiichi Sankyo: Other: Provide clinical trial testing services. Berisha:Daiichi Sankyo: Employment. Adams:Johns Hopkins University: Employment. Ding:Navigate BP: Employment; Daiichi Sankyo: Other: Provide clinical trial testing services. Nakamaru:Daiichi Sankyo: Employment. Lameh:Navigate BioPharma Inc,: Employment; Daiichi Sankyo: Other: Provide clinical trial testing services. Pollner:Navigate BioPharma Inc.: Employment. Kobayashi:Daiichi Sankyo: Employment.
Abstract
With the advent of multiple HER-2 targeting agents, the need for a diagnostic test that accurately predicts the levels of HER2 in newly diagnosed breast cancer patients has never been ...greater. The goal of this study was to develop an immunofluorescence based assay to objectively and reproducibly quantify HER-2 protein level in Formalin-Fixed Paraffin-Embedded (FFPE) specimens using AQUA Technology and to provide a binary readout, thus eliminating reflex testing and enabling quick treatment decisions.
Methods: Three well known HER-2 antibody clones, A0485, CB11, and SP3 were evaluated across a three-log dilution series under four different antigen retrieval conditions on a tissue microarray (TMA) containing 80 breast cancer cases with known central HER2 immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) amplification status. The top six immunofluorescence assay combinations out of 108 conditions were quantified by AQUA scoring based on the their ability to accurately separate negative and positive cases using receiver operating characteristic (ROC) analysis. These top six assay conditions were then assessed on an independent TMA containing 94 breast cancer cases and their AQUA scores were compared to HER2 protein levels determined by reverse phase protein microarray (RPMA®), HER2 mRNA levels assessed by RNAscope® as well as to the current “gold standard” IHC and FISH amplification.
Results: Top three conditions clearly segregating HER2 positive and HER2 negative breast cancer cases were selected and analyzed on an independent TMA. Conditions associated with both SP3 and A0485 antibodies demonstrated high correlations with both HER2 protein levels determined by RPMA analysis and HER2 mRNA levels assessed by RNAscope (spearman's rho >0.85).
Conclusion: Assessment of HER2 status using AQUA Technology was confirmed using two expression platforms, including both protein (RPMA) and RNA (RNAscope) supporting the utilization of the AQUA methodology for diagnostic test development.
Citation Format: Jennifer Bordeaux, Krupa Chandrasekaran, Sue Beruti, Mike Nerenberg, Corinne Ramos, David Rimm, Jelveh Lameh, Naveen Dakappagari. Evaluation of HER-2 RNA and protein levels in a large cohort of breast cancer specimens to support development of a diagnostic immunofluorescence assay quantified by AQUA® Technology. abstract. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2840. doi:10.1158/1538-7445.AM2014-2840
Abstract
Propelled by the advent of new technologies and an evolving regulatory landscape, the desire to personalize cancer treatments has never been greater. There is a critical need to reliably ...evaluate target inhibition and pharmacodynamic activity of investigational drugs in biologically relevant compartments. However, the ability to fulfill such a task in global clinical trials is complicated by varying technical expertise available at clinical sites and considerations about specimen stability for centralized laboratory analysis. We developed a novel formalin-based preservation method that enables specimen stabilization in a single step, requiring less than 20 minutes, without the need for specialized training or instrumentation. Stabilized specimens can be frozen for shipping and batched analysis at a later time point, in a specialized laboratory. Using this preservation method, we developed a flow assay enabling identification of multiple cell-types, and quantification of intracellular biomarkers in target cellular compartments, in both the peripheral blood (PB) and bone marrow (BM). We present pharmacodynamic (PD) data for a novel anticancer investigational agent intended for acute myeloid leukemia and multiple myeloma and its effect on multiple target biomarkers of the PI3K signaling pathway, including phosphorylation of the S6 ribosomal protein (S6). Our novel fixation method allowed detection of pS6 modulation in a dose dependent manner in both tumor cells and PB or BM in response to the novel investigational agent. Assay sensitivity and concordance were evaluated by comparing flow assay with western blot analysis, with each assay performed at a different site using the same batched frozen samples; exceptional preservation of phosphorylated proteins for more than 72 hours could be observed, when frozen immediately following fixation. In conclusion, our novel preservation method enables reliable quantification of signaling biomarkers in centralized laboratories at different time points post sample (PB or BM) acquisition requiring minimal processing at collection sites.
Citation Format: Anil Pahuja, Abdel Saci, Shyam Sarikonda, Armin Graber, Benjamin Lee, Jelveh Lameh, Shabnam Tangri, Naveen Dakappagari. A novel blood preservation system to study oncogenic signaling pathway biomarkers by flow cytometry in leukemia/lymphoma clinical trials. abstract. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-96. doi:10.1158/1538-7445.AM2014-LB-96
Abstract
Introduction: Gene amplification/deletion is a common tactic employed by tumor cells to proliferate uncontrollably. Multiple methods have been developed to determine gene copy numbers, such ...as FISH, qPCR, and, more recently, Next Generation Sequencing (NGS). However, these methods have limitations: FISH and NGS are very labor intensive; and FISH and qPCR can detect only a limited number of targets. Therefore, it is imperative to develop methods that are more user-friendly and have increased multiplexing capabilities to detect copy number variations (CNV). Using a novel digital barcode technology, NanoString Technologies (Seattle, WA) has developed the nCounter® technology to meet these needs. This new technology will be especially valuable after the recent FDA approval of the ProsignaTM assay, a breast cancer gene signature assay for subtype classification to help guide treatment decisions. In the BioPharma group at Genoptix, Inc., Carlsbad, CA, we have started to assess the nCounter technology for copy number determinations in a CLIA laboratory setting.
Methods: A cancer gene CNV panel composed of 55 genes was custom designed and evaluated. The CNV results of a subset of genes generated from our gene panel using the nCounter technology were compared with the results generated from qPCR, digital droplet PCR (ddPCR), FISH, publications, and NGS. Furthermore, assay robustness was tested by using different amounts and quality of input FFPE DNA. An initial comparison on the CNVs obtained from genomic DNA digested by Alu1 enzyme or sonicated by Bioruptor® Pico (Diagenode Inc, Denville, NJ). Finally, the effects of different DNA isolation methods on copy number determinations were also evaluated.
Results: Overall, the copy numbers generated using nCounter technology were concordant with results from the abovementioned methods as analyzed by comparing selected genes with known CNVs. A wide range of FFPE DNA input (200ng-4ug) could be tolerated by the assay. Based on a limited number of samples, DNA isolation methods, such as Qiagen, Promega Maxwell® CSC, or Roche Cobas®, have minimal effect on copy numbers.
Conclusions: With the short hands-on time, relative high level of automation, and no amplification requirements, nCounter technology is able to generate CNVs in good agreement with other established CNV methods. This new 55 cancer gene CNV assay may be useful in clinical trials to assist in patient stratification and care.
Citation Format: Tingdong Tang, Wenge Shi, Loretta Hipolito, Julie Mayer, Jelveh Lameh, Shabnam Tangri, Reinhold Pollner. Development of a nanostring copy number assay for a customized 55 gene panel using challenging formalin-fixed paraffin-embedded (ffpe) tumor samples. abstract. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3734. doi:10.1158/1538-7445.AM2014-3734
We examined the agonist‐dependent sequestration/internalization of dopamine D2 receptor (the long form D2L and short form D2S), which were transiently expressed in COS‐7 and HEK 293 cells with or ...without G‐protein‐coupled receptor kinases (GRK2 or GRK5). Sequestration was assessed quantitatively by loss of 3H sulpiride‐binding activity from the cell surface and by transfer of 3H spiperone‐binding activity from the membrane fraction to the light vesicle fraction in sucrose‐density gradients. In COS‐7 cells expressing D2 receptors alone, virtually no sequestration was observed with or without dopamine (< 4%). When GRK2 was coexpressed, 50% of D2S receptors and 36% of D2L receptors were sequestered by treatment with 10–4 m dopamine for 2 h, whereas no sequestration was observed in cells expressing the dominant negative form of GRK2 (DN‐GRK2). When GRK5 was coexpressed, 36% of D2S receptors were sequestered following the same treatment. The agonist‐dependent and GRK2‐dependent sequestration of D2S receptors was reduced markedly in the presence of hypertonic medium containing 0.45 m sucrose, suggesting that the sequestration follows the clathrin pathway. Internalization of D2S receptors was also assessed by immunofluorescence confocal microscopy. Translocation of D2 receptors from the cell membrane to intracellular vesicles was observed following the treatment with dopamine from HEK 293 cells only when GRK2 was coexpressed. D2S receptors expressed in HEK 293 cells were shown to be phosphorylated by GRK2 in an agonist‐dependent manner. These results indicate that the sequestration of D2 receptors occurs only through a GRK‐mediated pathway.
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