Ala F. Nassar

CyTOF supports efficient detection of immune cell subsets from small samples.

Analysis of immune cell states is paramount to our understanding of the pathogenesis of a broad range of human diseases. Immunologists rely on fluorescence cytometry for cellular analysis, and while detection of 8 markers is now well established, the overlap of fluorescent signals limits efficiency. Mass cytometry or CyTOF (Cytometry by Time-Of-Flight) is a new technology for multiparameter single cell analysis that overcomes many limitations of fluorescence-based flow cytometry and can routinely detect as many as 40 markers per sample. This technology provides tremendous detail for cellular analysis of multiple cell populations simultaneously and is a powerful technique for translational investigations. Here we present reproducible detection of immune cell subsets starting with as few as 10,000 cells. Our study provides methods to employ CyTOF for small samples, which is especially relevant for investigation of limited patient biopsies in translational and clinical research.

UPLC–MS for metabolomics: a giant step forward in support of pharmaceutical research

Metabolomics is a relatively new and rapidly growing area of post-genomic biological research. As use of metabolomics technology grows throughout the spectrum of drug discovery and development, and its applications broaden, its impact is expanding dramatically. This review seeks to provide the reader with a brief history of the development of metabolomics, its significance and strategies for conducting metabolomics studies. The most widely used analytical tools for metabolomics: NMR, LC-MS and GC-MS, are discussed along with considerations for their use. Herein, we will show how metabolomics can assist in pharmaceutical research studies, such as pharmacology and toxicology, and discuss some examples of the importance of metabolomics analysis in research and development.

Mass cytometry moving forward in support of clinical research: advantages and considerations

Since 1970, the invention of flow cytometry FACS has become widely used for single-cell measurements in medicine and biology [1]. In the last 30 years FACS has become the work horse to provide fast, objective and quantitative recording of fluorescent signals from individual cells as well as physical separation of cells of particular interest. A recent breakthrough, cytometry time-of-flight (CyTOF, mass cytometry), builds on flow cytometry and adds MS for unprecedented marker detection in biological samples [2,3]. CyTOF is a novel and exciting technology for real-time analysis of single cells using inductively coupled plasma TOF-MS. Current CyTOF technique allows the detection of 40 parameters at the single-cell level; this capacity will increase as more isotopes become available. CyTOF can resolve multiple metal conjugated antibodies for biomarkers detection on cells with minimal signal overlap, which maximizes the information obtained from each individual sample. To make the best use of mass cytometry to produce high-quality quantitative data from clinical samples, we must understand the experimental variables and how to minimize them. The ability to analyze multiplexed assays is of great importance for clinical diagnostic and other analytical applications. Mass cytometry is a significant advance for studies in medical fields including immunology, hematology and oncology. CyTOF has the ability to perform complex characterization at single-cell level both phenotypically and functionally from normal and diseased states. Single cell technologies have allowed researchers to measure the effects of a drug and better understand its mechanism of action. While research in drug companies is focused on the mechanism of drug–target interactions and its resulting pharmacology, CyTOF techniques may become a driving tool for biomarker discovery. There is increasing interest from a variety of disciplines for single-cell experimentation methods, such as cancer research, cardiovascular research, embryonic stem cells and development, gene expression profiling, hematopoietic stem cells and progenitors, immunity/ infectious disease, induced pluripotent stem cells, neural research and RNA sequencing [4–9]. The advantages of CyTOF are clear, and investigators employing best practices may achieve robust discovery in a routine bioanalytical setting.

Impact of recent innovations in the use of mass cytometry in support of drug development

Cytometry by time-of-flight (CyTOF) is a novel technology for the real-time analysis of single cells. CyTOF is a significant advance in fields including immunology, hematology, and oncology. It resolves multiple metal-conjugated probes per cell with minimal signal overlap, which maximizes the information obtained from each individual sample. CyTOF provides the ability to phenotypically and functionally profile cells from normal and diseased states. Single cell technologies enable researchers to measure the effects of a drug at the single cell level and better understand its mechanism of action. Here, we discuss novel instruments for the analysis of individual biological cells, the impact of recent innovations in support of drug development, and the important roles of CyTOF in drug profiling.

Rapid label‐free profiling of oral cancer biomarker proteins using nano‐UPLC‐Q‐TOF ion mobility mass spectrometry

It has become quite clear that single cancer biomarkers cannot in general provide high sensitivity and specificity for reliable clinical cancer diagnostics. This paper explores the feasibility of rapid detection of multiple biomarker proteins in model oral cancer samples using label‐free protein relative quantitation.

In vitro cleavage of diisocyanate-glutathione conjugates by human gamma-glutamyl transpeptidase-1.

Abstract Isocyanates differ from many other xenobiotics in their ability to form S-linked conjugates with glutathione (GSH) through direct nucleophilic addition reactions (e.g. without enzymatic “preactivation” and/or transferase activity), potentially predisposing them to metabolism via the mercapturic acid pathway. In vivo, mono-isocyanates are metabolized via the mercapturic acid pathway and excreted as N-acetylated cysteine conjugates, however, the metabolism of di-isocyanates remains unclear. We assessed the ability of purified human gamma-glutamyl transpeptidase-1 (GGT-1), a primary enzyme of the mercapturic acid pathway, to cleave S-linked GSH conjugates of 4,4′-methylene diphenyl diisocyanate (MDI) and 1,6-hexamethylene diisocyanate (HDI), two widely used industrial chemicals. A combination of liquid chromatography (LC), tandem mass spectrometry (MS/MS) and hydrogen-deuterium exchange studies confirmed GGT-1 mediated formation of the 607.2 and 525.2 m/z (M + H)+ ions corresponding to bis(cys-gly)-MDI and bis(cys-gly)-HDI, respectively, the cleavage products expected from the corresponding bis(GSH)-diisocyanate conjugates. Additional intermediate metabolites and mono(cys-gly)-conjugates with partially hydrolyzed diisocyanate were also observed. Consistent with GGT enzyme kinetics, metabolism proceeded more rapidly under conditions that favored transpeptidation versus hydrolytic mechanisms of cleavage. Together the data demonstrate the capacity of human GGT-1 to cleave GSH conjugates of both aromatic and aliphatic diisocyanates, suggesting a potential role in their metabolism.

Metabolic disposition of the anti-cancer agent [14C]laromustine in male rats

Abstract 1. Laromustine (VNP40101M, also known as Cloretazine) is a novel sulfonylhydrazine alkylating (anticancer) agent. This article describes the use of quantitative whole-body autoradiography (QWBA) and mass balance to study the tissue distribution, the excretion mass balance and pharmacokinetics after intravenous administration of [14C]VNP40101M to rats. A single 10 mg/kg IV bolus dose of [14C]VNP40101M was given to rats. 2. The recovery of radioactivity from the Group 1 animals over a 7-day period was an average of 92.1% of the administered dose, which was accounted for in the excreta and carcass. Most of the radioactivity was eliminated within 48 h via urine (48%), with less excreted in feces (5%) and expired air accounted for (11%). The plasma half-life of [14C]laromustine was approximately 62 min and the peak plasma concentration (Cmax) averaged 8.3 μg/mL. 3. The QWBA study indicated that the drug-derived radioactivity was widely distributed to tissues through 7 days post-dose after a single 10 mg/kg IV bolus dose of [14C]VNP40101M to male pigmented Long–Evans rats. The maximum concentrations were observed at 0.5 or 1 h post-dose for majority tissues (28 of 42). The highest concentrations of radioactivity were found in the small intestine contents at 0.5 h (112.137 µg equiv/g), urinary bladder contents at 3 h (89.636 µg equiv/g) and probably reflect excretion of drug and metabolites. The highest concentrations in specific organs were found in the renal cortex at 1 h (28.582 µg equiv/g), small intestine at 3 h (16.946 µg equiv/g), Harderian gland at 3 h (12.332 µg equiv/g) and pancreas at 3 h (12.635 µg equiv/g). Concentrations in the cerebrum (1.978 µg equiv/g), cerebellum (2.109 µg equiv/g), medulla (1.797 µg equiv/g) and spinal cord (1.510 µg equiv/g) were maximal at 0.5 h post-dose and persisted for 7 days. 4. The predicted total body and target organ exposures for humans given a single 100 µCi IV dose of [14C]VNP40101M were well within the medical guidelines for maximum radioactivity exposures in human subjects.

Reaction products of hexamethylene diisocyanate vapors with “self” molecules in the airways of rabbits exposed via tracheostomy

Abstract 1. Hexamethylenediisocyanate (HDI) is a widely used aliphatic diisocyanate and a well-recognized cause of occupational asthma. 2. “Self” molecules (peptides/proteins) in the lower airways, susceptible to chemical reactivity with HDI, have been hypothesized to play a role in asthma pathogenesis and/or chemical metabolism, but remain poorly characterized. 3. This study employed unique approaches to identify and characterize “self” targets of HDI reactivity in the lower airways. Anesthetized rabbits free breathed through a tracheostomy tube connected to chambers containing either, O2, or O2 plus ∼200 ppb HDI vapors. Following 60 minutes of exposure, the airways were lavaged and the fluid was analyzed by LC-MS and LC-MS/MS. 4. The low-molecular weight (<3 kDa) fraction of HDI exposed, but not control rabbit bronchoalveolar lavage (BAL) fluid identified 783.26 and 476.18 m/z [M+H]+ ions with high energy collision-induced dissociation (HCD) fragmentation patterns consistent with bis glutathione (GSH)-HDI and mono(GSH)-HDI. Proteomic analyses of the high molecular weight (>3 kDa) fraction of exposed rabbit BAL fluid identified HDI modification of specific lysines in uteroglobin (aka clara cell protein) and albumin. 5. In summary, this study utilized a unique approach to chemical vapor exposure in rabbits, to identify HDI reaction products with “self” molecules in the lower airways.

Population pharmacokinetic (PK) analysis of laromustine, an emerging alkylating agent, in cancer patients.

Abstract 1. Alkylating agents are capable of introducing an alkyl group into nucleophilic sites on DNA or RNA through covalent bond. Laromustine is an active member of a relatively new class of sulfonylhydrazine prodrugs under development as antineoplastic alkylating agents, and displays significant single-agent activity. 2. This is the first report of the population pharmacokinetic analysis of laromustine, 106 patients, 66 with hematologic malignancies and 40 with solid tumors, participated in five clinical trials worldwide. Of these, 104 patients were included in the final NONMEM analysis. 3. The population estimates for total clearance (CL) and volume of distribution of the central compartment (V1) were 96.3 L/h and 45.9 L, associated with high inter-patient variability of 52.9% and 79.8% and inter-occasion variability of 26.7% and 49.3%, respectively. The population estimates for Q and V2 were 73.2 L/h and 29.9 L, and inter-patient variability in V2 was 63.1%, respectively. 4. The estimate of Vss (75.8 L) exceeds total body water, indicating that laromustine is distributed to tissues. The half-life is short, less than 1 h, reflecting rapid clearance. Population PK analysis showed laromustine pharmacokinetics to be independent of dose and organ function with no effect on subsequent dosing cycles.

Abstract 1635: Multiplexed analysis of fixed tumor tissues using imaging mass cytometry

Introduction: Comprehensive evaluation of the tumor microenvironment with preservation of spatial context requires simultaneous in situ detection of multiple targets. Current fluorescence-based methods can accommodate up to 5-6 distinct markers and can be applied to formalin fixed, paraffin embedded tissue (FFPE). Imaging mass cytometry uses metal-conjugated antibodies and mass spectrometry to perform highly multiparametric, quantitative measurement of protein targets within tissue sections providing unprecedented detail of quantitative tissue labeling. To date, the IMC technology has not been widely available and its performance has not yet been compared with other validated methods. Methods: Using multiplexed quantitative immunofluorescence (QIF, AQUA/Genoptix) and imaging mass cytometry (IMC, Fluidigm) we validated and optimized metal-conjugated assays to detect HER2 (29D8-CST, 176Yb), CD3 (DAKO, 170Er), Ki-67 (B56-BD biosciences, 168Er), Histone H3 (D1H2-CST, 176Yb), pancytokeratin (C11-Biorad, 162Dy), Vimentin (RV202-BD Pharmingen, 156Gd), LipoR (Fluidigm, 115In) and DNA/nuclei (DNA intercalator, Fluidigm, 191/193Ir). The markers were applied to FFPE samples from tumor cell lines, human tonsil, lymph node; and breast and lung carcinomas represented in tissue microarray format. Data was visualized using MCD viewer and ImageJ software and results from QIF and IMC were compared. Results: Metal conjugation did not affect the performance of the primary antibodies studied here. The staining patterns of the epithelial/tumor and stromal compartments evaluated using the IMC platform and QIF were comparable. HER2 signal was higher in tumors and cell lines harboring HER2 amplification by FISH/IHC. The CD3 signal was higher in T-cell lymphoma cells and in the inter-follicular areas of human tonsil and lymph nodes. Ki-67 signal was nuclear, co-localized with Histone H3 and showed positivity in lymphoid germinal centers, cytokeratin-positive carcinoma cells and CD3+ tumor infiltrating lymphocytes. Results obtained using the IMC at different time points in serial tissue sections were comparable (P Conclusion: Quantitative and reproducible measurement of immune and non-immune targets with spatial resolution using the IMC platform is feasible in FFPE breast and lung carcinomas and the results are comparable to multiplexed QIF. Expansion of the current panel to include >30 markers for immune cell phenotypes/function and tumor markers is ongoing. Citation Format: Franz Villarroel-Espindola, Daniel Carvajal-Hausdorf, Ila Datar, Amanda Esch, Narges Rashidi, Ala Nassar, Shelly Ren, Ruth R. Montgomery, Roy S. Herbst, David L. Rimm, Kurt A. Schalper. Multiplexed analysis of fixed tumor tissues using imaging mass cytometry [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 1635. doi:10.1158/1538-7445.AM2017-1635