Using Flow and Mass Cytometry to Demonstrate Robust Tissue Processing to Query Molecular Heterogeneity in Phase 1 of the Accelerating Medicines Partnership (AMP)

Kevin Wei¹, Deepak Rao², Fan Zhang³, Chamith Fonseka³, Kamil Slowikowski³, Joshua Keegan⁴, Laura T. Donlin⁵, Jason Turner⁶, Mandy J. McGeachy⁷, Nida Meednu⁸, David Lieb⁹, Stephen Kelly¹⁰, Susan M. Goodman¹¹, David L. Boyle¹², William H. Robinson¹³, Paul J. Utz¹⁴, Gary S. Firestein¹⁵, Harris Perlman¹⁶, Edward F. DiCarlo¹⁷, Costantino Pitzalis¹⁰, Andrew Filer¹⁸, Brendan Boyce¹⁹, Ellen M. Gravallese²⁰, Chad Nusbaum²¹, James Lederer⁴, Nir Hacohen^22,23,24, Peter Gregersen²⁵, Larry W. Moreland²⁶, Michael Holers²⁷, Vivian P. Bykerk²⁸, Soumya Raychaudhuri³, Michael Brenner²⁹ and Jennifer H. Anolik⁸, ¹Division of Rheumatology, Immunology and Allergy, Brigham and Women's Hospital and Harvard Medical School, Boston, MA, ²Division of Rheumatology, Immunology, and Allergy, Brigham and Women's Hospital and Harvard Medical School, Boston, MA, ³Divisions of Genetics and Rheumatology, Brigham and Women's Hospital and Harvard Medical School, Boston, MA, ⁴Department of Surgery, Brigham and Women's Hospital and Harvard Medical School, Boston, MA, ⁵Arthritis and Tissue Degeneration Program and the David Z. Rosensweig Genomics Research Center, Hospital for Special Surgery, New York, NY, ⁶Institute of Inflammation and Ageing, University of Birmingham, Birmingham, United Kingdom, ⁷Medicine, University of Pittsburgh, Pittsburgh, PA, ⁸Medicine- Allergy, Immunology and Rheumatology, University of Rochester Medical Center, Rochester, NY, ⁹Broad Institute, Cambridge, MA, ¹⁰Centre for Experimental Medicine and Rheumatology, William Harvey Research Institute, Barts and The London, School of Medicine and Dentistry, Queen Mary University of London, London, United Kingdom, ¹¹Medicine, Hospital for Special Surgery/Weill Cornell Medicine, New York, NY, ¹²University of California San Diego, La Jolla, CA, ¹³Stanford University School of Medicine, Stanford, CA, ¹⁴Medicine, Stanford University School of Medicine, Stanford, CA, ¹⁵Medicine, University of California San Diego, La Jolla, CA, ¹⁶Department of Medicine, Division of Rheumatology, Northwestern University Feinberg School of Medicine, Northwestern University Feinberg School of Medicine,, Chicago, IL, ¹⁷Laboratory Medicine, Hospital for Special Surgery, New York, NY, ¹⁸Institute of Inflammation and Ageing (IIA), University of Birmingham, Birmingham, United Kingdom, ¹⁹University of Rochester Medical Center, Rochester, NY, ²⁰Lazare Research Bldg, University of Massachusetts Medical School, Worcester, MA, ²¹The Broad Institute and Harvard, Cambridge, MA, ²²Harvard Medical School, Boston, MA, ²³Massachusetts General Hospital, Charlestown, MA, ²⁴Broad Institute of MIT and Harvard, Cambridge, MA, ²⁵The Feinstein Institute for Medical Research, Northwell Health, Manhasset, NY, ²⁶Rheumatology & Clinical Immunology, University of Pittsburgh, Pittsburgh, PA, ²⁷Medicine, Division of Rheumatology, University of Colorado Denver, Aurora, CO, ²⁸Divison of Rheumatology, Hospital for Special Surgery, New York, NY, ²⁹Brigham and Women's Hospital and Harvard Medical School, Boston, MA

Meeting: 2017 ACR/ARHP Annual Meeting

Date of first publication: September 18, 2017

Keywords: CyTOF, rheumatoid arthritis (RA) and synovitis, Synovial Immune Biology

Session Information

Date: Monday, November 6, 2017

Title: Rheumatoid Arthritis – Human Etiology and Pathogenesis Poster II

Session Type: ACR Poster Session B

Session Time: 9:00AM-11:00AM

Background/Purpose:

Discovery and application of new therapies for rheumatoid arthritis (RA) has been hampered by multiple factors, including disease heterogeneity and the lack of well established approaches to analyze the synovial target tissue. The goal of the Accelerating Medicines Partnership (AMP) program is to deconstruct RA through cellular and molecular profiling of synovial tissue and matched peripheral blood from individuals with RA. During phase 1 of the AMP – RA program, we tested the feasibility of applying cytometric and transcriptomic analyses to synovial tissues from RA patients recruited from clinical sites across the network.

Methods:

Patient Enrollment. A multicenter cross-sectional study of individuals undergoing elective surgical procedures and a prospective observational study of synovial biopsy from RA ≥ age 18 with at least one inflamed joint were recruited from 10 contributing sites in the network.

Tissue Processing. Synovial tissues were cryopreserved on site then shipped to a central processing site for tissue disaggregation, cell sorting, mass cytometry and RNA-seq. Synovial tissue quality and grading of synovitis were evaluated via histologic analysis (H and E staining).

Analytic Pipelines. Cellular composition of RA and osteoarthritic (OA) synovial tissue was determined by flow cytometry and mass cytometry. RNA-seq was performed in parallel.

Results:

58 synovial tissues acquired from 22 synovial biopsies and 36 elective surgical procedures (20 RA and 16 OA) were analyzed by combination of flow cytometry, mass cytometry, and RNA-seq. Synovial cellular composition determined by flow cytometry and mass cytometry were highly consistent in T cells (r=0.98), B cells (r=0.96), myeloid cells (r=0.64), endothelial cells (r=0.81) and synovial fibroblasts (r=0.93), validating synovial analysis by mass cytometry of synovial tissue. Whereas RA synovial tissue obtained from elective surgical procedures exhibited varied degrees of inflammation with mean Krenn inflammation score (0-3) of 0.94 (S.D.=0.51), synovial tissues obtained from biopsies revealed significantly higher Krenn inflammation score than OA (1.68 vs 0.73; p=0.02) and higher abundance of lymphocytes by flow cytometry (67% vs 8%; p<0.001). Lymphocytic infiltration assessed by cytometry was significantly correlated with histologic inflammation score (p<0.001, r=0.58), further supporting the fidelity of flow cytometric assessment of cryopreserved synovial tissue. Principle component analysis of synovial cell flow cytometry data identified 3 RA arthroplasty samples with inflammatory features resembling that seen in RA biopsies. Consistent with this analysis, these arthroplasty samples also showed higher inflammation score of inflammation by histology (2.0 vs 0.77, p<0.001). Ongoing transcriptomic profiling of sorted synovial cells demonstrate molecular heterogeneity in RA synovium.

Conclusion:

In Phase 1 of the AMP-RA program, robust cell yield from synovial tissue enables mass and flow cytometric data. These results lay the ground work for the larger phase 2 of the Accelerating Medicines Partnership (AMP) – RA network, which will utilize >100 independent samples.

Disclosure: K. Wei, Roche Pharmaceuticals, 2; D. Rao, None; F. Zhang, None; C. Fonseka, None; K. Slowikowski, None; J. Keegan, None; L. T. Donlin, None; J. Turner, None; M. J. McGeachy, None; N. Meednu, None; D. Lieb, None; S. Kelly, None; S. M. Goodman, None; D. L. Boyle, None; W. H. Robinson, None; P. J. Utz, None; G. S. Firestein, None; H. Perlman, None; E. F. DiCarlo, None; C. Pitzalis, None; A. Filer, None; B. Boyce, None; E. M. Gravallese, Abbott Immunology Pharmaceuticals, 2,Lilly, Inc, 2,New England Journal of Medicine, 3,Up to Date, 7,Lilly Inc., 5,Sanofi/Genzyme, 5; C. Nusbaum, None; J. Lederer, None; N. Hacohen, None; P. Gregersen, None; L. W. Moreland, None; M. Holers, None; V. P. Bykerk, None; S. Raychaudhuri, Pfizer Inc, 2,Roche Pharmaceuticals, 2; M. Brenner, None; J. H. Anolik, None.

To cite this abstract in AMA style:

Wei K, Rao D, Zhang F, Fonseka C, Slowikowski K, Keegan J, Donlin LT, Turner J, McGeachy MJ, Meednu N, Lieb D, Kelly S, Goodman SM, Boyle DL, Robinson WH, Utz PJ, Firestein GS, Perlman H, DiCarlo EF, Pitzalis C, Filer A, Boyce B, Gravallese EM, Nusbaum C, Lederer J, Hacohen N, Gregersen P, Moreland LW, Holers M, Bykerk VP, Raychaudhuri S, Brenner M, Anolik JH. Using Flow and Mass Cytometry to Demonstrate Robust Tissue Processing to Query Molecular Heterogeneity in Phase 1 of the Accelerating Medicines Partnership (AMP) – RA Network [abstract]. Arthritis Rheumatol. 2017; 69 (suppl 10). https://acrabstracts.org/abstract/using-flow-and-mass-cytometry-to-demonstrate-robust-tissue-processing-to-query-molecular-heterogeneity-in-phase-1-of-the-accelerating-medicines-partnership-amp-ra-network/. Accessed .

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