The Immunophenotype of Mast Cells The Search for Diagnostic & Prognostic Features within Systemic Mastocytosis Author: A. Alheraky Supervisors: Dr. A.B. Mulder; Dr. K. Meijer & Ing. M.T. Nijk 2018 December 1 2 Word of thanks I am grateful for the opportunity that has been given to me to work on this project. I would like to thank my supervisors, Andre Mulder, Kees Meijer and Marije Nijk for their positive input and feedback and Rene Mulder for his help. I would like to thank my father, and my whole family for what they have given me. And all praise is due to my Lord. Hereby, I present to you a modest piece of research on the disease of mastocytosis. 3 4 Contents Abstract ......................................................................................................................................................... 7 Introduction ................................................................................................................................................... 8 Mast Cell Function .................................................................................................................................... 8 Mast Cell Maturation and Pathogenesis .................................................................................................... 8 Diagnosis of Mastocytosis ....................................................................................................................... 10 Classification of Systemic Mastocytosis ................................................................................................. 11 Provisional Subtypes of Systemic Mastocytosis ..................................................................................... 11 Prognosis of Systemic Mastocytosis ....................................................................................................... 12 Immunophenotype of Mast Cells ............................................................................................................ 12 Biomarker Function ................................................................................................................................. 13 Immunophenotype SM vs non-SM ......................................................................................................... 14 Association of Immunophenotype with Clinical Features ...................................................................... 15 Research Goals ........................................................................................................................................ 15 Materials & Methods ................................................................................................................................... 16 Principle of Flow Cytometry ................................................................................................................... 16 Data Assessment...................................................................................................................................... 16 Gating Methods ................................................................................................................................... 16 Laboratory and Clinical Data .............................................................................................................. 17 Statistical Methods .................................................................................................................................. 17 Variables .............................................................................................................................................. 17 Statistical Calculations ........................................................................................................................ 17 Results ......................................................................................................................................................... 19 Gating methods........................................................................................................................................ 19 Marker Correlations with cKIT ............................................................................................................... 21 Dendrogram ............................................................................................................................................. 27 Correlation of all Markers with in a Proximity Matrix ........................................................................... 28 Expression Patterns ................................................................................................................................. 29 Comparison Autofluorescence Signal with Marker Signal ..................................................................... 31 Discussion ................................................................................................................................................... 33 Gating Strategy ........................................................................................................................................ 33 Immunophenotype ................................................................................................................................... 34 Correlations and Associations for Future Research ............................................................................... 36 5 Bibliography ................................................................................................................................................ 37 Appendix ..................................................................................................................................................... 43 Annex 1: ...................................................................................................................................................... 43 Assessment of flowyctometric data with InfinicytTM .............................................................................. 43 Goal ..................................................................................................................................................... 43 Gating instructions .............................................................................................................................. 43 Acquiring statistical data ..................................................................................................................... 47 Annex 2: .................................................................................................................................................. 49 CD117 ................................................................................................................................................. 49 CD45 ................................................................................................................................................... 52 CD71 ................................................................................................................................................... 54 CD2 ..................................................................................................................................................... 56 CD30 ................................................................................................................................................... 58 CD13 ................................................................................................................................................... 60 CD25 ................................................................................................................................................... 62 CD52 ................................................................................................................................................... 64 FceRI ................................................................................................................................................... 66 CD33 ................................................................................................................................................... 68 CD56 ................................................................................................................................................... 70 CD123 ................................................................................................................................................. 72 CD22 ................................................................................................................................................... 74 CD34 ................................................................................................................................................... 76 CD138 ................................................................................................................................................. 78 6 Abstract Systemic mastocytosis is a clonal disorder that is defined by the neoplastic proliferation of MCs in one or more organ systems where the cKIT D816V activating point mutation plays a predominant role. Constitutive activity of the KIT receptor, excessive proliferation, increased cell survival and an aberrant expression of a series of proteins is induced. The WHO 2017 classification of SM is clinically subdivided into different forms; ie. indolent systemic mastocytosis (ISM), smouldering systemic mastocytosis (SSM), aggressive systemic mastocytosis (ASM), systemic mastocytosis with an associated