Phenotyping of Disease-Initiating CD34+/CD38- Stem Cells in BCR-ABL1- MPN Reveals Expression of Multiple Cytokine Receptors and Resistance-Related Antigens
Daniel Ivanov1,2, Jelena D. Milosevic Feenstra2, Alexandra Keller1,2, Gregor Eisenwort1,2, Irina Sadovnik1,2, Harald Herrmann2,3, Barbara Peter1,2, Michael Willmann2,4, Georg Greiner2,5, Gabriele Stefanzl1,2, Katharina Slavnitsch2,6, Gregor Hoermann2,5,7, Wolfgang R. Sperr1,2, Peter Bettelheim8, Klaus Geissler9, Elisabeth Koller10, Michael Fillitz10, Thamer Sliwa10, Michael Pfeilstöcker2,10, Felix Keil2,10, Thomas Rülicke2,6, Robert Kralovics5,11, Peter Valent1,2
1Department of Medicine I, Division of Hematology and Hemostaseology, Medical University of Vienna, Austria; 2Ludwig Boltzmann Institute for Hematology and Oncology, Medical University of Vienna, Austria; 3Department of Radiation Oncology, Medical University of Vienna, Austria; 4Department for Companion Animals & Horses, Clinic for Internal Medicine and Infectious Diseases, University of Veterinary Medicine Vienna, Austria; 5Department of Laboratory Medicine, Medical University of Vienna, Austria; 6Institute of Laboratory Animal Science, University of Veterinary Medicine Vienna, Austria; 7MLL Munich Leukemia Laboratory, Munich, Germany; Department of Internal Medicine I, Division of Hematology, Medical University of Graz, Austria; 8Elisabethinen Hospital Linz, Austria; 9Medical School, Sigmund Freud University, Vienna, Austria; 10Third Medical Department for Hematology and Oncology, Hanusch Hospital Vienna, Austria; 11CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria. Disclosures
Peter Valent, MD:
• Allcyte GmbH - Research Funding • Pfizer - Honoraria • Cellgene - Honoraria and Research Funding
Robert Kralovics, PhD
• AOP Orphan Pharmaceuticals AG - Honoraria • PharmaEssentia - Honoraria • Quiagen - Honoraria • Novartis – Honoraria • MyeloPro Diagnostics and Research – Current equity holder in private company
The other co-authors have nothing to disclose. Introduction
The classical BCR-ABL1-negative myeloproliferative neoplasms (MPN) are characterized by over-production of myeloid cells, disease-related mutations in certain driver-genes (JAK2, CALR, MPL) and an increased risk to transform to secondary acute myeloid leukemia (sAML). Although considered stem cell-derived neoplasms, little is known about the phenotype and functional properties of disease-initiating neoplastic stem cells (NSC) in MPN and sAML. These cells are considered most critical target populations to be examined for expression of molecular and immunological targets with the aim to develop improved or even curative NSC-eliminating therapies.
The aim of the present study is to characterize the immature CD34+/CD38− stem cells and CD34+/CD38+ progenitor cells from MPN patients in order to identify the expression pattern of surface markers and targets. Methods
Using a panel of monoclonal antibodies (n=40) and multicolor flow cytometry, we established the immunological phenotype and target expression profiles of putative CD34+/CD38- NSC and CD34+/CD38+ progenitor cells in patients with polycythemia vera (PV, n=18), essential thrombocythemia (ET, n=29), primary myelofibrosis (PMF, n=38) and post-MPN sAML (n=11).
CD34+/CD38+ Progenitor cells
CD34+/CD38- Stem cells
In order to define and analyze immature CD34+/CD38- stem cells and CD34+/CD38+ progenitor cells, a sequential gating strategy indicated by black arrows was applied using FlowJo software. Expression of novel and established stem cell antigens and lineage-related markers
Expression of cell surface antigens Antigen CD CD34+/CD38- CD25 CD34+/CD38+ on stem cells (CD34+/CD38-) CD34+/CD38- p<0.001 nBM ET PV PMF sAML p<0.01
IL-2RA CD25 - +/- - + +/-
DPPIV CD26 - - - +/- -
Hermes CD44 ++ ++ ++ ++ +
Thy-1 CD90 +/- - +/- +/- -
C1qR1 CD93 +/- +/- +/- + +/-
CD34+/CD38- CD26 CD34+/CD38+ ADGRE5 CD97 ++ ++ ++ ++ + CD34+/CD38-
MIC2 CD99 ++ ++ ++ ++ ++
Prominin-1 CD133 ++ + ++ + ++
CLL-1 CD371 - - - - n.t.
IL-1RAP n.c. - +/- - - +/-
Abbreviations: CD, cluster of differentiation; nBM, normal bone marrow; n.c., not Bone marrow aspirates were analyzed by flow cytometry for expression of the clustered; n.t., not tested. ++, strongly expressed in most cases; +, clear expression indicated surface markers. Staining index represents the ratio between median in most cases; +/−, expression in minority of cases or weak expression in most fluorescence intensity (MFI) for the marker and MFI for the isotype control. Dotted cases; −, no expression in a vast majority of cases. horizontal line represents the cut-off value for negativity (staining index <1.5). Expression of cytokine receptors on stem- and progenitor cells
Expression of cell surface antigens CD34+/CD38- CD34+/CD38+ Antigen CD CD117 on stem cells (CD34+/CD38-) CD34+/CD38- nBM ET PV PMF sAML
IL-2RA CD25 - +/- - + +/- Endoglin CD105 + + + + + TPOR CD110 +/- +/- +/- +/- +/- G-CSFR CD114 - - - - - M-CSFR CD115 - - - - - GM-CSFR CD116 +/- - - - - KIT CD117 ++ ++ ++ ++ ++
IL-3RA CD123 + + + + + CD34+/CD38- CD184 CD34+/CD38+ CD34+/CD38- IL-3RB CD131 - - - - - FLT3 CD135 +/- - +/- - +/- p<0.0001 p<0.01 CXCR4 CD184 - +/- +/- + +/- IGF-1R CD221 +/- +/- +/- +/- +/- NGF-R CD271 - - - - - VEGFR-2 CD309 - - - - - IL-1RAP n.c. - +/- - - +/- EPOR n.c. +/- - - - - MET n.c. - - - - - OSMRB n.c. - - - - - Expression of immune checkpoints on stem- and progenitor cells
Expression of cell surface antigens CD34+/CD38- CD274 CD34+/CD38+ Antigen CD CD34+/CD38- on stem cells (CD34+/CD38-)
nBM ET PV PMF sAML p<0.001 p<0.05 T44 CD28 - - - - -
IAP CD47 ++ ++ ++ ++ ++
Campath-1 CD52 + + +/- +/- +/-
B7-1 CD80 - - - - -
HB15 CD83 + +/- +/- +/- -
B7-2 CD86 - - - +/- -
Tactile CD96 - - - +/- n.t. CD34+/CD38- PD-L2 CD273 - - - - -
PD-L1 CD274 + + + + +
PD1 CD279 - - - - -
Abbreviations: CD, cluster of differentiation; nBM, normal bone marrow; n.c., not clustered; n.t., not tested. ++, strongly expressed in most cases; +, clear expression in most cases; +/−, expression in minority of cases or weak expression in most cases; −, no expression in a vast majority of cases. Expression of surface target antigens on stem- and progenitor cells
Expression of cell surface antigens CD34+/CD38- CD33 CD34+/CD38+ Antigen CD CD34+/CD38- on stem cells (CD34+/CD38-) nBM ET PV PMF sAML p<0.01 p<0.01 IL-2RA CD25 - +/- - + +/- DPPIV CD26 - - - +/- - Siglec-3 CD33 ++ + + ++ + Hermes CD44 ++ ++ ++ ++ + Campath-1 CD52 + + +/- +/- +/- C1qR1 CD93 +/- +/- +/- + +/- Tactile CD96 - - - +/- n.t. MIC2 CD99 ++ ++ ++ ++ ++ KIT CD117 ++ ++ ++ ++ ++ CD34+/CD38- IL-3RA CD123 + + + + + FLT3 CD135 +/- - +/- - +/- CXCR4 CD184 - +/- +/- + +/- PD-L2 CD273 - - - - - PD-L1 CD274 + + + + + PD1 CD279 - - - - - CLL-1 CD371 - - - - n.t. IL-1RAP n.c. - +/- - - +/- MPN donors´ MNCs containing CD34+ cells engraft in NSGS mice
2.4 Gy
BM aspirate
x 5 NSGS Flow cytometric analysis for the i.v injection engraftment of human cells MNC isolation 28 weeks 2.4 Gy
x 5 NSGS
Total human engraftment of the 3 MPN donors in the BM of NSGS mice. T-cell depleted bulk MNC or purified CD34– cells from donors with post-PV MF (#1) or PV (#2 and #3) were intravenously injected into sublethally irradiated NSGS mice. After 28 weeks, mice were sacrificed and engraftment was evaluated by flow cytometry analysis of the BM cells. The bars show the mean ± SD of the percentage of engrafted human CD45+ cells among all mice in the respective cohort. *, p<0.05. Conclusions
MPN stem- and progenitor cells display a unique phenotype, including cytokine receptors, immune checkpoint molecules and other target antigens, such as CD33, CD52, CD117, CD123 and PD-L1.
CD25, CXCR4 (CD184) and PD-L1 (CD274) were expressed at significantly higher levels on MPN NSC compared to normal SC.
High expression of CD25 on MPN NSC was particularly observed in patients with PMF.
MPN NSC expressed significantly lower levels of CD33 compared to healthy SC.
The stem cell phenotype in MPN did not change substantially during progression to sAML.
We showed that MPN stem cells reside in the CD34+ fraction of the malignant clone and the disease- initiating capacity of MPN stem- and progenitor cells could be confirmed using primary MPN cells in xenotransplantation experiments with NSGS mice.