Phenotyping Of Leukemic Stem Cells In Ph+ ALL and Ph- ALL Reveals Unique Profiles Of Markers and Targets In Distinct Disease Variants
Authors: Cerny-Reiterer Sabine , Blatt Katharina , Herrmann Harald , Eisenwort Gregor , Herndlhofer Susanne , Sill Heinz , Rabitsch Werner , Mannhalter Christine , Hauswirth Alexander , Sperr Wolfgang R , Jaeger Ulrich , Valent Peter 
Published: October 2019

Abstract
Acute lymphoblastic leukemia (ALL) is a life-threatening hematopoietic neoplasm characterized by abnormal growth and accumulation of lymphatic blast cells in various hematopoietic tissues. In a substantial number of patients, the Philadelphia (Ph) chromosome and the related oncoprotein BCR/ABL, are detectable. Despite recent advances in the management and therapy of patients with ALL, including the use of BCR/ABL1 tyrosine kinase inhibitors (TKI), the prognosis is still poor. Therefore, several attempts have been made to improve targeted treatment approaches in ALL. One strategy is to identify markers and targets expressed on leukemic stem cells (LSC) in these patients and to apply targeted drugs in order to eliminate LSC. In patients with Ph+ ALL, the leukemia-initiating cell-population is considered to reside within a CD34+/CD38- fraction of the clone. In the present study, we examined the expression of various stem cell markers and target antigens in CD34+/CD38- stem cells and in more mature CD34+/CD38+ progenitor cells in patients with Ph+ ALL (n=12), Ph- ALL (n=13), Ph+ CML (n=20), and in control bone marrow (BM) samples (unexplained cytopenia, n=10). Surface expression of target antigens was analyzed by multicolor flow cytometry, and mRNA expression levels by qPCR. As assessed by flow cytometry, CD34+/CD38- cells were found to co-express CD19, the stem cell-homing receptor CD44, the Campath-1 antigen (CD52), AC133 (CD133), FLT3 (CD135), and CXCR4 (CD184) in all ALL patients examined. In a majority of the ALL patients tested (14/25), LSC also expressed Siglec-3 (CD33). In CML, LSC were found to express a similar profile of antigens, including CD33, CD44, CD52, CD133, CD135, and CXCR4, but these cells did not express CD19. In control BM samples, CD34+/CD38- cells expressed a similar phenotype, but the levels of CD33 and CD52 were lower compared to LSC in ALL and CML. The IL-1RAP was found to be expressed on LSC in patients with Ph+ CML and Ph+ ALL, but not on LSC in Ph- ALL or in normal BM stem cells. By contrast, the SCF receptor KIT (CD117) was found to be expressed on LSC in Ph+ CML but was hardly detectable on LSC in patients with Ph+ ALL or Ph- ALL. The IL-2RA (CD25) and the SDF-1-degrading surface enzyme dipeptidyl-peptidase IV (DPPIV=CD26) were expressed on LSC in patients with CML and in all patients with Ph+ ALL exhibiting BCR/ABL-p210, whereas in Ph+ ALL with BCR/ABL-p190, LSC variably expressed CD25, and did not express CD26. In patients with Ph- ALL and in the normal BM, CD34+/CD38- cells did not express CD25 or CD26. The target receptor CD20 was detectable on ALL LSC in 7/18 patients examined. All target receptors tested were also detectable on more mature CD34+/CD38+ progenitor cells in patients with Ph+ ALL and Ph- ALL. In consecutive studies, expression of target antigens was confirmed at the mRNA level by qPCR analyses of highly enriched ALL LSC. Finally, we were able to show that the CD52-targeting drug alemtuzumab induces rapid lysis of CD34+/CD38- ALL LSC in all patients examined (Figure). In summary, our data show that LSC in Ph+ ALL and Ph- ALL express a unique phenotype, including clinically relevant cytokine receptors and cell surface target antigens, including the Campath-1 antigen, CD52. In Ph+ ALL with BCR/ABL-p210, the phenotype of ALL LSC largely resembles the phenotype of LSC in Ph+ CML, confirming the close relationship and similar pathogenesis of these two types of leukemias.
Ficoll-isolated MNC of 4 patients with Ph+ ALL were incubated in control medium (Co) or in various concentrations of alemtuzumab (10-300 µg/ml) in RPMI 1640 medium in the presence of 30% human serum at 37°C for 1 hour. After washing, cells were stained with fluorochrome-conjugated mAb against CD34, CD38 and CD45 for 15 minutes. DAPI-staining was used to evaluate the percentage of viable cells. Cells were analysed using a FACSCanto II and FlowJo software. Results show the numbers of viable CD34+/CD38- cells and are expressed as percent of control (Co). Values represent the mean±S.D. of four independent experiments. Asterisk (*): p<0.05 compared to control.

Disclosures:
Valent: Novartis: Consultancy, Honoraria, Research Funding.