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First AIDS cure progress 2010_Blood Journal

First AIDS cure progress 2010_Blood Journal

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Published by James Felton Keith
The novelAMLstem cell–associated antigen CLL-1 aids in discrimination
between normal and leukemic stem cells
The novelAMLstem cell–associated antigen CLL-1 aids in discrimination
between normal and leukemic stem cells

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Published by: James Felton Keith on Dec 18, 2010
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doi:10.1182/blood-2007-03-083048Prepublished online Jul 3, 2007;2007 110: 2659-2666SchuurhuisJanBijan Moshaver, Marijke Stigter-van Walsum, Sonja Zweegman, Gert J. Ossenkoppele and GerritAnna van Rhenen, Guus A. M. S. van Dongen, Angèle Kelder, Elwin J. Rombouts, Nicole Feller, 
discrimination between normal and leukemic stem cellsThe novel AML stem cellassociated antigen CLL-1 aids in
(2756 articles)Hematopoiesis and Stem Cells
Articles on similar topics may be found in the following 
Information about ordering reprints may be found online at: 
Information about subscriptions and ASH membership may be found online at:.Hematology; all rights reservedCopyright 2010 by The American Society of Washington DC 20036.by the American Society of Hematology, 2021 L St, NW, Suite 900,Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly
 For personal use only.by on December 17, 2010.www.bloodjournal.orgFrom
Anna van Rhenen,
GuusA. M. S. van Dongen,
Ange`le Kelder,
Elwin J. Rombouts,
Nicole Feller,
Bijan Moshaver,
Marijke Stigter-van Walsum,
Sonja Zweegman,
Gert J. Ossenkoppele,
and Gerrit Jan Schuurhuis
Department of Hematology and
Department of Otolaryngology, VU [Vrije Universiteit] University Medical Center,Amsterdam;
Department of Hematology,Erasmus University Medical Center, Rotterdam, the Netherlands
In CD34
acute myeloid leukemia (AML),the malignant stem cells reside in theCD38
compartment. We have shown be-fore that the frequency of suchCD34
cells at diagnosis corre-lateswithminimalresidualdisease(MRD)frequency after chemotherapy and withsurvival. Specific targeting ofCD34
cells might thus offer thera-peutic options. Previously, we found thatC-type lectin-like molecule-1 (CLL-1) hashigh expression on the whole blast com-partment in the majority of AML cases.We now show that CLL-1 expression isalso present on the CD34
stem-cell compartment inAML (77/89 patients).The CD34
population, containingthe CD34
cells, does en-graft in nonobese diabetic/severe com-binedimmunodeficiency(NOD/SCID)micewith outgrowth to CLL-1
blasts. CLL-1expression was not different between di-agnosisandrelapse(n
9).Inremission,both CLL-1
normal and CLL-1
malig-nant CD34
cells were present.A high CLL-1
fraction was associatedwith quick relapse. CLL-1 expression iscompletely absent both on CD34
cells in normal (n
11) and in regenerat-ing bone marrow controls (n
6). ThisAML stem-cell specificity of the anti-CLL-1 antibody under all conditions ofdisease and the leukemia-initiating prop-erties of CD34
cells indicate thatanti–CLL-1 antibody enables both AML-specific stem-cell detection and possiblyantigen-targeting in future. (Blood. 2007;110:2659-2666)
2007 by TheAmerican Society of Hematology
Despite high-dose chemotherapy, only 30% to 40% of patients withacute myeloid leukemia (AML) survive, which is due mainly torelapse of the disease.
AML is generally regarded as a stem-celldisease. However, there is debate whether normal stem cellsundergoingleukemogenicmutationsistheexplanationforleukemo-genesis.Alternatively, leukemogenic mutations occurring at a laterdevelopmental stage, resulting in stem cell–like behavior, might bean alternative or additional option.
For CD34
AML, severalauthors have shown that leukemic stem cells are present in theCD34
It has been proven in vitro thatthese stem cells are more resistant to chemotherapy, compared withthe progenitor CD34
In vivo, after chemotherapy,the residual malignant CD34
cells are thought to differen-tiate to a limited extent
producing leukemic cells with animmunophenotype, which usually reflects that at diagnosis. Sensi-tive techniques allow early detection of small numbers of thesedifferentiated leukemic cells, called minimal residual disease(MRD), which eventually causes relapse of the disease.
Since inthis concept the stem cell is the origin of MRD and relapse, stemcell–targeted therapy would be of potentially high benefit forAMLpatients. Moreover, early detection of leukemic stem cells afterchemotherapeutictreatmentmightofferprognosticvalueinpredict-ing relapse of the disease. Different options for stem-cell identifica-tion and/or targeted therapy have been described such as anti-CD123, anti-CD44, and anti-CD33, but all have some (potential)disadvantages, including expression on normal stem cells and/ornonhematologic tissues.
Since the bone marrow of a (chemo-therapy-) treated patient cannot be considered normal, it is ex-tremely important to study whether after treatment normal stemcells in such regenerating bone marrow remain negative for theantigen of interest. So far, this has not been examined for CD33,CD44, and CD123.In this paper, we focus on the newly discovered antigen CLL-1,which we have described to be present on the majority of CD34
aswell as CD34
AML cases.
In peripheral blood, both monocytesand granulocytes show some CLL-1 expression, while it is absentin other tissues.
The intracellular domain of CLL-1 contains bothan immunotyrosine-based inhibition motif as well as a YXXMmotif, suggesting a role for CLL-1 as a signaling receptor.Phosphorylation of immunotyrosine-based inhibition motif–containing receptors on a variety of cells leads to inhibition of activationpathwaysviarecruitmentoftheproteintyrosinephospha-tases SHP-1, SHP-2, and SHIP.
The YXXM motif, on the otherhand, encompasses a potential SH2 domain–binding site for thep85 subunit of phosphatidylinositol 3
an enzyme impli-cated in cellular activation pathways. Whether these properties canbe translated to CLL-1 is still unknown.We now demonstrate that CD34
cells of AML patientsshowed engraftment in nonobese diabetic/severe combined immunode-ficient (NOD/SCID) mice, indicating that CLL-1 is expressed on theleukemic stem-cell population in these patients.Also, we found CLL-1to be present on AML and absent on normal CD34
cells atdifferenttimepointsofdisease/treatment,whichholdspotentialtoserve
Submitted March 30, 2007; accepted June 29, 2007. Prepublished online as
First Edition paper, July 3, 2007; DOI 10.1182/blood-2007-03-083048.The publication costs of this article were defrayed in part by page chargepayment. Therefore, and solely to indicate this fact, this article is herebymarked ‘‘advertisement’’in accordance with 18 USC section 1734.© 2007 by TheAmerican Society of Hematology2659BLOOD, 1 OCTOBER 2007
 For personal use only.by on December 17, 2010.www.bloodjournal.orgFrom
as a tool to detect residual leukemic CD34
cells after therapyandasapossibletargetfortherapy.
Patients, materials, and methods
Patient and control samples
The AML clinical protocols and the biologic studies were approved by thescientific research committee and the medical ethics committee of the VUUniversity Medical Center (Amsterdam, the Netherlands). Leukemic cellsof 89 patients presenting with CD34
AML at our institute were obtainedafter informed consent in accordance with the Declaration of Helsinki, atdiagnosis, and after chemotherapeutic treatment. CD34
AML was definedas samples with a CD34 percentage more than 1 because we havepreviously shown that in samples with less than 1% CD34
cells, theseCD34
cells are in general of normal origin.
In 16 cases, bone marrow(BM) was not available at diagnosis and peripheral blood (PB) was usedinstead. After chemotherapeutic treatment, only BM was used. RelapseAMLBM samples were obtained from 9 patients. Diagnosis of patients wasbased on morphology using French-American-British (FAB) classification,immunophenotyping, and cytogenetics.
Control normal bone marrow(NBM) was obtained from 11 patients undergoing cardiac surgery afterinformed consent. Control regenerating bone marrow (RBM) was obtainedfrom 3 patients with acute lymphoblastic leukemia, one patient withnon-Hodgkin lymphoma, one patient with completely CD34
AML, andone patient with CLL-1
AML. Mobilized peripheral blood (MPB) wasobtained from 6 non-AML patients after granulocyte colony-stimulatingfactor (G-CSF) stimulation.Patient characteristics are shown in Table 1. AML samples wereanalyzed fresh (n
52) or after storage in liquid nitrogen (n
37). Forexpression studies after chemotherapy and in controls fresh material wasused. For relapse studies (n
9) in 4 cases frozen-thawed material wasused. NOD/SCID mice repopulation experiments were performed usingfrozen-thawed material. Procedures used in this study have previously beenvalidated for the use of both fresh and frozen-thawed material.
In freshsamples, red blood cells were lysed using a 10-minute red blood cell lysison ice with 10 mL lysis buffer (155 mM NH
Cl, 10 mM KHCO
, 0.1 mMNa
EDTA, pH 7.4) and washed twice with phosphate-buffered saline (PBS)with 0.1% human serum albumin (HSA) added. Frozen samples wereprepared using a Ficoll gradient (1.077 g/mL; Amersham Biosciences,Freiburg, Germany) and subsequent red blood cell lysis. Cells were thenfrozen in RPMI (Gibco, Paisley, United Kingdom) with 20% heat-inactivated fetal bovine serum (FBS; Greiner, Alphen aan den Rijn, theNetherlands) and 10% DMSO (Riedel-de Haen, Seelze, Germany) inisopropanol-filled containers and subsequently stored in liquid nitrogen.When needed for analysis, cells were thawed and suspended in prewarmedRPMI with 40% FCS at 37°C. Cells were washed and enabled to recover for45 minutes in RPMI with 40% FCS at 37°C. Cells were washed again andsuspended in PBS with 0.1% HSA.
Fluorescence-activated cell sorting analysisof CD34
Fluorescence-activated cell sorting (FACS) procedures have been described indetail before.
In short, fresh cells were incubated with monoclonal antibodiesfor15minutesatroomtemperature,washedonceinPBScontaining0.1%HSA,and analyzed by flow cytometry. Monoclonal antibody combinations containedfluoresceinisothiocyanate(FITC)–,phycoerythrin(PE)–,peridinylchlorophyllin(PerCP)–, or allophycocyanin (APC)–labeled monoclonal antibodies. Anti-CD34 FITC, anti-CD34 PerCP, anti-CD45 PerCP, anti-CD45 APC, anti-CD38APC,anti-CD123PE,anti-CD7PE,anti-CD19PE,anti-CD33FITC,anti-CD33APC, and Via-Probe (7-amino-actinomycin D, 7AAD) were all from BDBiosciences (Basel, Switzerland); anti-CD34 FITC, which was used in some of thesamples,wasfromImmunotech(Marseille,France);andannexinVFITCwasfrom Nexins Research (Kattendijke, the Netherlands). Anti–CLL-1 and isotypecontrols(GBSandDNP)werefromCrucell(Leiden,theNetherlands).When frozen-thawed cells were used, annexinV FITC was included inthe majority of samples to gate out apoptotic/dead cells before stem-cellassessment. In the remaining samples, this was done by Syto16 (MolecularProbes, Eugene, OR) together with 7AAD in one tube, which enabled togate out apoptotic/dead cells.
The scatter properties of the viable cellswere then used in the tube containing CLL-1.PBS was used as a negative control, since for these specific antibodiesisotype controls offered the same results.
Data acquisition was performed usingaFACScalibur(BDBiosciences)equippedwithanargonandreddiodelaser,andanalysiswasperformedusingCellquestsoftware(BDBiosciences).Blasts were identified by CD45
dim/low side scatter
characteristics accordingto Lacombe, taking into account that the CD34
population is aminor population.
cell selection
cellswereselectedforthecolony-formingunit(CFU)assaysfromNBMand for the fluorescent in situ hybridization (FISH) analysis of a sample from anAML patient in complete remission. After Ficoll separation and red blood celllysis, mononuclear cells were incubated in PBS containing 5 mM EDTA and0.1% HSA for 30 minutes at room temperature with CD34 Reagent (MiltenyiBiotec, Bergisch Gladbach, Germany), according to the manufacturer’s proce-dure.Cellswerewashedandallowedtoflowthroughapositive-selectioncolumnin a magnetic field (Automacs; Miltenyi Biotec). After 2 rounds of selection,CD34
cells were collected. Purity was checked using flow cytometry and wasmorethan95%inallcases.
FACS sorting of CD34
FACS-sorted cells were used for CFU assays, FISH analysis, and NOD/ SCID mice experiments. Cells were incubated for 15 minutes at roomtemperature with Via-Probe (7AAD), anti-CD34 FITC, anti-CD38 APC,and anti–CLL-1 PE and washed in PBS containing 0.1% HSA. Cells weresubsequently sorted using a FACS Vantage cell sorter with Turbo Sortupgrade (BD Biosciences) equipped with an argon (377G) and a helium-neon (127) laser, both from Spectra-Physics (Mountain View, CA) or aFACSAria equipped with solid-state lasers (red, blue, and violet; BDBiosciences). Cells were sorted based on viability (7AAD negative),
Table 1. Patient characteristics
Characteristic Quantity
Patients, no. 89Male/female 45/44Age at diagnosis, y, mean (range) 55 (16-79)WBC count at diagnosis,10
/L, mean (range) 38 (0.7-278)
FAB classification, no. (%)
M0 5 (6)M1 12 (13)M2 14 (16)M3 3 (3)M4 11 (12)M5 20 (23)M6 3 (3)Refractory anemia with excess blasts 10 (11)Refractory anemia with excess blasts in transformation 5 (6)Not classied 6 (7)
Cytogenetic risk group, no. (%)
Favorable 11 (12)Intermediate 49 (55)Poor 9 (10)No metaphases 20 (23)
Flt3 ITD, no. (%)
Present 22 (25)Absent 54 (61)Not analyzed 13 (14)WBC indicates white blood cell; FAB, French-American-British.
2660 VAN RHENEN et al BLOOD, 1 OCTOBER 2007
 For personal use only.by on December 17, 2010.www.bloodjournal.orgFrom

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