CD52) As Novel Drug Target in Neoplastic Stem Cells in 5Q-Patients with MDS and AML

Published OnlineFirst May 5, 2014; DOI: 10.1158/1078-0432.CCR-13-2811

Clinical Cancer Cancer Therapy: Clinical Research

Identiﬁcation of Campath-1 (CD52) as Novel Drug Target in Neoplastic Stem Cells in 5q-Patients with MDS and AML

Katharina Blatt1, Harald Herrmann2, Gregor Hoermann3, Michael Willmann4, Sabine Cerny-Reiterer1,2, Irina Sadovnik1, Susanne Herndlhofer1, Berthold Streubel5, Werner Rabitsch6, Wolfgang R. Sperr1,2, Matthias Mayerhofer7, Thomas Rulicke€ 8, and Peter Valent1,2

Abstract Purpose: The CD52-targeted antibody alemtuzumab induces major clinical responses in a group of patients with myelodysplastic syndromes (MDS). The mechanism underlying this drug effect remains unknown. Experimental Design: We asked whether neoplastic stem cells (NSC) in patients with MDS (n ¼ 29) or acute myelogenous leukemia (AML; n ¼ 62) express CD52. Results: As assessed by ﬂow cytometry, CD52 was found to be expressed on NSC-enriched þ CD34 /CD38 cells in 8/11 patients with MDS and isolated del(5q). In most other patients with MDS, þ CD52 was weakly expressed or not detectable on NSC. In AML, CD34 /CD38 cells displayed CD52 in 23/ 62 patients, including four with complex karyotype and del(5q) and one with del(5q) and t(1;17;X). In quantitative PCR (qPCR) analyses, puriﬁed NSC obtained from del(5q) patients expressed CD52 mRNA. We were also able to show that CD52 mRNA levels correlate with EVI1 expression and that NRAS induces the expression of CD52 in AML cells. The CD52-targeting drug alemtuzumab, was found to induce comple- þ þ ment-dependent lysis of CD34 /CD38 /CD52 NSC, but did not induce lysis in CD52 NSC. Alemtu- þ zumab also suppressed engraftment of CD52 NSC in NSG mice. Finally, CD52 expression on NSC was found to correlate with a poor survival in patients with MDS and AML. Conclusions: The cell surface target Campath-1 (CD52) is expressed on NSC in a group of patients with MDS and AML. CD52 is a novel prognostic NSC marker and a potential NSC target in a subset of patients with MDS and AML, which may have clinical implications and may explain clinical effects produced by alemtuzumab in these patients. Clin Cancer Res; 20(13); 3589–602. 2014 AACR.

Introduction plasms (1–3). Both conditions share pathogenetic and Myelodysplastic syndromes (MDS) and acute myeloge- clinical features; and many patients with MDS transform nous leukemia (AML) are stem cell-derived, myeloid neo- to overt AML during disease evolution. MDS and AML are classified according to blast cell counts as well as cytogenetic and molecular features (3–6). The prognosis in AML varies, depending on age, burden of neoplastic (stem) cells, and Authors' Affiliations: 1Department of Internal Medicine I, Division of Hematology & Hemostaseology, Medical University of Vienna, Austria; certain cytogenetic and molecular lesions. In patients with 2Ludwig Boltzmann Cluster Oncology, Medical University of Vienna, Aus- MDS, prognostically relevant cytogenetic subgroups of tria; 3Department of Laboratory Medicine, Medical University of Vienna, patients have also been identified (7–9). Although in Austria; 4Department for Companion Animals and Horses, Clinic for Small Animals, Clinical Unit of Internal Medicine, University of Veterinary Med- low-risk MDS, an "isolated del(5q)" is indicative of a good icine Vienna, Austria; 5Department of Obstetrics and Gynecology, Medical prognosis, the prognosis of del(5q) patients with advanced 6 University of Vienna, Austria; Department of Internal Medicine I, Bone MDS or AML is poor, especially when the clone exhibits Marrow Transplantation Unit, Medical University of Vienna, Austria; 7Lud- wig Boltzmann Institute of Osteology, Hanusch-Hospital, Vienna, Austria; additional cytogenetic defects (7–11). A complex karyotype and 8Institute of Laboratory Animal Science, University of Veterinary is almost always a bad prognostic sign, independent of age Medicine Vienna, Austria and the category of MDS or AML. Note: Supplementary data for this article are available at Clinical Cancer Clonal cells in MDS and AML are organized hierar- Research Online (http://clincancerres.aacrjournals.org/). chically similar to normal hematopoiesis (2, 12–16). In Corresponding Author: Peter Valent, Division of Hematology and Hemos- this hierarchy, only the most primitive progenitors, also taseology & Ludwig Boltzmann Cluster Oncology, Department of Internal Medicine I, Medical University of Vienna, Wahringer€ Gurtel€ 18-20, A-1090 termed neoplastic stem cells (NSC), or leukemic stem Vienna, Austria. Phone: 43-1-40400-44160; Fax: 43-1-40400-40300; cells (LSC) in AML, have the capacity of long-term self- E-mail: [email protected] renewal, and thus are responsible for unlimited prolifer- doi: 10.1158/1078-0432.CCR-13-2811 ation, clonal evolution, and relapse after therapy 2014 American Association for Cancer Research. (12–16). In both MDS and AML, NSC and LSC are

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addition, our data show that alemtuzumab can attack Translational Relevance þ CD52 NSC/LSC. Although neoplastic stem cells (NSC) represent a most critical target of therapy in myelodysplastic syndrome Materials and Methods (MDS) and acute myelogenous leukemia (AML), no Patients NSC-targeting treatment concept has been translated Sixty-two patients with AML (35 females, 27 males; into clinical practice so far, and only little is known median age: 62 years) and 29 with MDS (14 females, 15 about expression of molecular targets on NSC in MDS males; median age: 70 years) were examined. Diagnoses and AML. Our study demonstrates that in del(5q) þ were established according to French-American-British patients with MDS and AML, CD34 /CD38 and þ þ (FAB) and World Health Organization (WHO) criteria CD34 /CD38 stem and progenitor cells express CD52, (4–6). The patients’ characteristics are shown in Supple- and that this antigen, Campath-1, serves as a potential mentary Tables S1 (MDS) and S2 (AML). Bone marrow target of therapy. Moreover, our data show that expres- (BM) aspirates were obtained from the iliac crest. Control sion of CD52 on NSC may be of prognostic significance bone marrow cells were obtained from 9 patients with in MDS and AML. These observations may have clinical chronic myelomonocytic leukemia (CMML), 7 with chron- implications and may explain reported effects of alem- ic myelogenous leukemia (CML), 8 with myeloproliferative tuzumab seen in some patients with MDS and AML. neoplasms (MPN), 5 with unclassifiable MDS/MPN (MDS/ MPN-U), 10 with acute lymphoblastic leukemia (ALL), 19 with cytopenia of undetermined significance (ICUS), and þ 25 with normal bone marrow (staging for lymphoma, n ¼ considered to reside in the CD34 compartment of the 19, Ewing sarcoma, n ¼ 1; remission marrow, n ¼ 5; clone (12–16). Depending on the disease variant, þ þ Supplementary Tables S3 and S4). Bone marrow mononu- CD34 /Lin cells or CD34 /CD38 cells may exhibit clear cells (MNC) were isolated using Ficoll. Karyotyping long-term repopulating capacity in NOD/SCID or and molecular analyses were performed according to stan- NOD/SCID IL-2Rgammanull (NSG) mice and thus stem dard techniques (34, 35). All donors gave written informed cell function. However, at least in AML, NSG-repopulat- þ þ consent. The study was approved by the ethics committee of ing NSC may also reside in the CD34 /CD38 fraction or the Medical University of Vienna (Vienna, Austria). even in a CD34-negtive subset of the clone (17, 18). During the past few years, substantial efforts have been made to characterize target expression profiles in NSC/LSC Reagents in MDS and AML (15, 16, 19–24). Among these targets are RPMI-1640 medium and fetal calf serum (FCS) were several surface molecules that are recognized by targeted purchased from PAA laboratories, FITC-labeled CD34 antibodies or antibody-toxin-conjugates. Likewise, it has monoclonal antibody (mAb) 581, PerCP-labeled CD45 þ been described that NSC/LSC-enriched CD34 /CD38 mAb 2D1, APC-labeled CD38 mAb HIT2, phycoerythrin cells in AML frequently coexpress CD33, CD44, and CD123 (PE)-labeled anti-CLL1 mAb 50C1 from BD Biosciences, PE-labeled CD52 mAb HI186, PE-labeled CD123 mAb (19, 20, 23, 24). € The CD52 antigen, also known as Campath-1, is 32703 from R&D Systems, FITC-labeled CD14 mAb TUK4 expressed broadly in lymphatic cells as well as on blood from Dako, APC-labeled CD123 mAb AC145 from Miltenyi monocytes (25, 26). On the basis of its expression on Biotech, and PerCP/Cy5.5-labeled CD90 mAb 5E10 and lymphoid progenitors and B cells, CD52 has been devel- Pacific Blue-labeled CD45RA mAb HI100 from Biolegend. oped as a drug target in advanced chronic lymphocytic Alemtuzumab was purchased from Genzyme and IgG leukemia (CLL) (25, 27–29). Indeed, the CD52-targeting from Abcam. For measuring drug effects on NSC/LSC, the þ drug alemtuzumab, is capable of eliminating CD52 lym- CountBright absolute counting beads (Invitrogen), the PE- phocytes in most patients with CLL (28, 29). labeled CD34 mAb 581 (BD Biosciences), and the APC/ More recently, alemtuzumab has also been adminis- Cy7-labeled CD45 mAb HI30 (Biolegend) were used. tered in patients with MDS (30, 31). The primary rational of this approach has been the observation that T-cell– Cell lines targeting immunosuppressive agents, like anti-thymocyte The CD52 AML cell line HL60 was maintained in RPMI- globulin, are effective in a subgroup of patients, especially 1640 medium with 10% FCS (37C), and the CD52-pos- those with hypoplastic MDS (32, 33). Alemtuzumab was itive ALL cell line Raji in RPMI-1640 plus 20% FCS. HL60 also found to induce remarkable responses and even and Raji cells were obtained from the DSMZ Institute remission in a few patients with MDS (30, 31). Whether (Braunschweig, Germany). The biologic stability of these these effects of alemtuzumab resulted from its immuno- cell lines was checked by cell surface phenotyping (flow suppressive activity remains unknown. We explored an cytometry) and their identity was confirmed by reauthenti- alternative mode of action of alemtuzumab in MDS, cation in the DSMZ Institute. HL60 cells were engineered to namely a direct effect on NSC. The results of our study express mutated RAS (NRAS G12D; NRAS Q61K) by lenti- show that CD52 is expressed on NSC/LSC-enriched viral transduction essentially as described (36). In brief, the þ CD34 /CD38 cells in patients with MDS and AML. In coding sequences of RAS mutants were cloned into

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lentiviral pWPI vector kindly provided by Dr. D. Trono AML, n ¼ 1) and 6 with MDS and del(5q), using RNeasy (University of Geneva, Switzerland). The pWPI vector con- Micro-Cleanup Kit (Qiagen). cDNA was synthesized using tains an internal ribosome entry site and a GFP coding Moloney murine leukemia virus reverse transcriptase (Invi- region. Recombinant lentiviruses were produced as trogen), random primers, first-strand buffer, dNTPs (100 described previously (37). After one week, transduced cells mmol/L), and RNasin (all from Invitrogen) according to the were examined for expression of CD52 by flow cytometry. manufacturer’s instructions. qPCR was performed as described (24) using iTaq SYBR-Green-Supermix with ROX Flow cytometry and characterization of NSC/LSC– (Bio-Rad) and primers specific for CD52, EVI1, and CD300a þ enriched CD34 /CD38 cells (Supplementary Table S5). mRNA levels were expressed as A number of mAb were applied to characterize NSC/LSC, percentage of ABL transcript levels. monocytes, and blood basophils. NSC/LSC-rich cells (called NSC/LSC below throughout this manuscript) were Evaluation of cytotoxic effects of alemtuzumab on stem þ þ defined as CD34 /CD45 /CD38 cells, and progenitor- and progenitor cells þ þ þ enriched cells as CD34 /CD45 /CD38 cells (20, 24, 38). Bone marrow MNC (AML, n ¼ 6; MDS, n ¼ 6; control þ Monocytes were identified as CD14 cells and basophils by samples, n ¼ 6) were incubated in various concentrations of their characteristic side-scatter properties and expression of alemtuzumab (10–300 mg/mL) in RPMI-1640 medium plus CD123 (39, 40). In patients with CD34-negative AML, 30% complement-containing human serum at 37C for CD34 blasts were identified by their characteristic side- 1 hour. Then, 10 mL calibration beads were added. After scatter properties. In a subset of patients with MDS (n ¼ 6) washing, cells were stained with fluorochrome-conjugated þ þ and AML (n ¼ 6), we examined CD34 /CD38 /CD90 / mAb against CD34, CD45, and CD38 for 15 minutes. Cells þ CD45RA cells or CD34 /CD38 /CD90 /CD45RA cells, were then subjected to 40, 6—diamidino—2—phenylindole respectively. Heparinized bone marrow cells (30–100 mL) (DAPI) staining to count viable cells on a FACSCanto-II (BD were incubated with combinations of mAb for 15 minutes. Biosciences). HL60 cells, Raji cells, and HL60 cells trans- After erythrocyte lysis with BD lysing solution (BD Bios- fected with empty vector or NRAS Q61K, were incubated ciences), expression of cell surface antigens was examined with various concentrations of alemtuzumab (0.1–500 by multicolor flow cytometry on a FACSCalibur or on a mg/mL) in RPMI-1640 medium with 30% serum at 37C FACSCantoII (BD Biosciences). Antibody reactivity was for 1 hour. After incubation, cells were examined for via- controlled by isotype-matched antibodies. The staining bility by propidium iodide staining. In experiments per- index (SI) was calculated from median fluorescence inten- formed with transfected HL60 cells, 10 mL calibration beads sities (MFI) obtained with the CD52 antibody and an were added and cells were examined for viability by propi- isotype-matched control antibody (SI ¼ MFICD52: dium iodide staining. In control experiments, cells were MFIcontrol). incubated with alemtuzumab in medium containing either IgG (20 mg/mL) or 30% human serum. þ Purification of CD34 /CD38 stem cells in MDS and AML Repopulation of AML cells in NOD/SCID In 11 patients with AML and 6 with del(5q) MDS, the IL-2Rgammanull (NSG) mice þ þ þ CD34 /CD38 NSC/LSC and CD34 /CD38 progenitors Primary AML cells (n ¼ 3 patients) were incubated in were purified from bone marrow MNC by cell sorting on a control medium or in alemtuzumab (500 mg/mL) with 30% FACSAria (BD Biosciences) using a PE-labeled CD34 mAb human serum at 37C for 1 hour. After incubation, AML and an APC-conjugated CD38 mAb as described (24, 38). cells were viable without signs of cell death or apoptosis þ After sorting, the purity of CD34 /CD38 stem cells and (alemtuzumab: 75%–80% cells viable; vs. control medium: þ þ CD34 /CD38 was >95% in each case, and cell viability 80%–90% of cells viable by Trypan blue staining). Drug- was >80% in all samples. exposed cells and control cells were washed, resuspended in 0.15 mL PBS with 2% FCS, and injected into the tail vein of Fluorecence in situ hybridization (FISH) studies adult female NSG mice (2–5 106 per mouse, 4–5 mice per In 5 patients with MDS and del(5q), sorted group; The Jackson Laboratory). Twenty-four hours before þ þ þ CD34 /CD38 cells, sorted CD34 /CD38 cells, and total injection, mice were irradiated (2.4 Gy). After injection, MNC were examined by FISH using a dual color probe-set mice were inspected daily and sacrificed after 10 weeks. (Cytocell) with a red probe for EGR1 (chromosomal band Bone marrow cells were obtained from flushed femurs, 5q31.1) and a green (control) probe for TAS2R1 (chromo- tibias, and humeri. Human AML cells were detected in bone somal band 5p15.31). The presence of trisomy 8 was marrow samples by multicolor flow cytometry using mAb confirmed in xenotransplanted cells by FISH using a probe against CD19, CD33, and CD45. AML repopulation was þ for centromere 8, obtained from Kreatech. FISH was per- measured by determining the percentage of CD45 cells in formed according to the manufacturer’s instructions. mouse bone marrow samples by flow cytometry. Animal studies were approved by the ethics committee of the Quantitative PCR (qPCR) Medical University of Vienna and the University of Vet- Total RNA was isolated from MNC of 11 patients with erinary Medicine Vienna (Vienna, Austria), and carried AML (FAB M1, n ¼ 5; M2, n ¼ 1; M4, n ¼ 4; secondary out in accordance with guidelines for animal care and

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protection. Animal experiment license was granted under patients with AML without del(5q) (32.7%), LSC expressed no. GZ 66.009/0040-II/10b/2009. high levels of CD52. This difference was found to be statistically significant (P < 0.05). In a majority of patients þ Statistical analysis in whom CD34 /CD38 cells displayed CD52, the þ þ To analyze the significance of differences in expression of CD34 /CD38 cells also expressed CD52 homogenously CD52 on NSC/LSC in various subgroups of patients with without a negative subfraction (MDS: 9/15 ¼ 60%; AML, MDS and AML, the Mann–Whitney U test was applied. To 14/23 ¼ 60.9%). Overall, CD52 was found to be expressed þ þ þ determine correlations between surface and mRNA expres- at higher levels on CD34 /CD38 cells and CD34 /CD38 sion levels of CD52 in NSC/LSC, between EVI1 mRNA and cells in patients with MDS or AML compared with control CD52 mRNA expression, between CD300a mRNA and bone marrow samples (Fig. 1B and Supplementary Fig. S1). CD52 mRNA expression, and between CD52 expression In patients with AML, CD52 was also found to be expressed and cytotoxic effects of alemtuzumab on NSC/LSC, a linear on monocytes and basophils in most donors, although in regression model was applied. The probability of overall several patients, expression levels were low or even undetect- survival (OS) in our patients with MDS (n ¼ 29) and AML able, thereby contrasting MDS (Table 1). Recent data suggest (n ¼ 62), and the AML-free survival in our patients with MDS that in patients with NPM1-mutated AML and other AML were calculated by the product limit method of Kaplan and types, LSC may (also) reside in a CD34-negative fraction of Meier. The median follow-up in our patients with MDS and the clone (18). Therefore, we were interested to learn whether AML was 422 days and 382 days, respectively. Moreover the CD52 is expressed on CD34-negative blasts in our patients number of patients at risk was calculated at 0 to 84 months. with AML. We were able to detect CD34-negative blast cells in Statistical significances in differences among patients with or 41/62 patients with AML.In11ofthese41patients,including þ without CD52 stem cells concerning survival and AML-free 3carryingaNPM1 mutation, the CD34-negative blasts survival (patients with MDS) were determined by log-rank expressed substantial amounts of CD52 (Table 1). We þ test. For determining the level of significance in drug inhi- also found that CD52 is expressed on the CD34 /CD38 / bition experiments, the Student’s t test was applied. Differ- CD90 /CD45RA subset of LSC in our patients with þ þ ences were considered significant when P < 0.05. AML. These CD34 /CD38 /CD90 /CD45RA cells also expressed CD123, and in a subset of patients (2/6), these Results cells expressed CLL-1 (Supplementary Table S6). þ þ CD52 is expressed on CD34 /CD38 NSC in patients Expression of CD52 on CD34 /CD38 NSC in other with del(5q) MDS myeloid neoplasms and ALL As assessed by flow cytometry, CD52 was found to be þ In a next step, we examined the expression of CD52 on expressed at high levels on CD34 /CD38 cells in 8/11 NSC in various control cohorts, CML, CMML, and other patients with del(5q) MDS, including 6 with isolated MDS/MPN overlap syndromes. In 6/6 patients with chronic þ del (5q) (Fig. 1A; Table 1). In most other MDS patients phase CML, CD34 /CD38 cells expressed CD52, whereas examined, CD52 was weakly expressed or not expressed on þ þ in one patient with accelerated phase CML, CD34 /CD38 CD34 /CD38 NSC (Fig. 1A; Table 1). We also confirmed þ þ cells did not express CD52 (Supplementary Table S7). In 4/9 that CD52 is expressed on the CD34 /CD38 /CD90 / þ patients with CMML, CD34 /CD38 cells expressed sub- CD45RA subset of NSC in our patients with MDS. These þ þ stantial amounts of CD52. In the other patients with CMML, þ CD34 /CD38 /CD90 /CD45RA cells also coexpressed CD34 /CD38 NSC/LSC displayed low levels of CD52 CD123 but did not express CLL-1 (Supplementary Table (Supplementary Table S7). We also examined 7 patients þ S6). For control purpose, we also examined expression of with JAK2 V617F MPN and one with JAK2 V617F MPN. þ CD52 on monocytes and basophils in our patients with In two of these patients, CD34 /CD38 cells stained pos- MDS. As expected, CD52 was detected on both cell types, itive for CD52 (Supplementary Table S7). Finally, we exam- without substantial differences in expression levels among ined NSC in 2 patients with acute leukemia with mixed the subgroups of patients examined (Table 1). (lymphoid/myeloid) phenotype (acute undifferentiated leukemia; AUL). In one patient with AUL, a complex kar- þ þ Expression of CD52 on CD34 /CD38 LSC in AML yotype with del(5q) was detected. In this patient, CD34 / In 23/62 patients with AML, LSC were found to express CD38 NSC/LSC expressed CD52 (Supplementary Table CD52. Of these patients with AML, 4 had a complex S7). In the other patient with AUL, who had a normal þ karyotype including del(5q), one del(5q), 2 dysplasia and karyotype, CD34 /CD38 NSC/LSC did not express CD52 inv(3), 2 t(8;21), one isolated inv(16), one 13q- anomaly, (Supplementary Table S7). Finally, we were able to show þ three trisomy 8, one monosomy 7, and 8 patients with AML that in most patients with ALL (8/10 ¼ 80%), the CD34 / had a normal karyotype. The highest levels of CD52 on CD38 stem cells express CD52 (Supplementary Table S7). NSC/LSC were recorded in one patient with del(5q), one þ with monosomy 7, one with inv(16), and one with a Expression of CD52 on CD34 /CD38 stem cells in normal karyotype (Fig. 1A; Table 1). In 5/7 patients with ICUS and normal bone marrow þ þ AML exhibiting del(5q) (71.4%), the CD34 /CD38 LSC In 2/19 patients with ICUS, CD34 /CD38 stem cells expressed high levels of CD52, whereas in only 18/55 clearly expressed CD52, whereas in the other ICUS patients

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Figure 1. Expression of CD52 on þ CD34 /CD38 cells in MDS and AML. A, bone marrow cells of patients with MDS or AML were stained with antibodies against CD34, CD38, CD45, and CD52. Expression of CD52 on þ þ CD45 /CD34 /CD38 cells was analyzed by multicolor flow cytometry as described in the text. The black open histograms show the isotype control and the red histograms represent CD52 þ expression on CD34 /CD38 cells. B, mean staining index of þ CD52 expressed on CD34 /CD38 cells obtained from patients with MDS (n ¼ 29; #1–29, top) or AML (n ¼ 62; #30–91, bottom) and comparison with þ CD52 expression on CD34 /CD38 cells in lymphomas (n ¼ 20; #152–171), patients with ICUS (n ¼ 19; #133–151), and patients in CR after AML (n ¼ 5; #172–176; controls). Expression of CD52 on stem cells was quantified by multicolor flow cytometry and was expressed as staining index (SI: MFICD52:MFIcontrol). Results represent the mean SD of all donors. Asterisk, P < 0.05.

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Table 1. Expression of CD52 on CD34þ/CD38 stem cells, CD34þ/CD38þ progenitor cells, monocytes, and blood basophils, in patients with MDS and AML and CD34 blasts in patients with AML

CD52 expression on

CD34þ/CD38 CD34þ/CD38þ CD34 No FAB diagnosis Karyotype cells cells Monocytes Basophils blasts 1 RAEB Complex,del(5q) þþþþþn.t. 2 RA 46,XX,del(5q) þþ þ þ n.t. 3 RA 47,XX,del(5q),þ8 þþþn.t. 4 RA 46,XX,del(5q) þþ þ þ n.t. 5 RAEB 46,XY,del(5q) þþn.t. 6 RA 46,XX,del(5q) þþþn.t. 7 RA 46,XY,del(5q) þþþþþþn.t. 8 RA 46,XX,del(5q) þþþþn.t. 9 RA 46,XX,del(5q) þþþþn.t. 10 RA 46,XX,del(5q) n.t. 11 RA 46,XX,del(5q) þþþþþn.t. 12 RAEB 47,XY,þ8 þþn.t. n.t. n.t. 13 RAEB 47,XX,þ21 þþþþþþn.t. 14 RA 45,XY, 7 þn.t. 15 RAEB 46,XY,t(8;21) þþn.t. 16 RAEB Complex,del(20q) þþþþþn.t. 17 RAEB 46,XX,del(20q) þþn.t. 18 RAEB 46,XY,del(20q) þþn.t. 19 RAEB 46,XX,del(11q) þþþþþn.t. 20 RAEB 46,XY þþn.t. 21 RARS 46,XY n.t. 22 RARS 46,XX n.t. n.t. n.t. 23 RAEB 46,XY þþ þ n.t. n.t. n.t. 24 RAEB 46,XY n.t. n.t. n.t. 25 RA 46,XX n.t. 26 RAEB 46,XY þn.t. n.t. n.t. 27 RA 46,XY n.t. n.t. n.t. 28 RARS 46,XX n.t. 29 RA n.t. þn.t. n.t. n.t. 30 AML M4 Complex del(5q) þþn.t. n.t. þ 31 AML M4 Complex del(5q) þþ þþ n.t. n.t. 32 sec. AML Complex del(5q) þþþþ 33 AML M4 Complex del(5q) þn.t. 34 AML M1 46,XX,del(5q) þn.t. n.t. 35 AML M2 46,XX,del(5q) n.t. n.t. þ 36 AML M0 46,XX,del(5q) þn.t. n.t. 37 sec. AML 45,XX,inv(3),-7 þþþþþþn.t. 38 sec. AML 46,XY,inv(3),del(7q) þþþþn.t. 39 AML M4eo 46,XX,inv(16) þþ þþ þ þ þþ 40 AML M4eo 46,XY,inv(16) þ 41 AML M4 46,XX,inv(16) n.t. n.t. þþ 42 AML M4eo 46,XY,inv(16) þn.t. n.t. 43 AML M2 47,XY,þ8 n.t. 44 AML M3 47,XX,t(15;17),þ8 þþþ 45 AML M3 46,XY,t(15;17) n.t. n.t. 46 sec. AML 47,XY,þ8 þþþþ 47 AML M5a 47,XX,þ8 n.t. n.t. (Continued on the following page)

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Table 1. Expression of CD52 on CD34þ/CD38 stem cells, CD34þ/CD38þ progenitor cells, monocytes, and blood basophils, in patients with MDS and AML and CD34 blasts in patients with AML (Cont'd )

CD52 expression on

CD34þ/CD38 CD34þ/CD38þ CD34 No FAB diagnosis Karyotype cells cells Monocytes Basophils blasts 48 AML M1 47,XX,þ8 þn.t. n.t. n.t. 49 AML M1 47,XX,þ8 þþn.t. n.t. þ 50 AML M2 45,XY,7,der(6) n.t. n.t. n.t. 51 sec. AML 45,XY,7 þþ þ þ þ n.t. 52 AML M4 46,XY,þ11,16 n.t. n.t. 53 AML M0 46,XX,t(3;3) þn.t. n.t. 54 AML M2 46,XX,t(8;21) þn.t. n.t. 55 AML M2 46,XY,t(8;21) þn.t. n.t. n.t. 56 sec. AML 46,XX,del(13q) þþn.t. 57 AML M1 Complex n.t. n.t. n.t. 58 sec. AML Complex þ 59 AML M2 46,XX 60 AML M0 46,XY þþþþþþþn.t. 61 AML M1 46,XX þ 62 AML M2 46,XY þþn.t. 63 AML M1 46,XX þn.t. n.t. n.t. 64 AML M4 46,XX þþ þ n.t. n.t. þ 65 sec. AML 46,XX n.t. 66 AML M2 46,XX n.t. n.t. n.t. 67 sec. AML 46,XX n.t. 68 AML M1 46,XY n.t. n.t. n.t. 69 AML M1 46,XY n.t. n.t. 70 AML M6 46,XX þn.t. n.t. 71 sec. AML 46,XY n.t. n.t. n.t. 72 AML M2 46,XY þn.t. n.t. 73 AML M4 46,XX þþþn.t. n.t. þ 74 sec. AML 46,XY n.t. n.t. n.t. 75 AML M1 46,XY n.t. n.t. n.t. 76 AML M2 46,XX n.t. n.t. 77 AML M1 46,XX n.t. n.t. 78 AML M4 46,XX n.t. n.t. 79 AML M4 46,XY þþn.t. n.t. þ 80 AML M5 46,XY n.t. n.t. 81 sec. AML 46,XY 82 AML M4 46,XX n.t. n.t. 83 AML M4eo 46,XY þn.t. n.t. þ 84 sec. AML 46,XY n.t. 85 AML M1 46,XY þþþþn.t. 86 AML M1 46,XX þn.t. n.t. 87 AML M4 46,XX n.t. n.t. 88 sec. AML 46,XY n.t. n.t. 89 AML M1 46,XX n.t. n.t. 90 AML M2 46,XX n.t. n.t. n.t. 91 sec. AML 46,XY n.t. n.t.

NOTE: Score of reactivity: þþ, MFI ratio 10–100; þ, MFI ratio 3.01–9.99; , MFI ratio 1.51–3; , MFI ratio <1.5. Abbreviations: No, number; RA, refractory anemia; RAEB, RA with excess blasts; RARS, RA with ring sideroblasts; sec. AML, secondary AML; n.t., not tested.

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þ þ Figure 2. Expression of CD52 mRNA in puriﬁed CD34 /CD38 stem cells in MDS and AML. A, RNA isolation from CD34 /CD38 cells of patients with MDS (#1, #2, #4, #7, #9, #11) or AML (#30, #34, #41, #55, #57, #68, #74, #75, #77, #78, #79) and qPCR (to quantify CD52 mRNA expression) were performed as described in the text. The black bars show CD52 mRNA levels relative to (as percent of) Abl mRNA expression. The levels þ of cell surface CD52 on the same CD34 /CD38 cells is also shown (below x-axis) in a semiquantitative score (, , þ,orþþ;seeTable1). B, correlations between CD52 mRNA levels and EVI1 mRNA levels (left) and between CD52 mRNA levels and CD300a mRNA levels (right) of þ the same patients used in Fig. 2A. The R values and P values are also shown. C, FISH was performedoncytospinpreparationsofCD34 /CD38 cells obtained from a patient with del(5q) MDS (#1). Deletion of 5q was evaluated using a dual color probe-set, including a red probe for EGR1 (chromosomal band 5q31.1) and a green control probe for TAS2R1 (5p15.31). In this patient, the 5q signal was not detected in a vast majority of the 200 interphase cells counted. Similar results were obtained in 4 other del(5q) patients. D, expression of CD52 on HL60 cells transfected with empty vector, NRAS G12D,orNRAS Q61K. Expression of CD52 was analyzed by ﬂow cytometry as described in the text. The black open histograms show the isotype control and the red histograms represent CD52 expression.

tested, stem cells did not express substantial amounts of mentary Table S8, CD52 was detected on both cell types CD52 (Supplementary Table S8). In control bone marrow in all control cases tested. samples obtained from controls with normal blood þ counts (n ¼ 5), CD34 /CD38 stem cells did not express NSC/LSC in MDS and AML express CD52 mRNA þ any detectable CD52. However, in 6 of 19 lymphoma Highly enriched CD34 /CD38 NSC of patients with del patients without known bone marrow inﬁltration, (5q) MDS or AML were found to express CD52 mRNA. As þ CD34 /CD38 cells were found to stain positive for visible in Fig. 2A, there was a good correlation between CD52. In 5 of these patients, stem cells expressed low surface expression of CD52 and CD52 mRNA expression levels of CD52, and in 8 patients, stem cells stained levels detected in NSC/LSC. Recent data suggest that CD52 negative for CD52 (Supplementary Table S8). We also expression is associated with EVI1 and CD300a expression examined the expression of CD52 on monocytes and in myeloid leukemias (41). Therefore, we were interested to basophils in our control donors. As shown in Supple- learn whether EVI1 and CD300a transcripts are detectable

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þ in NSC/LSC. Indeed, in all samples tested, CD34 /CD38 NSC/LSC expressed EVI1 and CD300a mRNA (Fig. 2B). Although we found a correlation between EVI1- and CD52 þ mRNA expression in CD34 /CD38 cells (R ¼ 0.66; P < 0.05), no correlation between CD300a and CD52 mRNA levels was found (P > 0.05; Fig. 2B). The clonal nature of the sorted NSC populations was conﬁrmed by FISH (Fig. 2C and Supplementary Table S9). In particular, in all 5 patients with del(5q) MDS examined, FISH analysis revealed that þ þ þ nearly 100% of the CD34 /CD38 and CD34 /CD38 cell populations tested expressed this chromosome abnormal- ity (Supplementary Table S9). Similarly, in one patient with AML with monosomy 7, FISH conﬁrmed that 100% of the þ þ þ CD34 /CD38 and 100% of the CD34 /CD38 expressed the 7 anomaly (not shown).

Oncogenic RAS induces expression of CD52 in AML cells Because RAS activation has been associated with EVI1 expression, we asked whether activated RAS may play a role in CD52 expression in AML cells. As visible in Fig. 2D, two different oncogenic NRAS mutants tested induced the expression of CD52 in HL60 cells. Incubation of these þ CD52 HL60 cells with alemtuzumab induced rapid cell lysis and a dose-dependent decrease in cell numbers, whereas no drug effect was seen in empty vector-transduced control cells (Supplementary Fig. S2A). These data suggest that CD52 expression on AML cells can be triggered by RAS activation.

The anti-CD52 antibody alemtuzumab induces rapid þ cell lysis in CD34 /CD38 NSC/LSC in patients with MDS and AML To demonstrate functional signiﬁcance of expression of the target receptor CD52 on NSC/LSC, we performed experiments using alemtuzumab and various MDS and AML samples. Samples were selected on the basis of high-level expression of CD52 (MFI ratio >3.01) or lack of CD52 (negative controls, MFI ratio <1.5) in these experiments. As visible in the top panels of Fig. 3A, short-term exposure to alemtuzumab (10–300 mg/mL; 1 hour) resulted þ in a signiﬁcant decrease in the numbers of CD52 NSC/LSC in MDS and AML compared with medium control. When

incubated in various concentrations of alemtuzumab (10–300 mg/mL) in RPMI-1640 medium in the presence of 30% human serum at 37C(5% CO2) for 1 hour. Then 10 mL calibration beads were added. Cells were washed and then stained with mAb against CD34, CD45, and CD38 for 15 minutes. Cells were then subjected to DAPI staining to count viable cells on a FACSCanto II. The left panels (black bars) show the effects of þ alemtuzumab on the CD34 /CD38 progenitor cells in these patients and the right panels (open bars) show the effects of alemtuzumab on þ þ CD34 /CD38 cells. Results represent the mean SD from three independent experiments in each panel. Asterisk, P < 0.05. C, HL60 cells þ Figure 3. Effects of alemtuzumab on growth and survival of neoplastic (CD52 , left) and Raji cells (CD52 , right) were incubated in various cells. A and B, primary cells from patients with del(5q) MDS (#2, #9, #11; A, concentration of alemtuzumab (0.1–500 mg/mL) in RPMI-1640 medium top), AML (#30, #48, #73; A, middle), or control bone marrow samples with either 30% serum (white bars), 30% heat-inactivated serum (black þ containing either CD52 (MFI > 3.01) stem cells (#136, #140, #150; B, top) bars), or IgG (20 mg/mL; grey bars) at 37C for 1 hour. Thereafter, cells and primary cells from patients with MDS (#24, #27, #28; A, bottom), AML were stained with propidium iodide (PI) and analyzed for cell viability on a (#45, #68, #71; A, bottom) or control bone marrow samples containing FACSCalibur. Results represent the mean SD from three independent CD52 (MFI < 1.5) stem cells (controls #143, #151, #157; B, bottom) were experiments. Asterisk, P < 0.05.

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Figure 4. Engraftment of AML cells in NSG mice. Primary AML MNC[(n ¼ 3 patients; #73, AML M4, FLT3-ITD and NPM1 mutation (left); #79, AML M4, FLT3-ITD, and NPM1 mutation (middle); #49, AML M1, trisomy 8 (right)] with CD52 MFI > 3.01 were incubated in control medium (Co) or in medium containing alemtuzumab (500 mg/mL) and 30% human serum at 37C for 1 hour. After incubation, cells were washed, resuspended in 0.15 mL PBS with 2% FCS, and injected into the tail vein of adult irradiated NSG mice (2–5 106 per mouse, 4–5 mice per group). Mice were inspected daily and sacriﬁced after þ 10 weeks. AML repopulation was measured by determining the percentage of CD45 cells in mouse bone marrow samples by ﬂow cytometry. Results represent mean SD from all mice per group in three independent experiments (3 donors). Engraftment level reduction by alemtuzumab: #73: 66%; #79: 44%; #49: 47%). Asterisk, P < 0.05.

human serum was replaced by IgG in these experiments, no (#73, #79), a NPM1 mutation was found. In these two effect of alemtuzumab was seen, suggesting a comple- patients, the CD34-negative blasts stained positive for ment-dependent reaction (Supplementary Fig. S2B). In CD52, and in vivo engraftment of AML cells was blocked our patients with MDS and AML in whom only CD52 by alemtuzumab. qPCR confirmed the presence of the NSC/LSC was detected, no significant effects of alemtu- NPM1 mutation and the FLT3-ITD mutation in xeno- zumab were seen (Fig. 3A, bottom). In control bone transplanted leukemic cells (#73, #79) grown in NSG þ marrow samples containing CD52 NSC, alemtuzumab mice; and in the sample of patient #49, FISH analysis showed a slight effect on stem cell numbers, whereas in confirmed the presence of trisomy 8 (not shown). control bone marrow samples containing only CD52 þ stem cells, alemtuzumab showed no effects (Fig. 3B). Influence of CD52 expression on CD34 /CD38 stem Overall, we found a significant correlation between cells on survival and disease evolution in MDS and AML expression of CD52 on stem cells and the cytotoxic effects Recent data suggest that the numbers and phenotype of þ of alemtuzumab on these cells (R ¼ 0.66, P < 0.05; CD34 /CD38 stem cells in MDS and AML are of prog- Supplementary Fig. S2C). We also examined the effects nostic significance (42–45). In the present study, we þ of alemtuzumab on HL60 cells and Raji cells (control asked whether expression of CD52 on CD34 /CD38 experiments). As expected, alemtuzumab (in 30% serum) stem cells in MDS and AML would be of prognostic induced a rapid and dose-dependent decrease in the significance. In both groups of patients (MDS n ¼ 29 þ numbers of CD52 Raji cells but showed no effects on and AML n ¼ 62), expression of CD52 was found to CD52 HL60 cells (Fig. 3C). In all experiments per- correlate with survival (Fig. 5A and B). In AML, the impact formed, alemtuzumab induced rapid cell lysis rather than of CD52 expression on survivalwasfoundtobesignif- þ apoptosis in CD52 Raji cells (not shown). No effects of icant (P < 0.05), whereas in patients with MDS, the þ alemtuzumab on CD52 cells were seen in the presence difference was not significant, which may be explained of IgG or heat-inactivated serum (Fig. 3C). by the relatively small numbers of patients. We also attempted to correlate expression of CD52 on NSC/LSC Preincubation of AML cells with alemtuzumab blocks with the WHO type of the disease. As visible in Supple- engraftment in NSG mice mentary Fig. S3, expression of CD52 showed a good To confirm that alemtuzumab acts on primitive dis- correlation with the WHO classification in AML and ease-initiating cells (NSC/LSC), we determined the effects MDS. In MDS, an obvious correlation was found between of the drug on NSG engraftment of AML cells in a CD52 expression and del(5q). In contrast, no correlation xenotransplantation model. As shown in Fig. 4, preincu- between CD52 expression and Revised International bation of AML cells with alemtuzumab (500 mg/mL in Prognostic Scoring System (IPSS-R) subgroups was found 30% human serum for 1 hour) resulted in a decreased (not shown). In AML, LSC consistently expressed CD52 engraftment of AML cells in vivo in NSG mice in all three in several subgroups, including AML with inv16, whereas samples examined. The engraftment was reduced to sim- in other groups, such as acute monocytic leukemia, LSC ilar levels by alemtuzumab in the three donors, namely to were consistently CD52 negative. No correlation was 66% in patient #73; to 44% in patient #79; and to 47% in found between expression of CD52 and risk groups patient #49 compared with control. In two of the three defined by Southwest Oncology Group (SWOG) criteria samples used in these xenotransplantation experiments (not shown).

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Figure 5. Inﬂuence of expression of CD52 on LSC on survival in MDS and AML. The probability of survival in patients with MDS (A) and AML (B) was determined þ þ for subgroups of patients in whom (i) CD34 /CD38 stem cells expressed high levels of CD52 or (ii) CD34 /CD38 stem cells expressed either low levels or did not express any detectable CD52. For the analysis of overall survival (OS), we used all MDS patients (A, n ¼ 29; #1–29) and all AML patients (B, n ¼ 62; #30–91). The median follow-up of our patients with MDS was 422 days and the median follow-up of our patients with AML was 382 days. The probability of survival was calculated by the product limit method of Kaplan and Meier. The difference in OS in our patients with AML was found to be signiﬁcant (P < 0.05). The bottom panels of 5A (MDS) and 5B (AML) show the number of patients at risk in both groups of patient.

Discussion these disease categories. In fact, CD52 was also found to The target antigen CD52 (Campath-1) is expressed on B be expressed on NSC/LSC in MDS and AML cases without lymphocytes, monocytes, and basophils. During the past del(5q). In this regard it is noteworthy, that in patients 10 years, the CD52-targeting drug alemtuzumab has been with other myeloid neoplasms, such as CML or CMML, þ used successfully in patients with advanced CLL (25–29). and also in patients with ALL, the CD34 /CD38 (puta- More recently, alemtuzumab was also found to induce tive) NSC/LSC also expressed CD52. Even in a subset of hematologic responses in patients with low-risk MDS patients with lymphomas without visible bone marrow þ (31). Initially this effect was considered to be mediated by involvement, CD34 /CD38 cells expressed CD52. In the immunosuppressive activity of alemtuzumab. We here contrast, in normal control bone marrow samples and þ propose an alternative mode of drug action and show that almost all patients with ICUS, CD34 /CD38 stem cells the target antigen CD52 is expressed on immature stem cells did not express CD52. Overall, the levels of CD52 on þ in a group of patients with MDS and AML. In addition, we CD34 /CD38 NSCinpatientswithdel(5q)MDSare þ show that alemtuzumab induces rapid cell lysis in CD52 signiﬁcantly higher than that found on stem cells in NSC/LSC in these patients. These data suggest that CD52 is a normal bone marrow. potential therapeutic target in MDS and AML, and that A number of previous studies have shown that LSC in þ þ treatment effects of alemtuzumab in these patients may, in AML can also reside within a CD34 /CD38 fraction of the part, be explained by targeting disease-initiating cells via clone (17). We therefore extended our studies to these cells þ þ Campath-1 (CD52). and were able to show that CD34 /CD38 cells in patients Among patients with low-risk MDS reported to respond with AML or MDS exhibiting del(5q) express higher levels of þ þ clinically to alemtuzumab in a previous study, several CD52 compared with normal CD34 /CD38 cells in con- patients were found to exhibit the del(5q) anomaly (31). trol bone marrow samples. Moreover, we found that the þ þ This is of particular interest, because in our study, CD34 /CD38 cells stained positive for CD52 in all þ þ CD34 /CD38 NSC in patients with del(5q) MDS patients with AML in whom CD34 /CD38 cells expressed expressed high levels of CD52, thereby contrasting other CD52. In patients with NPM1-mutated AML and other AML patients with MDS or controls. In addition, we found that types, LSC may reside within a CD34-negative subfraction þ CD34 /CD38 stem cells in patients with AML exhibiting of the clone (18). Therefore, we were also interested to learn del(5q) express detectable (mostly high) levels of CD52. whether CD52 is expressed on CD34-negative blast cells in Together, marked expression of CD52 is usually seen in our patients with AML. In these experiments, a CD34- MDS and AML with del(5q), but is not a speciﬁc marker for negative blast cell population was detectable in 41 of our

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þ 62 patients with AML, and in 11 of these 41 patients, blast found that alemtuzumab induces cell lysis in CD34 / cells expressed CD52. CD38 cells in patients with del(5q) MDS. Moreover, þ In a next step, we confirmed expression of CD52 in AML alemtuzumab induced rapid cell lysis in CD34 /CD38 þ þ and MDS stem cells by qPCR. In these experiments, we were and CD34 /CD38 cells in all patients with AML in þ able to show that highly enriched (sorted) CD34 /CD38 whom these cells expressed CD52, whereas no drug effect NSC/LSC in patients with del(5q) MDS and del(5q) AML, was seen with CD52 NSC/LSC. Finally, we were able to þ express detectable CD52 transcripts, and that CD52 surface show that incubation of CD52 NSC/LSC with alemtu- expression correlates with CD52 mRNA levels. We also zumab reduces their leukemic engraftment in NSG mice. þ confirmed that the CD34 /CD38 cells, sorted from bone Whether these effects are responsible for the remarkable marrow samples of our patients with MDS and AML exhi- clinical effects of alemtuzumab seen in patients with MDS, biting del(5q), are clonal cells. In particular, in each case remains unknown. However, it is at least tempting to þ examined, the CD34 /CD38 fraction of neoplastic cells speculate that some of the effects of the drug are exerted expressed the del(5q) anomaly by FISH. through NSC/LSC elimination in these patients. Clinical So far, little is known about the regulation of expression trials correlating CD52 expression on NSC/LSC with of CD52 on NSC/LSC in MDS and AML. Recent data suggest responses to alemtuzumab are now warranted to clarify that expression of CD52 in AML cells is associated with EVI1 this question. and CD300a expression in myeloid leukemias (41). Treatment with alemtuzumab is sometimes accompanied Although the exact mechanism remains unknown, this by severe prolonged cytopenia (25–29). We therefore asked observation suggests that EVI1 may be involved in the whether CD52 is also expressed on normal bone marrow expression of CD52 in AML cells. In the present study, we stem cells. However, in normal bone marrow samples and þ þ were able to show that CD34 /CD38 stem cells in MDS in most patients with ICUS, CD34 /CD38 cells did not and AML with del(5q) express detectable levels of EVI1 and express CD52. In contrast, in some patients with non- CD300a mRNA, and that EVI1 transcript expression corre- Hodgkin lymphoma without histologically detectable bone lates with expression of CD52 mRNA. Because EVI1 expres- marrow involvement, CD52 was found to be expressed on þ sion has been described in the context of RAS activation, we CD34 /CD38 stem cells. In these patients, alemtuzumab þ þ also examined the effects of mutated NRAS variants on was found to induce cell lysis in CD34 /CD38 /CD52 expression of CD52 (46, 47). In these experiments, we stem cells. In contrast, no effects of alemtuzumab on nor- found that mutant NRAS induces the expression of CD52 mal CD52 stem cells were seen. þ in HL60 cells. These data suggest that RAS-dependent sig- In summary, our data show that CD34 /CD38 NSCs in naling may be involved in abnormal expression of CD52 in patients with del(5q) MDS and a group of AML, express the AML (stem) cells. target antigen CD52. Moreover, our data show that alem- Recent data suggest that the numbers and phenotype of tuzumab induces rapid, complement-dependent lysis of þ CD34 /CD38 stem cells in MDS and AML are of prog- NSC/LSC in these patients. In addition, CD52 may be a nostic significance (42–45). A clinically important ques- prognostic NSC/LSC marker in patients with MDS and tion in this study was whether expression of CD52 on AML. The exact value of CD52 as a NSC/LSC marker and þ CD34 /CD38 bone marrow stem cells in MDS and AML target remains to be determined in future studies. is associated with prognosis. The results of our study þ showthatexpressionofCD52onCD34 /CD38 cells Disclosure of Potential Conflicts of Interest in MDS and AML is indicative of poor survival. We also No potential conflicts of interest were disclosed. þ asked whether CD52 expression on CD34 /CD38 stem cells in MDS or AML correlates with other prognostic Authors' Contributions (clinical or laboratory) parameters. However, we were Conception and design: K. Blatt, P. Valent unable to substantiate any correlations between CD52 Development of methodology: G. Hoermann þ Acquisition of data (provided animals, acquired and managed patients, expression on CD34 /CD38 cells and other (potentially provided facilities, etc.): K. Blatt, H. Herrmann, G. Hoermann, M. Will- prognostic) parameters analyzed such as the number of mann, S. Cerny-Reiterer, I. Sadovnik, S. Herndlhofer, B. Streubel, W. Rabitsch, W.R. Sperr, M. Mayerhofer, T. Rulicke€ cytopenias, blast cells, or the IPSS category, which is best Analysis and interpretation of data (e.g., statistical analysis, biosta- explained by the low numbers of patients in each group. tistics, computational analysis): K. Blatt, H. Herrmann, G. Hoermann, Nevertheless, our observations suggest that expression of M. Willmann, S. Cerny-Reiterer, I. Sadovnik, S. Herndlhofer, B. Streubel, W.R. Sperr, M. Mayerhofer CD52 on NSC/LSC may be a prognostic feature in MDS Writing, review, and/or revision of the manuscript: K. Blatt, G. Hoer- and AML. However, prospective studies with more mann, M. Willmann, W. Rabitsch, T. Rulicke,€ P. Valent patients are required to confirm that CD52 is an inde- Administrative, technical, or material support (i.e., reporting or orga- nizing data, constructing databases): M. Willmann, S. Herndlhofer pendent risk factor concerning survival in patients with Study supervision: P. Valent MDS and AML. On the basis of the intriguing effects of alemtuzumab in Acknowledgments patients with MDS and AML (31, 48) and our flow The authors thank Gabriele Stefanzl (Department of Internal Medicine I, cytometry data, we were interested to learn whether Medical University of Vienna) as well as Gunther€ Hofbauer and Andreas þ þ Spittler (Cell Sorting Core Unit, Medical University of Vienna) and Tina alemtuzumab would attack CD34 /CD38 and CD34 / Bernthaler (University of Veterinary Medicine Vienna) for excellent technical þ CD38 cells in MDS and AML. In these experiments, we support.

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Grant Support advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate This work was supported by the Austrian Science Fund (FWF) SFB project this fact. #F4704-B20 (to P. Valent). The costs of publication of this article were defrayed in part by the Received October 15, 2013; revised March 13, 2014; accepted April 14, payment of page charges. This article must therefore be hereby marked 2014; published OnlineFirst May 5, 2014.

References 1. Corey SJ, Minden MD, Barber DL, Kantarjian H, Wang JC, Schimmer 19. Sperr WR, Hauswirth AW, Florian S, Ohler L, Geissler K, Valent P. AD. Myelodysplastic syndromes: the complexity of stem-cell dis- Human leukaemic stem cells: a novel target of therapy. Eur J Clin Invest eases. Nat Rev Cancer 2007;7:118–29. 2004;34:31–40. 2. Dick JE. Acute myeloid leukemia stem cells. Ann N Y Acad Sci 20. Florian S, Sonneck K, Hauswirth AW, Krauth MT, Schernthaner GH, 2005;1044:1–5. Sperr WR, et al. Detection of molecular targets on the surface of 3. Nimer SD. Myelodysplastic syndromes. Blood 2008;111:4841–51. CD34þ/CD38 stem cells in various myeloid malignancies. Leuk 4. Bennett JM, Catovsky D, Daniel MT, Flandrin G, Galton DA, Gralnick Lymphoma 2006;47:207–22. HR, et al. Proposals for the classification of the acute leukaemias. 21. Roboz GJ, Guzman M. Acute myeloid leukemia stem cells: seek and French-American-British (FAB) co-operative group. Br J Haematol destroy. Expert Rev Hematol 2009;2:663–72. 1976;33:451–8. 22. Krause A, Luciana M, Krause F, Rego EM. Targeting the acute myeloid 5. Bennett JM, Catovsky D, Daniel MT, Flandrin G, Galton DA, Gralnick leukemia stem cells. Anticancer Agents Med Chem 2010;10:104–10. HR, et al. Proposals for the classification of the myelodysplastic 23. Bernstein ID. CD33 as a target for selective ablation of acute myeloid syndromes. Br J Haematol 1982;51:189–99. leukemia. Clin Lymphoma 2002;2:S9–11. 6. Vardiman JW. The World Health Organization (WHO) classification of 24. Hauswirth AW, Florian S, Printz D, Sotlar K, Krauth MT, Fritsch G, et al. tumors of the hematopoietic and lymphoid tissues: an overview with Expression of the target receptor CD33 in CD34þ/CD38/CD123þ emphasis on the myeloid neoplasms. Chem Biol Interact 2010;184: AML stem cells. Eur J Clin Invest 2007;37:73–82. 16–20. 25. Domagała A, Kurpisz M. CD52 antigen—a review. Med Sci Monit 7. Greenberg P, Cox C, LeBeau MM, Fenaux P, Morel P, Sanz G, et al. 2001;7:325–31. International scoring system for evaluating prognosis in myelodys- 26. BugginsAG,MuftiGJ,SalisburyJ,CoddJ,WestwoodN,ArnoM, plastic syndromes. Blood 1997;89:2079–88. et al. Peripheral blood but not tissue dendritic cells express CD52 8. Haase D, Germing U, Schanz J, Pfeilstocker€ M, Nosslinger€ T, Hildeb- and are depleted by treatment with alemtuzumab. Blood randt B, et al. New insights into the prognostic impact of the karyotype 2002;100:1715–20. in MDS and correlation with subtypes: evidence from a core dataset of 27. Ravandi F, O'Brien S. Alemtuzumab. Expert Rev Anticancer Ther 2124 patients. Blood 2007;110:4385–95. 2005;5:39–51. 9. Schanz J, Steidl C, Fonatsch C, Pfeilstocker€ M, Nosslinger€ T, Tuechler 28. Gribben JG, Hallek M. Rediscovering alemtuzumab: current and H, et al. Coalesced multicentric analysis of 2,351 patients with mye- emerging therapeutic roles. Br J Haematol 2009;144:818–31. lodysplastic syndromes indicates an underestimation of poor-risk 29. Jaglowski SM, Alinari L, Lapalombella R, Muthusamy N, Byrd JC. The cytogenetics of myelodysplastic syndromes in the international prog- clinical application of monoclonal antibodies in chronic lymphocytic nostic scoring system. J Clin Oncol 2011;29:1963–70. leukemia. Blood 2010;116:3705–14. 10. Boultwood J, Lewis S, Wainscoat JS. The 5q-syndrome. Blood 30. Kim H, Min YJ, Baek JH, Shin SJ, Lee EH, Noh EK, et al. A pilot dose- 1994;84:3253–60. escalating study of alemtuzumab plus cyclosporine for patients with 11. Mallo M, Cervera J, Schanz J, Such E, García-Manero G, Luno~ E, et al. bone marrow failure syndrome. Leuk Res 2009;33:222–31. Impact of adjunct cytogenetic abnormalities for prognostic stratifica- 31. Sloand EM, Olnes MJ, Shenoy A, Weinstein B, Boss C, Loeliger K, et al. tion in patients with myelodysplastic syndrome and deletion 5q. Alemtuzumab treatment of intermediate-1 myelodysplasia patients is Leukemia 2011;25:110–20. associated with sustained improvement in blood counts and cyto- 12. Lapidot T, Sirard C, Vormoor J, Murdoch B, Hoang T, Caceres-Cortes genetic remissions. J Clin Oncol 2010;28:5166–73. J, et al. A cell initiating human acute myeloid leukaemia after trans- 32. Mufti GJ, Chen TL. Changing the treatment paradigm in myelodys- plantation into SCID mice. Nature 1994;367:645–8. plastic syndromes. Cancer Control 2008;15:14–28. 13. Bonnet D, Dick JE. Human acute myeloid leukemia is organized as a 33. Sloand EM, Barrett AJ. Immunosuppression for myelodysplastic syn- hierarchy that originates from a primitive hematopoietic cell. Nat Med drome: how bench to bedside to bench research led to success. 1997;3:730–7. Hematol Oncol Clin North Am 2010;24:331–41. 14. Thanopoulou E, Cashman J, Kakagianne T, Eaves A, Zoumbos N, 34. Brothman AR, Persons DL, Shaffer LG. Nomenclature evolution: Eaves C. Engraftment of NOD/SCID-beta2 microglobulin null mice changes in the ISCN from the 2005 to the 2009 edition. Cytogenet with multilineage neoplastic cells from patients with myelodysplastic Genome Res 2009;127:1–4. syndrome. Blood 2004;103:4285–93. 35. Haferlach T, Schnittger S, Kern W, Hiddemann W, Schoch C. Genetic 15. Taussig DC, Pearce DJ, Simpson C, Rohatiner AZ, Lister TA, Kelly G, classification of acute myeloid leukemia (AML). Ann Hematol et al. Hematopoietic stem cells express multiple myeloid markers: 2004;83:97–100. implications for the origin and targeted therapy of acute myeloid 36. Hoermann G, Cerny-Reiterer S, Herrmann H, Blatt K, Bilban M, Gis- leukemia. Blood 2005;106:4086–92. slinger H, et al. Identification of oncostatin M as a JAK2 V617F- 16. Valent P. Targeting of leukemia-initiating cells to develop curative drug dependent amplifier of cytokine production and bone marrow remo- therapies: straightforward but nontrivial concept. Curr Cancer Drug deling in myeloproliferative neoplasms. FASEB J 2012;26:894–906. Targets 2011;11:56–71. 37. Hoermann G, Cerny-Reiterer S, Perne A, Klauser M, Hoetzenecker K, 17. Taussig DC, Miraki-Moud F, Anjos-Afonso F, Pearce DJ, Allen K, Ridler Klein K, et al. Identification of oncostatin M as a STAT5-dependent C, et al. Anti-CD38 antibody-mediated clearance of human repopulat- mediator of bone marrow remodeling in KIT D816V-positive systemic ing cells masks the heterogeneity of leukemia-initiating cells. Blood mastocytosis. Am J Pathol 2011;178:2344–56. 2008;112:568–75. 38. Herrmann H, Kneidinger M, Cerny-Reiterer S, Rulicke€ T, Willmann M, 18. Taussig DC, Vargaftig J, Miraki-Moud F, Griessinger E, Sharrock K, Gleixner KV, et al. The Hsp32 inhibitors SMA-ZnPP and PEG-ZnPP Luke T, et al. Leukemia-initiating cells from some acute myeloid exert major growth-inhibitory effects on CD34(þ)/CD38(þ) and CD34 leukemia patients with mutated nucleophosmin reside in the CD34 (þ)/CD38() AML progenitor cells. Curr Cancer Drug Targets 2012; () fraction. Blood 2010;115:1976–84. 12:51–63.

www.aacrjournals.org Clin Cancer Res; 20(13) July 1, 2014 3601

Blatt et al.

39. Agis H, Fureder€ W, Bankl HC, Kundi M, Sperr WR, Willheim M, et al. 44. Ogata K, Nakamura K, Yokose N, Tamura H, Tachibana M, Taniguchi Comparative immunophenotypic analysis of human mast cells, blood O, et al. Clinical significance of phenotypic features of blasts in patients basophils and monocytes. Immunology 1996;87:535–43. with myelodysplastic syndrome. Blood 2002;100:3887–96. 40. Fureder€ W, Schernthaner GH, Ghannadan M, Hauswirth A, Sperr WR, 45. van de Loosdrecht AA, Westers TM, Westra AH, Drager€ AM, van der Semper H, et al. Quantitative, phenotypic, and functional evaluation of Velden VH, Ossenkoppele GJ. Identification of distinct prognostic basophils in myelodysplastic syndromes. Eur J Clin Invest subgroups in low- and intermediate-1-risk myelodysplastic syn- 2001;31:894–901. dromes by flow cytometry. Blood 2008;111:1067–77. 41. Saito Y, Nakahata S, Yamakawa N, Kaneda K, Ichihara E, Suekane A, 46. Li Q, Haigis KM, McDaniel A, Harding-Theobald E, Kogan SC, Akagi et al. CD52 as a molecular target for immunotherapy to treat acute K, et al.Hematopoiesis and leukemogenesis in mice expressing myeloid leukemia with high EVI1 expression. Leukemia 2011;25:921–31. oncogenic NrasG12D from the endogenous locus. Blood 2011;117: 42. van Rhenen A, Feller N, Kelder A, Westra AH, Rombouts E, Zweegman 2022–32. S, et al. High stem cell frequency in acute myeloid leukemia at 47. Wolf S, Rudolph C, Morgan M, Busche€ G, Salguero G, Stripecke R, diagnosis predicts high minimal residual disease and poor survival. et al. Selection for Evi1 activation in myelomonocytic leukemia induced Clin Cancer Res 2005;11:6520–7. by hyperactive signaling through wild-type NRas. Oncogene 43. Witte KE, Ahlers J, Schafer€ I, Andre M, Kerst G, Scheel-Walter HG, et al. 2013;32:3028–38 High proportion of leukemic stem cells at diagnosis is correlated with 48. Tibes R, Keating MJ, Ferrajoli A, Wierda W, Ravandi F, Garcia-Manero unfavorable prognosis in childhood acute myeloid leukemia. Pediatr G, et al. Activity of alemtuzumab in patients with CD52-positive acute Hematol Oncol 2011;28:91–9. leukemia. Cancer 2006;106:2645–51.

3602 Clin Cancer Res; 20(13) July 1, 2014 Clinical Cancer Research

Identification of Campath-1 (CD52) as Novel Drug Target in Neoplastic Stem Cells in 5q-Patients with MDS and AML

Katharina Blatt, Harald Herrmann, Gregor Hoermann, et al.

Clin Cancer Res 2014;20:3589-3602. Published OnlineFirst May 5, 2014.

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