A Leukemia-Associated CD34/CD123/CD25 ...clincancerres.aacrjournals.org/content/clincanres/21/17/...Biology of Human Tumors A Leukemia-Associated CD34/CD123/CD25/ CD99þ Immunophenotype

Biology of Human Tumors

A Leukemia-Associated CD34/CD123/CD25/CD99þ Immunophenotype Identifies FLT3-Mutated Clones in Acute Myeloid LeukemiaDaniela F. Angelini1, Tiziana Ottone2,3, Gisella Guerrera1, Serena Lavorgna2,3,Michela Cittadini2,3, Francesco Buccisano2, Marco De Bardi1, Francesca Gargano1,Luca Maurillo2, Mariadomenica Divona2, N�elida I. Noguera3,4, Maria Irno Consalvo2,Giovanna Borsellino1, Giorgio Bernardi5, Sergio Amadori2, Adriano Venditti2,Luca Battistini1, and Francesco Lo-Coco2,3

Abstract

Purpose: We evaluated leukemia-associated immunopheno-types (LAIP) and their correlation with fms-like tyrosine kinase 3(FLT3) and nucleophosmin (NPM1) gene mutational status inorder to contribute a better identificationof patients at highest riskof relapse in acute myeloid leukemia (AML).

Experimental Design: Bone marrow samples from 132patients with AML were analyzed by nine-color multiparametricflow cytometry. We confirmed the presence of the mutation indiagnostic samples and in sorted cells by conventional RT-PCRand by patient-specific RQ-PCR.

Results:Within theCD34þ cell fraction, we identified a discretepopulation expressing high levels of the IL3 receptor a-chain(CD123) and MIC-2 (CD99) in combination with the IL2 recep-tor a-chain (CD25). The presence of this population positively

correlated with the internal tandem duplications (ITD) mutationin the FLT3 gene (r ¼ 0.71). Receiver operating characteristicsshowed that, within the CD34þ cell fraction a percentage ofCD123/CD99/CD25þ cells �11.7% predicted FLT3–ITD muta-tions with a specificity and sensitivity of >90%. CD34/CD123/CD99/CD25þ clones were also detectable at presentation in 3patients with FLT3 wild-type/NPM1þ AML who relapsed withFLT3-ITD/NPM1þ AML. Quantitative real-time PCR designed atrelapse for each FLT3-ITD in these three cases confirmed thepresence of low copy numbers of the mutation in diagnosticsamples.

Conclusions: Our results suggest that the CD34/CD25/CD123/CD99þ LAIP is strictly associated with FLT3-ITD–positivecells. Clin Cancer Res; 21(17); 3977–85. �2015 AACR.

IntroductionImmunophenotyping by multiparametric flow cytometry

(MPFC) is a valuable and effective tool for diagnostic character-ization, classification, andminimal residual disease (MRD)mon-itoring in acute myeloid leukemia (AML). In fact, several studieshave defined surface and cytoplasmic markers that are aberrantlyexpressed on AML blasts at diagnosis (1, 2); these combinationsmay identify leukemia-associated immunophenotypes (LAIP),which allow sensitive monitoring of MRD during follow-up

(3–5). Compared with molecular evaluation of MRD throughPCR amplification of genetic AML lesions, MPFC offers theadvantage of being applicable to the vast majority (i.e., >90%)of cases (6, 7).

In addition to immunophenotype, both karyotypic andmolec-ular genetic characterization are essential parts of the diagnosticwork-up in AML. Although in some cases immunophenotypicprofiles have been correlated with AML genetic subsets, to date noantigenic signature has been associatedwith anAMLgenetic entitywith absolute specificity. For instance, expression of CD14, CD4,CD11b, and CD64 or CD36 (markers of monocytic and granu-locytic differentiation) combined with high levels of CD34 andCD117 and abnormal expression of CD2 characterizes—but isnot specific for—AML inv (16) or t(16;16) (8). Similarly, acutepromyelocytic leukemia (APL) with t(15;17) is characterized byvery low or absent expression of CD34 and HLA-DR, and posi-tivity for CD117/CD33; however, this antigenic profile is notcompletely specific for APL and some variant LAIPs have beendescribed (9–11).

Several studies have reported correlations of aberrantlyexpressed markers with clinical outcome in AML. For example,CD7 and CD25 expression has been associated with poor prog-nosis in normal karyotype (NK) AML (12–14). The IL3 receptor-a(CD123) is overexpressed in 45%ofAMLpatients, and this higherexpression has also been associated with poor outcome (15).Finally, overexpression of CD123 has been correlated in this

1Neuroimmunology and Flow Cytometry Units, Fondazione SantaLucia-I.R.C.C.S., Rome, Italy. 2Department of Biomedicine and Preven-tion, University of Tor Vergata, Rome, Italy. 3Laboratorio di Neuro-Oncoematologia, Fondazione Santa Lucia I.R.C.C.S, Rome, Italy.4Department of Chemical Biochemistry (Hematology), National Uni-versity of Rosario, Argentina. 5Experimental Neuroscience, Fonda-zione Santa Lucia, I.R.C.C.S., Rome, Italy.

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

D.F. Angelini and T. Ottone contributed equally to this article.

Corresponding Author: Francesco Lo-Coco, Department of Biomedicine andPrevention, University Tor Vergata, Via Montpellier 1, 00133 Rome, Italy. E-mail:[email protected].

doi: 10.1158/1078-0432.CCR-14-3186

�2015 American Association for Cancer Research.

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setting with mutations in the fms-like tyrosine kinase receptor(FLT3) gene (16).

A consistent antigenic profile with high CD33 expression hasalso been associated with AML with mutated nucleophosmin(NPM1; refs. 17 and 18). Patients withNPM1-mutated AML havefavorable prognosis except when this gene alteration is accom-panied by FLT3 lesions and in particular by internal tandemduplications (FLT3-ITD), which are universally regarded as pre-dictor of a poor outcome (19–22).

Screening by PCR for FLT3-ITD at diagnosis is relevant for riskstratification of AML patients; however, this technique is neithersufficiently sensitive nor it may be routinely applied to MRDmonitoring due to the instability of this alteration and its inter-patient variability. Recent studies suggest that FLT3-ITD AMLrelapse may occasionally originate from very small subclones,which are undetectable at diagnosis by routine screening (23).Wehave previously developed a patient-specific real-time quantita-tive PCR to implement FLT3-ITDdetection at time of relapse (24),but this approach is unfortunately not applicable at diagnosis tounravel minor FLT3-ITD subclones. Through this assay, we ana-lyzed paired diagnostic and relapse samples in AML patients whohad showed FLT3-ITD only at relapse by conventional RT-PCR.We found that small FLT3-ITD clones that were chemoresistantand eventually gave origin to disease relapse were already presentat diagnosis in these patients (24). These findings highlight theneed of improving diagnostic identification in AML of smallnumber of cells harboring genetic lesions associated with poorprognosis.

In this study,we sought to investigatewhether nine-colorMPFCcould be helpful to detect minor subclones potentially associatedwith resistance and disease recurrence in AML. We combinedtypical markers of early myeloid progenitors including CD34,CD117, CD38, with markers associated with FLT3 mutations,such as CD123, CD25, and CD7. This approach allowed us toidentify a subset of CD34þ cells whose presence is highly predic-tive of the FLT3 mutations in AML.

Materials and MethodsPatients

A total of 132 consecutive patients ages 19 to 85 years withnewly diagnosed AML observed and treated at the HematologyUnit of the Department of Biomedicine and Prevention of the

University of Tor Vergata during the period 2012 to 2014 wereincluded in the study. The vast majority of patients ages <75 yearsreceived intensive chemotherapy according to European Organi-zation of Research and Treatment of Cancer (EORTC)/GruppoItalianoMalattie EMatologiche dell'Adulto (GIMEMA) protocols,whereas patients ages >75 years received supportive care ortherapy with hypomethylating agents. According to the declara-tion of Helsinki, all patients gave informed consent for the studythat was approved by the Institutional Review Board of thePoliclinico Tor Vergata of Rome.

Diagnostic work-upRoutine morphologic, immunophenotypic, and genetic

analyses were carried out in all cases at presentation. Conven-tional karyotyping was performed on bone marrow (BM)diagnostic aspirates after short-term culture and analyzed afterG-banding. The description of the karyotypes was done accord-ing to the International System for Human CytogeneticNomenclature. As for molecular analysis, total RNA wasextracted from Ficoll-Hypaque isolated BM mononuclear cellscollected at diagnosis and for selected cases, during follow-upusing standard procedures (25) and reverse-transcribed withrandom hexamers as primers. DNA was extracted using acolumn-based Qiagen Kit protocol (Qiagen). RNA samplesextracted from sorted cells population were subjected toreverse-transcription using 200 ng of RNA with the SuperScriptIII One-Step RT-PCR system (Invitrogen) according to themanufacturer's instructions. Most common AML gene fusions,FLT3 status (both ITD and TKDmutations) and NPM1mutated(mut) or wild-type (wt) gene status were investigated using theprotocols reported elsewhere (19, 26, 27). For selected patients,the FLT3-ITD data are also represented as a ratio of mut/wt cellsobtained by a quantitative assay based on Genescan analysis(19). This assay compares the relative abundance of wt versusmutant FLT3 alleles.

MPFC studies and high-speed cell sortingImmunophenotypic studies were performed on BM samples at

diagnosis and, for selected cases, in samples obtained duringfollow-up and at disease relapse. Cells were acquired on a CyAnADP 9 Color equipped with three lasers (Beckman Coulter, Brea,CA). Briefly, BM samples were lysed with ammonium chloride(pH7.2) and stainedwith predefined optimal concentrations of 8antibodies and a dead cell stain dye (LIVE/DEAD Fixable AquaDead Cell Stain Kit, Life Technologies). Each sample at diagnosiswas stained with 10 distinct antibody combinations ("pre-mixes"); the markers CD45, CD34, CD117 and HLA-DR repre-sented the "backbone" and were present in all combinations, inaddition to a viability stain. The remaining 4 markers weredifferent in each mix. The number of events acquired for eachleukemic sample was determined as to have at least 500 eventsaccumulated in the gate of CD34þ cells. Events analyzed undergofurther back gating analysis (CD45 vs side scatter) and exclusionof doublets and dead cells. Following acquisition and analysis ofthe samples stained with the 10 pre-mixes, new antibody combi-nationswere designedon the spot to include allmarkers expressedby the leukemic cells, thus each sample was studied specificallyusing custom-designed panels. Data were analyzed using FlowJosoftware (TreeStar Inc.).

High-speed cell sorting was performed on a Moflo (BeckmanCoulter). Cells were isolated by Ficoll-Hypaque to prevent

Translational Relevance

Although it is well established that FLT3 mutations conferpoor prognosis in AML, the significance of minor clonesharboring this alteration is unclear. Furthermore, such clonesare not easily detectable through molecular assays. We showhere that minor FLT3-ITD–positive subclones are clinicallyrelevant in AML as they may emerge after therapy in patientslabeled as FLT3 wild-type at presentation. Identificationthrough multiparametric flow cytometry at diagnosis of animmunophenotypic fingerprint associated with these sub-clones is a novel and simplified tool with improved sensitivityto unravel these clones and allowing patient stratification andrisk-adapted treatment with potential impact on outcome ofthe disease.

Angelini et al.

Clin Cancer Res; 21(17) September 1, 2015 Clinical Cancer Research3978




granulocytic contamination, and then stained as previouslydescribed. The antibodies used are listed in SupplementaryTable S1.

FLT3-ITD patient-specific quantitative analysis by RQ-PCRFor FLT3-ITD quantification, we developed patient-specific

real-time assays (RQ-PCR). The strategy for FLT3-ITD patient-specific primers designing is reported elsewhere (24).On the basisof clinical outcome, molecular and immunophenotypic results, 4AML patients (UPN 104, 146, 213, and 241) were selected forfurther analysis by RQ-PCR for the patient-specific FLT3-ITDanalysis using several follow-up RNA samples. UPN 104 and146 were characterized by two FLT3-ITDs clones, whereas UPN213 and 241 by only one FLT3-ITD clone. The assays weredesigned to evaluate the copy number variation among differentclones carrying FLT3-ITDs. The RQ-PCR for FLT3-ITD quantifica-tion was performed using patient-specific FLT3-ITD forward pri-mers (FLT3-ITDs genomic coordinates and primer sequencesare available on request), a common reverse primer and probe(FLT3-ITD_reverse 50-AGACTCCTGTTTTGCTAATTCCATA-30,FLT3-ITD_Probe 50-FAM AAGGTACTAGGATCAGGTGCTTTTG-GAA-30-BHQ1). The reaction mixture of 25 mL contained1�Master Mix (Applied Biosystems), 300 nmol/L of each primer,200 nmol/L of Taqman probe, and 5 mL of cDNA (1/10 of RTproduct). Amplification conditions were: 2 minutes at 50�C, 10minutes at 95�C followed by 60 cycles at 95�C for 15 seconds, andat 60�C for 1 minute for UPN 146, 213, and 241, and 62�C forUPN 104. A threshold value of 0.2 was used and baseline was setto 3 to 15. Patient-specific FLT3-ITDs sensitivity and specificitytests were carried out for each patient with serial water dilutions ofRNA and with RNA from healthy donors.

Statistical analysisDifferences between categorical variables were evaluated by the

Pearsonx2 test, whereas differences between continuous variableswere analyzedby anunpaired t testwith 95%confidence intervals.Positive correlations between categorical and quantitative vari-ables were analyzed by the Spearman rank correlation coefficient.Receiver operating characteristic (ROC) analysis was performedwithMedCalc statistical software (Ostend Belgium). The standarderror was calculated using DeLong and colleagues (28). Theconfidence interval for the AUCwas calculated using the Binomialexact confidence interval.

ResultsFLT3 and NPM1 mutational status of patients at diagnosis

Out of 132 tested patients, 36 harbored in their leukemic cellsthe FLT3-ITD mutations and 48 NPM1 mutations. Consideringthe status of both genes, 9 patients tested FLT3-ITD/NPM1-wt, 21FLT3-ITD�/NPM1-mut, 27 FLT3-ITD/NPM1-mut, and 75 FLT3-ITD�/NPM1-wt. Karyotypic data were available for 70% ofpatients. The main demographic characteristics and karyotype ofpatients according to NPM1/FLT3 gene status are reported inSupplementary Table S2.

MPFC analysis in total BM and in the CD34þ compartmentAs shown in Supplementary Table S3, MPFC studies in the

total BM revealed a statistically significant difference in theexpression of CD123, CD25, CD99, CD7, and CD11b betweenFLT3-ITD and FLT3-ITD� patients (P ¼ 0.02, P ¼ 0.000006, P ¼0.03, P ¼ 0.02, P ¼ 0.005, respectively). The fraction of CD34þ

cells was significantly lower in patients with NPM1-mut, com-pared with patients who did not carry the mutation (Fig. 1A,top; Supplementary Table S4); accordingly, the bulk of theleukemic cell population in NPM1-mut patients was CD34�

/CD117þ (Fig. 1A, bottom). In addition, CD34þ cells derivedfrom the FLT3 and NPM1-mutated group expressed the lowestlevels of CD38 (Fig. 1B).

When focusing the analysis on CD34þ cells, FLT3-ITDpatients expressed CD123, CD25, and CD99 at significantlyhigher levels compared with patients with FLT3-ITD�, regard-less of NPM1 gene status (Supplementary Table S4; Fig. 1B).Expression of CD11b and CD7 was higher in samples with bothmutations, but did not discriminate this group from the othergenetic subsets. Thus, we investigated whether these threemarkers (CD123, CD25, and CD99) were coexpressed by thesame cells. Figure 1C shows MPFC analysis of BM samples frompatients with different NPM1/FLT3 gene status. Cumulativedata of the percentage of CD34þ cells that stained positive forCD25, CD123, and CD99 simultaneously showed that thisantigen combination is very rarely observed or barely detect-able in patients with FLT3-ITD� (Fig. 1C and D). Althoughpositivity for each marker measured separately was not suffi-cient to unequivocally discriminate between FLT3-ITD andFLT3-ITD� patients, the combination of all three markerssimultaneously reduced the variance within the group ofFLT3-ITD� patients. We then screened all patients for thepresence or absence of FLT3-TKD, and we identified eight casesharboring this mutation in diagnostic BM samples, five ofwhich were in the NPM1 wt group, whereas three carried alsoa mutation inNPM1. As shown in Fig. 1D (red circles), only onecase presented with a significant fraction of CD34/CD123/CD25/CD99þ cells. It should be noted, however, that thispatient also had a mutated karyotype. Interestingly, also the2 patients without identifiable FLT3 mutations and with theLAIP had mutated karyotype and were sent to allogeneic stemcell transplantation (SCT).

Figure 1D also shows that 3 patients with FLT3-ITD andNPM1wt presented a low percentage of CD34/CD123/CD25/CD99þ

cells. All these 3 patients had mutated karyotype at diagnosis andunderwent allogeneic SCT.

Five BM samples from healthy subjects were also analyzed forthe presence of CD34/CD123/CD25/CD99þ cells. Figure 2 showsa representative MPFC analysis and cumulative data of CD34þ

cells derived fromhealthy donors as comparedwith an FLT3-ITDþ

AML patient. This combination of markers is present in healthyindividuals at a rate ranging from 0.3% to 4% of CD34þ cells.

ROC curveWe found a strong positive correlation (r¼ 0.7136; P < 0.0001)

between the percentage of CD123/CD99/CD25þ populationwithin CD34þ cells and the presence of the FLT3-ITD mutation,as evaluated in 132 AML BM samples by conventional RT-PCR(Fig. 3A).

ROC curve analysis identified the value >11.7% of CD34/CD123/CD99/CD25þ cells within the total CD34þ fraction asthe best determinant for predicting the presence of FLT3-ITDmutation with a specificity and sensitivity of >90%. The areaunder the ROC curve (AUC) was calculated to be 0.96 with 95%confidence interval ranging from 0.91 to 0.99 and significancelevel of P < 0.0001 (Fig. 3B). The positive and negative predictivevalue calculated using 11.7% as threshold were 84.3931% and

Immunophenotype of FLT3-ITD–Mutated Cells

www.aacrjournals.org Clin Cancer Res; 21(17) September 1, 2015 3979




96.1488%, respectively (sensitivity ¼ 90%; specificity ¼ 93.75;disease prevalence ¼ 27.3).

To note, the fraction of CD123/CD99/CD25þ cells withinCD34þ cells is significantly higher in the group with mutationsinboth FLT3 andNPM1, as comparedwith samples that only havethe mutation in FLT3 (Fig. 1D).

The ROC analysis generated using only the markers CD123 andCD25 showed similar results, but with a sensitivity and specificitylower than that calculatedwithalsoCD99(SupplementaryFig. S1).

With respect to conventional RT-PCR,MPFC analysis proved tobe superior in the ability to detect the FLT3-ITD mutation insubclones. Indeed, blasts from 3 patients (UPN 241, 213, and104) who underwent FLT3-ITD AML relapse had been labeled asFLT3-wt by conventional RT-PCR at the time of diagnosis. These

patients showed at presentation a significant number of CD123/CD99/CD25þ cells within the CD34þ population: 13%, 11.8%,and 20.7%, respectively (Supplementary Fig. S2A, red circles).Patient-specific RQ-PCR with primers designed at relapseunveiled low copy numbers of FLT3-ITD in all three cases usingdiagnostic samples. Supplementary Figure S2B shows that whenthe percentage of the CD34/CD123/CD25/CD99þ cell subset ontotal sample was lower than 0.1%, conventional RT-PCR failed todetect the FLT3-ITD subclone.

Purified CD34/CD123/CD99/CD25þ cells carry the FLT3-ITDmutation

Based on the availability of samples, we proceeded to investi-gate the mutational profiles of cell-sorted subfractions within the

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Figure 1.MPFC analysis of CD34þ cells in AMLbone marrow samples according tothe mutational status of FLT3 andNPM1 genes. A, percentage of CD34þ

(top graph) and CD34� CD117þ cells(bottom graph) within the totalsample. B, percentage of CD123, CD25,CD99, CD7, CD11b within the CD34þ

CD117þ/� cell fraction according to theFLT3 and NPM1 gene status. CD38expression was measured as medianof thefluorescencewithin CD34þ cells.C, MPFC analysis of fourrepresentative AML samples showsthe expression of CD123 vs. CD25 andCD123 vs. CD99 within the CD34þ

CD117þ/� population. D, coexpressionof CD123, CD25, and CD99 within theCD34þ populationwas calculatedwithBoolean logic operator. The graphshows the percentage of CD34 cellsthat coexpress the three markersalongwith FLT3-TKDmutation (in red)and karyotype status: normalkaryotype (NK) or mutated karyotype(Mut-K).

Angelini et al.





CD34þ population. We identified two patients harboring FLT3-ITD (UPN 118 and 146) in whom MPFC had revealed 64% and36% of CD123/CD99/CD25þ within CD34þ cells (1.7% and1.9% of the total sample). Cells from both patients were purifiedby cell sorting according to CD34, CD117, CD123, CD99, CD25,CD7, and CD38 expression (Fig. 4) at diagnosis.

The isolated subfractions showed an enrichment of themutated allele within the cell population, expressing themarkers we have identified as LAIP for the FLT3-ITD muta-tion: in both patients, the CD34þ, CD117þ/�, CD38low,CD25þ, CD123þ, CD99þ, and CD7þ/� fraction was highlyenriched in cells carrying the mutation, with a FLT3 ratio of21.67 in one patient and of 8.46 in the other (Fig. 4, subset1). In patient UPN 118, we identified a second subpopulationof cells expressing high levels of CD38 and which contained aheterogeneous cell population with a FLT3 ratio similar to thewhole MNC (value ¼ 2.04). The ratio is an indirect quantify

of the number of FLT3-ITD–positive and –negative cells. Inparticular, Thiede and colleagues (19) found that a highFLT3-ITD allelic burden confers a poor prognosis for AMLpatients.

Within the CD34þ cells of patient UPN 146, we could identifyand isolate to purity another two subpopulations: one containingCD117þCD123þCD99�CD25�CD7þ/�CD38þ cells, mostly har-boring FLT3 wt (subset 2), and the other (subset 3) characterizedby expression of CD99 and negativity for CD38, which on thecontrary was highly enriched in mutated FLT3 (ratio 7.18).

By patient-specific RQ-PCR, we could define the copy numberof FLT3-ITDs clones in the three phenotypic subsets, which isreported in Supplementary Table S5.

These results confirm that cells carrying the FLT3-ITDmutationare contained within distinct subfractions of the CD34þ cellpopulation and canbe enrichedbasedon the expressionof surfacemarkers.

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Figure 2.MPFCanalysis of CD34þ cells in healthybone marrow samples. A, MPFCanalysis of a bonemarrow sample froma healthy donor. The sequential gatingstrategy shows the expression ofCD123 vs. CD25 and CD123 vs. CD99within CD34þCD117þ/� cell subset. B,cumulative data of the percentage ofCD123/CD25/CD99þ within CD34þ

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Figure 3.ROC curve analysis. A, Spearman rankcorrelation coefficient (r) between thepercentage of CD123þCD25þCD99þ

cells within the total CD34compartment and the FLT3-ITDmutation in 132 AML samples. Thecoefficient was calculated giving thevalue 1 to the presence of FLT3-ITD and�1 to the absence of FLT3-ITD. B, theROC curve is generated by plotting thevariables mentioned above. Statisticalanalysis was performed by "Med Calc"software.






Sequential immunophenotypic and RQ-PCR monitoringstudies through patient-specific FLT3-ITD

Four patients (UPN 241, 213, 146, and 104) were followedlongitudinally through post-induction, post-consolidation ther-apy, and at relapse by patient-specific RQ-PCR using RNA derivedfrom sequential BM samples (Supplementary Table S5). Follow-ing serial dilution experiments with patient-specific RQ-PCR forFLT3-ITD, the assay showed maximum reproducible sensitivityand specificity at 10�6 in UPN 104 and 213; and at 10�4 in UPN146 and 241. The coefficient of the standard curves for the 4samples ranged from 0.995 to 0.999 and the slope ranged from3.418 to 4.24.

A progressive decrease in FLT3-ITD copy number wasdetected in UPN 146 after induction therapy but unlike thesmaller FLT3-ITD clone (ITD, 30bp), the most representativeclone (ITD, 60bp) detected at the diagnosis in this case (Fig. 4B,top; Supplementary Table S5) remained unchanged after con-solidation therapy. Accordingly, MPFC analysis after post-con-solidation therapy detected a CD123/CD25/CD99þ cell subsetcomprising 20% of CD34þ cells and 0.6% of the total blastpopulation (Fig. 5A).

UPN 104, 213, and 241 displayed a normal karyotype andhad been labeled as FLT3-wt using the conventional RT-PCR;however, within the small fraction of CD34þ cells, the CD123/CD99/CD25þ population was detected by MPFC above thedefined threshold (Figs. 5B, top and 6A; SupplementaryFig. S2A and S3A). The FLT3-ITD patient-specific analysis byRQ-PCR performed on all samples at relapse allowed identi-fying retrospectively FLT3-ITD in the diagnostic sample (Sup-plementary Table S5).

In the case of UPN104, the FLT3-ITD–specific RQ-PCR showeda low expression of the two ITD clones at diagnosis, which werebelow the detection limit of conventional RT-PCR assays andwhich decreased after induction therapy. The FLT3-ITD copynumber remained undetectable after the second course of con-solidation therapy with Ara-C (Supplementary Table S5 and Fig.6B). This patient relapsed during follow-up and MPFC analysisshowed an expansion of the CD34/CD123/CD99/CD25þ subset,which at this time point represented over 65% of the total blastpopulation (Fig. 6C).

The FLT3-ITD patient-specific RQ-PCR of UPN 213 and 241showed a low expression of the ITD clone at diagnosis, whichdecreased after induction and post consolidation therapy. MPFCanalysis had initially shown a percentage of the aberrant popu-lation very close to the threshold defined by the ROC curve(11.8% and 13% of CD34þ cells, respectively). This percentagedecreased to 2% after post consolidation treatment for UPN 213(Fig. 5B, bottom) in parallel with FLT3-ITD copy number fluctua-tions, but unfortunately there is no MPFC data from the relapse.However, MPFC analysis for UPN 241 showed a 96% of CD123/CD25/CD99þ within CD34þ cells when the patient relapsed,reflecting the very high FLT3-ITD copy number (SupplementaryFigs. S3B–S3C).

Figure 6 and Supplementary Fig. S3 also show that, both atdiagnosis and relapse, the CD34/CD123/CD99/CD25þ subsetcells expressed low levels of CD38.

DiscussionUsing MPFC in leukemic blasts from AML patients, we iden-

tified a CD34þ cell subset characterized by CD25/123/99 coex-pression that, compared with the total mononuclear cell fraction,contained an enriched FLT3-ITD–positive population. Our estab-lished threshold of 11.7% cells staining positive for this antigenicprofile allowed to predict FLT3-ITDwith specificity and sensitivity>90% and enabled the identification of FLT3-mutated subclonesalso in cases labeled as FLT3 wt by conventional RT-PCR duringdiagnostic routine work-up. As reported previously by our groupand by others (29–31), these minor FLT3-mutated subclones,which escape diagnostic recognition, may undergo clonal evolu-tion and ultimately result in overt disease relapse, hence theirimproved identification at diagnosis throughMPFC appears to beclinically relevant.

CD45+CD34+CD117+ CD123+CD99+CD25+

CD7+/– CD38low/–

Subset 1

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Subset 3

CD45+CD34+CD117+ CD123+CD99–CD25–

CD7+/– CD38+

CD45+CD34+CD117–

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CD7+/– CD38–

395 bp365 bp

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R = 2.66 Whole MNC

365 bp425 bp

R = 8.46

395 bp

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intensity(arbitrary

units)

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Figure 4.RT-PCR of FLT3-ITD mutation on whole MNC and sorted cell subsets of AMLsamples. A, diagnostic leukemic cells from UPN 118 AML were analyzedby RT-PCR for FLT3-ITD in whole MNC (top) and using two different cellsubsets (bottom) sorted on the basis of expression of CD45, CD34, CD117,CD123, CD99, CD25, CD7, and CD38. R represents the ratio betweenfluorescence intensity of the FLT3-ITD and FLT3-wt peaks. B, UPN 146 AMLsample at diagnosis (top) and three different cell subsets (bottom) sortedbased on expression of the same markers described above.

Angelini et al.





Our results suggest that the CD34/CD25/CD123/CD99þ

LAIP is strictly associated with FLT3-ITD mutations in thecontext of NK-NPM1–mutated AML. A total of six cases in thisstudy showed discordant results with respect to the correlationbetween the established LAIP threshold and FLT3 mutationalstatus (Fig. 1D). Of these, three patients with FLT3-ITD hadCD34/CD25/CD123/CD99þ cells below the ROC-assessed11.7% threshold, and 3 patients with FLT3 wt configurationdisclosed a CD34/CD25/CD123/CD99 LAIP above the thresh-old; interestingly, all these six cases showed mutated karyotypeand NPM1 wt gene configuration. Although our findingsrequire confirmation in other independent and larger studies,we outline the clinical relevance of the precise diagnosticdefinition of NK-NPM1 mutated AML—a subset commonlyassociated with a favorable prognostic outcome (32)—for thepossible concomitant presence of small subclones harboringthe FLT3-ITD mutation.

Other authors have correlated the expression of CD25 andCD123 with FLT3-ITD mutation in AML (13). In particular, thestudy by Gonen and colleagues (13) showed a significant asso-ciation of CD25 and CD123 with FLT3mutation but did not finda correlation of CD25 expression with the putative stem cellmarkers like CD34. Our data have revealed high copy numberof FLT3-ITD in the CD34/CD123/CD99þCD38�CD117� popu-lation, suggesting thatCD25 expressionmayappear at a later stagefrom CD34/CD123/CD99þ subset. However, an ROC analysisperformed on this phenotype (AUC ¼ 0.89) indicates that thisLAIP is not optimal for the identification of samples with FLT3-ITD.

Rollins-Raval and colleagues (33) described the associationof CD123 in AML patients with both FLT3 and NPM1 muta-tions, in keeping with our observations. Moreover, we reporteda higher CD123 expression in FLT3 mutated AML patients as

compared with those with germline FLT3 (16). In the study byEhninger and colleagues (34), the analysis of CD33 and CD123restricted to the CD34þ compartment allowed to detect anassociation with FLT3 mutations in up to 73.1% of AMLpatients.

Our study shows that AML blasts with FLT3 mutations alsocoexpress CD99. The addition of CD99 to CD123 and CD25and the restriction of the analysis to the CD34 populationallowed to increase the specificity and sensitivity of the asso-ciation of immunophenotypes with FLT3-ITD from 73.1%, asdetected by Ehninger and colleagues (34), to more than 90%.Notably, in this study, the CD34/CD123/CD25/CD99þ cellfraction was detectable in healthy marrow samples at levels<4%, that is far below the 11.7% ROC-generated threshold forthe association with FLT3-ITD. CD99, also known as MIC2 orsingle-chain type-1 glycoprotein, is a O-glycosylated transmem-brane antigen expressed on all leukocytes and is involved in theregulation of apoptosis and adhesive properties of T cells (35,36). The expression level of this antigen in normal hemato-poiesis is related to distinct maturational stages: It is high inbone marrow CD34þ cells, and then declines during the dif-ferentiation process (37). In normal BM, we detect CD99expression on CD34þ cells, but it does not correlate withCD123 or CD25. Interestingly, CD99 expression by tumor cellshas been described in Ewing Sarcoma, where this antigen is thepotential target of immunotherapy (38).

Bachas and colleagues (29, 31) have studied in detail thesubclones detectable within the leukemic blasts of AML patientsand analyzed their role in the development of relapse. By ana-lyzing AML cases with FLT3mutations collected both at diagnosisand at relapse, these authors found amost significant enrichmentof cells carrying the FLT3 mutation in the CD34þ/CD38� com-partment. Our results further improve the identification of these

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Figure 5.MPFC analysis of CD34þ cells at diagnosisand post consolidation AML samples.MPFC analysis of UPN 146 AML bonemarrow sample (A) and of UPN 213 (B) atdiagnosis and post consolidation therapy.Gate and arrows in blue show theexpression of CD25, CD123, CD99 withinthe CD34þCD117þ/� population. The lastplot shows how the selected populations(in blue) match with CD45 and sidescatter in back gating analysis of the totalsample (in red).






clones by amore accurate LAIP, which strengthens the associationwith FLT3 mutations.

As to the potential clinical use of our findings, we observe thatsearch of this LAIP at diagnosis may complement routine geneticcharacterization. In particular, we recommend that samples fromAML patients with normal karyotype and NPM1-mutated/FLT3wt genotype by routine RT-PCR, which show a CD34/CD123/CD25/CD99 population above the indicated threshold be strictlymonitored by bothmolecular andMPFCduring follow-up for thepossible emergence of FLT3-mutated clones.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: D.F. Angelini, T. Ottone, G. Borsellino, L. Battistini,F. Lo-CocoDevelopment of methodology: D.F. AngeliniAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): G. Guerrera, S. Lavorgna, M. Cittadini, F. Buccisano,F. Gargano, M. Divona, N.I. Noguera

Analysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis):D.F. Angelini, T. Ottone, F. Buccisano,M.I. Consalvo,G. BorsellinoWriting, review, and/or revision of the manuscript: D.F. Angelini,F. Buccisano, L. Maurillo, G. Borsellino, G. Bernardi, S. Amadori, A. Venditti,L. Battistini, F. Lo-CocoAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): M. De BardiStudy supervision: S. Amadori, L. BattistiniOther (performed the experiments): G. Guerrera

Grant SupportThis study was partially supported by the Associazione Italiana per la Ricerca

sul Cancro (IG 11586) to F. Lo-Coco and Associazione Italiana contro leLeucemie (AIL).

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received December 11, 2014; revised April 6, 2015; accepted April 25, 2015;published OnlineFirst May 8, 2015.

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1

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FLT3-ITD_33bpFLT3-ITD_66bpDiagnosis

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2015;21:3977-3985. Published OnlineFirst May 8, 2015.Clin Cancer Res Daniela F. Angelini, Tiziana Ottone, Gisella Guerrera, et al. Myeloid Leukemia

-Mutated Clones in AcuteFLT3Immunophenotype Identifies +A Leukemia-Associated CD34/CD123/CD25/CD99

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