Overexpression of CD123 correlates with the hyperdiploid genotype in acute lymphoblastic leukemia

We evaluated CD123 expression in 95 pediatric and 24 adult ALLpatients and compared the results with the CD123 expressionin normal B-cell precursors. Early B-cell precursors were negativewhile intermediate precursors and mature B cells showed weakCD123 expression. Leukemic blasts in 31% of precursor-B ALLsamples exhibited strong expression of CD123, 61% had moderateCD123 expression and 8% were negative; 81.5% of ALL with hyperdiploidkaryotype ( $≥$ 52 chromosomes) showed strong CD123 overexpression.In contrast, cases with ETV6/RUNX1 rearrangement had weak CD123expression. Our study suggests that overexpression of CD123is an aberrant phenotype present in a subset of precursor-BALL with hyperdiploid genotype, and represents an additionalmarker of good prognosis in pediatric precursor-B ALL. Moreover,aberrant CD123 expression in ALL is a good marker for monitoringof minimal residual disease.

Key words: overexpression, CD123, hyperdiploid genotype.

Introduction

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Introduction

CD123 is an ${alpha}$ subunit of a heterodimer IL-3 receptor, a memberof the cytokine receptor superfamily.^1,2 It is a 70 kDa protein,which binds IL-3 with high specificity but with low affinity.³CD123 is encoded by the IL-3a gene, located in the pseudo-autosomalregion (PAR) at the ends of the short arms of the X and Y chromosomes(Xp22.3 and Yp11.3).⁴ B-lymphoid blasts from acute lymphoblasticleukemia (ALL) have been shown to express functional CD123 andCD40 ligand stimulates proliferation of precursor-B (pre-B)ALLby upregulating CD123 expression.^5,6 However, the prevalenceof CD123 expression in pre-B ALL is not clear: one study of13 B-ALL cases reported CD123 expression in all cases as significantlyhigher than normal B-cell precursors.⁷ Another study (25 pre-BALL cases) found 40% of pre-B ALL to have high CD123 expressionin comparison to human myeloid and lymphoid progenitors.⁸

Our goal was to characterize the expression of CD123 in differentmaturational stages of normal B-cell precursors in the bonemarrow by comparison to leukemic blasts of pre-B ALL, usingmultiparameter flow cytometry (FC). We also sought to establishif the level of CD123 expression in ALL correlated with anyrecurrent genetic abnormality. Finally, we investigated whetherthe overexpression of CD123 on residual leukemic blasts canbe applied for follow-up of minimal residual disease (MRD).

Design and Methods

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Design and Methods

A total of 95 consecutive diagnostic samples of childhood ALL(89 newly diagnosed, 6 at relapse, mean age six years, range0–18) were collected from December 2001 to October 2004.Samples from 24 adults with newly diagnosed ALL (mean age 48years, range 21–78) were collected between December 2002and March 2005. The diagnosis of ALL was established accordingto WHO 2001 criteria.⁹ Immunophenotype was classified accordingto EGIL:¹⁰ among pediatric ALL cases there were 5 BI, 60 BII,20 BIII and 10 T-ALL, and among adults 2 BI, 14 BII, 5 BIIIand 3 TALL.

All children were karyotyped, and in most cases, additionalgenetic analyses, including interphase FISH, SKY, or PCR forBCR/ABL1, ETV6/RUNX1, PBX1/TCF3 and MLL rearrangements, weremade as part of the routine investigations.¹¹ Genetic abnormalitiesin childhood pre-B-ALL cases included 26 cases with hyperdiploidkaryotype, 18 with ETV6/RUNX1, 2 with BCR/ABL1, 2 with PBX1/TCF3,4 with MLL gene rearrangement, and 11 with normal karyotype;18 cases showed abnormal karyotype with non-recurrent cytogeneticabnormalities. Karyotypes were available for 13 of 21 adultpatients with pre-B ALL: 3 with BCR/ABL1, 1 with MLL gene rearrangement,1 hyperdiploid, 3 with normal karyotype and 5 cases with non-recurrentgenetic abnormalities.

Hospital control (HC) BM samples for studies of non-leukemicB-cell precursors were obtained from 28 patients: 15 children(median age seven years, range 0–18) and 13 adults (medianage 63 years, range 20–84) with normal patterns of B-celldifferentiation in the bone marrow. HC patients were investigateddue to anemia, leukopenia, reactive lymphocytosis, staging ofmalignant lymphoma, suspect but not confirmed myelodysplasticsyndrome or myeloma, and included children who were at leastsix months post-maintenance therapy for leukemia with no evidenceof disease recurrence. In addition, the expression of CD123on regenerating B-precursors was analyzed in 42 BM samples takenduring follow-up of patients who were a part of the childhoodALL cohort, but as shown by the extensive FC analysis (see below)did not have any MRD after induction therapy (8 follow-up sampleswere taken at day 50 post-induction therapy, 29 samples weretaken at day 103, 2 samples were taken six months post-inductionand 3 samples were taken 12 months post-induction therapy. Non-leukemicB-cell progenitors were categorized as early, intermediate,and mature B cells by expression of CD10, CD20 and/or CD34 onCD19-gated cells as previously described.¹²

Immunophenotypic studies were performed by standard four-colorFC¹³ and included the combination: CD66c-FITC/CD123-PE/CD19-PerCP/CD34-APC.

All studied patients were uniformly treated by NOPHO 2000 protocoland followed by FC for detection of MRD.¹³ MRD follow-up panelswere individually tailored depending on aberrant findings atdiagnosis.

The most commonly applied antibody combinations with CD123 were:CD66c/CD123/CD19/CD45, CD34/CD123/CD19/CD10, CD19/CD123/CD20/CD33,TdT/CD123/CD19/CD10, CD56/CD123/CD19/CD45 and CD19/CD123/HLADR/CD10.MRD was defined as a cluster of at least 10-20 events with aberrantphenotype.

The clustered events had forward and side scatter characteristicsidentical to those of leukemic blasts at diagnosis.

Overall, samples for immunophenotypic studies included bonemarrow (BM) aspirate (n=189), peripheral blood (n=16), and cellsuspension from lymph node biopsy or fine needle aspirate (n=3).The CD123 expression was quantified by mean fluorescence intensity(MFI) in each gated cell population. A significant change inCD123 and other antigen expression in gated leukemic cells infollow-up samples were defined as a 1 SD increase or decreaseof MFI by comparison to the MFI at diagnosis as calculated bythe analysis software (Paint-a-Gate Pro, BD).

Descriptive statistical values including arithmetic mean, median,SD and range were used. Sensitivity and specificity were calculatedaccording to the formula shown at http://www.cebm.utoronto.ca/glossary/spsn.htm).Comparisons of different cell populations were done by the Mann-WhitneyU test and two-tailed Student’s t-test. All reported pvalues are two-sided. All calculations were performed usingSPSS 12.0 (SPSS, Chicago, IL, USA) software.

The study was approved by the Ethics Committee of KarolinskaUniversity Hospital in accordance with institutional researchguidelines.

Results and Discussion

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Results and Discussion

Hospital control samples (n=28) showed median values of 0.23%(range 0.01–1.03), 1.00% (range 0.04–20.50) and1.72% (range 0.26–17.27) of early, intermediate and matureB cells in the bone marrow, respectively (representative patternin a child is shown in Figure 1 A–D). The CD123 expressionin normal B cells was low and showed median MFI values of 8(range 4–26), 18 (range 5–36), and 9 (range 3–48),in three stages of maturation, respectively. CD34-positive B-cellprecursors had a median CD123 MFI of 10 (range 4–39),while CD34-negative B-cell precursors had a median CD123 MFIof 13 (range 3–65). There was no significant differencein CD123 expression between studied subpopulations of B cellsin HC BM samples and in regenerating (post-chemotherapy) B cells.

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Figure 1. CD123 expression in non-leukemic B-precursors and precursor-B ALL in a bone marrow sample from a 5-year old child from the Hospital Control group (investigated for low-grade fever and lymphadenopathy, and found not to have any aberrant lymphoid population). (A) CD10 vs. CD20 expression in CD19 cells: CD19⁺ cells with high CD10 and no CD20 expression are early B-cell precursors (red); CD19⁺ cells with moderate CD10 and variable CD20 expression are intermediate B cell precursors (green); CD19⁺ cells with no CD10 and high CD20 expression are mature B cells (blue). (B–D) histograms showing CD123 expression by early precursors (red), intermediate precursors (green) and mature B cells (blue). (E–F) diagnostic sample from a patient with B-precursor ALL shows high expression of CD123 on CD19⁺/CD34⁺ leukemic B-cells; CD19⁺ B-cells are painted red. (G–H) high CD123 expression identifies the residual leukemic population which shows reduced CD34 expression in day 15 bone marrow sample.

CD123 expression showed considerable variability in childhoodpreprecursor-B ALL. Six samples (7%) had no CD123 expression(MFI <10), 50 (59%) had intermediate expression (MFI 10–100),and 29 (34%) had strong expression of CD123 (MFI> 100), (Figure 1 E and F).Adult preprecursor-B ALL showed similar heterogeneity: 2 samples(10%) had no CD123 expression, 15 (71%) had intermediate expression,and 4 (19%) had a high CD123 expression (MFI>100). Therewas no correlation between expression of CD123 and other myeloid-associatedmarkers, such as CD13 or CD33. Only a single case of T-precursorALL showed CD123 expression with MFI=33 (7.7%). The immunophenotypiccharacteristics of this case were consistent with the most immatureT-ALL recently shown to express CD123.¹⁴

When EGIL categories of B-precursor ALL were considered, thehighest CD123 expression was found in EGIL B-II category (sc.common ALL) (p>0.001). This was probably due to a strongcorrelation found between high CD123 expression and hyperdiploidgenotype. The hyperdiploid precursor-B ALL showed significantlyhigher CD123 expression than other major subcategories includingETV6/RUNX1, BCR/ABL1, MLL, PBX/TCF3 and B-precursor ALL withnormal karyotype (Figure 2, p<0.0001). Twenty-two of 27 samplesfrom patients with hyper-diploid ALL displayed CD123 MFI >100,but only 6 of 71 non-hyperdiploid precursor-B ALL showed similarhigh expression. The calculated sensitivity and specificityof high CD123 expression for hyperdiploid genotype are 81.5%and 91.5%, respectively, with a positive predictive value of78.6%. Of interest, all cases with ETV6/RUNX1 showed uniformlylow expression of CD123. However, some precursor-B ALL geneticcategories (such as BCR/ABL1 and MLL rearrangements) were notfrequent in our material and require further studies.

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Figure 2. CD123 Expression in B-cell precursors, B-ALL and T-ALL. Dots for CD34⁺ and CD34^– B cells demonstrate CD123 MFI for the two populations of precursors in non-leukemic (HC) samples. Dots for B-ALL genetic subtypes and for T-ALL represent CD123 MFI of the leukemic population. Thirteen (of total 21) adult leukemia cases for which karyotypes were available are plotted.

The overall CD123 expression in precursor-B ALL was significantlyhigher than in normal B cells (p<0.0001) of any maturationstage (Figure 2). Therefore, high expression of CD123 in preprecursor-BALL can be defined as an aberrant or leukemia-related phenotype.CD123 overexpression could be used as leukemia-related aberrantphenotype for MRD follow-up in 60 of 106 (57%) of pre-B ALLpatients in our cohort. High CD123 expression was one of theaberrant features confirming residual leukemia found in 22 samples(from 21 patients) taken at day 15 and/or day 29 post-therapy(see Figure 1 G and H). The CD123 showed stable overexpressionin 77% samples with residual leukemia. By comparison, MFI ofCD34 and TdT was stable in 32% and 31% of samples, respectively,as previously described by Gaipa et al.¹⁵

In conclusion, we found that CD123 expression is generally lowin B-cell precursors and mature B cells both in HC BM samplesand in post-chemotherapy regenerating B cells. The CD123 overexpressionin B-precursor ALL correlated with hyperdiploid genotype, thesecond most common genetic abnormality in childhood ALL. Incontrast, B-ALL samples with ETV6/RUNX1 rearrangement showedexpression similar to normal B-cell precursors. The biologicalsignificance of CD123 expression in early precursor-B ALL withhyperdiploid genotype remains unclear. Analogous to the situationin acute myeloid leukemia, it may provide a proliferative advantageto leukemic blasts.¹⁶ The mechanism of overexpression is notknown. However, 88% of children with high hyperdiploid karyotypehave an extra X chromosome, on which the IL-3R ${alpha}$ gene is located.¹⁷Whether a gene dosage effect may be responsible for the overexpressionof this gene is not clear. The gene dosage hypothesis may besupported by a recent study of gene expression profiles in acutelymphoblastic leukemia, where Kuchinskaya et al. found thathigh expression of the IL-3R ${alpha}$ mRNA was one of the ten significantpredictors of hyperdiploid phenotype (predictive strength 8.3).Besides the IL-3R ${alpha}$ gene, three other genes in this group werelocated on chromosome X.¹¹

A remarkably stable expression of CD123 in residual leukemiccells during early therapy confirmed its reliability as a markerof MRD. It showed much less variation of expression than othercommonly used markers for MRD including CD34, TdT, and CD10.

The high expression of CD123 in a subset of ALL may providean important therapeutic target as has recently been suggestedby Lhermitte et al.¹⁴ Diphtheria toxin fused to human interleukin-3was shown to induce cytotoxicity in acute myeloid leukemia withCD123 expression.¹⁸ Our data suggest that CD123 expression shouldbe evaluated in acute lymphoblastic leukemia for (i) its prognosticvalue as a predictor of hyperdiploid genotype, (ii) monitoringminimal residual disease at follow-up and (iii) to prospectivelyselect patients who may benefit from the novel therapies.

Footnotes

^* Current address: Department of Pathology, University of PittsburghMedical Center, Pittsburgh, PA, USA

Authorship and Disclosures

All authors contributed to conception, design, analysis andinterpretation of data, drafting and critically revising thearticle for important intellectual content, and final approvalof the version to be published.

The authors reported no potential conflicts of interest.

Received for publication September 9, 2008. Revision received February 13, 2009. Accepted for publication February 17, 2009.

References

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