Elisabeth Paietta, Laboratory DMonteore Medical Center-North Division, Immunolo

Keywords:immunophenotypesgenetic lesionssurrogate markerstargeted therapy

Although it makes perfect sense to hypothesize that genotypes control the phenotypes of leukemiccells, it has been difcult and sometimes impossible to establish concordances between phenotypicand genotypic categories. Rather than abandoning the valid assumption that antigen patterns on thesurface of leukemic cells are caused by and thus reect genotypic characteristics [1], it is more

* Corresponding author. Tel.: 1 718 920 9520; fax: 1 718 920 1161.E-mail address: epaietta@earthlink.net.

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Best Practice & Research Clinical

Best Practice & Research Clinical Haematology 23 (2010) 359368therapeutic targets grows, the role of surrogate antigen proleswill grow, as they can predict for the efcacy of targetedapproaches in lieu of expensive, time-consuming and not alwaysaccessible genetic analyses.

2010 Elsevier Ltd. All rights reserved.

Introduction1521-6926/$ see front matter 2010 Elsevier Ltdoi:10.1016/j.beha.2010.08.001irector, Professor of Medicine *

gy Laboratory, Cancer Center, 600 East 233rd Street, Bronx, NY 10466, USA

The basic hypothesis of surrogate marker proles is that individualgenetic lesions result in characteristic distortions of the cellularphenotype with some predictable consistency that can be exploi-ted by sophisticated immunophenotyping. While cytogenetic andmolecular aberrancies currently are accepted prognostic predictorsin acute leukemias, single antigen expression and even antigenicproles rarely impact on prognosis. However, increasingly,phenotypes are delineated which can serve as surrogates forunderlying genetic aberrations of clinical importance. This devel-opment is of particular signicance as antileukemic therapybecomes available that targets any component of the disturbedmolecular pathways associated with these genetic lesions. Thischapter will focus on established surrogate marker proles, such asthose for PML/RARa, AML1/ETO, FLT3-gene mutated acutelymphocytic leukemia (ALL), and BCR/ABLPOS ALL. As the list ofleukemias5

Surrogate marker proles for genetic lesions in acute

Haematologyjournal homepage: www.elsevier .com/locate/behad. All rights reserved.

E. Paietta / Best Practice & Research Clinical Haematology 23 (2010) 359368360reasonable to blame our limitations with respect to antigen recognition. Sophisticated immunophe-notyping reliably allows for identifying the lineage afliation of leukemia cells and the determinationof the degree of maturation [2]. Since myeloid and lymphoid leukemias receive distinct therapies,this function of ow cytometry is essential in the work-up for any leukemia patient and, in fact, manylaboratories have restricted their antibody panels to those useful in lineage differentiation. In itsextreme, one cell aliquot stained for intracytoplasmic CD3 (cCD3), intracytoplasmic CD22 (cCD22) andmyeloperoxidase can yield the necessary information regarding lineage afliation with the T-, or B-lymphoid, and myeloid lineage, respectively. While cost-effective, this approach can hardly beexpected to yield new data with respect to prognostic novel antigens or antigen expression patternsassociated with genetic lesions.

To date, cytogenetic and molecular aberrations dominate prognosis both in acute myeloid (AML)and acute lymphoblastic leukemia (ALL). On the other hand, few single antigens are unequivocallypredictive of outcome. The signicance of single antigens for prognosis may derive from the availabilityof targeted antibody therapy [3], such as rituximab and variants thereof, which target CD20 irrespectiveof the diagnostic characterization of the CD20 expressing abnormal cells. A single candidate antigenwith apparent negative prognostic signicance is the neural-cell adhesion molecule, CD56, in acutepromyelocytic leukemia (APL) [4,5] and in AML with the 8;21 translocation [6]. In t(8;21) AML, theCD56 expression is associated with a disproportionate incidence of granulocytic sarcoma, a tendencythatmay be responsible for the inferior outcome in these patients [7,8]. On the other hand, in APL, CD56expression is commonly associated with the S-form of the PML/RARa transcript [9], a disease subtypewith frequent expression of CD2, a T-lineage afliated antigen, andmutation of the FLT3 gene rev [10]. Ithas been hypothesized that CD2POS, FLT3-mutated APLmay be derived from a distinct [11], presumablyearlier level of differentiation [12]. Thus, the same antigen may have prognostic signicance in variousleukemia subtypes without a shared justication or apparent biologic reason.

While not surrogates per se, single antigens can serve as diagnostic pointers for genotypes. Thending of CD2 expression on the surface of otherwise unequivocally myeloid leukemia cells is rare andshould raise a ag in themind of everyowcytometrist. Two differential diagnosesmust be considered,either S-form PML/RARaPOS APL [9] or AML with inv(16)(p13q22)/t(16;16)(p13;q22) [13]. In general,these two subsets can be easily distinguished given the characteristic APL immunophenotype that willbe discussed in detail below (HLA-DRNEGCD11aNEGCD18NEG). Until nownot appreciated, however, is theobservation that CD2POS APL occasionally may lack these APL-typical antigenic features, somethingnever seen in L- or V-form APL, thus oftenmisleading laboratory investigators or pathologists Paietta E,unpublished. My recommendation for any case of CD2POS myeloid leukemia is the immediate prepa-ration of RNA for polymerase chain reaction (PCR) assays to detect PML/RARa or CBFb/MYH11 for inv(16)(p13q22)/t(16;16)(p13;q22), especially since these leukemia subdiagnoses require distinct therapies.

The major caveat associated with the description of surrogate marker proles is a reliance oncommonly occurring antigens. For instance, the initial specic marker prole for PML/RARaPOS APLwas simple double negativity for HLA-DR and CD34. We now know that a much more accuratedenition of the APL immunophenotype is required and that variability exists in the antigen prolewith respect to morphologic and molecular subtypes of PML/RARaPOS APL [9]. A similar mistake wasmade in BCR/ABLPOS acute lymphocytic leukemia (ALL) for which a specic immunophenotype wasoriginally based on strong expression of CD34 and CD10, weak expression of CD38, and aberrantreactivity for CD13 [14]. Analysis of the Eastern Cooperative Oncology Group (ECOG) phase III trial,E2993, revealed that the true surrogate marker prole for BCR/ABL positivity in ALL consisted ofexpression of CD25 and dual expression of the myeloid-afliated antigens, CD33 and CD13, while theexpression levels of CD34, CD10 and CD38 were of inferior statistical signicance [15]. The associationof CD33/CD13 with BCR/ABLPOS ALL was independent of the maturation stage of the B-lymphoblasts.Among all B-lineage ALL, CD33 and/or CD13 are found preferentially in Early Pre-B ALL (CD10POS),while the more mature myeloid antigens, CD65(S) and CD15(S), are found on the surface of moreimmature Pro-B ALL (CD10NEG) lymphoblasts rev [2]. The majority of MLL/AF4POS ALL cases, derivedfrom t(4;11)(q21;q23), lack CD10rev [16]. The tendency of MLL/AF4POS lymphoblasts to express CD65and CD15 is, therefore, not surprising and may simply be related to the CD10NEG Pro-B maturationstage. However, in trial E2993, we found that MLL/AF4POS lymphoblasts co-expressed CD65 and CD15

POSwhether associated with Pro-B or Early Pre-B maturation stage. These observations in BCR/ABL and

MLL/AF4POS ALL suggest that myeloid antigen expression in B-lineage ALL is determined by theunderlying genetic defect rather than B-lymphoid developmental stage.

In the absence of genotype-specic therapy (e.g., ATRA for APL, tyrosine kinase inhibitors for BCR/ABLPOS ALL), marker proles for genotypes may only have the potential of a targeted approach in the(maybe near) future. Until then, correct lineage afliation remains the determinant factor in the choiceof leukemia treatment. That identication of the dominant lineage afliation can be more challengingthan the characterization of the underlying genotype is exemplied by the new leukemia subtype withmutations in NOTCH1 and silenced CEBPA gene. Wouters et al. [17] described that this novel AMLsubtype expressed CD7, CD34, TdT, CD33 and CD13; remarkably there were high levels of mRNA forCD3, weak expression of surface CD3 in one of 6 specimens, rearrangements of T-cell receptor genes,and expression of the T-cell-specic Src-family kinase LCK. Proof of an afliationwith the T- and/or themyeloid lineage, by testing for cCD3 and myeloperoxidase ow cytometrically, unfortunately, was notobtained. Subsequently, Terriou et al. [18] clearly demonstrated that cases with methylated CEBPA andNOTCH1 mutation belonged to the T-cell lineage based on cCD3 expression and negativity formyeloperoxidase.

Reliable surrogate marker proles for genetic lesions are of clinical interest when the associatedgenotype is prognostically signicant and/or when targeted therapy for that genotype is available.Outcome-based classication of the leukemias nowadays is based predominantly on cytogeneticaberrations and their molecular derivatives (e.g., fusion genes). Increasingly, however, genetic andepigenetic proling data are the major source of information regarding perturbed pathogeneticpathways and potential therapeutic targets. The identication of antigen proles which can predict forthe existence of, for example, gene mutation or gene silencing events by applying rather inexpensiveand quick multiparameter ow cytometry would be an important step in the translation of sophisti-cated laboratory studies into clinically relevant leukemia characterization.

PML/RARaPOS acute promyelocytic leukemia (APL) with t(15;17)(q22;q21)

APL is unique in that the morphologic M3 (and M3v) FAB class (FrenchAmericanBritish classi-cation) is interchangeablewith characteristic immunophenotypic features [10]. Given the unmistakablyhypergranular features of M3 leukemic promyelocytes, which rarely cause disparity among morphol-ogists, itwas tempting todescribe a characteristic immunophenotype for this disease subtypeearlyon inthe use ofowcytometry in leukemia diagnosis. The initial denition of an APLmarker prole consistedof negativity for CD34 and HLA-DR, which, unfortunately, is still considered sufcient for APL diagnosisbymanypathologists. Subsequently, the prolewas renedbyadding thedifferential reactivity of CD15/CD15S antibodies, caused by the sialylation of the CD15 carbohydrate antigen in APL, weak expression ofCD38 and CD45, expression of CD9, and lack of P-glycoprotein rev [10]. Still, this prole had only limitedability to distinguishAPL fromotherHLA-DRNEG andCD34NEGAML subtypes, such as natural-killer (NK)-cell AML [19], an important distinction considering that NK-AML is unresponsive to ATRA. The newestand most comprehensive denition [9] reliably identies t(15;17)(q22;q21) APL based predominantlyon: (1) lack of HLA-DR and CD133, two antigens expressed at differentiation levelsmore immature thanthat of promyelocytes during normal myelopoiesis; (2) absence of several adhesive molecules, such asCD11a (aL subunit of the leukocyte integrin LFA-1), CD18 (b2 subunit of LFA-1), and CD11b (aM subunit ofMac-1 integrin); (3) lowexpression of carbohydrate structures (CD65S, CD15),which serve as ligands forother adhesionmolecules, and (4) faint expressionof CD45, the common leukocyte antigen, andof CD38,a bifunctional ectoenzyme catalyzing involved in cell adhesion to endothelium. In summary, APL ischaracterized by absence or weak expression of adhesion molecules.

The question whether CD117 is expressed by APL cells is an important one, especially for minimalresidual disease (MRD) monitoring. CD117, the stem cell factor receptor KIT, is expressed by normalhematopoietic precursor cells and is lost prior tomaturation of normalmyeloblasts into promyelocytes.There exists the erroneous view that leukemic promyelocytes are equally negative for CD117. Theintensity of CD117 staining of leukemic promyelocytes is sometimes very low and can be recognizedonly by comparison with negative staining of normal cells contaminating the leukemic sample [10].Due to its invariable presence in APL, CD117 is an excellent tool for the monitoring of residual APL cells

E. Paietta / Best Practice & Research Clinical Haematology 23 (2010) 359368 361post treatment.

The hypogranular variant of APL (FAB M3v) is frequently associated with the S-form of the PML/RARa transcript, a molecular variant derived from the most 50 breakpoint in the PML gene. In APL withthe S-isoform, there is increased occurrence of CD34, HLA-DR, and/or CD56 expression and of FLT3 genemutations, when compared with the L- or V-isoform [10]. Furthermore, S-form APL uniquely expressesCD2, a T-lineage afliated antigen never found in L- or V-form APL [9]. We recently noted that CD2expression in APL can obviate the nding of the characteristic marker prole and present with weak,though measurable expression of HLA-DR, CD34, CD11a and CD18 [20]. Given that CD2 expression inAML is rare, the nding of CD2POS non-lymphoid leukemic cells must prompt a suspicion of APL andPCR analysis for PML/RARa. This is particularly important in hypogranular cases, inwhich the expectedcharacteristic scattergram forM3 APL, reecting cells of large size (high forward angle light scatter, FSC)with a high degree of granularity (high 90 degree angle scatter or side scatter, SSC) is not seen [10].

AML1/ETOPOS AML with t(8;21)(q22;q22)

Translocation (8;21)(q22;q22) is a common recurring cytogenetic abnormality in AML [21]. The twogenes involved in this translocation are the AML1 transcription factor, now called RUNX1, on chro-mosome 21q22.3, and the Eight-Twenty-One oncoprotein (ETO) (also called MTG8) on chromosome8q22 [22]. For the purpose of this chapter, wewill use the term AML1/ETO for the t(8;21) fusion gene.This AML subtype is an example of Core Binding Factor (CBF) leukemias. AML1/ETO AML not onlycarries a favorable prognosis, [23] but also demonstrates unique sensitivity to multiple sequentialcycles of high-dose cytosine arabinoside [24].

t(8;21) AML has a characteristic immunophenotype, which was originally based on expression ofCD19, a B-lineage associated antigen, and of CD56, the neural-cell adhesion molecule, by CD34POS

myeloblasts [25]. While all myeloblasts with the CD19/CD56 marker prole invariably contain AML1/ETO transcripts, a substantial fraction of t(8;21) patients lack either one or both of these discriminatingantigens. Of note, CD19 expression can be of very weak intensity [26] and, in fact, may not be recog-nized if a uorescein isothiocyanate (FITC)-conjugated CD19 antibody is used. Evenwith phycoerythrin(PE) [26] or other uorochrome-conjugated antibodies, CD19 staining can easily be missed, unlessother antigenic characteristics suggest an AML1/ETOPOS AML (Paietta E, unpublished observation). Onecharacteristic feature of t(8;21) AML is a striking myeloid immaturity, such as weak or absentexpression of CD33 [27], and failure to detect myeloperoxidase by antibody binding [28], consistentwith the suggestion that the AML1/ETO fusion event occurs at an early stem/progenitor cell stage [22].This lack of myeloid maturation has mostly gone unnoticed, possibly due to the frequent association oft(8;21) AML with FAB M2 and the continuing tendency of pathologists to place more weight onmorphology rather than immunophenotype, particularly in AML. Furthermore, low expression of theintegrins, CD11a and CD18, is a very helpful diagnostic tool in this AML subtype. With the exception of t(8;21) and t(15;17), >90% of AML demonstrate strong expression of CD11a and 100% express CD18. Thelow expression of CD11a in t(8;21) AML is explained by the inhibition of CD11a promoter activitythrough the AML1/ETO fusion product [29].

Due to the high degree of myeloid immaturity, CD19POS t(8;21) cases can be misdiagnosed as ALL[2,28,30]. It is imperative, therefore, to test all cases of acute leukemia simultaneously for cCD22 (or cCD3)and myeloperoxidase in the same cell aliquot. The dual absence of myeloperoxidase and cCD22 ina questionable case is consistentwith AML. Thismarker combination is particularly relevant since CD19POS

t(8;21) myeloblasts frequently co-express PAX5, a B-lineage associated transcription factor [31]. Themarkers HLA-DR, CD34 and CD133 discriminate between CD11aNEG/LOW, CD18NEG t(8;21) AML and APL.

BCR/ABLpos ALL with t(9;22)(q34;q11)

ALL is a genetically heterogeneous disease with some major genotypes associated with recurrentcytogenetic abnormalities. In adult ALL, the Philadelphia chromosome, t(9;22)(q32;q11), resulting inthe formation of the BCR/ABL fusion transcript, is the major poor prognostic indicator [32]. Its inci-dence increases with age, ranging from 2% to 4% in children versus 2530% in adults. The BCR/ABL generesides in the Philadelphia chromosome, the derivative chromosome 22 formed by the t(9;22)

E. Paietta / Best Practice & Research Clinical Haematology 23 (2010) 359368362(q34;q11) [33]. It encodes a fusion protein in which part of the SH3 domain of c-ABL is replaced by

portions of the BCR protein; thus all BCR/ABL proteins share a similar carboxy terminal ABL tyrosinekinase domain, but differ in the BCR portion. Inw30% of BCR/ABLPOS ALL, the breakwithin the BCR geneoccurs in the major breakpoint cluster region (M-bcr), resulting, when joined with a portion of c-ABLfrom chromosome 9, in a b2a2 or b3a2 fusion transcript encoding a protein of 210,000 daltonmolecularweight (p210BCR-ABL). In the majority of BCR/ABLPOS ALL cases, a break in the minor breakpoint clusterregion (m-bcr) forms the e1a2 transcript encoding a 190,000 dalton protein (p190BCR-ABL). The chimericproteins encoded by the BCR/ABL gene have constitutive tyrosine kinase activity.

The disappointing response of BCR/ABLPOS patients to standard therapy [32] and the availability ofspecic tyrosine kinase inhibitors for the treatment of BCR/ABLPOS disease [34] make it imperative thatthese patients are recognized accurately. Unfortunately, cytogenetic analyses in ALL are hampered bysuboptimal chromosome structure [35]. Furthermore, approximately 10% of BCR/ABLPOS ALL lackevidence of the t(9;22) by chromosome banding [36]. Until recently, the detection of BCR/ABL reliedexclusively on uorescence-in situ hybridization (FISH) or PCR, which required sufcient cellularmaterial and access to amolecular diagnostic laboratory. Lately, a bead-based owcytometric assay hasbeen developed, which allows for the demonstration of BCR/ABL proteins in diagnostic ALL specimensreliably and rapidly, within 4 h after sample receipt [37]. Among 65 ALL specimens tested at the time ofpresentation, the ECOG Leukemia Translational Studies Laboratory found 100% concordance betweenthis bead assay and semiquantitative PCR for BCR/ABL. Furthermore, the recommended cell count of 1million mononuclear cells [37] could be reduced to 250,000 without negative impact on the quality ofthe assay (Paietta E, unpublished observation). Caveats associated with this assay are the increasedprotease activity in BCR/ABL transcript-containing cells from chronic myelogenous leukemia anda lower limit of sensitivity of 0.1%, making this assay unsuitable for MRD monitoring [37].

In the beginning, alleged surrogatemarker proles for BCR/ABLPOS ALL relied solely on differences instaining intensity of antigens commonly found in B-lineage ALL, such as strong expression of CD10 andCD34 and weak expression of CD38 [1,14,38]. The serendipitous nding of expression of CD25 in 4patients with BCR/ABLPOS ALL by Nakase et al. in 1992 did not nd any acclaim [39]. However, in 1997,a preliminary analysis of 144 patients enrolled in ECOGs phase III adult ALL trial, E2993, conrmedwithout question the association between BCR/ABL positivity and expression of CD25, the a-chain ofthe interleukin-2 receptor [15]. The nal analysis of E2993 [40] demonstrated that CD25 expressioncorrelated with increased expression of stem cell markers, such as CD133, CD105, CD109, CD135, andCD123, in BCR/ABLPOS lymphoblasts suggesting that CD25POS BCR/ABLpos lymphoblasts are arrested atan immature, potential stem cell level of B-cell differentiation. This may also explain the strongexpression of CD34 and the frequent nding of dual expression of the myeloid antigens CD33/CD13 byBCR/ABLPOS lymphoblasts. Because BCR/ABLPOS lymphoblasts belong unequivocally to the B-celllineage (cCD22POS, myeloperoxidaseNEG), this phenotype, despite dual or single myeloid antigenexpression, must not be considered representative of a biphenotype. Primo et al. [41] reported thatpatients with del(9)(p21), in addition to the t(9;22), lacked both CD33 and CD13, an interestingobservation that warrants conrmation.

The most striking observation with respect to CD25 and BCR/ABLPOS ALL is that expression levels ofCD25 are of prognostic signicance predicting for a lower likelihood to achieve complete remission[40,42] and shorter overall (OS) [40] or event-free survival [42] among BCR/ABLPOS patients. In otherwords, CD25 is unique in its dual function as a dependable marker of BCR/ABLPOS ALL and an inde-pendent prognostic factor for outcome in this disease.

FLT3-gene mutated T-lineage ALL

The rare T-ALL subset with activating FLT3 gene mutations [43,44] belongs to the few knownexamplesof genemutations,whicharepredictedby thepresenceof auniqueantigenexpressionpattern.

Typically, T-lineage lymphoblasts express cCD3, and are invariably surface positive for CD7.Attempts to stratify patients according to their maturation stage, based on expression of surface CD3,CD2, or CD34, as suggested by the WHO classication [21], does not provide subsets associated withoutcome or other clinico-biological features [44]. ECOG recently described two major subclasses of T-ALL with prognostic signicance and insubstantial immunophenotypic overlap: myeloid antigenNEG

POS POS pos

E. Paietta / Best Practice & Research Clinical Haematology 23 (2010) 359368 363CD1a T-ALL and CD13/CD33 T-ALL [44]. CD1a T-ALL represents the commonly accepted cortical

T-cell maturation stage rev [2] which lacks CD34 and myeloid antigens, and is frequently positive forsurface CD3, CD4 and/or CD8. With respect to clinical signicance, CD1a expression was benecial interms of prolonged OS. The other subtype with prognostic signicance was T-ALL with expression ofCD13, for which OS was signicantly inferior to that of CD13NEG T-ALL patients. Whenever present,additional expression of CD33 did not add to the inferior prognostic effect of CD13. The majority ofCD13POS T-ALL lacked surface CD3, CD4, CD8, and often CD2, while CD34 was present [44], charac-terizing CD13/CD33pos T-ALL as most immature T-ALL, analogous to the pro-T maturation stage rev [2].

The stem cell factor (SCF) receptor, c-Kit or CD117, is consideredmore specic for themyeloid lineagethan CD33 or CD13 in leukemia immunophenotyping rev [2]. During normal lymphopoiesis, CD117 isexpressedbya fractionof CD34POS, CD3NEG, CD4NEG, CD8NEG (triple-negative) thymocytes [45]. Innormalintrathymic triple-negative T-cell precursors, expression of CD117 coincideswith that of CD135, the FLT3receptor tyrosinekinase that is activatedby theFLT3 ligand [46]. The combinationof SCFand interleukin-7 causes progressive differentiation of these progenitors intomature T-lymphocytes,while SCFand FLT3ligand induce differentiation of triple-negative thymocytes along the myeloid pathway [47].

CD117 expression by leukemic lymphoblasts is rare and mostly, albeit not exclusively, associatedwith the T-cell phenotype [16,45,48-50]. Recently, CD117POS T-lineage ALL was characterized as animmunophenotypically distinct subtype frequently associated with myeloid morphologic features,including the presence of Auer rods. CD117POS lymphoblasts are surface CD3NEG, CD34POS, CD62LPOS,CD56NEG, CD2POS, CD7POS, CD1aNEG, CD5NEG, CD4/CD8NEG, CD56NEG, expressing one myeloid antigen,CD13 [43]. The lack of CD5 expressionmust be particularly emphasized since it is unique to this subtypeof T-lineage ALL. A close association of CD13 with CD117 in T-ALL has been previously postulated, basedsolely on c-kit gene expression [51]. Occasionally, myeloperoxidase could be demonstrated by antibodystaining in some CD117POS lymphoblasts which invariably expressed cCD3. The simultaneous expres-sion of T-lineage and myeloid lineage-specic antigens qualies this immunophenotype as trulybiphenotypic. This antigen prole, which suggests dual T and myeloid differentiation potential,resembles multipotent thymic precursors [52].

Strikingly, CD117POS ALL cases show a high frequency of FLT3 gene mutations, either internaltandem duplications (ITDs) or point mutations [43,44]. Activating mutations of the FLT3 gene are themost common known genetic abnormalities in AML, but they rarely occur in ALL [46]. B-lineage ALLscontaining MLL gene rearrangements have previously been found to demonstrate FLT3 mutations [53].In further support of a T-lineage afliation, CD117POS FLT3-mutated lymphoblasts overexpress LYL1 andLMO2 oncogenes [43], transcription factors, which have been associated with an early CD34pos

thymocyte phenotype in pediatric T-ALL [54]. In pediatric T-ALL, these cases demonstrate relativeresistance to standard chemotherapy [54]. However, despite CD13 expression, CD117POS/CD13POS T-ALLdid not carry the same inferior prognosis as CD117NEGCD13POS T-ALL [43,44]. Despite the low frequencyof FLT3-gene mutations in adult ALL, the availability of a variety of FLT3-kinase inhibitors suggestsa potential targeted approach in the treatment of this small patient ALL cohort.

Caremust be taken, though,whenassessingCD117POS T-ALL as a surrogatemarker prole for FLT3genemutations.Wehave recently found thatanysignicantdeviation fromourpreviouslypublished [43]classicsurface CD3NEG, CD34POS, CD62LPOS, CD56NEG, CD2POS, CD7POS, CD1aNEG, CD5NEG, CD4/CD8NEG, CD13POS

phenotype can obviate the association with FLT3 gene mutations. The most important such antigenmodications are expression of CD5, CD56 or of CD33 instead of CD13 [16]. This observation stresses onceagain the importance of establishing entire antigen proles as surrogate immunophenotypes for geneticlesions; single antigens can serve in this role only in exceptional cases (e.g., CD25 in B-lineage ALL).

Conclusions and future perspectives

Acute leukemias are heterogeneous diseases. Among all cases with similar morphologic featuresthat lack comparable antigen expression patterns and do not share genotypic characteristics, hyper-granular APL is the exception. FAB M3 APL presents with a unique antigen prole, characterized bya lack of immature stem cell antigens (e.g., CD34, CD133), and a lack of adhesion molecules (e.g.,integrins CD11a, CD11b, CD18). But even in the case of APL, distinct morphologic (FAB M3v, hypo-granular APL) and molecular features (S-form of PML/RARa transcript) are reected in changes in the

E. Paietta / Best Practice & Research Clinical Haematology 23 (2010) 359368364PML/RARa-specic surrogate marker prole, which include the expression of the T-lineage afliated

antigen, CD2. Thus, surrogate antigen combinations are incredibly precise and any variation of theglobal genetic lesion, such as various isoforms of the same transcript, is reected in a distinct alterationof the immunophenotype. This is demonstrated in APL, and is also suggested by preliminary data inBCR/ABLPOS ALL, where striking differences in the expression of CD25 and myeloid antigens betweenthe e1a2 and b2a2/b3a2 transcript forms were detected [55]. This is of particular interest given thepublished difculties in establishing specic gene signatures for BCR/ABL isoforms [5659]. The recentdiscovery of frequent deletions in IKZF1, a gene that encodes the B-lymphoid transcription factorIKAROS, in BCR/ABLPOS pediatric [60] and adult ALL [61] introduces a new variable into the denition ofsurrogate markers. Since IKZF1 deletions are frequent in pediatric high-risk ALL overall [60], it will beinteresting to assess whether the currently accepted surrogate antigen prole for BCR/ABLPOS ALL is atall affected by the presence of IKZF1 deletions. Thus, surrogate antigen patterns should be re-evaluatedfor newassociations and predictive powerwhenever novel relevant gene expression, genemutation, orepigenetic studies become available.

Surrogate markers are very useful as bioinformatics tools in the identication of genes whoseexpression patterns correlate with different phenotypes or sample characteristics of interest but whichwere not known to be associated with these characteristics. On the other hand, gene expression andmutation analyses can aid signicantly in the search for hitherto unrecognized surrogate antigen

E. Paietta / Best Practice & Research Clinical Haematology 23 (2010) 359368 365proles. The efcacy of ow cytometry is limited due to its inability to test all antibodies on hand in aneffort to reveal novel surrogate antigens. Therefore, it will be more practical in the future to selectantigens of potential genotypic relevance based on available gene expression data for already char-acterized genotypes (e.g., FLT3 gene mutations) or prognostically dened genetic or epigenetic proleswith unknown biological correlates (e.g., within the group of normal karyotype leukemias).

While surrogate marker proles are not meant to replace conrmatory molecular analyses, theyprovide clinicians with an early diagnostic clue. A suspicion of APL in a newly admitted patient derivedfrom ow cytometry results will allow the immediate initiation of all-trans retinoic acid without harmshould the diagnosis of APL not be conrmed in subsequent analyses. In BCR/ABLPOSALL, unsuccessful ornormal cytogeneticsmaynot prompt amolecular analysis for BCR/ABL, unless the immunophenotype issuggestive of that diagnosis. The increasing availability of therapies targeted at specic genetic lesionswill boost interest in the denition of surrogate marker proles for targeted genotypes. For instance, inFLT3-gene mutated AML, the use of inhibitors of FLT3-kinase activity currently must await the result ofmolecular analysis. To complicatematters, cell signalingdatahave recentlyshownthat a fractionof FLT3-mutated blast cells cannot respond to kinase inhibitors in vitro, while apparently unmutated leukemiasshow the characteristic cellular response of FLT3-mutated disease [62]. Surrogate antigens that canpredict a functional FLT3 mutation status would be of great clinical impact. This challenge furtherexemplies thatmultiparameterowcytometry can contribute to leukemiadiagnosis far beyond its rolein the identication of lineage afliation.

Practice points

1. The basic concept behind the value of surrogatemarker proles is that antigen patterns on thesurface of leukemic cells are associated with and thus reective of genotypic characteristics.

2. Accepted surrogate marker proles available to date are those for PML/RARaPOS APL, AML1/ETOPOS AML, BCR/ABLPOS ALL, and FLT3 gene mutated T-ALL.

3. The major caveat associated with the description of surrogate marker proles is a reliance oncommonlyoccurring antigens,merelycharacteristic of the leukemias hematopoietic cell lineage.

4. Surrogatemarker proles are not meant to replace conrmatorymolecular analyses, but theycan provide clinicians with an early diagnostic suggestion.

5. Reliable surrogate marker proles for genetic lesions are of particular clinical interest whenthe associated genotype is prognostically signicant and/or when targeted therapy for thatgenotype is available.

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[6] Baer MR, Stewart CC, Lawrence D, et al. Expression of the neural cell adhesion molecule CD56 is associated with short

E. Paietta / Best Practice & Research Clinical Haematology 23 (2010) 359368366remission duration and survival in acute myeloid leukemia with t(8;21)(q22;q22). Blood 1997;90:164350.[7] Tallman MS, Hakimian D, Shaw JM, et al. Granulocytic sarcoma is associated with the 8;21 translocation in acute myeloid

leukemia. J Clin Oncol 1993;11:6907.[8] Krishnan K, Ross CW, Adams PT, et al. Neural cell-adhesion molecule (CD 56)-positive, t(8;21) acute myeloid leukemia

(AML, M-2) and granulocytic sarcoma. Ann Hematol 1994;69:3213.Conict of interest statement

No conicts of interest to declare.

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Research agenda

1. More emphasis needs to be placed on the characterization of surrogate marker proles forgenotypes of clinical interest, such as FLT3-gene mutations in AML.

2. Advantage should be taken of known surrogate antigens in the supervised analysis of geneticdata.

3. Rather than continuing to test blast cells from large cohorts of leukemia patients with asmany antibodies as available, indications regarding antigens of signicance could be derivedfrom gene expression arrays.

4. At least at the present time, a collaborative effort between molecular biologists and owcytometrists is necessary to introduce the genetic characterization of the leukemias intopatient diagnosis in real time to affect treatment decisions.[9] Paietta E, Goloubeva O, Neuberg D, et al. A surrogate marker prole for PML/RARa expressing acute promyelocyticleukemia and the association of immunophenotypic markers with morphologic and molecular subtypes. Cytometry BClin Cytom 2004;59B:19.

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E. Paietta / Best Practice & Research Clinical Haematology 23 (2010) 359368368

Surrogate marker profiles for genetic lesions in acute leukemiasIntroductionPML/RARPOS acute promyelocytic leukemia (APL) with t(15;17)(q22;q21)AML1/ETOPOS AML with t(8;21)(q22;q22)BCR/ABLpos ALL with t(9;22)(q34;q11)FLT3-gene mutated T-lineage ALLConclusions and future perspectivesConflict of interest statementReferences