Best Practice & Research Clinical Haematology 23 (2010) 359368

Contents lists available at ScienceDirect

Best Practice & Research Clinical

Haematology
journal homepage: www.elsevier.com/locate/beha

Surrogate marker proles for genetic lesions in acute

leukemias
Elisabeth Paietta, Laboratory Director, Professor of Medicine *
Monteore Medical Center-North Division, Immunology Laboratory, Cancer Center, 600 East 233rd Street, Bronx, NY 10466, USA

Keywords:
immunophenotypes
genetic lesions
surrogate markers
targeted therapy

The basic hypothesis of surrogate marker proles is that individual

genetic lesions result in characteristic distortions of the cellular
phenotype with some predictable consistency that can be exploited by sophisticated immunophenotyping. While cytogenetic and
molecular aberrancies currently are accepted prognostic predictors
in acute leukemias, single antigen expression and even antigenic
proles rarely impact on prognosis. However, increasingly,
phenotypes are delineated which can serve as surrogates for
underlying genetic aberrations of clinical importance. This development is of particular signicance as antileukemic therapy
becomes available that targets any component of the disturbed
molecular pathways associated with these genetic lesions. This
chapter will focus on established surrogate marker proles, such as
those for PML/RARa, AML1/ETO, FLT3-gene mutated acute
lymphocytic leukemia (ALL), and BCR/ABLPOS ALL. As the list of
therapeutic targets grows, the role of surrogate antigen proles
will grow, as they can predict for the efcacy of targeted
approaches in lieu of expensive, time-consuming and not always
accessible genetic analyses.
2010 Elsevier Ltd. All rights reserved.

Introduction
Although it makes perfect sense to hypothesize that genotypes control the phenotypes of leukemic
cells, it has been difcult and sometimes impossible to establish concordances between phenotypic
and genotypic categories. Rather than abandoning the valid assumption that antigen patterns on the
surface 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.
1521-6926/$ see front matter 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.beha.2010.08.001

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reasonable to blame our limitations with respect to antigen recognition. Sophisticated immunophenotyping reliably allows for identifying the lineage afliation of leukemia cells and the determination
of 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, many
laboratories have restricted their antibody panels to those useful in lineage differentiation. In its
extreme, one cell aliquot stained for intracytoplasmic CD3 (cCD3), intracytoplasmic CD22 (cCD22) and
myeloperoxidase can yield the necessary information regarding lineage afliation with the T-, or Blymphoid, and myeloid lineage, respectively. While cost-effective, this approach can hardly be
expected to yield new data with respect to prognostic novel antigens or antigen expression patterns
associated 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 unequivocally
predictive of outcome. The signicance of single antigens for prognosis may derive from the availability
of targeted antibody therapy [3], such as rituximab and variants thereof, which target CD20 irrespective
of the diagnostic characterization of the CD20 expressing abnormal cells. A single candidate antigen
with apparent negative prognostic signicance is the neural-cell adhesion molecule, CD56, in acute
promyelocytic leukemia (APL) [4,5] and in AML with the 8;21 translocation [6]. In t(8;21) AML, the
CD56 expression is associated with a disproportionate incidence of granulocytic sarcoma, a tendency
that may be responsible for the inferior outcome in these patients [7,8]. On the other hand, in APL, CD56
expression is commonly associated with the S-form of the PML/RARa transcript [9], a disease subtype
with frequent expression of CD2, a T-lineage afliated antigen, and mutation of the FLT3 gene rev [10]. It
has been hypothesized that CD2POS, FLT3-mutated APL may be derived from a distinct [11], presumably
earlier level of differentiation [12]. Thus, the same antigen may have prognostic signicance in various
leukemia subtypes without a shared justication or apparent biologic reason.
While not surrogates per se, single antigens can serve as diagnostic pointers for genotypes. The
nding of CD2 expression on the surface of otherwise unequivocally myeloid leukemia cells is rare and
should raise a ag in the mind of every ow cytometrist. Two differential diagnoses must 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 will
be discussed in detail below (HLA-DRNEGCD11aNEGCD18NEG). Until now not appreciated, however, is the
observation that CD2POS APL occasionally may lack these APL-typical antigenic features, something
never seen in L- or V-form APL, thus often misleading laboratory investigators or pathologists Paietta E,
unpublished. My recommendation for any case of CD2POS myeloid leukemia is the immediate preparation 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 on
commonly occurring antigens. For instance, the initial specic marker prole for PML/RARaPOS APL
was simple double negativity for HLA-DR and CD34. We now know that a much more accurate
denition of the APL immunophenotype is required and that variability exists in the antigen prole
with respect to morphologic and molecular subtypes of PML/RARaPOS APL [9]. A similar mistake was
made in BCR/ABLPOS acute lymphocytic leukemia (ALL) for which a specic immunophenotype was
originally based on strong expression of CD34 and CD10, weak expression of CD38, and aberrant
reactivity 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 of
expression of CD25 and dual expression of the myeloid-afliated antigens, CD33 and CD13, while the
expression levels of CD34, CD10 and CD38 were of inferior statistical signicance [15]. The association
of 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 more
immature Pro-B ALL (CD10NEG) lymphoblasts rev [2]. The majority of MLL/AF4POS ALL cases, derived
from t(4;11)(q21;q23), lack CD10rev [16]. The tendency of MLL/AF4POS lymphoblasts to express CD65
and CD15 is, therefore, not surprising and may simply be related to the CD10NEG Pro-B maturation
stage. However, in trial E2993, we found that MLL/AF4POS lymphoblasts co-expressed CD65 and CD15
whether associated with Pro-B or Early Pre-B maturation stage. These observations in BCR/ABLPOS and

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MLL/AF4POS ALL suggest that myeloid antigen expression in B-lineage ALL is determined by the
underlying 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 choice
of leukemia treatment. That identication of the dominant lineage afliation can be more challenging
than the characterization of the underlying genotype is exemplied by the new leukemia subtype with
mutations in NOTCH1 and silenced CEBPA gene. Wouters et al. [17] described that this novel AML
subtype expressed CD7, CD34, TdT, CD33 and CD13; remarkably there were high levels of mRNA for
CD3, 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 afliation with the T- and/or the
myeloid lineage, by testing for cCD3 and myeloperoxidase ow cytometrically, unfortunately, was not
obtained. Subsequently, Terriou et al. [18] clearly demonstrated that cases with methylated CEBPA and
NOTCH1 mutation belonged to the T-cell lineage based on cCD3 expression and negativity for
myeloperoxidase.
Reliable surrogate marker proles for genetic lesions are of clinical interest when the associated
genotype is prognostically signicant and/or when targeted therapy for that genotype is available.
Outcome-based classication of the leukemias nowadays is based predominantly on cytogenetic
aberrations and their molecular derivatives (e.g., fusion genes). Increasingly, however, genetic and
epigenetic proling data are the major source of information regarding perturbed pathogenetic
pathways and potential therapeutic targets. The identication of antigen proles which can predict for
the existence of, for example, gene mutation or gene silencing events by applying rather inexpensive
and quick multiparameter ow cytometry would be an important step in the translation of sophisticated 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 classication) is interchangeable with characteristic immunophenotypic features [10]. Given the unmistakably
hypergranular features of M3 leukemic promyelocytes, which rarely cause disparity among morphologists, it was tempting to describe a characteristic immunophenotype for this disease subtype early on in
the use of ow cytometry in leukemia diagnosis. The initial denition of an APL marker prole consisted
of negativity for CD34 and HLA-DR, which, unfortunately, is still considered sufcient for APL diagnosis
by many pathologists. Subsequently, the prole was rened by adding the differential reactivity of CD15/
CD15S antibodies, caused by the sialylation of the CD15 carbohydrate antigen in APL, weak expression of
CD38 and CD45, expression of CD9, and lack of P-glycoprotein rev [10]. Still, this prole had only limited
ability to distinguish APL from other HLA-DRNEG and CD34NEG AML subtypes, such as natural-killer (NK)cell AML [19], an important distinction considering that NK-AML is unresponsive to ATRA. The newest
and most comprehensive denition [9] reliably identies t(15;17)(q22;q21) APL based predominantly
on: (1) lack of HLA-DR and CD133, two antigens expressed at differentiation levels more immature than
that of promyelocytes during normal myelopoiesis; (2) absence of several adhesive molecules, such as
CD11a (aL subunit of the leukocyte integrin LFA-1), CD18 (b2 subunit of LFA-1), and CD11b (aM subunit of
Mac-1 integrin); (3) low expression of carbohydrate structures (CD65S, CD15), which serve as ligands for
other adhesion molecules, and (4) faint expression of CD45, the common leukocyte antigen, and of CD38,
a bifunctional ectoenzyme catalyzing involved in cell adhesion to endothelium. In summary, APL is
characterized by absence or weak expression of adhesion molecules.
The question whether CD117 is expressed by APL cells is an important one, especially for minimal
residual disease (MRD) monitoring. CD117, the stem cell factor receptor KIT, is expressed by normal
hematopoietic precursor cells and is lost prior to maturation of normal myeloblasts into promyelocytes.
There exists the erroneous view that leukemic promyelocytes are equally negative for CD117. The
intensity of CD117 staining of leukemic promyelocytes is sometimes very low and can be recognized
only 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
post treatment.

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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 with
the S-isoform, there is increased occurrence of CD34, HLA-DR, and/or CD56 expression and of FLT3 gene
mutations, when compared with the L- or V-isoform [10]. Furthermore, S-form APL uniquely expresses
CD2, a T-lineage afliated antigen never found in L- or V-form APL [9]. We recently noted that CD2
expression 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 in
AML is rare, the nding of CD2POS non-lymphoid leukemic cells must prompt a suspicion of APL and
PCR analysis for PML/RARa. This is particularly important in hypogranular cases, in which the expected
characteristic scattergram for M3 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 two
genes involved in this translocation are the AML1 transcription factor, now called RUNX1, on chromosome 21q22.3, and the Eight-Twenty-One oncoprotein (ETO) (also called MTG8) on chromosome
8q22 [22]. For the purpose of this chapter, we will 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 only
carries a favorable prognosis, [23] but also demonstrates unique sensitivity to multiple sequential
cycles of high-dose cytosine arabinoside [24].
t(8;21) AML has a characteristic immunophenotype, which was originally based on expression of
CD19, 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 discriminating
antigens. Of note, CD19 expression can be of very weak intensity [26] and, in fact, may not be recognized if a uorescein isothiocyanate (FITC)-conjugated CD19 antibody is used. Even with phycoerythrin
(PE) [26] or other uorochrome-conjugated antibodies, CD19 staining can easily be missed, unless
other antigenic characteristics suggest an AML1/ETOPOS AML (Paietta E, unpublished observation). One
characteristic feature of t(8;21) AML is a striking myeloid immaturity, such as weak or absent
expression of CD33 [27], and failure to detect myeloperoxidase by antibody binding [28], consistent
with 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 of
t(8;21) AML with FAB M2 and the continuing tendency of pathologists to place more weight on
morphology rather than immunophenotype, particularly in AML. Furthermore, low expression of the
integrins, 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. The
low expression of CD11a in t(8;21) AML is explained by the inhibition of CD11a promoter activity
through 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 in
a questionable case is consistent with AML. This marker combination is particularly relevant since CD19POS
t(8;21) myeloblasts frequently co-express PAX5, a B-lineage associated transcription factor [31]. The
markers 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 recurrent
cytogenetic abnormalities. In adult ALL, the Philadelphia chromosome, t(9;22)(q32;q11), resulting in
the formation of the BCR/ABL fusion transcript, is the major poor prognostic indicator [32]. Its incidence increases with age, ranging from 2% to 4% in children versus 2530% in adults. The BCR/ABL gene
resides in the Philadelphia chromosome, the derivative chromosome 22 formed by the t(9;22)
(q34;q11) [33]. It encodes a fusion protein in which part of the SH3 domain of c-ABL is replaced by

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portions of the BCR protein; thus all BCR/ABL proteins share a similar carboxy terminal ABL tyrosine
kinase domain, but differ in the BCR portion. In w30% of BCR/ABLPOS ALL, the break within the BCR gene
occurs in the major breakpoint cluster region (M-bcr), resulting, when joined with a portion of c-ABL
from chromosome 9, in a b2a2 or b3a2 fusion transcript encoding a protein of 210,000 dalton molecular
weight (p210BCR-ABL). In the majority of BCR/ABLPOS ALL cases, a break in the minor breakpoint cluster
region (m-bcr) forms the e1a2 transcript encoding a 190,000 dalton protein (p190BCR-ABL). The chimeric
proteins 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 of
specic tyrosine kinase inhibitors for the treatment of BCR/ABLPOS disease [34] make it imperative that
these patients are recognized accurately. Unfortunately, cytogenetic analyses in ALL are hampered by
suboptimal chromosome structure [35]. Furthermore, approximately 10% of BCR/ABLPOS ALL lack
evidence of the t(9;22) by chromosome banding [36]. Until recently, the detection of BCR/ABL relied
exclusively on uorescence-in situ hybridization (FISH) or PCR, which required sufcient cellular
material and access to a molecular diagnostic laboratory. Lately, a bead-based ow cytometric assay has
been developed, which allows for the demonstration of BCR/ABL proteins in diagnostic ALL specimens
reliably and rapidly, within 4 h after sample receipt [37]. Among 65 ALL specimens tested at the time of
presentation, the ECOG Leukemia Translational Studies Laboratory found 100% concordance between
this bead assay and semiquantitative PCR for BCR/ABL. Furthermore, the recommended cell count of 1
million mononuclear cells [37] could be reduced to 250,000 without negative impact on the quality of
the assay (Paietta E, unpublished observation). Caveats associated with this assay are the increased
protease activity in BCR/ABL transcript-containing cells from chronic myelogenous leukemia and
a lower limit of sensitivity of 0.1%, making this assay unsuitable for MRD monitoring [37].
In the beginning, alleged surrogate marker proles for BCR/ABLPOS ALL relied solely on differences in
staining intensity of antigens commonly found in B-lineage ALL, such as strong expression of CD10 and
CD34 and weak expression of CD38 [1,14,38]. The serendipitous nding of expression of CD25 in 4
patients 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, conrmed
without question the association between BCR/ABL positivity and expression of CD25, the a-chain of
the interleukin-2 receptor [15]. The nal analysis of E2993 [40] demonstrated that CD25 expression
correlated with increased expression of stem cell markers, such as CD133, CD105, CD109, CD135, and
CD123, in BCR/ABLPOS lymphoblasts suggesting that CD25POS BCR/ABLpos lymphoblasts are arrested at
an immature, potential stem cell level of B-cell differentiation. This may also explain the strong
expression of CD34 and the frequent nding of dual expression of the myeloid antigens CD33/CD13 by
BCR/ABLPOS lymphoblasts. Because BCR/ABLPOS lymphoblasts belong unequivocally to the B-cell
lineage (cCD22POS, myeloperoxidaseNEG), this phenotype, despite dual or single myeloid antigen
expression, must not be considered representative of a biphenotype. Primo et al. [41] reported that
patients with del(9)(p21), in addition to the t(9;22), lacked both CD33 and CD13, an interesting
observation that warrants conrmation.
The most striking observation with respect to CD25 and BCR/ABLPOS ALL is that expression levels of
CD25 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 other
words, CD25 is unique in its dual function as a dependable marker of BCR/ABLPOS ALL and an independent 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 known
examples of gene mutations, which are predicted by the presence of a unique antigen expression pattern.
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 with
outcome or other clinico-biological features [44]. ECOG recently described two major subclasses of TALL with prognostic signicance and insubstantial immunophenotypic overlap: myeloid antigenNEG
CD1aPOS T-ALL and CD13/CD33POS T-ALL [44]. CD1apos T-ALL represents the commonly accepted cortical

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T-cell maturation stage rev [2] which lacks CD34 and myeloid antigens, and is frequently positive for
surface CD3, CD4 and/or CD8. With respect to clinical signicance, CD1a expression was benecial in
terms of prolonged OS. The other subtype with prognostic signicance was T-ALL with expression of
CD13, 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 of
CD13POS T-ALL lacked surface CD3, CD4, CD8, and often CD2, while CD34 was present [44], characterizing 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 considered more specic for the myeloid lineage
than CD33 or CD13 in leukemia immunophenotyping rev [2]. During normal lymphopoiesis, CD117 is
expressed by a fraction of CD34POS, CD3NEG, CD4NEG, CD8NEG (triple-negative) thymocytes [45]. In normal
intrathymic triple-negative T-cell precursors, expression of CD117 coincides with that of CD135, the FLT3
receptor tyrosine kinase that is activated by the FLT3 ligand [46]. The combination of SCF and interleukin7 causes progressive differentiation of these progenitors into mature T-lymphocytes, while SCF and FLT3
ligand induce differentiation of triple-negative thymocytes along the myeloid pathway [47].
CD117 expression by leukemic lymphoblasts is rare and mostly, albeit not exclusively, associated
with the T-cell phenotype [16,45,48-50]. Recently, CD117POS T-lineage ALL was characterized as an
immunophenotypically 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 expression must be particularly emphasized since it is unique to this subtype
of T-lineage ALL. A close association of CD13 with CD117 in T-ALL has been previously postulated, based
solely on c-kit gene expression [51]. Occasionally, myeloperoxidase could be demonstrated by antibody
staining in some CD117POS lymphoblasts which invariably expressed cCD3. The simultaneous expression of T-lineage and myeloid lineage-specic antigens qualies this immunophenotype as truly
biphenotypic. 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 internal
tandem duplications (ITDs) or point mutations [43,44]. Activating mutations of the FLT3 gene are the
most common known genetic abnormalities in AML, but they rarely occur in ALL [46]. B-lineage ALLs
containing 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 and
LMO2 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 relative
resistance to standard chemotherapy [54]. However, despite CD13 expression, CD117POS/CD13POS T-ALL
did not carry the same inferior prognosis as CD117NEGCD13POS T-ALL [43,44]. Despite the low frequency
of FLT3-gene mutations in adult ALL, the availability of a variety of FLT3-kinase inhibitors suggests
a potential targeted approach in the treatment of this small patient ALL cohort.
Care must be taken, though, when assessing CD117POS T-ALL as a surrogate marker prole for FLT3 gene
mutations. We have recently found that any signicant deviation from our previously published [43] classic
surface CD3NEG, CD34POS, CD62LPOS, CD56NEG, CD2POS, CD7POS, CD1aNEG, CD5NEG, CD4/CD8NEG, CD13POS
phenotype can obviate the association with FLT3 gene mutations. The most important such antigen
modications are expression of CD5, CD56 or of CD33 instead of CD13 [16]. This observation stresses once
again the importance of establishing entire antigen proles as surrogate immunophenotypes for genetic
lesions; 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 features
that lack comparable antigen expression patterns and do not share genotypic characteristics, hypergranular APL is the exception. FAB M3 APL presents with a unique antigen prole, characterized by
a 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, hypogranular APL) and molecular features (S-form of PML/RARa transcript) are reected in changes in the
PML/RARa-specic surrogate marker prole, which include the expression of the T-lineage afliated

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antigen, CD2. Thus, surrogate antigen combinations are incredibly precise and any variation of the
global genetic lesion, such as various isoforms of the same transcript, is reected in a distinct alteration
of the immunophenotype. This is demonstrated in APL, and is also suggested by preliminary data in
BCR/ABLPOS ALL, where striking differences in the expression of CD25 and myeloid antigens between
the e1a2 and b2a2/b3a2 transcript forms were detected [55]. This is of particular interest given the
published difculties in establishing specic gene signatures for BCR/ABL isoforms [5659]. The recent
discovery of frequent deletions in IKZF1, a gene that encodes the B-lymphoid transcription factor
IKAROS, in BCR/ABLPOS pediatric [60] and adult ALL [61] introduces a new variable into the denition of
surrogate markers. Since IKZF1 deletions are frequent in pediatric high-risk ALL overall [60], it will be
interesting to assess whether the currently accepted surrogate antigen prole for BCR/ABLPOS ALL is at
all affected by the presence of IKZF1 deletions. Thus, surrogate antigen patterns should be re-evaluated
for new associations and predictive power whenever novel relevant gene expression, gene mutation, or
epigenetic studies become available.
Surrogate markers are very useful as bioinformatics tools in the identication of genes whose
expression patterns correlate with different phenotypes or sample characteristics of interest but which
were not known to be associated with these characteristics. On the other hand, gene expression and
mutation analyses can aid signicantly in the search for hitherto unrecognized surrogate antigen
proles. The efcacy of ow cytometry is limited due to its inability to test all antibodies on hand in an
effort to reveal novel surrogate antigens. Therefore, it will be more practical in the future to select
antigens of potential genotypic relevance based on available gene expression data for already characterized genotypes (e.g., FLT3 gene mutations) or prognostically dened genetic or epigenetic proles
with unknown biological correlates (e.g., within the group of normal karyotype leukemias).
While surrogate marker proles are not meant to replace conrmatory molecular analyses, they
provide clinicians with an early diagnostic clue. A suspicion of APL in a newly admitted patient derived
from ow cytometry results will allow the immediate initiation of all-trans retinoic acid without harm
should the diagnosis of APL not be conrmed in subsequent analyses. In BCR/ABLPOS ALL, unsuccessful or
normal cytogenetics may not prompt a molecular analysis for BCR/ABL, unless the immunophenotype is
suggestive of that diagnosis. The increasing availability of therapies targeted at specic genetic lesions
will boost interest in the denition of surrogate marker proles for targeted genotypes. For instance, in
FLT3-gene mutated AML, the use of inhibitors of FLT3-kinase activity currently must await the result of
molecular analysis. To complicate matters, cell signaling data have recently shown that a fraction of FLT3mutated blast cells cannot respond to kinase inhibitors in vitro, while apparently unmutated leukemias
show the characteristic cellular response of FLT3-mutated disease [62]. Surrogate antigens that can
predict a functional FLT3 mutation status would be of great clinical impact. This challenge further
exemplies that multiparameter ow cytometry can contribute to leukemia diagnosis far beyond its role
in the identication of lineage afliation.

Practice points
1. The basic concept behind the value of surrogate marker proles is that antigen patterns on the
surface 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 on
commonly occurring antigens, merely characteristic of the leukemias hematopoietic cell lineage.
4. Surrogate marker proles are not meant to replace conrmatory molecular analyses, but they
can provide clinicians with an early diagnostic suggestion.
5. Reliable surrogate marker proles for genetic lesions are of particular clinical interest when
the associated genotype is prognostically signicant and/or when targeted therapy for that
genotype is available.

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Research agenda
1. More emphasis needs to be placed on the characterization of surrogate marker proles for
genotypes of clinical interest, such as FLT3-gene mutations in AML.
2. Advantage should be taken of known surrogate antigens in the supervised analysis of genetic
data.
3. Rather than continuing to test blast cells from large cohorts of leukemia patients with as
many antibodies as available, indications regarding antigens of signicance could be derived
from gene expression arrays.
4. At least at the present time, a collaborative effort between molecular biologists and ow
cytometrists is necessary to introduce the genetic characterization of the leukemias into
patient diagnosis in real time to affect treatment decisions.

Conict of interest statement

No conicts of interest to declare.

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