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Acute Leukemias of
Ambiguous Lineage

T he addition of cytochemical and immunophenotypic Group for the Immunologic Classification of Leukemia
(EGIL) for the lineage assignments in acute leukemias.
techniques to the standard morphologic evaluation of
leukemic blast cells has led to the growing recognition l Myeloid Lineage: Myeloperoxidase (cytochemistry, flow
of acute leukemias with ambiguous lineage assignment. cytometry, or immunohistochemistry). For monocytic differen-
According to the WHO classification, these leukemias fall tiation at least two of the following markers: NSE, lysozyme,
into the following categories (Figure 25.1). CD11c, CD14, and CD64.
l B-Cell Lineage: Strong CD19 expression with:
1. Acute undifferentiated leukemia (AUL) which lacks sufficient
l Strong coexpression of at least one of the following markers:
evidence (such as morphologic, cytochemical, and immuno-
phenotypic features) of lineage differentiation. CD79a, cytoplasmic CD22, and CD10, or
l Weak CD19 expression with strong coexpression of at least
2. Mixed phenotype acute leukemia:
a. Acute bilineage (or multilineage) leukemia which repre- two of the following markers: CD79a, cytoplasmic CD22,
sents an acute leukemia with more than one population of and CD10.
l T-Cell Lineage: Cytoplasmic CD3 (with an intensity approach-
blast cells. The total number of blasts from both lineages
should be ≥20% of marrow or blood cells. ing that of normal T cells) or surface CD3.
b. Acute biphenotypic (multi-phenotypic) leukemia in which
the population of leukemic cells coexpress more than one
lineage-specific marker, such as a combination of myeloid-
specific and lymphoid-specific, or B- and T-specific mol-
ecules. In rare occasions, the leukemic blasts may express Acute Undifferentiated Leukemia
a combination of myeloid-, B-cell- and T-cell-associated
markers.
Acute undifferentiated leukemia (AUL) is defined as a
The following WHO criteria for lineage assignment in leukemia with no morphologic, cytochemical, or specific
acute leukemia with ambiguous lineage have replaced immunophenotypic features of lymphoid or myeloid dif-
the scoring system which was proposed by the European ferentiation. AUL is extremely rare and probably accounts
for <1% of acute leukemias.

MORPHOLOGY
l Bone marrow is often hypercellular with increased number of
uniform, undifferentiated immature cells.
l Blasts consist of primitive undifferentiated cells with scant dark

blue cytoplasm, no cytoplasmic granules or Auer rods, round


or oval nucleus and fine nuclear chromatin (Figure 25.2). The
nucleoli are often conspicuous, but in some cases are promi-
nent. Blasts account for >20% of the bone marrow or periph-
eral blood differential counts.
l Blood examination may show anemia, thrombocytopenia,

and/or leukopenia.
FIGURE 25.1  Scheme of clonal development of acute leukemias
of undifferentiated, bilineal, and biphenotypic types.

Atlas of Hematopathology. DOI: http://dx.doi.org/10.1016/B978-0-12-385183-3.00025-5


© 2013 Elsevier Inc. All rights reserved.
318 Acute Leukemias of Ambiguous Lineage

FIGURE 25.3  Flow cytometric findings of undifferentiated acute


leukemia. The CD45-dim gate reveals a prominent population
of blasts that are dimly positive for CD45. In addition, the blasts
express CD34 and partial CD7, but are negative for CD3, CD5,
CD10, CD13, CD14, CD19, CD33, CD41, CD64, and CD117. The
blasts are negative for CD4 and CD56 (not shown).

l Variable expression of myeloid or lymphoid associated


antigens can be seen, such as CD7, CD13 or CD33,
B and CD19, but the expression is insufficient for lineage
assignment.

MOLECULAR AND CYTOGENETIC STUDIES


l By definition these leukemias are “undifferentiated” and
therefore would not be expected to exhibit B-lymphoid,
T-lymphoid, or myeloid markers. However, this can be a mat-
ter of semantics, dependant upon which markers one is using
to arrive at such definitions. While protein markers may be
absent based on immunophenotypic methods, a cell of pre-B
lineage, for example, might show rearrangement of the immu-
noglobulin genes by sensitive PCR methods.
l Nevertheless, these leukemias are too rare to make any gener-

alizations about particular molecular findings that might give


a clue as to cell of origin.
l No recurrent or specific cytogenetic abnormalities are noted
C
by karyotyping or FISH.
FIGURE 25.2  Undifferentiated acute leukemia. Bone marrow biopsy
section (A) and marrow smear (B, low power; C, high power)
demonstrating undifferentiated blasts (see Figure 25.3).
DIFFERENTIAL DIAGNOSIS
If only a limited panel of antigens is tested, morphologic
IMMUNOPHENOTYPE (FIGURE 25.3)
and immunophenotypic features can overlap with those
l Blasts do not express lineage-specific antigens. seen in blastic plasmacytoid dendritic cell (BPDC) neo-
l Blasts are commonly positive for dim CD45. plasm. Other differential diagnoses include ALL, AML,
l Blasts are often positive for antigens that are associated with
minimally differentiated and without maturation, pure
early hematopoiesis, such as CD34, CD38, HLA-DR, and occa- erythroid leukemia, and megakaryoblastic leukemia.
sionally TdT.
Mixed Phenotype Acute Leukemia 319

A A

B
FIGURE 25.4  Acute bilineal leukemia. Bone marrow (A) and B
peripheral blood (B) smears show two distinct populations of FIGURE 25.5  Acute bilineal leukemia. Bone marrow (A) and
leukemic blast cells (larger and smaller). peripheral blood (B) smears show two distinct populations of
leukemic blast cells (larger and smaller).

with 11q23 abnormalities or the t(9;22) (Philadelphia


Mixed Phenotype Acute Leukemia chromosome). The B-cell/myeloid phenotype is the most
frequent subtype.
Mixed phenotype acute leukemia (MPAL) includes the fol-
lowing subtypes: (a) acute bilineal (or multilineage) leuke-
mia consisting of more than one population of blast cells, MORPHOLOGY
and (b) acute biphenotypic (multi-phenotypic) leukemia l Acute bilineal leukemia may consist of two distinct morpho-
in which the blast population coexpresses more than one logic populations of blast cells. In most cases these two pop-
lineage-specific marker, such as a combination of myeloid- ulations represent lymphoid and myeloid lineages (Figures
specific and lymphoid-specific, or B- and T-specific mol- 25.4 and 25.5).
l Lymphoblasts are usually smaller with scant cytoplasm, no
ecules. In rare instances, the leukemic blasts may express
a combination of myeloid-, B-cell- and T-cell-associated cytoplasmic granules, and less prominent nucleoli, while
blasts of myeloid origin (myeloblasts, monoblasts) are larger
markers, or represent a combination of (a) and (b).
with more abundant cytoplasm, variable amounts of cytoplas-
Biphenotypic acute leukemias probably represent less
mic granules, and prominent nucleoli.
than 10% of all acute leukemias. The bilineal acute leuke- l The bilineal nature of an acute leukemia may not be distin-
mias are less frequent. Mixed phenotype acute leukemia guished on a morphologic basis, but the two populations of
can occur at any age, but is more frequent in adults. They blast cells demonstrate distinct cytochemical and immuno-
usually have an aggressive clinical course, particularly those phenotypic properties (see below).
320 Acute Leukemias of Ambiguous Lineage

FIGURE 25.6  Acute bilineal leukemia. Flow cytometric studies


reveal two distinct populations of large and small blast cells (left
panel). The large blasts (right panel, in purple) express myeloid-
associated antigens, while the small blasts (right panel, in red)
reveal features seen in B lymphoblastic leukemia.

IMMUNOPHENOTYPE AND CYTOCHEMICAL STAINS


Bilineal Acute Leukemia (Figure 25.6)
l Two distinct populations of blasts can be detectable by either
common gating strategies (e.g., CD45 gating and scatter gat-
B
ing) or selected back gating approaches of target populations.
l Each population expresses antigens of one specific lineage.
l Both blastic populations can also express non-lineage specific

markers, such as CD34, CD38, HLA-DR, etc.


l The combination of lymphoid and myeloid phenotype bilin-

eal acute leukemia is more common than that of B- and T-cell


phenotype.
l The presence of two populations of blast cells in bilineal acute

leukemia is often synchronous; both populations of blast cells


are present at the same time. In rare instances, there may be
two simultaneous neoplastic processes in two separate sites.
For example, the bone marrow may be involved with ALL and
the CNS infiltrated by AML.
l Asynchronous bilineal acute leukemias are more frequently

reported and are those in which one lineage switches to


another during the disease process. The predominant pattern
in most studies is ALL switching to AML in relapse. The fre-
quency of lineage switch in relapse is about 7%. Lineage switch C
may represent relapse of the original clone or development of FIGURE 25.7  Acute biphenotypic leukemia (myeloid/B-lymphoid).
a second new clone. An increased risk of development of AML Bone marrow smear shows blast cells with variable amount of
has been reported in patients with ALL who receive intensive non-granular cytoplasm (A). These cells are myeloperoxidase-
chemotherapy, particularly in patients with 11q23 abnormali- positive (B, cytochemical stain; C, immunohistochemical stain).
ties or those with t(9;22) (Philadelphia chromosome). The blast cells show a mixed phenotype (myeloid/B-lymphoid) by
l There are also occasional reports of switching from bilineal to flow cytometry; see Figure 25.8).
biphenotypic acute leukemias and vice versa.
l Cytochemical stains such as MPO, Sudan Black B, and NSE are

helpful in distinguishing blasts of myeloid origin. Myeloblasts


are often MPO and Sudan Black B positive and monoblasts/
promonocytes usually express NSE.
Mixed Phenotype Acute Leukemia 321

FIGURE 25.8  Flow cytometric findings of acute biphenotypic leukemia FIGURE 25.10  Flow cytometric findings of acute biphenotypic
(myeloid/B-lymphoid). Open gate display by CD45 gating (in green) leukemia (myeloid/T-lymphoid). Open gate display by CD45 gating
reveals excess blasts (25% of the total) that are dimly positive (in blue) shows excess abnormal blasts (94% of the total) that
for CD45. The blast-enriched gate (in magenta) demonstrates are CD45 dimly positive. Density plots of the blast-enriched
abnormal blasts expressing both B- and myeloid-lineage-specific gate (in magenta) demonstrate that the blasts express T-lineage
markers. The positive B-lineage antigens include CD19 (moderate markers CD2, CD5 (partial, dim; not shown), CD7, and intracellular
to bright), CD22 (dim), and intracellular CD79a. The positive CD3. In addition, the blasts are positive for myeloid antigens
myeloid markers include CD11b (not shown), CD13 (dim), CD15 CD13, CD15 (partial), CD117 (heterogeneous), and intracellular
(partial), CD33, and intracellular myeloperoxidase (partial). myeloperoxidase (partial, heterogeneous). Positivity for
myeloperoxidase is further confirmed by immunohistochemical
studies. The blasts are also positive for CD34, HLA-DR (partial; not
shown), and intracellular TdT.

Biphenotypic Acute Leukemia (Figures 25.7 to 25.10)


l Blasts express antigens that are specific for more than one
lineage.
l Common forms include mixed B- and myeloid or T- and

myeloid biphenotypes.
l Rarely, blasts may express B- and T-cell biphenotype, or mixed

B-, T-, and myeloid triphenotype.

MOLECULAR AND CYTOGENETIC STUDIES


A Based on molecular and cytogenetic studies, we can divide
mixed phenotype acute leukemias into two major groups:
1. Mixed phenotype acute leukemia with recurrent genetic
abnormalities
2. Mixed phenotype acute leukemia, otherwise not specified.

Mixed Phenotype Acute Leukemia with Recurrent


Genetic Abnormalities
l These include:
l MPAL with t(9;22)(q34;q11.2) (BCR-ABL1)
l MPAL with t(v;11q23) (MLL rearranged)
l MPAL B/myeloid, not otherwise specified
l MPAL T/myeloid, not otherwise specified.
B l Translocation of 11q23 and the t(9;22) are the most frequent
FIGURE 25.9  Acute biphenotypic leukemia (myeloid/T-lymphoid). cytogenetic abnormalities observed in biphenotypic and bilin-
Bone marrow smear shows blast cells with scanty cytoplasm (A). eal acute leukemias of B-precursor/myeloid type.
These cells show coarse PAS-positive cytoplasmic granules (B). l More than 70 partner genes have been identified in associa-
The blast cells show a mixed phenotype (myeloid/T-lymphoid by tion with 11q23 translocations, such as t(4;11), t(9;11), and
flow cytometry; see Figure 25.10).
t(11;19) (Figures 25.11 and 25.12).
322 Acute Leukemias of Ambiguous Lineage

FIGURE 25.11  A karyotype with deletion of


11q23 and trisomy 10 in a biphenotypic acute
leukemia.

FIGURE 25.12  Karyotype with a t(11;19)


(q23;p13) and dup 1q in a biphenotypic acute
leukemia.

l The Philadelphia chromosome may be a part of a complex set l Since a small proportion of T-cell leukemias can show sec-
of cytogenetic abnormalities, such as combination of t(9;22) ondary rearrangement of immunoglobulin genes, and vice
and del(7) or t(2;9;22) (Figure 25.13). versa for B-cell leukemias, care must be taken not to overcall a
l The T-precursor/myeloid biphenotypic or bilineal acute leukemias biphenotypic diagnosis based on such findings.
may be associated with t(5;18)(q31;q23) and t(3;12)(p25;q24.3). l Cyclin A1 and HOXA9 gene expression have also been
l Also, in some cases trisomy 10 has been reported in associa- reported in these lesions.
tion with acute biphenotypic leukemia (see Figure 25.11).
l Some cases of acute biphenotypic leukemia may show com-

plex chromosomal aberrations (Figure 25.14).


l IgH and TCRG clonal gene rearrangements may both be seen Mixed Phenotype Acute Leukemia, Otherwise
in biphenotypic acute leukemias. Not Specified
l However, unlike the markers identified by flow cytometry or

immunohistochemistry, it is not possible by PCR to distinguish In this category the blast cells show coexpression of
whether these gene rearrangements are present in two distinct myeloid-specific and lymphoid-specific molecules, but lack
sets of malignant cells, as opposed to coexisting in the same cells. recurrent genetic abnormalities.
Differential Diagnosis 323

FIGURE 25.13  A karyotype with a three-way t(2;9;22) in a biphenotypic acute leukemia.

FIGURE 25.14  A complex karyotype in a biphenotypic acute leukemia: 46,XX,t(4;7)(p12;p11.2),t(6;7)(q13;q36),add(7)(p12),inv(10)


(p12p15),t(11;17)(q23;q21),del(15)(q22q22),add(20)(q13.1).

with aberrant expression of myeloid-associated markers.


Differential Diagnosis Bilineal acute leukemias may show morphologic evidence
of two separate leukemia populations, such as larger and
The diagnosis of biphenotypic acute leukemia is estab- smaller blasts. But diagnosis is confirmed by the evi-
lished by immunophenotypic studies. These leukemias dence of two populations of blast cells distinctly express-
should be distinguished from AMLs with aberrant expres- ing molecules representing more than one hematopoietic
sion of lymphoid-associated markers and from ALLs lineage.
324 Acute Leukemias of Ambiguous Lineage

Additional Resources
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tics and outcome of children with biphenotypic acute leukemia, ous lineage, biphenotype, without CD34, TdT or TCR-rearrangement,
Haematologica 94:1682–1690, 2009. Intern Med 48:1437–1441, 2009.
Béné MC: Biphenotypic, bilineal, ambiguous or mixed lineage: Owaidah TM, Al Beihany A, Iqbal MA, Elkum N, Roberts GT:
strange leukemias! Haematologica 94:891–893, 2009. Cytogenetics, molecular and ultrastructural characteristics of biphe-
Gerr H, Zimmermann M, Schrappe M, et  al: Acute leukaemias of notypic acute leukemia identified by the EGIL scoring system,
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Gluzman DF, Nadgornaya VA, Sklyarenko LM, et al: Immunocytochemical tumours of haematopoietic and lymphoid tissues, ed 4, Lyon, 2008,
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Naghashpour M, Lancet J, Moscinski L, et  al: Mixed phenotype van den Ancker W, Terwijn M, Westers TM, et  al: Acute leukemias
acute leukemia with t(11;19)(q23;p13.3)/ MLL-MLLT1(ENL) B/T- of ambiguous lineage: diagnostic consequences of the WHO 2008
lymphoid type: a first case report, Am J Hematol 85:451–454, 2010. classification, Leukemia 24:1392–1396, 2010.

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