Acute leukemias of ambiguous lineage
Article Outline
- Abstract
- Diagnosis of acute leukemia
- Biphenotypic acute leukemias (BAL) as defined by the European Group for Immunophenotyping of Leukemia (EGIL)
- Mixed phenotype acute leukemias (MPAL)
- MPAL natural history and genetic studies
- Other leukemias of ambiguous lineage
- References
- Copyright
The 2008 edition of the WHO Classification of Tumors of Haematopoietic and Lymphoid Tissues recognizes a special category called “leukemias of ambiguous lineage.” The vast majority of these rare leukemias are classified as mixed phenotype acute leukemia (MPAL), although acute undifferentiated leukemias and natural killer lymphoblastic leukemias are also included. The major immunophenotypic markers used by the WHO 2008 to determine the lineage for these proliferations are myeloperoxidase, CD19, and cytoplasmic CD3. However, extensive immunophenotyping is necessary to confirm that the cells indeed belong to 2 different lineages or coexpress differentiation antigens of more than 1 lineage. Specific subsets of MPAL are defined by chromosomal anomalies such as the t(9;22) Philadelphia chromosome BCR-ABL1 or involvement of the MLL gene on chromosome 11q23. Other MPAL are divided into B/myeloid NOS, T/myeloid NOS, B/T NOS, and B/T/myeloid NOS. MPAL are usually of dire prognosis, respond variably to chemotherapy of acute lymphoblastic or acute myeloblastic type, and benefit most from rapid allogeneic hematopoietic stem cell transplantation.
Keywords: Acute leukemia , Biphenotypic , Undifferentiated , Natural killer (NK) , Immunophenotype , Immunohistochemistry
Diagnosis of acute leukemia
Beside elevated peripheral white blood cell counts, which are not always present, the initial diagnosis of acute leukemia (AL) relies strongly on morphology and immunophenotyping of a bone marrow sample. In the vast majority of cases, a homogeneous infiltration of the bone marrow with sheets of morphologically similar cells displaying a homogeneous immunophenotype is observed. Further classification and lineage assignment depends on cytologic features as the first approach and then on the immunophenotype determined by a selection of antibodies applied by flow cytometry or immunohistochemistry.
The French–American–British classification has long used only morphologic and cytochemical criteria to discriminate among the various subtypes of acute myeloid leukemias (AML) and the 3 cytologic categories of acute lymphoblastic leukemia (ALL).1
Immunophenotyping allows a more precise characterization of AL by detecting lineage-associated markers expressed by the leukemic cells and has become an indispensable part of the integrated hematopathological diagnostics of leukemia. This methodology relies on the use of an antibody panel, to be chosen among the more than 300 clusters of differentiation (CD), defining human leukocyte antigens.2 The development of new fluorochromes, instruments with 2-3 lasers, and new software for analysis has made multiparameter (also called multidimensional) flow cytometry an excellent immunophenotyping tool. Over the years, several consensus panels have been proposed, allowing a proper identification of the lineage and differentiation stage of the malignant clone. One of the most recent has been published by the members of the Work Package 10 (WP10, Diagnostics) of the European Leukemia Net Consortium, European Union–funded organization of physicians, scientists, and patients with interest in leukemia (http://www.leukemia-net.org).3
Biphenotypic acute leukemias (BAL) as defined by the European Group for Immunophenotyping of Leukemia (EGIL)
The first attempt to standardize the diagnostics of BAL was provided by the EGIL group in 1995.4 Fine morphologists had repeatedly recognized leukemia cases in which there seemed to be 2 different clones among the proliferating cells. Later, observations of aberrant coexpressions of immunophenotypic markers considered to have an association with B, T, or myeloid lineages added to the complexity, even in morphologically homogeneous proliferations. To describe these rare leukemias, the acronym BAL has been used, encompassing both proliferations exhibiting 2 different clones (also called biclonal or bilineage leukemias) and cases with coexpression of markers from 2 or even sometimes 3 lineages on blast cells (also called biphenotypic leukemias). Several definitions were proposed by various authors, usually in case reports or small series.
The scoring system proposed by the EGIL group was designed to define lineage assignment of bona fide BAL cases and was later integrated in the 2001 WHO classification.5 As presented in Table 1, this system attributed different weights or scores to the expression of selective markers classically associated with the B, T, or myeloid lineage. To be properly applied, this scoring system required that scores above 2 were observed for more than 1 lineage. The panel of antibodies recommended by the EGIL is well within the range of what is now recommended. Yet many laboratories have long relied on limited strategies, oriented toward 1 lineage suggested by morphology, and did not explore markers associated with other lineages. This may have resulted over the years in an underestimation of the frequency of biphenotypic leukemias. Also, the EGIL scoring system for BAL has often been misused by accepting that scores equal to 2 were high enough for lineage assignment, whereas in the original publication it was stressed that only scores above 2 should be accepted.4 EGIL scores ≤2 for 1 of the lymphoid or myeloid lineages (so-called lineage infidelity or aberrant or variant marker expression) can often be observed in both ALL and AML cases.6, 7 The most frequent among ALL cases with aberrant expression of myeloid markers are leukemias corresponding to the earliest differentiation stages of B and T lymphocytes (B-I or T-I groups in the EGIL classification, also called pro-B and pro-T in the WHO classification). These cases are also often associated with cytogenetic anomalies such as the Philadelphia chromosome t(9;22), t(12;21) or with involvement of the mixed lineage leukemia (MLL) associated gene on chromosome 11 such as t(4;11).8, 9 In AML, expression of various lymphoid markers is often seen in cases with recurrent translocations (reviewed in Ref. 10). Nearly all reports concerning the clinical course of BAL, albeit difficult to really compare or assess because they seldom fulfilled all EGIL criteria, have pointed toward the poor prognosis of these leukemias unless hematopoietic stem cell transplantation can be performed. Their management uses alternately AML or ALL schedules of chemotherapy, but although complete remission is often observed, it usually is of short duration.11
Table 1. European Group for Immunophenotyping of Leukemia scoring system for biphenotypic⁎ acute leukemias (BAL)4
| B lineage | T lineage | Myeloid | |
|---|---|---|---|
| 2 points | CD79 cμ cCD22 | CD3 T cell receptor | Myeloperoxidase |
| 1 point | CD19 CD10 CD20 | CD2 CD5 CD8 CD10 | CD13 CD33 CDw65 CD117 |
| 0.5 points | TdT CD24 | TdT CD1a CD7 | CD14 CD15a CD64 |
⁎ To establish BAL diagnosis, a score >2 must be established for at least 2 lineages. |
Mixed phenotype acute leukemias (MPAL)
A major change in the 2008 revision of the WHO classification was to simplify the definition of BAL.12 A group of experts reviewed available data from the literature and their own laboratories and defined the new entity of MPAL among leukemias of ambiguous lineage (Table 2).12 It was pointed out that AL cases carrying recurrent AML-associated translocations and cases with FGFR1 mutations should not be considered MPAL. MPAL associated with t(9;22)(q34;q11) BCR-ABL1 and those with MLL rearrangement have been considered separate entities, provided, however, that they fulfilled the immunophenotypic criteria of MPAL definition.
Table 2. World Health Organization 2008 classification of acute leukemias of ambiguous origin and expression of lineage assignment markers for mixed phenotype acute leukemia (MPAL)12
| Category | B lineage: CD19⁎ | T lineage: CD3† | Myeloid lineage: myeloperoxidase‡ | Monocytic lineage§: CD14, CDD11c, CD14, CD64, lysozyme, nonspecific esterase |
|---|---|---|---|---|
| Acute undifferentiated leukemia | — | — | — | — |
| MPAL with t(9;22)(q34;q11.2) BCR-ABL1 | + | ±¶ | + | — |
| MPAL with t(v;11q23); mixed lineage leukemia rearranged | + | — | ± | ± |
| MPAL, not otherwise specified (NOS) B/myeloid | + | — | ± | ± |
| MPAL, NOS T/myeloid | — | + | ± | ± |
| MPAL, NOS other | + | + | + | ± |
| Natural killer (NK) cell lymphoblastic leukemia/lymphoma∥ | — | — | — | — |
⁎ Must be corroborated by expression of 1 (if CD19 bright) or 2 (if CD19 dim) B cell markers. |
† Cytoplasmic and/or membrane by flow cytometry. Because of cross-reactivity with CD3ε, cytoplasmic CD3 can also be detected by immunohistochemistry in NK cells. |
‡ Detected by cytochemistry and/or immunohistochemistry and/or flow cytometry. |
§ Two monocytic markers are needed to establish monocytic lineage. |
¶ Very rare. |
∥ Provisional entity. |
In this new proposal, only markers considered most specifically lineage-associated have been retained for lineage assignment (Table 2). Of note, in MPAL cases a comprehensive description of the immunophenotypic anomalies displayed by the blasts should be performed. EGIL scoring can therefore still be applied to provide the case with documentation of potentially valuable leukemia-associated immunophenotypic patterns. The latter may prove extremely important to follow minimal residual disease,11 even after allogeneic hematopoietic stem cell transplantation.
For the myeloid lineage, the detection of cytoplasmic myeloperoxidase (MPO) by cytochemistry, immunohistochemistry, or flow cytometry with an anti-MPO antibody is considered the most significant marker. Flow cytometry allows for the detection of myeloperoxidase, even in some cases of undifferentiated AML.13 In cases more oriented toward the monocytic lineage, which may lack MPO, the presence of nonspecific esterase by cytochemistry or the detection of surface CD14, CD11c, CD36, or CD64 can be used. Another possible positive marker for the monocytic lineage is the intracytoplasmic lysozyme that can be detected by immunohistochemistry or flow cytometry.14
B lineage assignment is based on CD19 expression and two different situations have been considered. If the CD19 labeling is bright, the presence of another B lymphocyte marker is considered enough to establish B lineage. If CD19 expression is of low intensity, the presence of two other B lineage–associated markers will be necessary. The markers corroborating B cell lineage can be chosen from among cytoplasmic CD79a, CD22, CD24, intracytoplasmic mu chains, or (less frequently expressed in MPAL) CD20 or CD21. The expression of CD10 can also be considered in this context as a B lineage ALL-related marker.
The strongest marker indicating T lineage engagement is the cytoplasmic expression of CD3, which must be investigated with a bright fluorochrome such as phycoerythrin or allophycocyanin and appear as a strong labeling. The presence of other T cell–associated markers such as CD2, CD5, or CD7 can add to the diagnosis of MPAL, although some of these markers can be seen on myeloid cells in AML and MDS3, 7, 15 and therefore should only be considered when associated with cytoplasmic CD3 for MPAL diagnosis.
A marker combination that can suggest MPAL is the intracytoplasmic (ie, performed on permeabilized cells) panel consisting of MPO, CD79a, and CD3, ideally combined with surface CD45 (labeled before permeabilization) for a better definition of the blast cell population (Figure 1).13, 16 Of note, the lone expression of CD79a can be seen in a fraction of T-ALL, which do not prove to be MPAL,17 because of the lack of CD19 expression.
Figure 1.
Example of the usefulness for lineage assignment of the combination myeloperoxidase (MPO), cCD13, cCD3, and cCD79a on a permeabilized sample of normal whole bone marrow (A–K) and a leukemic sample (L). Color code: red, polymorphonuclears; green, monocytes; purple, lymphocytes; cyan, immature cells defined as “not polymorphonuclears, not monocytes, not lymphocytes,” called the “bermudes” area by the GTLLF.27 (A) Whole populations on a side scatter/CD45 “cartography” scattergram. (B) Whole populations on an MPO/cCD13 scattergram. (C) Whole populations on an MPO/cCD3 scattergram. (D) Whole populations on an MPO/cCD13 scattergram. (E) Polymorphonuclear gating of the MPO/cCD13 scattergram showing MPO gradient of expression. (F) Monocyte gating of the MPO/cCD13 scattergram showing weak MPPO labeling. (G) Lymphocyte gating of the MPO/cCD3 scattergram showing MPO-negative cCD3-positive T cells. (H) Lymphocyte gating of the MPO/cCD79a scattergram showing MPO-negative cCD79a-positive B cells. (I) Bermudes gating of the MPO/cCD79a scattergram showing hematogones. (J) Gating of the hematogones population in dark blue. (K) Backgating of the hematogones defined in J on the initial “cartography” scattergram. The same strategy would be used to identify the blastic population in a leukemic sample as shown in L for MPO positivity backgating. Mixed phenotype acute leukemia would show the coexpression of markers of different lineages such as cCD79 and MPO or cCD3 and MPO.
The flow cytometry patterns that will be observed in most cases demonstrate coexpression of the markers on the same cells (Figure 2). In cases of biclonal/bilineal proliferations, 2 different blast populations can be detected, usually with a different back-gating on the CD45/SSC canonical scattergram. In other cases, transitional patterns with apparently only part of the blast population being biphenotypic can be observed.
Figure 2.
Example of a case of T/myeloid mixed phenotype acute leukemia. The 4 top scattergrams show the same combinations as in Figure 1. There is large population of blasts in the “bermudes” area, very few neutrophils, almost no monocytes, and a normal amount of lymphocytes. The latter mostly express CD79a. The blastic population coexpresses myeloperoxidase (with a spreading pattern) and cyt.CD3 as highlighted in the backgating illustrated in the middle panels. In another tube, these cells are shown to coexpress CD34 and faintly CD13 (bottom scattergram).
The diagnosis of MPAL without an access to fresh tissue and flow cytometry studies may be a challenge because CD19 detection in paraffin sections is technically critical and double stainings involving 2 cytoplasmic markers may be difficult to evaluate. However, a suspicion of MPAL can be raised using immunohistochemical staining of a bone marrow biopsy or extramedullary infiltrate in tissue sections if blastic populations express CD3 and/or MPO or MPO and/or more than 1 other B cell–associated marker such as CD79a, PAX-5, CD22, and CD10 (Figure 3).
Figure 3.
Example of mixed phenotype acute leukemia, not otherwise specified, T/myeloid diagnosed in immunochemistry. The material studied was a mediastinal biopsy from a young male patient. It was received formalin fixed. Immunostainings showed a diffuse infiltration of blasts with strong positivity for CD7, variable expression of CD3, weak positivity for CD33, and a fraction of blasts positive for myeloperoxidase. The blast population was also positive for TdT, CD2, and CD5 (weaker than normal T cells), as well as partly for CD117 and CD123 (not shown). There was some positivity for CD79a, which may be seen in some T lineage acute lymphoblastic leukemia. CD34, PAX-5, CD10, CD4, CD8, and CD1a were negative. A similar immunophenotype was found in a bone marrow biopsy that was diffusely infiltrated by blasts. There was no peripheral blood involvement. Cytogenetic studies showed a hyperdiploid karyotype.
MPAL natural history and genetic studies
MPAL represent less than 5% of AL and display myeloid morphology in about 60% of cases. Four possible types of immunophenotype combinations have been reported, namely myeloid/B, myeloid/T, B/T, and the occasional trilineage myeloid/B/T.11 Among other markers classically observed in AL, MPAL often express terminal deoxynucleotidyl transferase (TdT), CD34, and HLA-DR.
Molecular studies of MPAL have exhibited rearrangements of immunoglobulin and/or TCR genes in My/B cases.18 MLL gene rearrangements can be present in about 25% of cases.
An interesting study by Rubnitz et al19 described microarray analysis on a series on childhood AL. Although B-ALL, T-ALL, and AML were clearly segregated by gene expression profiles, the 13 cases of MPAL tested in this series formed a distinct cluster among these 3 entities. These results suggest that the morphologic and immunophenotypic anomalies, which allowed us to define BAL/MPAL, may indeed reflect specific, as yet not defined, molecular characteristics.
It remains unclear whether MPAL arise from a normal extremely plastic early progenitor or because of severe genomic aberrations. Observations of a large array of cytogenetic anomalies have been reported in clinical case reports. The most frequent cytogenetic finding in MPAL is the Philadelphia chromosome, BCR-ABL1 fusion gene. This aberration is observed in chronic myelocytic leukemia BCR-ABL1, in B-precursor ALL (often expressing myeloid antigens but not fulfilling MPAL criteria), and in some cases of de novo AML hinting at such possible phenotypic plasticity as will be discussed below. However, not all MPAL cases display t(9;22) and there is as yet no real explanation for their development.
MPAL could arise from a diseased myeloid progenitor, a lymphoid precursor becoming aberrant, or the proliferation of an early progenitor not definitely committed. In favor of the first alternative, it may be noted that some MPAL cases have been reported to develop after myelodysplasia or myeloproliferative neoplasms.20, 21 Also, leukemias with AML-related specific translocations such as t(8;21) or t(15;17) may display lymphoid markers.10, 22
A lymphoid origin is suggested by ALL morphology observed in about 40% the cases and by some chromosomal anomalies more characteristic of ALL such as t(4;11).23 The persistence and proliferation of MPAL cells was demonstrated in a small series of 8 patients to be associated with resistance to apoptosis mediated by overexpression of members of the IAP family.24 The coexistence of FLT3-ITD and MLL rearrangements was found in the biphenotypic leukemic cell line MV4-11.22 Biphenotypic leukemogenesis could also be obtained by manipulating MLL/SEPT6 and FTL3-ITD in mouse models of myeloproliferative disease.25 In SCID mice, human CD34+cells with the MLL/AF9 fusion gene, t(9;11), were demonstrated to be able to give rise to ALL, AML, or MPAL depending on growth factors in the recipient mouse.26 Therefore, the bone marrow microenvironment could also be crucial in orienting a multipotent leukemic stem cell toward overt differentiation or a mixed phenotype.
Other leukemias of ambiguous lineage
The acute leukemias of ambiguous lineage defined by the WHO 2008 classification include, in addition to MPAL, 2 other rare types of leukemias.12 The first, called acute undifferentiated leukemia, can be identified only after an extensive immunophenotyping. The expression of classical myeloid and lymphoid markers and also markers associated with plasmacytoid dendritic cells (CD4/CD56), basophils, mast cells, and natural killer (NK) cells must be excluded. Thus, cases of acute undifferentiated leukemia may express only HLA-DR, CD34, and/or CD38 and/or TdT. The other rare type of leukemias in this classification is the provisional entity of NK cell lymphoblastic leukemia/lymphoma. These still rather poorly defined cases may share some T cell markers such as CD2 or CD7 and can express CD56, rarely CD16, and sometimes CD94 or CD161. Panels of anti-KIR antibodies are required to better characterize these cells.
In conclusion, MPAL is a rare form of acute leukemia that could become better recognized if implementation of the biologically better justified and strict classification criteria is integrated in immunophenotyping panels. However, a comprehensive immunophenotyping remains necessary, especially to recognize aberrant patterns useful in tracking the persistent malignant clone as minimal residual disease. A better definition of MPAL cases, together with further molecular explorations, might help provide a better understanding of the origin of these peculiar leukemias.
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PII: S0740-2570(11)00103-1
doi:10.1053/j.semdp.2011.08.004
© 2012 Elsevier Inc. All rights reserved.
