Mixed Phenotype Acute Leukemias

April 25, 2012

[N A J Med Sci. 2012;5(2):119-122.] PDF File

Chen Gao, MD, PhD; Amy M. Sands, MD; Jianlan Sun, MD, PhD*

Mixed phenotype acute leukemia represents a small  subset of acute leukemia that cannot be simply assigned as myeloid or lymphoid lineage,  because of the ambiguous phenotype the leukemic cells exhibit. It encompasses leukemias containing  separate populations of blasts of more than one lineage, or a single population of blasts  co-expressing antigens of more than one lineage. The 2008 World Health Organization classification  established strict criteria for diagnosis of mixed phenotype acute leukemia, emphasizing myeloperoxidase  for myeloid lineage assignment, cytoplasmic CD3 for T lineage assignment, and CD19 and other  B markers for B lineage assignment. A variety of cytogenetic lesions have been identified  in this group of diseases, two of which, the t(9;22)(q34;q11) BCR-ABL1 translocation,  and t(v;11q23) with MLL rearrangement are considered separate entities. Other categories  include T/myeloid NOS, B/myeloid NOS and other rare types. Mixed phenotype acute leukemia  is associated with poor outcome compared with other types of acute leukemias, particularly  in those with Philadelphia chromosome, and clinically presents challenges in diagnosis and  treatment. 

Key Words: acute leukemia,  acute leukemia of ambiguous lineage, acute leukemia, acute leukemia of ambiguous lineage,  cytogenetics, diagnosis, mixed phenotype acute leukemia (MPAL) 

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INTRODUCTION 

Most cases of acute leukemia can be classified  based on the lineage of the leukemic cells as myeloid, B-lymphoblastic (B-ALL) or T-lymphoblastic  leukemia (T-ALL). However, there are uncommon cases in which the blasts show differentiation  towards more than one lineage. In the 2008 World Health Organization (WHO) classification  these cases are identified as mixed phenotype acute leukemias (MPAL), under the category of  acute leukemias of ambiguous lineage.1 MPAL encompasses leukemias containing separate populations of blasts of more than one lineage  (bilineal or bilineage), and a single population of blasts co-expressing antigens of more  than one lineage (biphenotypic). Cases that can be classified in another category are excluded,  including acute myeloid leukemia (AML) with recurrent translocations t(8;21), t(15;17) or  inv(16), leukemias with FGFR1 mutations, chronic myelogenous leukemia (CML) in blast crisis,  myelodysplastic syndrome (MDS)-related AML and therapy-related AML, even if they have MPAL  immunophenotype.1 Of note, a diagnosis of MPAL should be reserved for patients who present with de novo acute  leukemia. 

Mixed phenotype acute leukemia is a rare disease,  representing only 3 – 5% of acute leukemias of all age groups, and 2.4 – 3.7% in children.2,3 However, the true incidence is difficult to establish due to problems with definition,  and perhaps variation between different laboratories. It affects both adults and children,  more frequently adults and has slight male preference.4 The prognosis for MPAL is poor comparing to other acute leukemias, with an overall survival  of 18 months.1,4

DIAGNOSIS 

Before the publication of the 2008 WHO classification,  the diagnosis and classification of MPAL were based on the scoring system proposed by the  European Group for the Immunological Classification of Leukemias (EGIL).5 The EGIL classification scheme assigns score points to major antigens to determine if certain  lineage is present. According to the original EGIL scoring system MPAL is defined when scores  are over two points for both myeloid and T- or B- lymphoid lineages (Table 1). CD117 was assigned  for 0.5 point in the original EGIL scoring system and later considered as a reliable marker  for myeloid commitment, and scored higher (1 point).6 The EGIL criteria was very helpful in classification of MPAL. However, even the revised  EGIL criteria can sometimes lead to an inaccurate classification. For instance, classical  AML cases with t(8;21) frequently express multiple B-cell markers (CD19, CD79a and CD20),  and cytoplasmic CD79a, considered to be specific in B-lineage determination, is positive  in a significant percentage of T-ALL cases. 


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Table 1. The European Group for the Immunological Classification of Leukemias (EGIL) scoring system.
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Table 2. 2008 WHO classification of acute leukemias of ambiguous lineage.
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The most recent 2008 WHO classification has established  new and strict criteria for the diagnosis of MPAL (Table 2). The T lineage is recognized by  the presence of specific T-lymphoid antigens, cytoplasmic CD3 (cCD3) or surface CD3.  Cytoplasmic CD3 expression is best demonstrated by flow cytometry with antibodies to CD3  epsilon chain. It should be noted that polyclonal CD3 antibodies used in immunohistochemistry  also react with the T-cell receptor zeta chain present in NK cells, and therefore considered  not specific for T lineage. Surface CD3 is rare but indicative of the T-lineage.  The myeloid lineage is demonstrated with the presence of myeloperoxidase (MPO) by flow cytometry,  immunohistochemistry or cytochemistry, or monocytic differentiation (requiring at least two  of the following: non-specific esterase (NSE), CD11c, CD14, CD64, lysozyme. Since there is  no single marker sufficiently specific for B-cell lineage, multiple antigens are required,  including strong expression of CD19 with one of the other B-cell markers (CD79a,  cytoplasmic CD22, CD10), or weak CD19 expression with at least two of the other B-cell markers.1

Compared with the EGIL scoring system, the current  2008 WHO criteria applied less but more specific markers to define the lineage of the blasts,  and incorporated the intensity of markers expression into the diagnostic algorithm.  The 2008 WHO criteria also emphasize MPO in the diagnosis of MPAL. MPO can be negative in  an AML with minimal differentiation, but it has to be positive in MPAL with myeloid lineage,  unless the myeloid lineage proven to be monocytic differentiation by expressing at least  two of the monocytic markers. Practically, diagnosis of MPAL largely relies on flow cytometric  immunophenotyping. Other diagnostic methods such as immunohistochemistry and cytochemistry  can be helpful. 

The B/myeloid MPAL is the most common among MPALs,  followed by T/myeloid, and other rare types of MPALs. A cohort of 100 patients diagnosed  as MPAL using the 2008 WHO criteria showed 59% of cases are B/myeloid immunophenotype,  and 35% are T/myeloid immunophenotype. A small portion of the cases are B+T-lymphoid imminophenotype  or trilineage (B+T+myeloid) immunophenotype.4

Morphologically most cases of MPAL display a  single population of leukemic cell, with 43% showing ALL morphology, and 42% showing AML morphology  in a large cohort study.4 Majority of the cases with AML morphology are M1 (AML without maturation) or M5 (acute monoblastic  and monocytic leukemia) according to the French-American-British (FAB) classification system,  and rarely M2 (AML with maturation) or M4 (acute myelomonocytic leukemia).4

The new WHO classification emphasizes MPO expression  in myeloid lineage assignment. This was supported by the report by Bene, et al. showing 98%  of the MPAL cases expressing MPO in at least 5% of blasts, 76% of the cases with more than  20% of blasts expressing MPO. Majority of the cases have variable populations of blasts coexpressing  MPO and lymphoid markers. Other myeloid markers are variably expressed, including CD13 (74%),  CD33 (66%), and CD117 (52%), and frequently coexpressed with MPO. Expression of monocyte-associated  markers include lysozyme (31%), CD15 (12%), and CD14 (8%).4

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There are four major categories listed under  MPAL in the 2008 WHO classification: B/myeloid, NOS; T/myeloid, NOS; MPAL with t(9;22)(q34;q11. 2); BCR-ABL1; and MPAL with t(v;11q23); MLL rearranged. 


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Figure 1. Wright-Giemsa-stained bone marrow smear from a patient with T/myeloid leukemia. Dimorphic populations of blasts are observed, one is small lymphoid appearing, the other has dispersed chromatin, prominent nucleoli and a moderate amount of pale cytoplasm, resembling myeloblasts, and positive for MPO.
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MPAL WITH t(9;22)(q34;q11.2); BCR-ABL1 

This is the most frequent recurrent genetic abnormality  occurring in MPAL and considered a distinctive entity. It accounts for 20% of all MPAL.1,4 It is a leukemia meeting the diagnostic criteria for MPAL with the blasts bearing the t(9;22)(q34;q11. 2) translocation or BCR-ABL1 rearrangement (Ph+) in patients with no history of CML.  It occurs more often in adults than in children.4 Clinically, the patients present similarly as other patients with acute leukemias,  with white blood cell counts likely to be high, resembling Ph+ ALL. 

Majority of the cases occurring in adults have  B/myeloid phenotype, while some show T/myeloid, B and T lineage, or trilineage leukemias.  Morphologically many cases show a dimorphic blast population, one resembling myeloblasts  and the other lymphoblasts. Some cases do not have distinguishing phenotypes.4,7,8

Cytogenetic abnormalities are identified by conventional  karyotyping for t(9;22), or FISH or PCR for BCR-ABL1 translocation. Additional cytogenetic  abnormalities are shown in many cases, including complex karyotypes. Ph+ is a poor prognostic  factor for MPAL, with a reported median survival of 8 months in 12 patients, significantly  worse than patients of all other types of MPAL.4


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Figure 2. T/myeloid leukemia with FLT3 mutation. Bone marrow section shows 100% cellularity with extensive replacement of marrow by sheets of blasts (A). Immunohistochemistry stains show a subset of blasts positive for CD3 (B), MPO (C), CD34 (D), TdT (E) and CD117 (F).
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MPAL WITH t(v;11q23); MLL REARRANGED 

This is a leukemia meeting the diagnostic criteria  for MPAL with blasts bearing a translocation involving the 11q23 breakpoint (MLL gene).  MLL rearrangement is more often seen in patients with a precursor B-ALL with aberrant expression  of myeloid markers, especially CD15 and CD65 but not MPO. These cases should not be considered  MPAL.1,9

MPAL with MLL rearranged is rare and accounts  for 8% of all patients with MPAL. It is more often seen in children and relatively common  in infancy.1,4 The clinical presentation is similar to other patients with acute leukemias. High white  blood cell counts are common as with other leukemia patients with MLL translocations.  Commonly these leukemias display a biphenotypic blast population, with one resembling monoblasts  and the other resembling lymphoblasts. The lymphoblast population often shows a CD19-positive,  CD10-negative B precursor immunophenotype, frequently positive for CD15. Expression of other  B markers is usually weak.1

The translocations involving MLL gene include  t(4;11)(q21;q23), t(11;19)(q23;p13), and t(9;11)(p22;q23), with confirmed partner genes being  AF4 on chromosome 4q21 and AF9 on 9p22.4 However, cases with chromosome 11q23 deletion should not be classified in this category.  The prognosis for this type of leukemia is poor.1,4

MPAL B/MYELOID, NOS 

This type of leukemia meets the diagnostic criteria  for assignment to both B and myeloid lineages and lacks the above mentioned recurrent cytogenetic  abnormalities. B/myeloid acute leukemia accounts for 59% of all MPAL4 cases and about 1% of all leukemias.1 It more commonly occurs in adults, but can be seen in children as well. 

Morphologically, the blasts have no distinguishing  features in most cases, with dimorphic populations, resembling lymphoblasts and myeloblasts,  or a single population resembling ALL. CD19 is strongly expressed in greater than 90% of  the cases, with majority positive in greater than 50% of the blasts. The blasts are also positive  for CD10, cytCD22, and/or cytCD79a.4 Multiple different cytogenetic changes have been demonstrated, however none is proven to  be specific in this subtype (will be discussed below). 

MPAL T/MYELOID, NOS 

This category meets the requirements for assignment  to both T-lymphoid and myeloid lineages without recurrent cytogenetic abnormalities.  It accounts for one third of MPAL and less than 1% of overall leukemias.1,4 It can occurs in both children and adults, but more commonly in children. 

There are no distinctive clinical features in  patients with T/myeloid acute leukemia. The blasts are composed of either a single population  or dimorphic populations (Figure 1). Cytoplasmic CD3 is expressed in virtually all cases,  with more than 20% of the blasts positive in majority of the cases. Other commonly expressed  T-lineage markers include CD2, positive in 27-98% of the blasts in 67% of the cases,  and CD7, positive in 24-99% of the blasts in 91% of the cases.4 Interestingly, the FMS-like tyrosine kinase 3 gene (FLT3) mutations is demonstrated to be  specifically associated with T/myeloid lineage. The immunophenotypic profile of CD117(bright),  terminal deoxynucleotidyl transferase (TdT), CD7, CD13 and CD34 is reported to be highly  sensitive (100%) and specific (94%) for predicting FLT3 mutation in T-ALL and T/myeloid acute  leukemia (Figure 2).10 Targeted therapy with FLT3 inhibitors has been developed and undergone clinical trials.11

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CYTOGENETICS 

The incidence of cytogenetic abnormalities is  high in MPAL, with only 13% of the cases showing a normal karyotype. About one third of the  cases have a complex karyotype with three or more structural chromosome abnormalities,  and 27% have other abnormalities. The most commonly involved chromosomal abnormalities include  del(6)(q11-21), 7q-, -7, t(2;7), del(5q) or -5, trisomy 4, and hyperdiploid karyotype.  ETV-6-RUNX1 rearrangement has been reported.12 Except for t(9;22), MLL rearrangement, and FLT3 mutation, there is no significant correlation  between the other cytogenetic abnormalities with age, sex, morphology, FAB subtype or immunophenotype.4

PROGNOSIS 

Young age, normal karyotype and ALL induction  therapy are associated with favorable survival, and Ph+ is a predictor for poor prognosis.  Study shows median survival is 139 months for children versus 11 months for adults,  139 months for patients with normal karyotype, versus 8 months for Ph+, and 139 months for  patients receiving ALL regimens, versus 11 months for those receiving AML schedules.4

In summary, MPAL is a rare disease with poor  prognosis. In order to establish the diagnosis, a panel of markers are suggested including:  MPO, CD3 (cytoplasmic and surface), CD19 plus three other B-lineage markers (CD22,  CD79a, CD10), and two or three monocytic markers. Adult and Ph+ patients have worse prognosis,  and bone marrow transplantation should be considered in first remission in these patients.1,4

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Chen Gao, MD, PhD; Amy M. Sands, MD; Jianlan  Sun, MD, PhD* 

Department of Pathology, Buffalo General Hospital,  State University of New York at Buffalo, 100 High Street, Buffalo, NY B 

*Corresponding Author: Department  of Pathology, Buffalo General Hospital, State University of New York at Buffalo,  100 High Street, Buffalo, NY 14203. Tel: 716-859-2140.
(Email address: jianlan. sun@gmail.com) 

CONFLICT OF INTEREST 

None. 

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REFERENCES  

1. Borowitz M, Bene MC, Harris NL, Porwit A, Matutes E. Acute leukemias of ambiguous lineage. In: Swerdlow SH, Campo E, Harris NL, et al, eds. World Health Organization Classification of Tumours: Pathology and Genetics of Tumours of Haematopoietic and Lymphoid Tissues. Lyon, France: IARC Press; 2008:150-155.
2. Rubnitz JE, Onciu M, Pounds S, et al. Acute mixed lineage leukemia in children: the experience of St Jude Children’s Research Hospital. Blood. 2009;113(21):5083-5089.
3. Al-Seraihy AS, Owaidah TM, Ayas M, et al. Clinical characteristics and outcome of biphenotypic acute leukemia in children. Haematologica. 2009; 94(12):1682-1690.
4. Matutes E, Pickl WF, Van’t Veer M, et al. Mixed phenotype acute leukemia (MPAL): clinical and laboratory features and outcome in 100 patients defined according to the WHO 2008 classification. Blood. 2011;117(11):3163-3171.
5. Bene MC, Castoldi G, Knapp W, et al. Proposals for the immunological classification of acute leukemias. European Group for the Immunological Characterization of Leukemias (EGIL). Leukemia. 1995;9(10):1783-1786.
6. Bene MC, Bernier M, Casasnovas RO, et al. The reliability and specificity of c-kit for the diagnosis of acute myeloid leukemias and undifferentiated leukemias. The European Group for the Immunological Classification of Leukemias (EGIL). Blood. 1998;92(2):596-599.
7. Carbonell F, Swansbury J, Min T, et al. Cytogenetic findings in acute biphenotypic leukaemia. Leukemia. 1996;10(8):1283-1287.
8. Killick S, Matutes E, Powles RL, et al. Outcome of biphenotypic acute leukemia. Haematologica. 1999;84(8):699-706.
9. Attarbaschi A, Mann G, Konig M, et al. Mixed lineage leukemia-rearranged childhood pro-B and CD10 negative pre-B acute lymphoblastic leukemia constitute a distinct clinical entity. Clin Cancer Res. 2006;12(10):2988-2994.
10. Hoehn D, Medeiros LJ, Chen SS, et al. CD117 expression is a sensitive but nonspecific predictor of FLT3 mutation in T acute lymphoblastic leukemia and T/myeloid acute leukemia. Am J Clin Pathol. 2012;137(2):213-219.
11. Levis M, Ravandi F, Wang ES, et al. Results from a randomized trial of salvage chemotherapy followed by lestaurtinib for patients with FLT3 mutant AML in first relapse. Blood. 2011;117(12):3294-3301.
12. Gerr H, Zimmermann M, Schrappe M, et al. Acute leukaemias of ambiguous lineage in children: characterization, prognosis and therapy recommendations. Br J Haematol. 2010;149(1):84-92.
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