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Year : 2017  |  Volume : 60  |  Issue : 1  |  Page : 43-49
Mixed-phenotypic acute leukemia series from tertiary care center

1 Department of Laboratory Haematology and Molecular Genetics, Tata Medical Centre, Kolkata, West Bengal, India
2 Department of Clinical Haematology, Tata Medical Centre, Kolkata, West Bengal, India
3 Department of Pediatric Oncology, Tata Medical Centre, Kolkata, West Bengal, India
4 Department of Laboratory Haematology and Cytognetics, Tata Medical Centre, Kolkata, West Bengal, India

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Date of Web Publication14-Feb-2017


Introduction: Mixed-phenotype acute leukemias (MPALs) are a heterogeneous group of rare leukemias constituting approximately 2%–5% of all leukemias, in which assigning a single lineage of origin is not possible. They are diagnosed by either the presence of antigens of more than one lineage or by the presence of dual population of blasts belonging to two or more lineages. We highlight the clinicopathological, immunophenotype, and genetic data of a cohort (n = 14) of patients diagnosed and treated at our center. Materials and Methods: We retrospectively analyzed consecutive cases of MPAL diagnosed in our flow cytometry laboratory from May 2012 to August 2015. These cases were diagnosed based on immunophenotyping of peripheral blood/bone marrow aspirates and morphology/genetics wherever available as per the World Health Organization (WHO) 2008 guideline. Results: Among 628 consecutive acute leukemia (AL) cases diagnosed and evaluated during this period, we identified 14 (2.2%) patients with MPAL fulfilling WHO 2008/EGIL criteria for immunological characterizing of AL criteria. Majority of these were males (n = 8, male:female ratio 1.3:1) and adults (n = 11, 78.5%). The median age of this cohort was 41 years (range 2–80). These cases were further classified as: B/myeloid (n = 9), T/myeloid (n = 4), and B/T MPAL (n = 1). Cytogenetics was available in 12 out of 14 cases, out of which, three cases had normal karyotype, three with t(9;22)(q34;q11), and two cases with complex karyotype. We also came across a rare case of B + T lymphoid MPAL who had mixed-lineage leukemia gene t(v; 11q23) rearrangement. Conclusion: MPAL is a complex entity with heterogeneous clinical, immunophenotypic, cytogenetic, and molecular features. Multiparametric flowcytometry by using comprehensive antibody panels is a valuable tool for diagnosis. Subsequent cytogenetic and molecular analysis for further prognostic stratification and treatment modalities are important.

Keywords: Cytogenetics, immunophenotype, mixed-phenotype acute leukemias

How to cite this article:
Pawar RN, Banerjee S, Bramha S, Krishnan S, Bhattacharya A, Saha V, Chakrapani A, Bhave S, Chandy M, Nair R, Parihar M, Arora N, Mishra D K. Mixed-phenotypic acute leukemia series from tertiary care center. Indian J Pathol Microbiol 2017;60:43-9

How to cite this URL:
Pawar RN, Banerjee S, Bramha S, Krishnan S, Bhattacharya A, Saha V, Chakrapani A, Bhave S, Chandy M, Nair R, Parihar M, Arora N, Mishra D K. Mixed-phenotypic acute leukemia series from tertiary care center. Indian J Pathol Microbiol [serial online] 2017 [cited 2020 Aug 12];60:43-9. Available from: http://www.ijpmonline.org/text.asp?2017/60/1/43/200057

   Introduction Top

Acute leukemia (AL) is a clonal hematopoietic stem cells disorder characterized by increase in immature cells (≥20%) in peripheral blood (PB) and/or bone marrow (BM). Diagnosis and subclassification of AL depend on multiparametric approaches which that include morphological assessment, immunophenotyping, karyotyping, and molecular genetics analyses. Most cases of ALs are classified as either myeloid or lymphoid lineage based on the expression of a set of antigens. A rare subgroup of AL, in which the blasts exhibit the antigens of more than one lineage is known as mixed-phenotype acute leukemia (MPAL).[1]

European group for immunological characterizing of acute leukemia (EGIL) in 1995 presented guidelines for classification of AL with biphenotypic marker expression. These criteria were subsequently integrated in the World Health Organization (WHO) 2001 guidelines for classifying ALs of ambiguous lineages. For many years, the EGIL guidelines were used for the diagnosis of MPAL. In 2008, new WHO criteria were proposed for the classification of ALs of ambiguous lineages. As per WHO 2008, MPAL now includes both biphenotypic and bilineal ALs. In biphenotypic ALs, usually, there is a single population of blasts that express the antigens of more than one lineage and in bilineal AL, there is separate population of blasts of more than one lineage. Hence, flowcytometric immunophenotyping is now mandatory for the diagnosis of MPAL.[2]

Careful interpretation of flowcytometry is important as aberrant antigen expression is a common phenomenon in ALs. The WHO 2008 classification of AL defined strict criteria for the diagnosis of MPAL. B-cell lineage assignment in MPAL relies on the strong expression of CD19 together with another B-cell associated marker or in cases with weak CD19 along with the expression of at least two B-lineage markers.[1] Myeloid lineage is characterized by the expression of myeloperoxidase either on cytochemistry/flowcytometry/immunohistochemistry or evidence of monocytic differentiation. T-cell lineage requires the presence of cytoplasmic CD3. The WHO 2008 excludes ALs with certain recurrent cytogenetic aberrancies or clinical presentations from MPAL: AL with t (8;21), t (15;17), or inv (16) is classified as AML with recurrent cytogenetic abnormalities despite their immunophenotypic marker expression. Furthermore, AL with FGFR1 mutations, CML-blast crisis, AL with MDS pre-phase, and therapy-related AL are separate entities.[3] In this study, we highlight the clinicopathological, immunophenotypic, and genetic data of a cohort of 14 such patients diagnosed and treated at our center.

   Materials and Methods Top

This is an audit of MPAL disorders diagnosed at the flow cytometry laboratory of the Tata Medical Center, Kolkata from May 2012 to August 2015. Clinical details, morphological evaluation, immunophenotyping, cytogenetic data, and relevant molecular data were were retrieved from the EMR. The diagnosis was based mainly on the WHO 2008 classification with a single case diagnosed on the basis of EGIL criteria.[1]

Case selection

Six hundred twenty-eight consecutive patients were analyzed as described. In all patients, air-dried PB smears and BM aspirates were stained with Giemsa stain. Myeloperoxidase was performed in all smears; other cytochemical stains-periodic acid-Schiff, nonspecific esterase, and specific esterase were done as suggested by the blast morphology. Cases were classified as ALL or AML by morphology and cytochemistry, according to the French-American-British criteria.[4]

Flow cytometry immunophenotyping

Flowcytometric immunophenotyping was done in all cases either on BM or PB. Six-color flow cytometry analysis was performed using FACS canto II (Becton Dickinson, San Jose, CA, USA). Fluorochromes include fluorescein isothiocyanate, phycoerythrin, PerCPCy5.5, allophycocyanin, PE-Cy7, and APCH7. Standard technique of stain, lyse then wash procedure was used followed by incubation at room temperature for 15–20 min. Cells were characterized into B-cells, T-cells, and myeloid lineage using the following markers CD19, CD10, CD20, CD34, HLA-DR, CD3, CD1a, CD2, CD3, CD4, CD5, CD7, CD8, CD13, CD33, CD117, CD11b, CD11c, CD14, CD15, CD45, CD56, CD71, CD235a, CD41, and CD61. Cytoplasmic markers used for the study (cCD3, cMPO, cCD79a, cCD22 and TdT) were permeabilized with Perm 2 Solution (Becton Dickenson, San Jose, CA, USA). All antibodies were obtained from: Becton Dickenson, except MPO (Beckman Coulter, Brea, USA). Results were analyzed using BD FACS Diva software version 6.1.3. Blasts were characterized by low side scatter, diminished or low CD45 expression and CD34 positivity. In case with negative CD34 in blast window, gating was done by using other markers of immaturity such as CD117 and CD19/CD10 co-expression. For interpretation we used a cut off of 20% positivity for surface markers and 10% for cytoplasmic markers as mentioned in the EGIL criteria.[1]


Conventional karyotypic analysis was done on bone marrow samples after 24-–48 h culture in tissue culture medium according to standard techniques. A complex karyotype was defined when three or more clonal structural chromosomal abnormalities were present. Fluorescence in situ hybridization (FISH) analysis was performed in selected cases for the confirmation of the translocations using a locus-specific dual color, break apart mixed-lineage leukemia (MLL; Abbot Vysis, Illinois, USA.) probe and dual color dual fusion BCR-ABL1 (Abbot Vysis, Illinois, USA) probe according to manufacturer's instructions, and following standard techniques.

   Results Top

Of the 628 newly diagnosed cases of AL seen during this period, 13 fulfilled the WHO-2008 and one EGIL criteria (8 males and 6 females). Three were under 15 years of age, and the median age was 41 years (range 2–80 years). The details of laboratory findings of total leukocytes count (TLC), hemoglobin (Hb), platelet count, and lactate dehydrogenase (LDH) in these cases are summarized in [Table 1]. Morphological evaluation of PB/BM did not reveal any characteristic morphological appearance of the blasts in any of these cases.
Table 1: Clinical and laboratory findings in mixed-phenotypic acute leukemia cases

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Immunophenotyping and cytogenetic details of all 14 MPAL cases are shown in [Table 2]. Out of 14 cases of MPAL, nine had combined B/myeloid phenotype [Figure 1], four cases had combined T/myeloid [Figure 2], and one patient had combined B/T Immunophenotype [Figure 3]. Of the nine MPAL cases with B/myeloid immunophenotype, five were males (5/9, 55.5%). All these nine cases had moderate CD19/10 co-expression except three which were CD10 negative. MPO positivity was seen in only six (66.6%) of the nine cases. Of the three, MPO negative cases, two cases had monocytic differentiation, and one was diagnosed based on EGIL criteria (UPN8). All four T/myeloid cases had cytoplasmic CD3 and MPO positivity as specific lineage-defining marker. CD34 expression was seen in 12/14 cases (85.7%).
Table 2: Immunophenotyping and cytogenetic details of mixed-phenotypic acute leukemia

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Figure 1: Flowcytometry plots for B/myeloid showing expression of CD19, CD10, cCD79a, TdT, and cMPO (population in Green) (UPN7)

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Figure 2: Flowcytometry plots for T/myeloid showing two population with expression of CD117, CD34, cMPO, TdT, and cCD3 (blue) and CD7, CD34, TdT, and cCD3 (green) (UPN10)

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Figure 3: Flowcytometry plots for B/T MPAL showing single population expression of CD19, CD20, CD7, cCD79a, and cCD3 (green population) (UPN14)

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As shown in [Table 2], cytogenetic correlation was available in 12 cases. Of the nine B/myeloid MPAL, three had normal karyotype and three had translocation t(9;22)(q34;q11) (2 on FISH, 1 karyotype). Of the four T/myeloid MPAL, one had inversion 16, and further subcategorized as AML with recurrent genetic abnormality. Two of four cases of T/myeloid had nonrecurrent cytogenetic abnormalities and one had a normal karyotype. Two out of three, CD10 negative MPAL cases were further evaluated for MLL gene rearrangement by FISH. One of these showed t(11;21)(q23;p11.2) resulting in MLL gene rearrangement. This was a case of B/T lymphoid MPAL (UPN14) rarely described in the literature.

Treatment and outcome

[Table 3] gives the summary of the treatment protocols used. In our study, half of the cases (n = 7) were advised only supportive therapy along with hydroxyurea (n = 1) or decitabine (n = 1) who did not follow-up further at our center. Hydroxyurea/decitabine treatment did not show any response and in view of the deteriorating general condition both patients also opted for supportive care. Intensive leukemia treatment was used in the remaining seven patients to achieve remission. All achieved morphological complete remission. 3/7 are on maintenance therapy, two patients have relapsed and died, and two are in long-term remission. The median follow-up for patients treated was 10 months (range 3–38 months). Two patients underwent transplant, of which one remains in long-term remission and the second patient relapsed.
Table 3: Management and follow-up details

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   Discussion Top

Multicolor and multiparametric flow cytometry are available in many centers in India, and increasing number of antibodies are routinely used to diagnose and subcategorize AL. This has led to increased the documentation of cross-lineage antigen expression, thereby creating confusion between true MPAL and aberrant expression of antigens. The most recent 2008 WHO classification has established new and strict criteria for the diagnosis of MPAL. For all practical purposes, diagnosis of MPAL mainly relies on flow cytometric immunophenotyping. Other additional methodology includes immunohistochemistry and cytochemistry which can at times be helpful. Most of our cases were diagnosed on flow cytometry.[1] The frequency of MPAL in our study was found to be 2.2% This is in concordance with published data, which documents the frequency ranging from 2.2 to 2.6%.[5],[6],[7],[8],[9] MPAL are thought to arise from multipotent progenitor stem cells capable of differentiating into myeloid and lymphoid lineages. Most of the MPAL cells are derived from early stages of hematopoietic differentiation. Stages of normal hematopoietic differentiation are seen because of lineage inconsistency exhibited by leukemic blasts. CD34 is a marker of early hematopoietic cells and its expression was seen in 85.7% (n = 12/14 cases).[6],[10],[11],[12]

Using the WHO 2008 criteria of MPAL, we observed B/myeloid immunophenotype in 64.2% (n = 9/14), T/myeloid immunophenotype in 28.5% (n = 4/14) and single case of B + T lymphoid immunophenotype MPAL. A similar study by Matutes E, et al. consisting of a larger cohort of 100 MPAL, diagnosed using the 2008 WHO criteria showed that 59% of cases are B/myeloid immunophenotype (n = 59), 35% T/myeloid immunophenotype (n = 35), and B/T immunophenotype 4% (n = 4).[5] Another study by Yan et al. of a cohort of 117 patients found that most of their biphenotypic cases 54.7% (n = 64) co-expressed myeloid and B-lymphoid antigens while 32.4% (n = 38) were myeloid and T lineage.[6]

In WHO 2008 MPAL guidelines, MPO positivity plays key role in the diagnosis of MPAL. MPO can be negative in an AML with minimal differentiation, but it has to be positive in myeloid lineage MPAL, unless the myeloid lineage is proven to be of monocytic differentiation. Out of nine cases of B/myeloid in this study, MPO positivity was seen in six cases, two out of the three MPO negative B/myeloid cases had monocytic differentiation, and one satisfied the EGIL criteria. All four T/myeloid cases had cytoplasmic CD3 and MPO positivity. This illustrates the importance of MPO in the assignment of myeloid lineage in MPAL.[1]

There is no single chromosomal abnormality that is uniquely associated with MPAL. In their study, of 23 biphenotypic (EGIL) cases, Owaidah et al. found that 68% had a clonal abnormality whereas 32% had a normal karyotype. The most common abnormalities included rearrangement of the MLL gene followed by the Philadelphia chromosome t(9;22)(q34;q11.2). Additional abnormalities included deletion of 6q, 5q, and 12p.[5],[13] Cytogenetic analysis was available in 12/14 cases in our study, seven B/myeloid immunophenotype, four T/myeloid immunophenotype, and one case of B/T immunophenotype. Normal karyotype was seen in only in four cases (33%). Philadelphia positivity t(9;22) was observed in three (25%). All these were adults, had B/myeloid immunophenotype, high TLCtotal leucocyte count and high serum lactose dehydrogenase levels. This finding is supported by Matutes et al. where they describe Philadelphia positivity in approximately 20% of MPALs in their study.[1],[5],[6],[14],[15] Two out of three, CD10 negative MPAL were further evaluated for MLL gene rearrangement by FISH. One of these had MLL gene rearrangement. This was a case of B + T lymphoid MPAL (UPN14) which is rarely described in the literature. She was a 3-year-old child who presented with high counts and high serum LDH levels.

One case of T/myeloid immunophenotype (UPN12) with inv (16) was noted in this study and was reclassified as AML with recurrent genetic abnormality. Two out of the remaining three cases of T/myeloid had nonrecurrent cytogenetic abnormalities like t(6;8) and del1p, and one case had normal karyotype. In the nine cases of T/myeloid, MPAL described by Weir et al. two had 2p13 translocations, two had complex karyotypes, and del(12p), add(7), and inv(1) were seen in one patient each. Normal karyotypes were found in only two T/myeloid patients. Similar to our findings they also describe that T/myeloid cases have frequent but generally nonrecurring abnormalities.[6],[15],[16] Cytogenetic abnormalities documented in four cases of B/T immunophenotype described by Matutes et al. included one with Philadelphia positivity, one with complex karyotype, one with other abnormalities, and one having normal karyotype. None of the cases showed MLL gene rearrangement.[5]

There have been few case reports and to the best of our knowledge, only two large studies described from India. Both these studies document the associated immunophenotypic abnormalities. In the largest study from India, Gujral et al. described 37 cases (1.5%) cases of MAPL.[17] Sukumaran et al. studied 506 cases of AL; out of them only 15 cases (2.9%) showed features of MPAL. This study described immunophenotype and treatment outcome in MPAL.[2] Extensive data on genetics profile, response to therapy, and clinical outcome of MPAL is not available. The rarity of these cases is one of the limitations to establish firm guidelines on treatment and prognostic stratification.

There are no established treatment protocols for patients with MPAL, which makes evaluation of outcome difficult in studies of this entity. Biphenotypic leukemias are associated with poor prognosis and are difficult to treat as described by Aribi, Ahmed, et al. They reviewed the clinical data for 31 biphenotypic ALs predominantly B/myeloid. Most of them were treated with ALL regimens while 7 patients received AML like regimens. The overall survival probability in their study at 2 years was only 60%.[18]

   Conclusion Top

MPALs are a rare subset of stem cell disorder with poor prognosis and are difficult to treat. It is important to diagnose them with the help of multiparametric flowcytometry using comprehensive antibody panels. Even though cytogenetics and molecular analysis for further prognostic stratification and treatment, are available, treating such patients remain a challenge even today.

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Conflicts of interest

There are no conflicts of interest.

   References Top

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Sukumaran R, Nair RA, Jacob PM, Nair Anila KA, Prem S, Binitha R, et al. Flow cytometric analysis of mixed phenotype acute leukemia: Experience from a tertiary oncology center. Indian J Pathol Microbiol 2015;58:181-6.  Back to cited text no. 2
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Correspondence Address:
Dr Neeraj Arora
Department of Laboratory Haematology and Molecular Genetics, Tata Medical Centre, Kolkata, West Bengal
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/0377-4929.200057

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  [Table 1], [Table 2], [Table 3]


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