Immunophenotyping pattern of mixed phenotypic acute leukemia : historical review and diagnostic pitfalls

Flowcytometry; Immunophenotyping; Mixed Phenotypic; Acute Leukemia; Background: Multiparametric approaches are used for the diagnosis and classification of acute leukemia. Even after extensive immunophenotyping, a rare and heterogeneous subgroup that cannot be readily classified as Mixed Phenotypic Acute leukemia in which the blasts exhibit the antigens of more than one lineage. This study aimed to analyze the incidence of Mixed Phenotypic Acute Leukemia and correlate the clinicopathological and immunophenotypic characteristics.


INTRODUCTION
Acute leukemia (AL) is a clonal hematopoietic stem cells disorder characterized by an increase in immature cells (>20%) in the bone marrow (BM) and/or peripheral blood (PB). Multiparametric approaches are being used for the diagnosis and sub-classification of AL like morphological assessment, immunophenotyping, karyotyping, and molecular genetics analyses. The first step in the classification of AL is to assign lineage by the resemblance to normal progenitor cells. Most cases are unequivocally assigned either myeloid or lymphoid lineage based on the expression of a set of antigens. 1,2 However, even after extensive immunophenotyping, a rare and heterogeneous subgroup of AL cannot be readily classified. Leukemias falling into this category have been given many different names, including acute mixed lineage leukemia, bi-phenotypic leukemias, hybrid leukemias, undifferentiated leukemias, and, most recently, leukemia of ambiguous lineage. 3 One of this subgroup of ALis Mixed Phenotypic Acute leukemia(MPAL)in which the blasts exhibit the antigens of more than one lineage. MPAL can be B/Myeloid, T/Myeloid, or B/T. The mixed phenotype in T/ myeloid or B/myeloid MPAL can occur in three ways: It is a rare disease and comprises 2-5% of AL. 5 This retrospective study aimed to analyze the incidence of MPAL and correlate the clinicopathological and immunophenotypical characteristics.

MATERIALS AND METHODS
This study was conducted in PT. B.D. Sharma Institute of Health Sciences, Rohtak from2016 to June 2020. Clinical details, morphological evaluation, immunophenotyping data were retrieved. The diagnosis of MPAL was based mainly on the WHO 2008 classification. 4 A serial number was assigned to each one to ensure confidentiality. Ethical approval for this study was obtained from the institutional ethics committee.
During this period 256 cases of AL were analyzed. In all patients, air-dried peripheral blood smears and bone marrow aspirates were stained with Giemsa stain. To study the blast morphology, cytochemical stains like MPO, Sudan, periodic acid Schiff (PAS), nonspecific esterase were performed. Cases were classified as ALL or AML by morphology and cytochemistry.
Listmode data were acquired on FACS Canto II Flowcytometer (Becton Dickinson, San Jose, CA) and analyzed by FACS Diva software. Blasts were characterized by low side scatter, diminished or low CD45 expression, and CD34 positivity. In case CD34 is negative in the blast window, gating was done by using other immaturity markers as CD117 and CD 19/10 co-expression. For interpretation, we used a cut-off of 20% for surface markers and 10% for cytoplasmic markers.
Morphological evaluation of PB/BM did not reveal any characteristic morphological appearance of the blasts. Six   1).
On flow cytometry, all cases were positive for cMPO while B/Myeloid cases were positive for CD19 and at least on B cell marker. T/Myeloid cases were positive for cCD3 as well as CD7 in all cases besides other myeloid and T cell markers (Table 3).
In our study, all 7 patients started with chemotherapy. As all of them were adult patients Hyper-fractionated cyclophosphamide, vincristine, doxorubicin, and dexamethasone(Hyper CVAD) regimen was given. Only two patients achieved complete remission after one month while one patient achieved late complete remission. Four patients were given palliative chemotherapy because of age and financial constraints. All these four patients expired during treatment.

DISCUSSION
The first published reports on biphenotypicAL occurred in the 1980s when monoclonal antibodies were first being used to characterize leukemic cells. A defined scoring system for biphenotypicAL was proposed by Catovsky et al. 6 The European Group for the immunological characterization of leukemias (EGIL) later proposed an immunological classification and characterization of AL, which included a definition for biphenotypicAL. BiphenotypicAL was defined by EGIL when a score over 2 points was achieved for the myeloid as well as one of the lymphoid lineages. At least 20% of cells staining with a monoclonal antibody was chosen as a cut-off point to consider a marker as positive. 7 An exception was made for CD3, MPO, CD 79a, and Tdt because of their high degree of specificity, this is not adopted by World Health Organization (WHO). 1 (Table 4)WHO classification proposed a simpler diagnostic algorithm based on more specific lineage markers. New consensus criteria for MPAL were published in the 4th edition of the WHO classification of tumors of hematopoietic and lymphoid tissues and remain essentially unchanged in the 2016 update to the classification WHO criteria. They emphasize a few key lineage-defining markers with particular emphasis on CD19 for B lineage, CD3 for T lineage, and MPO for myeloid lineage4 (Table 5).
For all practical purposes, the WHO approach in the diagnosis of MPAL mainly relies on a few markers on flow cytometric immunophenotyping. Other additional methodology includes immunohistochemistry and cytochemistry which can at times be helpful. 8 MPAL is thought to arise from a multipotential hemopoietic stem cell that has the potential to differentiate into any lineage. Most of the reported cases of MPAL express early hematopoietic markers CD34 and HLA-DR suggesting an early precursor stem cell origin. Another explanation for the development of MPAL is that the blasts originate from a lymphoid precursor that has reactivated a myeloid differentiation program. 5,10 BCR-ABL1 fusion and KMT2A translocation are two genetically defined categories in MPAL. KMT2A translocations are more common in pediatric MPALwhile BCR-ABL1 is more common in adults. 2,9 The WHO classification of MPAL specifically excludes the entities which are covered in the proposed AML   Most of our cases were diagnosed on flow cytometry. The frequency of MPAL in our study was found to be 2.7% (7/256). This is in concordance with published data, which documents the frequency ranging from 2.2 to 2.6%. [11][12][13][14][15] There was a slight female preponderance with M: F ratio of 0 MPAL is derived from the early stages of hematopoietic differentiation which are capable of differentiating into myeloid and lymphoid lineages. CD34 and HLA-DR is a marker of early hematopoietic cells and their expression was seen in 100% of case in our study. These findings were in concordance with the studies done by Charles et al 2 and Pawar et al. 8 Pawar et al 8 reported higher mean Hb and platelet count while TLC and peripheral blast count were lower than our study. The difference may be due to the fewer number of patients in the present observation.
The most important concern for the diagnostic pathologist approaching a new case of AL is to provide necessary and sufficient information for initial treatment at the earliest. Outcomes for MPAL were generally worse than for comparison cohorts of patients with AML and ALL due to high-risk genetics. Most reports show a better initial response to ALL-directed chemotherapy than AML directed therapies. 2,16,17 This makes sense because most AL that fit the current WHO classification of MPAL more closely resembles ALL because of genetics and immunophenotype.
There were a few limitations of this study like a small number of patients, lack of cytogenetics and molecular studies (PCR) in these cases to draw an important conclusion. However, a comprehensive panel of antibodies is a must for the correct diagnosis of MPAL.

CONCLUSIONS
MPALisa rare leukemia with diagnostic and therapeutic challenge and worse prognosis due to origin from primitive multipotent progenitors which are resistant to treatment and lineage plasticity. Strictdiagnostic criteria should be followed in the diagnosis. Immunophenotyping is essential and should include lineage-specific markers along with the cytoplasmic markers for proper categorization of MPAL. Even though cytogenetics and molecular analysis for further prognostic stratification and treatment, are available, treating such patients remain a challenge even today.