Pathology of Acute Leukemias of Ambiguous Lineage

Acute leukemias in general have a distinct lineage, either lymphoid (ie, acute lymphoblastic leukemia [ALL]) or myeloid (ie, acute myeloid leukemia [AML]). However, in a small subset of patients who present with acute leukemia, a specific lineage cannot be assigned. These cases are characterized as acute leukemias of ambiguous lineage (ALAL). ^{[1, 2, 3]}

ALALs include immature hematopoietic neoplasms that show no distinct evidence of specific lineage differentiation (ie, acute undifferentiated leukemia [AUL]) as well as leukemias that express markers of more than one lineage (ie, mixed phenotype acute leukemia [MPAL]). The MPAL further includes two subtypes: (1) those in which there is more than one malignant (blast) population, each of which represents a different lineage (formerly known as bilineal leukemia); and (2) those in which the malignant (blast) clone coexpresses lineage-specific markers (formerly known as biphenotypic acute leukemias).

The 2016 revision (revised 4th edition) of the World Health Organization (WHO) Classification includes further refinements to the diagnostic criteria for ALAL, including molecular characterization of MPALs. ^[2] In the updated WHO Classification, the category of acute leukemias of ambiguous lineage includes the following ^[1] :

• AULs
• MPAL with t(9;22)(q34.1;q11.2); BCR-ABL1 rearranged
• MPAL with t(v;11q23.3); KMT2A ( MLL) rearranged
• MPAL, B/myeloid, not otherwise specified (NOS)
• MPAL, T/myeloid, NOS
• ALAL, NOS

Various scoring systems have been used previously; however, the 2016 WHO criteria for lineage assessment in MPAL are presented in Table 1. ^{[1, 4, 5]} It is important to recognize that these recommendations only apply to MPAL; these criteria do not apply to straightforward cases of AML or ALL in which MPAL is not a diagnostic consideration. The 2016 WHO update also emphasizes that for bilineal MPAL, it is more important that each individual blast population would meet the criteria for B, T, or myeloid leukemia, than that the specific markers below be present. ^[1]

Table 1. MPAL Lineage Assessment Criteria ^{[1, 4]} (Open Table in a new window)

Of note, ALALs exclude distinct cases of AML that may express lymphoid-associated markers or ALL that may express myeloid-associated markers. For example, low-intensity myeloperoxidase (MPO) expression has been noted in otherwise typical cases of B-cell ALL (B-ALL), and the 2016 WHO guidelines caution against making a diagnosis of B/myeloid MPAL in the absence of other evidence of myeloid differentiation. ^[1] It is also important that flow cytometry antibodies against the CD3 epsilon chain be used, as immunohistochemical antibodies may detect the zeta chain of CD3, which lack specificity. ^[4] Cases of acute leukemia that may be classified in another category based on genetic or clinical features are also excluded (eg, AML with translocation t(8;21) and expression of multiple B-cell markers). Similarly, cases of AML with myelodysplasia-related changes and therapy-related AML should be classified accordingly with a comment that leukemic blasts harbor a mixed phenotype, if one is present.

See also the following:

• Pathology of Acute Myeloid Leukemia With Myelodysplasia-Related Changes

• Pathology of Acute Myeloid Leukemia Not Otherwise Specified

• Pathology of Myelodysplastic Syndromes Associated With Isolated Del (5q)

• Pathology of Myeloid Proliferations Related to Down Syndrome

• Pathology of Blastic Plasmacytoid Dendritic Cell Neoplasm

• Pathology of Therapy-Related Myeloid Neoplasms (t-MNs)

• Pathology of Unclassifiable Myelodysplastic Syndromes

• Pediatric Myelodysplastic Syndrome

Acute undifferentiated leukemia (AUL) is extremely rare, and little is known about the frequency of this disease.

Mixed phenotype acute leukemia (MPAL) with t(9;22)(q34;q11.2) (or BCR-ABL1 rearrangement) is also rare, accounting for less than 1% of acute leukemias. It is the most common form of MPAL that is associated with a recurrent cytogenetic abnormality. This diagnosis should not be made in patients with known chronic myeloid leukemia (CML) who progress to the blast phase with features of MPAL. Although this condition does occur in children, it is more common in adults.

MPAL with t(v;11q23) (or KMT2A (MLL) rearrangement) is more common in children and infants than older individuals. The MPALs (B/myeloid and T/myeloid types) include those that are biphenotypic or of mixed lineage. They account for less than 1% of acute leukemias.

There are no unique clinical features associated with the various types of acute leukemias of ambiguous lineage (ALALs). Generally, patients with ALAL have a poor prognosis. Factors that increase the mortality risk in the geriatric population include age older than 80 years and not receiving chemotherapy. ^[6]

The blasts of acute undifferentiated leukemia (AUL) clearly have no morphologic features of myeloid or lymphoid differentiation (see the image below).

Pathology of Acute Leukemias of Ambiguous Lineage. Blasts of acute undifferentiated leukemia.

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The MPALs with t(9;22) q34;q11.2) (or BCR-ABL1 rearrangement) and the MPALs with t(v;11q23) (or KMT2A (MLL) rearrangement) commonly have a dimorphic blast population. One such population resembles lymphoblasts, and the other resembles myeloblasts or monoblasts (see the following image).

Pathology of Acute Leukemias of Ambiguous Lineage. Blasts of mixed phenotype acute leukemia with t(4;11q23).

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As mentioned above, the MPALs (B/myeloid and T/myeloid) are biphenotypic or of mixed lineage. As such, in most cases, either the blasts have no distinguishing morphologic features (ie, undifferentiated blasts), or the blast populations are dimorphic, with features of lymphoblasts and myeloblasts.

The diagnosis of acute leukemia of ambiguous lineage (ALAL) is based on immunophenotyping—primarily, flow cytometric immunophenotyping, although immunohistochemistry and/or cytochemistry may also play important roles in characterization (see Table 1 under the Overview section and Figures below). ^{[1, 7, 8]}

Acute undifferentiated leukemias (AULs) typically express no more than one surface membrane antigen of any given lineage. By definition, they lack T-cell-specific, myeloid-specific, and B-lineage-specific markers, as well as other lineage-specific markers (eg, those for plasmacytoid dendritic cells, erythroid precursors, and megakaryocytes). AULs are negative for MPO and the esterases by enzyme cytochemistry. The blasts often express CD34, CD38, and/or HLA-DR, and may express TdT and/or CD7, none of which are considered lineage-specific.

The mixed phenotype acute leukemias (MPALs) with t(9;22)(q34;q11.2) (or BCR-ABL1 rearrangement) are most often composed of myeloblasts and B-lymphoblasts, although some have myeloblasts and T-lymphoblasts, and others even have three components (ie, myeloblasts, B-cell lymphoblasts, and T-cell lymphoblasts). The MPALs with t(v;11q23) (or KMT2A (MLL) rearrangement) are most often composed of lymphoblasts with the following immunophenotypes: CD19+, CD15+, CD20-, CD10-, and HLA-DR+, as well as a myeloblast component with monocytic differentiation (ie, monoblasts). In rare cases, a more mature immunophenotype with surface light chain expression may be seen; in these cases, there is no evidence of a c-myc rearrangement. ^{[9, 10]} The MPALs (B/myeloid and T/myeloid) may be biphenotypic or of mixed lineage (see the following figures).

Pathology of Acute Leukemias of Ambiguous Lineage. Immunohistochemical features of mixed phenotype acute leukemia (T/myeloid). Blasts (upper left, hematoxylin and eosin [H&E] staining) are monomorphous, medium-sized cells with dispersed chromatin and small prominent nucleoli that stain positively for terminal deoxynucleotidyl transferase (TdT) (upper right). Blasts coexpress CD3 (lower left) and myeloperoxidase (MPO, lower right) with weak intensity staining, indicative of cytoplasmic localization.

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Pathology of Acute Leukemias of Ambiguous Lineage. Flow cytometry of mixed phenotype acute leukemia (T/myeloid). Top row: Light scatter analysis of the same case depicted on histology in the previous image reveals a single population of blasts (gold population) with increased forward scatter (FSC) and side scatter (SSC), indicative of large cell size (left) and dim expression of CD45 (leukocyte common antigen, right) in comparison to normal lymphocytes (red population, right). Middle row: T-cell antigen analysis shows the blasts to be negative for surface CD3 but positive for cytoplasmic CD3 (green population, left), confirming T-cell linage, and negative for CD4 and CD8 (green population, center). The blast population dimly expresses additional T-cell antigens, CD2 and CD7 (gold population, right). Note normal T cells (red population) positive for surface and cytoplasmic CD3 (left), CD2 and CD7 (right), and for either CD4 or CD8 (middle). Bottom row: Myeloid antigen analysis shows the same blast population (gold) to be dimly positive for CD13 (left), variably positive for CD33 (left), positive for CD34 (middle) and CD117 (middle and right), and dimly positive for MPO (right). The histologic and immunophenotypic findings together support a diagnosis of mixed phenotype acute leukemia (T/myeloid) with biphenotypic features.

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There are too few cases of acute undifferentiated leukemia (AUL) to determine whether there is an associated consistent genetic abnormality, although expression of some genes associated with an unfavorable prognosis in AML (eg, BAALC, ERG, MN1) may be seen.

As described with regard to mixed phenotype acute leukemia (MPAL) with t(9;22)(q34;q11.2) (or BCR-ABL1 rearrangement) and MPAL with t(v;11q23) (or KMT2A (MLL) rearrangement), these leukemias are defined by the consistent presence of their respective genetic abnormalities. Of note, it appears that patients with MPAL with t(9;22) may respond favorably to treatment with tyrosine kinase inhibitors (TKI). ^[11]  The most common partner gene in the KMT2A (MLL) rearrangement is AF4 on chromosome 4, band q21. ^{[12, 13]} Translocations t(9;11) and t(11;19) are also encountered.

MPALs (B/myeloid and T/myeloid) commonly have clonal cytogenetic abnormalities. In the B/myeloid type, complex karyotypes are often seen, and abnormalities encountered in more than one single case include the following: del(6p), 12p11.2 abnormalities, del(5q), structural abnormalities of 7, and numeric abnormalities, including near tetraploidy. ^{[12, 13]} Moreover, alterations have been identified in the following genes that are also associated with either acute myeloid leukemia (AML) or acute lymphoblastic leukemia (ALL): ASXL1, TET1, TET2, IDH1, IDH2, DNMT3A, NOTCH1, ETV6, and IKZF1. Despite these genetic characteristics, there are insufficient data to designate any of these changes as recurrent.