FINAL DIAGNOSIS - Acute Undifferentiated Leukemia
Acute undifferentiated leukemias fall under the 2008 WHO classification of acute leukemias of ambiguous lineage. This category also includes mixed phenotype acute leukemias. Essentially, the leukemias classified into this category do not have a phenotype of a single cell lineage that allows for a simple, clear-cut classification and are therefore, a diagnosis of exclusion.
The acute undifferentiated leukemias are recognized as such because the leukemic cells do not express any lineage-specific markers that allow for their categorization into myeloid or lymphoid lineage leukemia. As a result, these leukemias must be subjected to comprehensive flow cytometric immunophenotypic analyses to assess for expression of markers of uncommon lineages, including plasmacytoid dendritic cell precursors, natural killer precursors, basophils, and even non-hematopoietic tumors. If none of these more uncommon lineage specific markers are present, the diagnosis of acute undifferentiated leukemia may be made.
Acute undifferentiated leukemias are rare; therefore, much has not been elucidated about this hematopoietic neoplasm. Their frequency, predilection for involvement of sites other than blood and bone marrow, genetic abnormalities, and progression/prognosis are not clearly known. In addition, the morphologic features of the blasts are indistinct and show no distinguishing features from blasts of other classifiable leukemias. By flow cytometry, the blasts are usually found to express no more than one lineage-associated marker, but specifically lack the lineage-specific markers required for classification. [2 ]
Classification of a blast population into a specific lineage is based upon a number of requirements that include analysis by cytochemisty, immunohistochemistry and flow cytometry. These requirements have changed over the years, but the most current 2008 WHO requirements are defined in the following table (Fig. 18). In order to be classified into a myeloid leukemia, blasts must exhibit myeloperoxidase expression by cytochemistry, immunohistochemistry, or flow cytometry. If myeloperoxidase expression cannot be established by any one of these methods, a blast population may still be classified as myeloid in origin by displaying monocytic differentiation through expression of two of the following markers: NSE, CD11c, CD14, CD64, or lysozyme. If either of these two requirements are not met, a myeloid categorization cannot be made. In order to establish a T-lineage leukemia, flow cytometry and/or immunohistochemistry must be used to determine if the blast population exhibits cytoplasmic CD3 expression or surface CD3 expression. Either the cytoplasmic or surface expression of this marker is sufficient for categorization as a T-lineage leukemia. Assignment of B-lineage differentiation requires the expression of multiple antigens; however, expression of CD19 is essential for this classification. The level of CD19 expression must be correlated with the strong co-expression of at least one or more of the following markers: CD79a, cytoplasmic CD22, and/or CD10. These guidelines have been established for a more uniform sub-classification of acute leukemias into a specific lineage or under the umbrella of acute leukemias of ambiguous lineage. 
As mentioned above, other acute leukemias of ambiguous lineage that exist include mixed phenotype acute leukemias (MPAL). Like acute undifferentiated leukemias, these mixed phenotype acute leukemias are rare and both subtypes account for less than 4% of all cases of acute leukemia.3 MPALs have been more thoroughly studied than the acute undifferentiated leukemias. These leukemias have been found to occur in both children and adults, with some subtypes more common in children. This entity was first described in the 1980s and referred to as biphenotypic or bilineal acute leukemia.  As more studies were conducted, better classification of these acute leukemias was established.
There are two general situations that can lead to an acute leukemia having a mixed phenotype. First, a single blast population may be present that co-expresses surface markers of two different lineages, and is in effect biphenotypic. Second, the leukemia may be composed of two distinct blast populations each of which expresses lineage-specific markers sufficient to have two distinct acute leukemias present. [1,2] Microscopic evaluation may be helpful in suggesting a mixed phenotype acute leukemia before immunophenotypic analysis is performed. The presence of both a large blast population and a small blast population may increase one's awareness to the possibility of a mixed phenotype. Also, blasts with an uninformative appearance may help to suggest an ambiguous lineage. However, it is important to realize that comprehensive immunophenotyping must be pursued as the lineage of many of these cases will turn out to be classifiable. 
MPALs generally fall into the categories of B/myeloid (B/MY) leukemias and T/myeloid (T/MY) leukemias, irrespective of whether one or more blast population is present. However, in the most recent WHO classification, MPALs can be further sub-classified based upon the chromosomal aberrations that are reported to be present through cytogenetic analysis. The most common aberration noted to be present is the Philadelphia chromosome (BCR-ABL1) or t(9;22)(q34;q11.2). It is important to recognize that some patients with chronic myelogenous leukemia may develop a leukemia that fits the definition of a MPAL. However, the diagnosis of mixed phenotype acute leukemia should not be made in these patients as it is best considered to represent chronic myelogeneous leukemia in blast phase. MPAL with BCR-ABL1 is rare, accounting for less than 1% of all acute leukemias and is more common in adults. It appears to have a worse prognosis than other MPALs; however, it has not been clarified how the prognosis compares to Philadelphia chromosome-positive acute lymphoblastic leukemia. Another commonly reported chromosomal aberration in MPALs is a translocation involving 11q23 (MLL gene). Again, it is a rare acute leukemia that appears to be more common in children, specifically in infancy. Interestingly, this acute leukemia usually presents as a dimorphic blast population with both monoblasts and lymphoblasts. This acute leukemia carries a poor prognosis. In the absence of either of these well-recognized chromosomal aberrations, MPALs can be classified as mixed phenotype acute leukemia, B/myeloid, NOS; mixed phenotype acute leukemia, T/myeloid, NOS; or mixed phenotype acute leukemia, NOS-rare types. Despite these many categories, there still exist ambiguous lineage leukemias that do not fit into these definitions, especially with the advancement of flow cytometric immunophenotypic panels. [2,4]
This case exhibited a blast population that had an indistinct morphology that was not suggestive of a myeloid or lymphoid origin. Through flow cytometric immunophenotypic studies, we demonstrated that the blasts did show some positivity of markers associated with various lineages, but did not meet the requirements for classification as myeloid, T-cell or B-cell lineage as outlined in the 2008 WHO classification. As a result, our diagnosis of an acute leukemia of ambiguous lineage, most specifically, acute undifferentiated leukemia was rendered.
Contributed by Stacey Barron, MD and Sarah Gibson, MD