Lineage Assignment in Acute Leukemia

Phenotypic analysis of a bone marrow that is completely replaced by blasts is an almost elementary problem. Multiparameter flow cytometry, however, can dissect and categorize all populations in bone marrow, and most important, it can distinguish leukemic cells from normal cells, even when the leukemic cells are not the majority population. The antibody panel used to study patients with acute leukemia typically contains representative markers of all lineages, with some redundancy to allow recognition and classification because many antigens may be aberrantly lost or acquired in leukemic cells.2,3 No standard combination of antibodies is used by all laboratories, but certain combinations have proved particularly useful not only for the most economical classification of leukemia, but also to demonstrate characteristic aberrant patterns that may be extremely useful for the detection of residual disease in follow-up samples, as discussed later in this chapter.

Difficulties encountered in the interpretation of flow cytometry data derive largely from two problems. First, in cases in which leukemic cells are not an obviously dominant population, it is important to ensure that the cells of interest are analyzed. The most useful general approach for this difficulty takes advantage of the fact that the common leukocyte antigen CD45 is differentially expressed on different types of hematopoietic cells and, when combined with side scatter, produces a display in which blasts occupy a unique position not occupied by normal cells (Fig. 15-1).⁴ Thus combining CD45 in one color with multiple combinations of antibodies in additional colors allows detailed characterization of blast populations in marrow even when they are present only in low numbers.

Failure to select the leukemic cell population for analysis, or including a mixture of leukemic and normal cells in a gate, may cause confusion in the reporting of flow cytometry results. It is best to identify the leukemic population visually and to provide a detailed description of the antigens expressed on the leukemic population, especially in myeloid leukemias, in which the dynamic patterns of maturation associated with morphologic variation in acute myeloid leukemia (AML) are reflected in changes in both light scatter and antigen expression as the leukemic cells mature. Tabular arrays of “percentage positive” are not recommended as part of a flow cytometry report, because they cannot reflect this complexity and may cause confusion.⁵

The second problem in interpreting flow cytometry results in leukemia derives from the fact that most of the reagents used in classifying leukemia are only relatively rather than absolutely specific. Thus correct classification requires not only a panel with some redundancy but also an understanding of patterns of reactivity of the antibodies. Markers such as CD13 and CD33, which are considered myeloid antigens because they originally were produced against myeloid leukemia cells, are found in up to 50% of cases of lymphoid leukemia,⁶ and interpretation of leukemias positive for these markers continues to be a cause of confusion. Generally speaking, the most specific markers of a given lineage are not highly sensitive, and the most sensitive ones are not specific. This finding is true more of myeloid markers than of lymphoid markers, and thus lymphoid leukemias usually can be recognized precisely, whereas poorly differentiated myeloid leukemias usually are defined by the presence of myeloid markers in the absence of specific lymphoid antigens.

Acute Leukemias of Indeterminate or Ambiguous Lineage

Although almost all cases of acute leukemia can be categorized easily regarding lineage, the lack of absolute specificity of most markers, and the promiscuity of their expression, implies that some cases cannot be resolved easily. Unfortunately, considerable controversy exists about the use of the terms mixed lineage, bilineal, and biphenotypic leukemia, and the new World Health Organization 2008 classification has recommended a more descriptive term: “mixed phenotype acute leukemia” (MPAL).⁷ These leukemias, which express myeloid- and lymphoid-associated markers in various combinations, represent a heterogeneous group of diseases. Previously, these leukemias were identified using a scoring system that assigned various point values to different antigens, with a diagnosis of “biphenotypic leukemia” rendered if the score were high for more than one lineage.⁸ The new World Health Organization classification recommends a different approach, recognizing the limitations of defining lineage with arbitrary scores. In this system, MPAL is defined using fewer, more specific markers, and, most significantly, cases of MPAL associated with specific genetic lesions are considered specific entities.

Association of Immunophenotype and Molecular Abnormalities

Many phenotypes in both acute lymphoblastic leukemia (ALL) and AML are highly associated with characteristic cytogenetic abnormalities.⁹ In ALL, these include cases associated with mixed lineage leukemia rearrangements, TEL-AML1 or E2A-PBX1. In AML, the most important link is in promyelocytic leukemia with the t(15;17) translocation. Whereas lack of human leukocyte antigen-DR subregion (HLA-DR) is the best-known abnormality, only about half of cases of DR-negative AML turn out to be acute promyelocytic leukemia, and other combinations of marker expression are much more sensitive and specific for acute promyelocytic leukemia.¹⁰ AML associated with the t(8;21) translocation also shows a characteristic phenotype.¹¹

Minimal Residual Disease Detection in Acute Leukemia

Several studies have demonstrated that the presence of residual leukemic cells in the marrow of patients who are in clinical and morphologic remission is a very strong adverse prognostic factor.12–20 Although the most extensive data exist in cases of childhood ALL,^13–17,19 the principle has also been shown to apply to persons with adult ALL^21,22 and to persons with AML.^18,20

Both molecular and flow-based methods have been used to detect minimal residual disease (MRD). Flow-based assays of MRD are based on the principle that nearly all leukemias show a pattern of expression of antigens that is aberrant compared with the pattern seen in normal differentiation.15,23–29 This aberrancy can take several forms. Some leukemic cells can abnormally express antigens of a different lineage or show loss of expression of a normal lineage marker. A more common finding is expression of normal differentiation antigens, but at an intensity that is different from that expected for a particular stage of differentiation. This latter attribute makes flow MRD analysis applicable to most cases of leukemia. Nonetheless, recognizing these deviations requires a clear understanding of patterns of maturation in normal differentiation, including marrow regeneration, as viewed in multiparameter space.

The pattern of antigen acquisition and loss during B-cell maturation in the bone marrow has been very well characterized, and certain markers are particularly useful for distinguishing normal and leukemic maturation. Consideration of markers including intensity of CD45, CD34, CD10, CD58, and CD38 or aberrant coexpression of myeloid or other unexpected antigens can allow detection of as few as 1 in 10⁴ leukemic cells, even when normal B-cell precursors are present in significant numbers (Fig. 15-2). Marrow T-cell acute lymphoblastic leukemia (T-ALL) also can be distinguished from normal T cells, in part because T-ALL will typically coexpress cytoplasmic CD3 and TdT, which is never seen on any normal cell.¹⁵ However, after treatment with steroid-based therapies, T-cell leukemias may undergo therapy-induced maturation and lose expression of TdT and other markers useful for distinguishing immature and mature T cells.³⁰ Thus T-ALL MRD detection often relies on detecting the same type of phenotypic abnormalities as seen in B-cell acute lymphoblastic leukemia (B-ALL). Detection of myeloid MRD usually is a more elaborate process because of the greater phenotypic heterogeneity in AML. Certain aberrant combinations, including coexpression of CD34 and CD56, CD117 and CD15, or CD7 and myeloid antigens occur with sufficient frequency to be useful in a large number of cases.^24,28,31 Achieving 10⁻⁴ sensitivity on a consistent basis is difficult, and sensitivities are more typically closer to 10⁻³. Because abnormal populations at diagnosis may not persist at recurrence, monitoring patients with acute leukemia is best done by looking for phenotypic differences from normal, rather than for a specific abnormal phenotype present at diagnosis.