Wallach's Interpretation of Diagnostic Tests: Pathways to Arriving at a Clinical Diagnosis (336 page)

   Cytochemistry, although extremely useful in the past, is taking a secondary role in the era of cytogenetic/genetic and immunophenotyping diagnosis and classification. It plays a role when a rapid result is beneficial, such as rapidly differentiating AML from ALL. The most commonly used stains are as follows:

   Myeloperoxidase or Sudan Black B: positive in AML with maturation, in myelomonocytic leukemia, and in erythroleukemia; strongly positive in APL, negative in ALL, minimally differentiated AML, monoblastic leukemia without differentiation and in megakaryocytic leukemia.

   Chloroacetate esterase: positive in AML with differentiation and in acute myelomonocytic leukemia; negative in ALL, AML without differentiation, and in acute monoblastic and erythroleukemia.

   Nonspecific esterase: positive (and inhibited by sodium fluoride) in acute myelomonocytic or monoblastic leukemia with or without differentiation; negative in ALL, and AML with granulocytic line as the main component.

   Periodic acid–Schiff (PAS): the pattern of granules staining with PAS may differentiate lymphoid from myeloid precursors (e.g., very coarse granules in ALL lymphoblasts).

   Lysozyme is positive in AML with monocytic differentiation.

   
Immunophenotype
: Most cases of AML are characterized by their complex immunophenotypes. There is great variation in immunophenotype depending on the leukemic subtype. Blasts are positive for CD34 (except for APL and some cases with monocytic differentiation, where CD34 may be weakly expressed or absent), and in some cases HLA-DR (except for APL) and CD117. The AML variants with differentiation toward the granulocytic phenotype express CD13, CD33, CD15, and CD65. Those with monocytic characteristics are positive for CD14, CD4, CD11b, CD11c, CD64, and CD36. The megakaryoblastic leukemias express platelet antigens, such as CD41 and/or CD61. CD2 is expressed in subsets of APL, more often in microgranular variant. CD19 is expressed in AML with RUNX1-RUNX1T1. Other T- or B-cell antigens are expressed in acute leukemia of ambiguous lineage (mixed phenotype acute leukemia). Because of this possibility and its impact on prognosis (poor), their panel of antigens used at the time of diagnosis must contain multiple myeloid, B- and T-cell markers.

   
Cytogenetic/molecular genetic
investigations determine to a great extent prognosis and therapeutic protocols and have become the major criteria the WHO uses for subclassification of AML. Cytogenetics is also critical in distinguishing AML from chronic myeloid leukemia in blast crisis. There are specific cytogenetic abnormalities seen only in AML, example t(1;22)(p13;q13). Complex karyotype has consistently been associated with poor outcome. Although cytogenetic studies are essential for diagnosis and classification, many of the variant translocations can be detected by real-time polymerase chain reaction (RT-PCR) that has higher sensitivity and as such is useful for residual disease monitoring. Abnormalities in certain genes, such as mutations in FLT3, nucleophosphin (NMP1), KIT of CEBPA, as well as gene expression profiles confer prognostic significance. Gene expression profiling leads to further subclassifications of AML, with prognostic and therapeutic implications. Ultimately, it is expected that a proteomic-based classification will emerge.

   AML with t(8;21)(q22;q22) with RUNX1-RUNX1T1 fusion is found in approximately 5% of AML cases. Generally shows maturation in the neutrophilic lineage, occurs in a younger population, and may present as myeloid sarcomas. Good response to chemotherapy.

   AML with inv(16)(p13.1q22) or t(16;16)(p13.1;q22) with fusion of the CBFB, and MYH11 genes shows monocytic and granulocytic differentiation and abnormal eosinophils in the bone marrow. This rearrangement may be difficult to detect without FISH or PCR. It is important to alert the cytogenetics laboratory if this variant is suspected. Myeloid sarcomas may be present at diagnosis or at relapse. This variant constitutes 5–8% of AML cases. Patients respond well to chemotherapy.