Establishing a Diagnosis of AML

Morphological analysis of peripheral blood and bone marrow smears stained with Wright Giemsa or May-

Figure 30-1. Rapid diagnosis of acute promyelocyte leukemia with the PML-RARA fusion by PML immunostaining. In leukemic blasts from cases of PML-RARA-negative AML and in normal cells, PML protein is localized within nuclear body structures (PML nuclear bodies). In such cells PML antisera detect a wild-type staining pattern comprising less than 30 (typically 5-20) discrete nuclear dots, e.g., a case of AML Ml (upper left panel). Whereas in APL cases with the PML-RARA fusion, PML nuclear bodies are disrupted, leading to a characteristic microspeck-led/microparticulate nuclear staining pattern (>30 nuclear dots) with PML antisera, which detects PML-RARA and wild-type PML proteins (lower left panel). Nuclear integrity is confirmed by a nuclear stain or in this case by phase contrast microscopy (right-hand panels). These studies were performed with a PML polyclonal antibody, with FITC conjugated secondary antibody.

Grunwald Giemsa is clearly the first step in establishing a diagnosis for patients with suspected leukemia. After confirmation that a patient has acute leukemia on the basis of morphology, cytochemistry, and immunophenotyping, patients with AML may be further classified according to blast characteristics and degree of differentiation in the marrow.7 This initial workup may reveal features that are helpful in predicting the presence of particular molecular lesions (see Table 30-2), and may be especially valuable in situations in which the expected cytogenetic abnormality is lacking, yet the predicted fusion gene is formed as a result of a cryptic rearrangement, for example, insertion event, which may be detected by fluorescence in situ hybridization (FISH) or reverse transcription polymerase chain reaction (RT-PCR).16,17

It is critical to identify patients who could possibly have APL, since these patients are at high risk of sudden induction death due to hemorrhage if not treated promptly and appropriately. Furthermore, this group requires early introduction of molecularly targeted therapy in the form of alltrans retinoic acid (ATRA) in order to achieve optimal treatment results. For the rapid diagnosis of APL, immuno-fluorescent methods using antibodies directed against the PML protein are of value, with the conversion from a normal pattern of 5 to 20 discrete dots to a microparticu-late nuclear staining pattern (>30 microspeckles; see Figure 30-1) being indicative of the presence of the PML-RARA fusion gene. Alternatively, RT-PCR for detection of the PML-

RARA fusion transcript may be used to confirm the diagnosis of APL.

Immunophenotyping is a valuable component of the routine diagnostic workup for patients with AML for a number of reasons, including (1) establishing, confirming, and further refining a diagnosis of AML, (2) identification of potential prognostic markers, and (3) establishing expression profiles suitable for subsequent MRD monitoring. Although immunophenotypic studies are assuming a more important role in the investigation of AML (reviewed in Reference 18), they are not discussed in any great detail in this chapter, which is restricted to the role of molecular genetic analyses.

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