Acute Promyelocytic Leukemia
Among the subtypes of AML with recurrent cytogenetic abnormalities, acute promyelocytic leukemia (APL) is unique for several reasons. First, because of the high risk of catastrophic hemorrhage, the rapid diagnosis of APL is essentially an emergency for pathologists. This hemorrhagic risk is the result of several contributing factors, including the failure of adequate platelet production due to bone marrow effacement, compounded by platelet consumption secondary to the activation of both coagulation and fibrinolytic pathways from granule release by hypergranular promyelocytes. A second reason that expeditious diagnosis of APL is essential relates to the need for rapid initiation of targeted therapy, which eventually reduces the risk of hemorrhage. This targeted therapy consists of either all-trans retinoic acid (ATRA) or ATRA plus arsenic trioxide (ATO).
The mechanism of action of ATRA in APL is well delineated in that ATRA deconstructs the DNA binding PML-RARA fusion protein, thus releasing the maturation block allowing leukemic promyelocytes to mature to neutrophils. ATO stimulates the addition of small modifier molecules to the PML moiety, resulting in oncoprotein degradation. Recent studies suggest that patients with APL may be potentially cured of leukemia with just ATRA and ATO without the need for toxic chemotherapy.
What New Testing Strategies are Available for the Rapid Diagnosis of APL?
The range of diagnostic modalities for APL and usual turnaround times are listed in Table 3. Note that turnaround times are estimates and vary significantly based on whether the test is performed in house or is sent out. Morphology is still highly relevant in APL diagnosis and is a very rapid modality, particularly in clinical practice situations optimized for stat CBC counts and pathologist review. Because of the high risk from a delay in diagnosis, the pathologist should always consider APL when reviewing a blood smear for possible AML, especially if the leukemic cells have abundant granules, have readily identifiable Auer rods, or appear "monocytic." The diagnostician must always have a high index of suspicion for APL in cases with very low WBC counts and marked thrombocytopenia. In this circumstance, careful screening of the blood smear for rare hypergranular promyelocytes is critical. Because of their size, these hypergranular promyelocytes may be more apparent at the feather edge or within the thick part of the smear. The sensitivity and specificity of morphologic assessment for classic APL and microgranular APL improve with the integration of the myeloperoxidase (MPO) cytochemical stain. Rapid communications to the clinician about a presumptive diagnosis of APL can be based on morphology ideally with cytochemical MPO. However, some cases of APL will not exhibit the typical morphology of APL as highlighted by workshop case 106 in which neither morphology nor flow cytometry was typical for APL, which was not detected until cytogenetic studies were completed Image 1.
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Image 1.
Bone marrow aspirate smear from a patient with a final diagnosis of acute promyelocytic leukemia that showed no morphologic clues to this diagnosis by morphology review. Note predominance of blasts with little maturation (Wright, ×400). (Courtesy of M. A. Yared, MD.)
Flow cytometry immunophenotyping also provides clues to APL, but multiple features must be assessed, including high side scatter in addition to the overall immunophenotypic profile (Table 3). If flow cytometry features prompt consideration for APL, cytospin Wright stain review along with MPO cytochemical staining should be performed Image 2.
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Image 2.
Flow cytometric histogram illustrates marked increase in side scatter typical of acute promyelocytic leukemia (APL). In addition, the leukemic cells express CD13, CD33, CD117, and CD65, while they are negative for CD34 and HLA-DR typical of classic APL. (Courtesy of A. Ruano, MD.)
Although both FISH and karyotyping typically provide definitive confirmation of APL, six workshop cases (cases 9, 110, 179, 247, 258, and 435) highlight that FISH and karyotyping can fail to identify some cases of APL due to cryptic t(15;17) with PML-RARA. In these six cases, the morphology (sometimes supported by cytochemical MPO) strongly suggested APL, which was cryptic by FISH/karyotyping. In all cases, the diagnosis of APL was eventually confirmed by polymerase chain reaction (PCR) Image 3, Image 4, and Image 5.
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Image 3.
This composite highlights morphologic and myeloperoxidase cytochemical features of classic hypergranular acute promyelocytic leukemia in which both cytogenetics and fluorescence in situ hybridization failed to detect t(15;17) (Wright, ×500). (Courtesy of D. S. Chabot-Richards, MD.)
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Image 4.
This composite highlights the morphologic features of classic hypergranular acute promyelocytic leukemia (APL) including the abundance of Auer rods. The definitive diagnosis of APL was based on molecular testing because the translocation was cryptic by both fluorescence in situ hybridization and cytogenetics (Wright-Giemsa, ×1,000). (Courtesy of S. C. Acree, MD.)
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Image 5.
Real-time polymerase chain reaction confirms PML-RARA (BCR3) in a case in which both fluorescence in situ hybridization and conventional cytogenetics were negative. (Courtesy of W. Wei, MD.)
Proximity ligation assay is a potential new tool for the rapid diagnosis of APL, as highlighted by workshop case 291. In this case, the patient had generalized petechial rash, hematuria, and rectal bleeding. Disseminated intravascular coagulation was confirmed by laboratory testing. Morphology, cytochemical stains, flow cytometry, FISH, and karyotype were all characteristic for APL Image 6, Image 7, and Image 8. The case submitters provided information and results of the proximity ligation assay method for rapid confirmation (6 hours of analytical time) of APL diagnosis (Image 8). This method has the potential to complement morphology, cytochemistry, and flow cytometry in the "same-day" diagnosis of APL, although off-hours availability is unlikely.
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Image 6.
This composite of peripheral blood smear and bone marrow showing spectrum of leukemic cells in a patient with acute promyelocytic leukemia (Wright-Giemsa, ×1,000). (Courtesy of A. Ruano, MD.)
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Image 7.
Bone marrow core biopsy section showing effacement by acute promyelocytic leukemia. Inset shows bone marrow aspirate smear (H&E, ×100). (Courtesy of A. Ruano, MD.)
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Image 8.
Composite of proximity ligation for PML-RARA for case 291 showing patient (left) and positive/negative controls (right) (×500). (Courtesy of A. Ruano, MD.)
Key Tips and Strategies for APL Diagnosis
Recognize value and rapidity of morphologic and cytochemical MPO staining in APL, which is the most rapid diagnostic modality and is available essentially 24/7.
Typical morphology for either classic or microgranular APL (ideally with cytochemical MPO) is sufficient for alerting clinicians with a presumptive diagnosis of APL prior to flow cytometry or genetic studies.
Maintain a high index of suspicion for APL, especially in cases in which the WBC is low with easily overlooked rare hypergranular promyelocytes.
APL should also be considered in leukemia cases with a monocytic appearance.
Recognize that APL cases can manifest "atypical" morphology; cytochemical MPO can prompt consideration of APL in this circumstance.
Recognize that t(15;17) of APL may be cryptic by FISH and cytogenetics (<10% of cases).
If morphology strongly supports APL, negative FISH/cytogenetics should prompt molecular testing before APL is excluded.
Maintain clinical laboratory testing systems to promote rapid identification of potential APL cases in conjunction with rapid communication to providers.
Adopt new testing modalities, as needed, to facilitate expeditious APL diagnosis.
Consider performing immunofluorescence PML antibody staining. APL shows a characteristic nuclear multigranular pattern and nucleolar exclusion.