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Acute Promyelocytic Leukemia with t(15;17)(q24;q21); PML-RARA

Optimizing Laboratory Testing for Hematologic Disorders Series


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Published: May 2013

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Dr. Reichard discusses the appropriate utilization of laboratory testing in diagnosis of acute promyelocytic leukemia (APL) with t(15;17)(q24;q21); PML-RARA. APL can often be differentiated from other types of AML based on bone marrow morphology, but the genetic aberration is diagnostic. Important laboratory assays include cytochemistry, flow cytometry, conventional cytogenetics, and PML-RARA identification by RT-PCR and FISH.

Presenter: Kaaren Reichard, MD

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My talk today is entitled optimizing laboratory testing for hematology disorders, acute promyelocytic leukemia with t(15;17)(q24;q21); PML-RARA fusion.

I have no conflicts of interest or financial disclosures to make.

This talk is 1 in a continuing series of hot topics that will address the issue of appropriate laboratory test utilization in hematologic disorders. This is a combined effort of the divisions of hematopathology and laboratory genetics, with the primary focus being on the identification of the correct algorithmic testing approach for various hematologic diseases. The second focus is to demonstrate how the laboratory can and should be engaged in reducing unnecessary testing.

Today we will be talking about acute promyelocytic leukemia. Our goals today include understanding the laboratory approach for evaluating the patient with possible acute promyelocytic leukemia. Second, to understand the appropriate utilization of cytochemistry, flow cytometry, cytogenetics, fluorescence in situ hybridization (FISH) and RT-PCR in the diagnosis and follow up of acute promyelocytic leukemia.

So why do we have test utilization issues?  First of all, laboratories typically do not provide sufficient guidance on how to use assays in hematologic diseases. Instead, laboratories just a listing of available tests. As new technologies and tests emerge, there can be a lack of understanding as to how these tests should be utilized in the context of other pre-existing assays.  Also, laboratories frequently do not have processes to either review test requests or to sequentially add or delete tests after initial results are determined.  Laboratory information systems don’t make it easy for clinicians to efficiently order assays and get information. And finally, laboratory reports do not always transmit the intended information and often come across as just lists of results instead of a correlation and diagnosis based on all the available results for that episode of care.

Test utilization issues also come about because of the realities within today’s clinical practices.  There are certainly varying levels of understanding of how to use hematologic associated assays amongst clinicians, even hematologists, as today’s practice has gotten significantly more complex. Also realize that patients with particular hematologic diseases may see a hematologist or internist who may not have experience with that particular disease.  Probably most important is that clinical knowledge at the time of test ordering is incomplete, and a clinician is compelled to order everything because it may be the only chance to get that piece of information.  Finally, we must realize that initial laboratory studies can help narrow the diagnostic choices and the specific testing needs. If laboratories don’t have a review and ordering process in place, clinicians have no choice but to order excess testing.

So what should we do? We think that it’s appropriate for laboratories and pathologists to develop laboratory utilization guidelines and algorithms for ancillary studies in hematologic diseases, to have test review processes in place, and to be sure that reports contain appropriate content and clinical relevance, all with the goal of reducing unnecessary testing and performing appropriate testing.

Today’s talk is on acute promyelocytic leukemia. This slide shows the current World Health Organization or WHO 2008 classification of acute myeloid leukemia involving the peripheral blood and bone marrow. As you can see there are 4 main categories which are highlights in bold.  Acute promyelocytic leukemia, our topic for today, is categorized within the subgroup of acute myeloid leukemias with a recurring genetic abnormality (translocation 15;17) (q24;q21);PML-RARA fusion

Important laboratory assays in the evaluation of suspected acute promyelocytic leukemia or APL include the complete blood cell and differential count, peripheral blood and bone marrow morphology, cytochemistry, flow cytometry, conventional cytogenetics and detection of the PML-RARA genetic fusion by FISH and/or RT-PCR. The focus of today’s presentation will be on the appropriate utilization of these tests in the diagnostic and follow-up evaluation of suspected APL.

APL comprises approximately 5 to 10% of total pediatric and adult acute myeloid leukemia cases. Thus, one is likely to encounter this disease in clinical practice. The diagnosis of acute promyelocytic leukemia with t(15;17); PML-RARA fusion is established by specifically detecting this recurrent molecular genetic abnormality. Importantly, although most cases of acute myeloid leukemia require the presence of greater than or equal to 20% blasts in the peripheral blood or bone marrow, this is not a requirement in the case of acute promyelocytic leukemia where the diagnosis can be made with fewer than 20% blasts if the PML/RARA genetic fusion is present. Acute promyelocytic leukemia may be associated with a significant bleeding diathesis, therefore, the clinical team should be promptly notified when a diagnosis of APL is suspected. Finally, although there may be a higher initial upfront morbidity associated with APL, the long-term overall prognosis is favorable for these patients, and in fact, is the most favorable of all AML categories.  After a diagnosis of acute promyelocytic leukemia is suspected based on morphologic review, a series of laboratory tests are available which can further refine, and ultimately allow, a definitive diagnosis to be made. The first test available in this series of ancillary laboratory testing options is cytochemical stains. Cytochemical stains, particularly myeloperoxidase, play a helpful role in the upfront evaluation of a suspected acute myeloid leukemia. Unfortunately however, cytochemical stains are perhaps not as widely used or available as they were in previous years. Cytochemical stains detect the presence of a specific protein or enzyme within a cell via a chemical reaction and must be performed on an unfixed specimen. Myeloperoxidase positivity in blasts is indicative of granulocytic lineage, while alpha naphthyl butyrate esterase or BE is useful in identifying cells of monocytic lineage.

This slide shows 2 photographic images of the utility of cytochemical stains in identifying blast lineage in some cases of acute myeloid leukemia.  In cases of suspected acute promyelocytic leukemia, the blasts and blast equivalents show abundant cytoplasmic granules positive by cytochemical myeloperoxidase, seen as a dark green or blue color in this example, on the left hand side of the screen. This staining intensity distinctly contrasts all other AML subtypes which typically show just a few, scattered myeloperoxidase-positive granules. For comparison, the photomicrograph on the right hand side of the screen shows an example of an acute myeloid leukemia with monocytic differentiation in which the blasts exhibit cytoplasmic butyrate esterase positivity, evident as a brown color in the cytoplasm.

Flow cytometry is the second laboratory tool available in the evaluation of acute leukemia. Multiparametric flow cytometric immunophenotyping has become routinely available in many laboratories and is considered to be standard-of-care in the evaluation of all new cases of acute leukemia. Flow cytometry can be performed on either a peripheral blood or bone marrow specimen provided that there are sufficient tumor cells present to ensure immunophenotypic accuracy.  In a case of acute promyelocytic leukemia, the diagnosis can be strongly suspected by flow cytometric studies when a CD34-negative, HLA-DR-negative, CD117- positive and CD33-bright positive immunophenotypic profile is observed. However, it must be emphasized that this immunophenotype is neither specific nor sensitive for a diagnosis of APL. For example, the microgranular variant of acute promyelocytic leukemia is often CD34 positive and acute myeloid leukemias with monocytic differentiation are often CD34 negative. Therefore, integration of the immunophenotypic findings with the other laboratory investigations is critical to provide an initial strong suspicion of APL.  

This slide shows an example of the classic immunophenotypic findings in a case of APL. In this composite schematic of 5 dot-plots, the blast population is depicted in red and demonstrates the typical immunophenotype of APL; CD34 is negative, CD33 bright positive, HLA-DR negative and CD117 positive. As shown by this example, a diagnosis of APL can be strongly suggested. Based on this finding, in conjunction with the morphologic and possible cytochemical results, a preliminary diagnosis of suspected acute promyelocytic leukemia could be communicated to the treating clinical team. The definitive diagnosis of APL will, however, require the detection of the translocation (15;17) PML-RARA fusion by one or more molecular techniques which we will discuss next.

  There are 3 complementary molecular techniques that can and should be utilized to assess for the typical recurring genetic abnormality associated with APL; namely, the translocation (15;17) (q24;q21) and the PML/RARA fusion. These molecular techniques include conventional cytogenetic analysis, FISH and RT-PCR studies. Although we emphasize an integrated approach to the diagnosis of acute myeloid leukemia in general, the final diagnosis of APL is specifically established by demonstrating the presence of the PML-RARA fusion. All 3 of these molecular testing modalities should be performed in a newly suspected case of APL for reasons that we will cover in the next few slides.

Similar to flow cytometry, conventional cytogenetics is considered standard-of-care in the workup of a patient with acute myeloid leukemia. Karyotyping is typically and optimally performed on an adequate bone marrow aspirate specimen although peripheral blood may be used if necessary if there are sufficient circulating blasts. In APL, conventional karyotypic analysis detects the reciprocal translocation 15;17 in the majority of cases. This classic translocation is highlighted in this representative karyogram by the 2 arrows. Conventional cytogenetics, in contrast to FISH and RT-PCR, shows the complete genomic complement and is able to demonstrate the presence of any additional chromosomal abnormalities that may be present. In rare cases of bona fide APL, the conventional cytogenetic study will reveal a normal karyotype. In these instances, FISH and RT-PCR are necessary to detect the cryptic PML-RARA fusion diagnostic of APL.  

In addition to conventional karyotyping, FISH for PML-RARA should be performed when a diagnosis of APL is suspected. FISH testing has a rapid turnaround time which facilitates the prompt rendering of this diagnosis.   In terms of the follow-up evaluation, however, in patients with previously diagnosed APL, FISH studies play a less important role in evaluating for residual disease as the positive detection sensitivity of FISH is only 1% versus that of quantitative RT-PCR which is 0.01 to 0.001%. 

RT-PCR for PML-RARA is the third assay in our molecular repertoire used in the diagnosis and follow-up of APL.  At the time of diagnosis, either a qualitative or quantitative assay can be performed.  This testing, much like FISH, often has a rapid turnaround time and is helpful in quickly establishing the diagnosis of APL.  Importantly, this assay will detect those very rare cases of APL, which are PML-RARA positive, which are cryptic by both cytogenetic and FISH studies.  By contrast however, this assay is not useful in detecting any cases of acute promyelocytic leukemia that have an alternative RARA translocation partner.  When monitoring a patient with APL on therapy, a quantitative RT-PCR assay for PML-RARA is needed. These studies are typically optimally performed on a bone marrow specimen because they have improved sensitivity compared with peripheral blood specimens.

Having now discussed the key laboratory tests that can be optimally utilized in the diagnosis and follow-up of patients with APL, in the next 2 slides, I would like to introduce to you an algorithm that is available on the Mayo Medical Laboratories Web page that summarizes our approach to the diagnosis and follow-up of acute promyelocytic leukemia.

Let’s begin with our approach to the diagnostic workup of acute promyelocytic leukemia.  Initial testing, after clinical or morphologic suspicion for APL, would consist of flow cytometric immunophenotyping and cytochemical stains as determined by the reviewing pathologist.  If the bone marrow morphology and/or flow cytometric results suggest a possible diagnosis of APL, additional testing on a bone marrow specimen would then include conventional cytogenetics, quantitative RT-PCR for PML-RARA and FISH for PML-RARA. No molecular studies need to be performed on the peripheral blood.   If 1 or more of the molecular results is positive, then a diagnosis of acute promyelocytic leukemia is established.  If however, the initial flow cytometric or morphologic results are not suspicious for APL, then the RT-PCR and FISH assays for PML-RARA do not need to be performed. Conventional cytogenetics, however, should be performed as usual in a new case of acute leukemia.

This slide shows our approach to the routine follow-up of patients previously diagnosed with PML-RARA- positive acute promyelocytic leukemia.  When the bone marrow shows no morphologic features of residual APL, conventional cytogenetic studies and quantitative RT-PCR for PML-RARA should be performed. At this point, no studies are needed on the peripheral blood and additionally, FISH is not indicated.  If the PCR and/or cytogenetic studies find molecular evidence of the translocation(15;17) or PML-RARA fusion, then residual disease, based on the positive genetic findings, can be reported.  Alternatively, if the PCR and cytogenetic findings are normal, then this would be reported as no molecular evidence of residual APL.   

In summary, the key laboratory assays that should be performed in a case of suspected acute promyelocytic leukemia include flow cytometry, cytochemistry (as available and determined by the pathologist) and conventional cytogenetic studies, FISH, and RT-PCR for PML-RARA if the morphology and flow cytometry are strongly suspicious.

For the follow-up bone marrow evaluation for patients on therapy for APL, key laboratory assays to be performed include conventional cytogenetics and quantitative RT-PCR. Flow cytometry, cytochemical stains, and FISH are not generally indicated.

Thank you for the opportunity to speak to you today about APL and our algorithmic approach to this disease. Hopefully, I have been able to introduce you to key concepts of how we, as practicing pathologists, and as directors of the laboratory, can work together with our clinical colleagues and the entire health care team to help control utilization of ancillary testing in the diagnosis and follow-up evaluation of acute promyelocytic leukemia.