A novel flow cytometric method for enhancing acute promyelocytic leukemia screening by multidimensional dot-plots

Genetic alterations determine the biological behavior of acute myeloid leukemia (AML); therefore, these are the most effective independent prognostic factors and serve as the basis of classifications and guidelines [1, 2]. The recommendation of the World Health Organization (WHO) classifies AML according to recurrent genetic abnormalities, which are associated with specific clinicopathological features. Acute promyelocytic leukemia (APL) is mostly caused by the PML-RARA fusion protein [3]. The promyelocytic leukemia (PML) gene is located on chromosome 15, while retinoic acid receptor alpha (RARA) on chromosome 17 [1]. The balanced reciprocal classic t(15;17)(q24;q21) translocation is detected in 90–92% of APL patients [4, 5]. In the remaining cases, complex translocations involving chromosomes 15 and 17 or a submicroscopic insertion result in the rise of PML-RARA transcript [1]. On the basis of the morphological appearances of aberrant promyelocytes, APL can be classified as hypergranular or microgranular [1]. The release of procoagulant mediators from the leukemic cells is likely the most important mechanism, which is responsible for APL-associated coagulopathy. Disseminated intravascular coagulopathy (DIC) and systemic fibrinolysis, which usually occurs at the time of diagnosis, are the major causes of early death [6‐8]. The immediate administration of retinoid differentiating agents, such as all-trans retinoic acid (ATRA) and arsenic trioxide, reduce the hemorrhagic complications of APL; moreover, chemotherapy completed with ATRA improves overall survival [9‐13]. Therefore, rapid diagnosis and prompt treatment are indispensable. The diagnostic algorithm of APL starts with morphology and immunophenotype examinations [2]. Classic APL is characterized by a distinct morphology; yet, the microgranular type can mimic acute monoblastic leukemia, where the clinical history also resembles APL regarding coagulopathy [1, 14]. The common immunophenotypic alterations in APL, such as CD117, CD64, cytoplasmic MPO, CD33 bright expression, and loss or only weak intensity of CD34 and HLA-DR expression, have been known for decades; however, these are not specific for APL [1]. The morphology and immunophenotype examination serve as screening, and the detection of t(15;17) confirms the diagnosis. Fluorescence in situ hybridization (FISH) is the most commonly used method for the identification of t(15;17). In rare cases of APL that do not harbor the classic cytogenetically visible translocation but still possess the PML-RARA rearrangement, the polymerase chain reaction (PCR) is crucial for detecting the fusion gene [4, 5, 15, 16].

Due to the use of multiple lasers and an increasing number of fluorochromes, more information can be obtained from cells by flow cytometric examinations, which lead to the increasing significance of this method. Our aim was to exploit the opportunities afforded by recent improvements in analysis software, which can handle such large amounts of data. We wanted to assess a new analysis protocol based on multidimensional radar dot-plot that was designed to expand the effectiveness of flow cytometric examination in the screening of APL. To test this protocol, we compared the results of an APL AML group to those of a non-APL AML group with the help of predefined gates around the blasts characterized by the most common immunophenotype in APL.

The major finding of our study was that with the help of predefined gates in a multidimensional radar dot-plot, an effective, reproducible, and quick protocol can be created for the screening of APL. Recently, routinely used flow cytometric analyzing software (e.g., Kaluza and Infinicyt) allows integrated data visualization, which improves the interpretation of a large amount of data generated by an increasing number of fluorochromes. The comprehensive dot-plots were used first in the identification of normal cell populations during maturation [24, 25]. When the position of normal cell types was mapped, on the one hand, the detection of alterations from the normal pattern permit the identification of characteristic dysplastic signs referred to as myeloproliferative disorders [24]. On the other hand, several studies have demonstrated that the appearance of aberrant cells on integrated dot-plots was associated with a distinct malignancy, which can be utilized in the detection of minimal residual disease [26‐28].

To the best of our knowledge, we were the first to examine APL with multidimensional dot-plots based on a wide range of markers. We used not only the markers the expression of which was frequently altered in APL (CD34, CD117, HLA-DR, CD15, cyMPO, CD13, CD33, CD64) but also the prognostic markers, such as CD56, CD7, CD2, and cyFXIII-A [22, 29‐32]. The expression of CD56 or CD7 marker is associated with poor prognosis [29], while the presence of cyFXIII-A or CD2 with good prognosis [22, 31].

We found that the aberrant promyelocytes can be characterized by unique patterns and positions on radar dot-plots regardless of the mechanism leading to the formation of PML-RARA fusion protein. Therefore, the results of these dot-plots can support the diagnosis of APL even in FISH-negative—cryptic—cases and indicate the performance of PCR examination as early as possible to confirm the diagnosis.

In addition, with the help of multidimensional dot-plots, more information can be obtained from blast cells with a single examination; therefore, APL cases can be differentiated from all types of AML, including CD34-negative cases. Earlier several studies confirmed the usefulness of bivariate dot-plots in the screening of APL cases within AML. These were based only on three or four markers, usually CD34, HLA-DR, and a marker indicative of maturation, such as CD15 or CD11b [30, 33, 34]. These bivariate dot-plots, however, were characterized by high specificity and sensitivity (> 90%) only with respect to all types of AML, most of which are CD34-positive cases. Our novel protocol based on multivariate dot-plots, in turn, brings the most value where the applicability of bivariate dot-plots is limited: differentiating between APL and AML with CD34- and/or HLA-DR-negative immunophenotype. More and more information has been accumulated over the past decade about this less common type of AML. It is characterized by normal karyotype, NPM1 mutation, and morphologically by “cup-like” blasts and M2 or M4 morphology as defined by FAB [17‐20, 35, 36]. Despite these common denominators, morphology suggests that this is not a homogeneous subtype of AML, and yet, another advantage of our protocol is that it can detect various patterns among CD34-negative non-APL cases. Thus radar dot-plots enable the more precise identification of similarly behaving AML cases.

It must be acknowledged that there are some limitations to this study. For three reasons, we were able to examine only a relatively small number of APL cases. First, APL is relatively uncommon in Hungary; secondly, radar dot-plots can be used to compare cases where the same antibodies and fluorochromes have been used; and thirdly, the eight-color staining method has been used in Hungary only since 2014. Therefore, a control study on a larger population would be necessary in order to validate our results. In addition, we examined only APL cases with PML-RARA fusion, but RARA may have other fusion partners, such as ZBTB16, NUMA1, STAT5B, or NPM1. The variant fusion partner is important because it can influence the prognosis through the response to ATRA. Sainty et al., who examined a large number of cases with APL lacking t(15;17), found that ZBTB16-RARA cases were associated with CD56 expression, and there were no other immunophenotypic differences from t(15;17) APL [37], which suggests that our multidimensional screening protocol could be used in those cases as well.

In conclusion, multidimensional (radar) dot-plots can be used for screening APL even in cryptic APL cases. Based on only four multidimensional dot-plots, our protocol examined 6–9 markers per tube at the same time, thus increasing the efficiency and effectiveness of the FC examination. This method is reproducible (within the same laboratory), easy-to-use, and quick regardless of the percentage of blasts size and can differentiate squarely between APL and non-APL AML cases even when the FC results based on bivariate dot-plots would be uncertain and suggest APL falsely. This differentiation is crucial, because APL requires the prompt administration of special treatment to achieve a favorable prognosis.