Where diagnosis for myelodysplastic neoplasms (MDS) stands today and where it will go: The role of flow cytometry in evaluation of MDS

Abstract

Myelodysplastic neoplasms (MDS) are clonal hematopoietic neoplasms defined by cytopenias and morphologic dysplasia. This definition distinguishes MDS from clonal hematopoiesis of indeterminate potential (CHIP) and clonal cytopenia of undetermined significance (CCUS) wherein a patient harbors a somatic mutation involving a myeloid malignancy-associated gene in the absence of cytopenia and, in case of CCUS, dysplasia (Khoury et al., 2022). As such, the diagnostic framework for MDS relies on the detection of morphologic dysplasia in bone marrow cells coupled with evidence of clonality in patients with cytopenia.

While the features of dysplasia in hematopoietic lineages have been long established, assessment is subjective and limited by inter-observer variability particularly in cases with mild morphologic changes. In addition, several non-neoplastic conditions give rise to dysplastic changes that can overlap markedly with those seen in MDS. Examples of such conditions include nutritional deficiencies, toxins, and exposure to various treatments. In view of the challenges inherent in dysplasia assessment, demonstration of clonality in a patient with cytopenia is critical to establishing a diagnosis of MDS, especially in lower-risk disease with no excess of blasts. Establishing clonality has long rested on detection of cytogenetic abnormalities using conventional karyotyping and/or fluorescence in situ hybridization (FISH). Beyond establishing evidence of clonality, cytogenetic studies also play an important role in risk stratification (Greenberg et al., 2012), and some cytogenetic abnormalities correlate with distinct morphological and clinical features such as those of MDS with low blasts and 5q deletion. Yet, the problem of using cytogenetic abnormalities in the diagnostic workup of MDS is that they are detected in only up to 50% of MDS cases. In cases with normal cytogenetic findings, other tools are needed to demonstrate clonality and establish the diagnosis of MDS. Mutation profiling using next-generation sequencing (NGS) demonstrates somatic mutations in 80%–90% of MDS cases (Ogawa, 2019), providing substantial value in diagnostic assessment and establishing the presence of clonality. Here too, beyond establishing evidence of clonality, mutation profiling studies also play an important role in risk stratification, and some gene abnormalities are required to diagnose certain MDS types with defining genetic abnormalities such as MDS with low blasts and SF3B1 mutation and MDS with biallelic TP53 inactivation (Bernard et al., 2022; Khoury et al., 2022; Malcovati et al., 2020). However, a persistent caveat is that CHIP and CCUS can also occur in otherwise healthy elderly individuals (Jaiswal et al., 2014), requiring caution in interpreting the significance of certain mutations, especially those that are well known age-related mutations (such as DNMT3A, TET2, and ASXL1). On that basis, MDS-associated somatic mutations alone are not per se diagnostic of MDS. Against this background, flow cytometry analysis has emerged in recent years as another valuable tool for MDS workup and the evaluation of cytopenic patients, as recommended in the European LeukemiaNet guidelines (van de Loosdrecht et al., 2023).

Although no universal consensus on the optimal flow cytometry approach for MDS has been established to date, the diagnostic utility of flow cytometry in the evaluation of MDS patients has been the subject of several systematic analyses. In a recent study by Oelschlaegel et al., the analytic performance characteristics of five flow cytometry scoring systems were assessed and showed different specificity and sensitivity (Oelschlaegel et al., 2021). When using flow cytometry to evaluate for MDS, various cell subsets can be interrogated to assess aberrant antigen expression and/or maturation patterns, including myeloid progenitors, B-cell progenitors, as well as maturing granulocytic, monocytic and erythroid cells. Among these, based on our experience (Sanz-De Pedro et al., 2018) and the experience of others (van de Loosdrecht et al., 2023), the evaluation of CD34+ myeloid precursors offers more reliable and specific results than the evaluation of other cell types. Conceptually, this is not unexpected given that MDS is inherently a hematopoietic stem cell disease, and such cells are enriched within the CD34+ myeloid precursor compartment. Further adding to their utility is the fact that CD34+ myeloid precursors tend to have a more stable immunophenotype in comparison to maturing cells and are less affected by reactive conditions or pre-analytic factors. This contrasts with granulocytes and monocytes whose antigen expression and maturation patterns can be impacted by aging samples, sample hemodilution, reactive conditions, or the presence of a paroxysmal nocturnal hemoglobinuria (PNH) clone (van der Velden et al., 2023; Westers et al., 2021). In the appropriate clinical setting and appropriate sample quality control, however, the evaluation of maturing myelomonocytic and erythroid components provides valuable information especially in cases with limited number of CD34+ cells or cases with subtle phenotypic abnormalities in the CD34+ compartment. In summary, although it is widely recognized that flow cytometry findings alone are not sufficient to establish a definitive diagnosis of MDS, the detection of immunophenotypic aberrancies in CD34-positive myeloid precursors and maturing hematopoietic cells provides valuable ancillary support in conjunction with morphologic, cytogenetic, and molecular assessment in patients presenting with cytopenia.

Immunophenotypic alterations in the CD34+ myeloid compartment commonly used to indicate aberrancy include: (1) altered intensities of expression, such as increased expression of CD13, CD117 and CD123, and decreased expression of CD38 and HLA-DR; (2) altered patterns of maturation/differentiation, such as high expression of CD13 or CD117 in CD38 + dim very early precursors; (3) aberrant expression of lymphoid antigens, such as CD5, CD7, and CD56; and (4) asynchronous expression of mature myelomonocytic antigens, such as CD10, CD15, and CD64 in CD38 + dim very early precursors. Although increased numbers of CD34+ myeloid precursors and/or decreased numbers of early B-cell precursors (stage 1 hematogones) have been used to indicate abnormality in the evaluation of MDS (Oelschlaegel et al., 2021), such findings should be interpreted with caution as some patients, especially those with SF3B1 mutation, can have preserved hematogones (Chen et al., 2020). Similarly, CD34+ myeloid precursors can be increased in non-neoplastic conditions and due to growth factor administration. Other limitations—none of which are exclusive to flow cytometry—include the absence of detectable aberrations in a small subset of MDS cases, reliance on live cells, and the need for interpretation expertise.

In summary, flow cytometry has contributed significantly to the workup of MDS patients, with demonstrable value, reliability, and accuracy in clinical practice (Kern et al., 2023; Oelschlaegel et al., 2021; Westers et al., 2023). Compared to cytogenetic studies and mutational profiling, flow cytometric analysis has a much shorter turnaround time and can provide diagnostic value in cases with normal cytogenetics and equivocal molecular findings. Thus, we recommend that flow cytometric analysis should be included as a component of the evaluation of patients with cytopenias alongside cytogenetic studies and NGS-based mutation profiling.