Diagnostic Work-up Of Myelodysplastic Syndromes Research Articles

Automated Detection of Dysplasia: Data Mining from Our Hematology Analyzers.

Myelodysplastic syndromes (MDSs) are clonal hematopoietic diseases of the elderly, characterized by chronic cytopenia, ineffective and dysplastic hematopoiesis, recurrent genetic abnormalities and increased risk of progression to acute myeloid leukemia. Diagnosis on a complete blood count (CBC) can be challenging due to numerous other non-neoplastic causes of cytopenias. New generations of hematology analyzers provide cell population data (CPD) that can be exploited to reliably detect MDSs from a routine CBC. In this review, we first describe the different technologies used to obtain CPD. We then give an overview of the currently available data regarding the performance of CPD for each lineage in the diagnostic workup of MDSs. Adequate exploitation of CPD can yield very strong diagnostic performances allowing for faster diagnosis and reduction of time-consuming slide reviews in the hematology laboratory.

Analysis of erythroid maturation in the nonlysed bone marrow with help of radar plots facilitates detection of flow cytometric aberrations in myelodysplastic syndromes

Accumulating data support the role of flow cytometry (FCM) in diagnostic work-up of myelodysplastic syndromes (MDS). Changes in erythropoiesis are less documented than in granulopoiesis. However, most studies were performed on bone marrow samples (BMSs) after red blood cell lysis. We have established a FCM protocol for erythropoiesis, following a no-lysis approach and live gate acquisition of nucleated cells using DNA dye DRAQ5. The ERY tube consisted of CD36, CD71, CD105, CD117, CD13, and CD45. Comparison with cytomorphological differential counts was carried out in a learning cohort of 80 BMS. To detect aberrations, we analyzed 208 BMS from 135 patients and five normal donors, divided into three cohorts: MDS (n = 68), nonclonal cytopenia (n = 43), and normal controls (n = 29). Radar plot (RP) was created for an overview of normal and aberrant patterns. The proportion of erythropoiesis in the ERY tube showed better agreement with the cytomorphology, compared to FCM panels on lysed BMS. We confirmed that aberrations in coefficient of variation (CV) of CD36 fluorescence intensity (p < .001), mean fluorescence intensity of CD36 (p = .012), and CV of CD105 (p < .001) can distinguish between MDS and nonclonal cytopenia. RP facilitated evaluation of erythropoietic maturation patterns and aberrant patterns were identified in 85% of MDS patients. This study provides evidence that a no-lysis approach and RP analysis allow a more reliable evaluation of erythropoiesis and erythroid dysplasia, supporting the integration of FCM erythroid panels in the standard work-up of MDS.

The fraction of CD117/c-KIT-expressing erythroid precursors predicts ESA response in low-risk myelodysplastic syndromes.

Compelling evidence has emerged for the relevance of flow cytometry (FC) in the diagnostic work-up of myelodysplastic syndromes (MDS) but due to technical issues, the erythroid lineage has been under investigated, specifically in the therapeutic context. Using the "no red cell lysis" method developed to set up the RED-score, we specifically quantified the fraction of CD117/c-KIT-expressing erythroid precursors in a cohort of 144 MDS patients and studied the correlation with response to erythropoiesis-stimulating agents (ESA) in a sub cohort of 63 low-risk MDS patients. We confirmed the previously reported increase in CD117/c-KIT-expressing erythroid precursors in a subset of MDS patients and demonstrated a strong association between a cut off of CD117/c-KIT-expressing erythroid precursors ≥3% and ESA response (P = 0.001), independent of red blood cell requirement. From our observations, we hypothesized that a decrease in CD117/c-KIT-expressing erythroid precursors could be a mechanism of ESA failure. Moreover, the fraction of CD117/c-KIT-expressing erythroid precursors was correlated with progression-free survival in low-risk MDS patients (P = 0.018). In vitro, we demonstrated in an EPO dependent cell line that CD117/c-KIT expression is necessary for cell survival under EPO stimulation. The quantification of the CD117/c-KIT-expressing erythroid precursors could be proposed as a new theranostic and prognostic marker in MDS treated by ESA. Future studies will be required to determine whether modulating CD117/c-KIT expression and signaling could be used to improve anemia in MDS. © 2019 International Clinical Cytometry Society.

Flow Cytometric Immunophenotyping in Myelodysplasia: Discovery and Diagnosis

Diagnosis of myelodysplastic syndromes (MDS) is based on the integration of results from diagnostic tools. Initial laboratory assessments in patients with suspected MDS comprise analysis of peripheral blood and bone marrow with the gold standard of cytomorphology, conventional cytogenetics and interphase fluorescence-in-situ hybridization (FISH). Single nucleotide polymorphism (SNP) array and next-generation sequencing (NGS) are emerging techniques. The pathological hallmark of MDS is dysplasia. Flow cytometry (FC) can identify aberrancies in antigen expression and differentiation patterns that are indicative of dysplasia. FC is regarded instrumental and now even recommended in the diagnostic work-up of (suspected) MDS, especially when dysplasia by cytomorphology is minimal and cytogenetics shows no (typical) abnormalities. No single FC marker has been identified that is specific for MDS. The WHO-2008 classification recommends the presence of three or more FC aberrancies as highly suggestive for MDS. Minimal requirements to analyze dysplasia by FC have been proposed by the European LeukemiaNet (ELNet) working group (ELNet iMDS-Flow. ELNet recommendations should enable a categorization of FC results in cytopenic patients as "normal", "suggestive of", or “high probability of” MDS. FC as a single technique is not sufficient for the diagnosis of MDS and should be part of an integrated diagnostic work-up. Within the ELNet-iMDS-Flow group, a score based on four cardinal parameters was validated in MDS patients with <5% blasts and non-clonal cytopenic controls. This diagnostic score comprises: a) SSC of granulocytes (reflecting granularity) as ratio to lymphocytes; b) percentage of CD34+ myeloid progenitors of nucleated cells; c) percentage of B cell progenitors within the total CD34+ compartment; and d) expression of CD45 on CD34+ myeloid progenitors as ratio to lymphocytes. An abnormal value for every single parameter is assigned 1 point; MDS is highly suggestive when a score of 2 or more is obtained. Sensitivity of this diagnostic score was 70% and specificity 92%. It was demonstrated that this score also separates distinct subgroups with respect to prognosis within IPSS-R risk groups. Most flow scores mainly incorporate markers that cover the myelomonocytic lineage, e.g. the flow cytometric scoring system (FCSS). The latter separates patients with no and mild-to-moderate dysplasia from those with severe dysplasia. The FCSS has recently been shown to add significantly in separating patients into low or high risk disease in the revised IPSS subgroups. Finally, FCSS originally designed as a prognostic score, but can also be applied as a diagnostic score as combined with the 4-parameter FC diagnostic score to further improve sensitivity and specificity. Flow profiles based on the myelomonocytic lineage may fail to recognize MDS patients that exclusively show erythroid and/or megakaryocytic dysplasia. Analysis of megakaryocytes is hampered by their paucity. Recent advances shows that when adding erythroid markers such as CD71, CD36 and CD117/CD105 may add significantly to the diagnostic score of MDS by FC. Interestingly, aberrancies in the myelomonocytic lineage have been shown in patients with solely erythroid dysplasia with impact on prognosis. In addition, aberrant FC profile of myeloid progenitors has been associated with high transfusion requirements and disease progression as well as with a short duration of response or even lack of response to growth factor and azacitidine treatment. In conclusion, FC is a useful tool in the diagnostic work-up of MDS. Further studies on the value of FC in diagnosis, prognosis and predicting response to treatment are ongoing, as well as correlation of FC results with data obtained by SNP and NGS within prospective multicenter clinical trials in low and high risk MDS. DisclosuresVan de Loosdrecht:celgene: Honoraria, Research Funding; alexion: Research Funding.

Revisiting guidelines for integration of flow cytometry results in the WHO classification of myelodysplastic syndromes-proposal from the International/European LeukemiaNet Working Group for Flow Cytometry in MDS.

Definite progress has been made in the exploration of myelodysplastic syndromes (MDS) by flow cytometry (FCM) since the publication of the World Health Organization 2008 classification of myeloid neoplasms. An international working party initiated within the European LeukemiaNet and extended to include members from Australia, Canada, Japan, Taiwan and the United States has, through several workshops, developed and subsequently published consensus recommendations. The latter deal with preanalytical precautions, and propose small and large panels, which allow evaluating immunophenotypic anomalies and calculating myelodysplasia scores. The current paper provides guidelines that strongly recommend the integration of FCM data with other diagnostic tools in the diagnostic work-up of MDS.

Cutting Edge: Flow Cytometry in Myelodysplastic Syndromes

Flow cytometry (FC) is recognized as one of the important tools in the diagnosis of myelodysplastic syndromes (MDS), especially when cytomorphology and cytogenetics are inconclusive. Minimal requirements to analyze dysplasia using FC have been proposed by the International Workshops from the European LeukemiaNet on Standardization of FC in MDS. These should enable a categorization of FC results in cytopenic patients as "normal," "suggestive of," or "indicative of" MDS. Notably, FC should be considered as part of an integrated diagnosis, rather than as a separate technique. This article highlights tools to implement FC in the diagnostic workup of MDS. Current advances in FC for diagnosis and prognosis of MDS are also introduced.

Altered immunophenotypic features of peripheral blood platelets in myelodysplastic syndromes

Multiparameter flow cytometric analysis of bone marrow and peripheral blood cells has proven to be of help in the diagnostic workup of myelodysplastic syndromes. However, the usefulness of flow cytometry for the detection of megakaryocytic and platelet dysplasia has not yet been investigated. The aim of this pilot study was to evaluate by flow cytometry the diagnostic and prognostic value of platelet dysplasia in myelodysplastic syndromes. We investigated the pattern of expression of distinct surface glycoproteins on peripheral blood platelets from a series of 44 myelodysplastic syndrome patients, 20 healthy subjects and 19 patients with platelet alterations associated to disease conditions other than myelodysplastic syndromes. Quantitative expression of CD31, CD34, CD36, CD41a, CD41b, CD42a, CD42b and CD61 glycoproteins together with the PAC-1, CD62-P, fibrinogen and CD63 platelet activation-associated markers and platelet light scatter properties were systematically evaluated. Overall, flow cytometry identified multiple immunophenotypic abnormalities on platelets of myelodysplastic syndrome patients, including altered light scatter characteristics, over-and under expression of specific platelet glycoproteins and asynchronous expression of CD34; decreased expression of CD36 (n = 5), CD42a (n = 1) and CD61 (n = 2), together with reactivity for CD34 (n = 1) were only observed among myelodysplastic syndrome cases, while other alterations were also found in other platelet disorders. Based on the overall platelet alterations detected for each patient, an immunophenotypic score was built which identified a subgroup of myelodysplastic syndrome patients with a high rate of moderate to severe alterations (score>1.5; n = 16) who more frequently showed thrombocytopenia, megakaryocytic dysplasia and high-risk disease, together with a shorter overall survival. Our results show the presence of altered phenotypes by flow cytometry on platelets from around half of the myelodysplastic syndrome patients studied. If confirmed in larger series of patients, these findings may help refine the diagnostic and prognostic assessment of this group of disorders.

Flow Cytometric Assessment of Myeloid Nuclear Differentiation Antigen (MNDA) Expression in Myelodysplastic Syndromes As a Diagnostic Marker,

Abstract 3805 Background:Detection of aberrant antigen expression by multiparameter flow cytometry (MFC) is increasingly used in the diagnostic work-up of myelodysplastic syndromes (MDS). Myeloid nuclear differentiation antigen (MNDA) has been described to be strongly expressed in mature myeloid cells while myeloid progenitor cells show a weak expression only. MNDA has been suggested to be aberrantly expressed in MDS and therefore may improve the diagnostic capabilities of MFC. Aim: To analyze differences in the expression of MNDA between non-MDS cytopenias, MDS, and AML. Patients and methods: Bone marrow samples from a total of 269 patients with cytopenias and suspected MDS were analyzed by cytomorphology and MFC in parallel for diagnostic purposes. MNDA expression was determined by MFC in granulocytes, monocytes, and myeloid progenitor cells using the antibody clone 3C1 (provided by Trillium Diagnostics, Bangor, ME). MNDA expression was compared between patients grouped according to cytomorphology as: no MDS (n=103), MDS (n=85), CMML (n=9), AML (n=19), and possible MDS (n=53, based on cytomorphologic features of dysplasia but not sufficient to diagnose MDS). In addition, MNDA expression was compared between patients with aberrant and normal karyotype (cytogenetics was available in 238/269 patients, an aberrant karyotype was present in 59 patients). Results: A higher percentage of granulocytes (G) and monocytes (M) with weak expression of MNDA (%dimG and %dimM) was consistently found in MDS/AML samples as compared to samples without MDS. In detail, %dimG (mean±SD, 20±20% vs. 8±10%, p<0.001) and %dimM (31±24% vs. 16±11%, p<0.001) were significantly higher in cases with MDS as compared to those without. Furthermore, in patients with MDS, as compared to those without, the mean fluorescence intensity (MFI) of MNDA expression was found to be lower in monocytes (mean MFI, 71±36 vs. 85±27, p=0.004) as well as in myeloid progenitor cells with bright (67±24 vs. 86±66, p=0.007) or dim (2.5±0.9 vs. 2.8±1.2, p=0.037) expression of MNDA. In AML cases, as compared to cases with no MDS, similar figures were observed with higher values for %dimG (27±27% vs. 8±10%, p=0.007) and for %dimM (45±31% vs. 16±11%, p=0.001). In addition, in AML cases MFI of MNDA expression was lower in monocytes (55±39 vs. 85±27, p<0.001). Considering the diagnostically challenging cases with dysplastic features by cytomorphology but not sufficient to diagnose MDS similar although smaller differences were seen as compared to cases with no MDS with higher values for both %dimG (15±22% vs. 8±10%, p=0.026) and %dimM (25±19% vs. 16±11%, p=0.002) as well as a lower MFI of MNDA expression in monocytes (73±26 vs. 85±27, p=0.010). Interestingly, in CMML cases no reduced expression of MNDA could be found in any of the cell lines analyzed which might argue in favour of regarding CMML as myeloproliferative rather than myelodysplastic disease. From the cytogenetic point of view cases with an aberrant karyotype had higher values for both %dimG (22±20% vs. 12±18%, p=0.001) and %dimM (33±24% vs. 23±21%, p=0.004) as compared to those with a normal karyotype. Conclusions: These data suggests that MDS, as compared to non-MDS bone marrow, is associated with a reduced expression of MNDA in different cell lines which in part also applies to AML. Further analyses should define the potential of MNDA assessment to improve the MFC-based approach to diagnose MDS. Disclosures:Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Bellos:MLL Munich Leukemia Laboratory: Employment. Alpermann:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Implementation of flow cytometry in the diagnostic work-up of myelodysplastic syndromes in a multicenter approach: Report from the Dutch Working Party on Flow Cytometry in MDS

Flow cytometry (FC) is recognized as an important tool in the diagnosis of myelodysplastic syndromes (MDS) especially when standard criteria fail. A working group within the Dutch Society of Cytometry aimed to implement FC in the diagnostic work-up of MDS. Hereto, guidelines for data acquisition, analysis and interpretation were formulated. Based on discussions on analyses of list mode data files and fresh MDS bone marrow samples and recent literature, the guidelines were modified. Over the years (2005–2011), the concordance between the participating centers increased indicating that the proposed guidelines contributed to a more objective, standardized FC analysis, thereby ratifying the implementation of FC in MDS.

Flow cytometry in myelodyplastic syndromes

Myelodysplastic syndromes (MDS) are a heterogeneous group of myeloid neoplasms with abnormal maturation and differentiation of one or more cell lineage, resulting in bone marrow failure and a predisposition to leukaemic transformation. Diagnosis is made by evaluation and assimilation of clinical features, peripheral blood, bone marrow biopsy and cytogenetics/FISH investigations. Criteria for the diagnosis and classification have been refined and proposed in a consensus publication from the International MDS Working Group, with immunophenotyping recommended as a co-criterion for diagnosis.¹ This has not been incorporated in WHO diagnostic criteria to date. Utilising our understanding and delineation of normal antigen maturation and expression patterns of myelomonocytic precursors enables the detection of aberrancy in mature and immature populations. There is a large body of literature supporting the routine utility of immunophenotyping in the diagnostic workup of MDS and provocative data in patients treated serially with hypomethylating agents. However, further validation and consensus of antibody panels, definitions of aberrancy and flow scoring systems are required to enable the introduction of immunophenotyping into mainstream diagnostic laboratories. Standardisation efforts are underway within the European LeukemiaNet.

The impact of cytomorphology, cytogenetics, molecular genetics, and immunophenotyping in a comprehensive diagnostic workup of myelodysplastic syndromes

Because of limited reproducibility of morphologic features, the morphological categorization of initial myelodysplastic syndromes (MDS) cases remains a major task in a diagnostic setting. To further evaluate the role of additional diagnostic methods for suspected early MDS, the authors analyzed 1965 cases with unclear cytopenia where at least cytomorphology and immunophenotyping were performed in parallel, combined with cytogenetics and molecular genetics. In 353 patients, both methods diagnosed malignant/nonmalignant disease other than MDS, and 557 patients had MDS-refractory anemia with excess of blasts/chronic myelomonocytic leukemia. The remaining 1055 patients (53.7%), where early MDS/reactive cytopenia had to be assumed, were categorized into 6 groups depending on cytomorphology/immunophenotyping results for or against MDS. In 659 of 1055 cases (62.4%) with suspected initial MDS, cytomorphology and immunophenotyping were concordant in the categorization of MDS/non-MDS. Cytogenetics, available in 951 of 1055 patients, revealed the highest frequency of aberrant karyotypes when both cytomorphology and immunophenotyping proposed MDS (63 of 227; 27.8%). But also in the groups where either cytomorphology or immunophenotyping showed evidence of MDS, aberrant karyotypes were found in 6% to 14% of patients. Even when both morphology and immunophenotyping showed no MDS, 11 of 208 (5.3%) had cytogenetic aberrations. RUNX1/AML1 mutation screening was positive in 15% in the latter group. NRAS, MLL-PTD, NPM1, and JAK2V617F were detected in low frequencies, confirming MDS diagnosis in the respective cases. This report outlines the power of a combined diagnostic approach for suspected initial cases of MDS including immunophenotyping, cytogenetics, and molecular genetics with selected markers in addition to cytomorphology. Diagnostic algorithms should be developed, and immunophenotyping should be further validated for this specific indication.

Application of Multiparameter Flow Cytometry for the Identification of Dysplasia in Granulocytic, Monocytic, and Erythrocytic Lineages and Blasts in Patients with Myelodysplastic Syndromes.

The diagnosis and classification of myelodysplastic syndromes (MDS) are based on cytomorphology (CM) and cytogenetics. A high degree of experience in CM is required to allow the accurate identification of dysmyelopoiesis and quantification of bone marrow blasts. The identification of dysplastic features in all lineages by multiparameter flow cytometry (MFC) has been shown feasible. To further analyze the potential role of MFC in the diagnostic work-up of MDS we analyzed 224 bone marrow samples from patients with suspected of proven MDS by MFC, CM, and cytogenetics in parallel. Blast counts as determined by CM and MFC, respectively, ranged from 0% to 21% (median, 5%) and from 0% to 33% (median, 4%; correlation: r=0.192, p=0.018). The median number of aberrant features detected by MFC were 0 for blasts (range, 0 to 4), 2 for granulocytes (0 to 7), 1 for monocytes (0 to 5), and 0 for erythrocytes (0 to 2). The most frequent dysplastic features observed in the blast populations included aberrant coexpression of CD11b (20.5%), CD15 (14.3%) and CD64 (14.3%). The most frequent dysplastic features observed in the granulocytic cell populations included reduced side-scatter signal corresponding to hypogranulation (71.4%), aberrant coexpression of CD56 (29.0%), aberrant pattern of CD13/CD16 expression (26.3%), aberrant pattern of CD11b/CD16 expression (25.9%), reduced expression of CD64 (17.0%), and aberrant expression of HLA-DR (14.7%). The most frequent dysplastic features observed in the monocytic cell populations included aberrant coexpression of CD56 (31.3%), aberrant coexpression of CD16 (26.3%), an aberrant pattern of CD11b/HLA-DR expression (6.7%), and aberrant coexpression of CD2 (5.8%). The most frequent dysplastic features observed in the erythroid cell populations included an aberrantly strong expression of CD71 and CD235a (23.7%), a lack of CD71 expression (10.7%), and an aberratly homogeneous expression of CD71 (7.1%). The presence of dysplastic features by CM as well as the presence of cytogenetic aberrations tended to be associated with a higher number of dysplastic features by MFC. These data suggest that the identification of dysplastic features by MFC is feasible although there is a large heterogeneity in aberrantly expressed antigens. Thus, a comprehensive panel of antibodies must be applied to allow the detection of dysplasia. Future studies will define the role of MFC in optimizing the diagnosis of MDS in cooperation with CM and cytogenetics.