Dysplasia and PNH-type cells in bone marrow aspirates of myelodysplastic syndromes.

BACKGROUND: Flow cytometry is increasingly applied in cytopenic patients suspected for myelodysplastic syndromes (MDS). Analysis includes evaluation of antigen expression patterns in granulocytes of which, for example, partial lack of CD16 may indicate dysplasia, but presence of paroxysmal nocturnal hemoglobinuria (PNH)-type cells should be considered. However, diagnostic bone marrow (BM) samples hamper PNH analysis because immature stages in the granulo-/monocytic compartment lack expression of certain glycophosphatidyl-inositol-anchored proteins. In this prospective study, we evaluated the presence of PNH-type cells in BM next to aberrancies from routine MDS immunophenotyping. METHODS: We combined antibodies defining maturation trajectories with FLAER. Validation of the designed method against routine PNH analysis and parallel analysis of BM and blood samples revealed similar results (granulocytes: Wilcoxon p = 0.25 and p = 0.82, respectively). We analyzed BM samples from 134 MDS, 17 chronic myelomonocytic leukemia, 15 aplastic anemia (AA), 1 PNH, 51 non-clonal cytopenic controls, and 12 normal controls. RESULTS: Most AA/PNH-BM samples showed clear PNH clones: median 1.1% (0%-35%); CD16 loss on mature neutrophils paralleled PNH-clone sizes. In MDS-BM, only 3.7% of cases showed ≥0.1% PNH-type cells, whereas partial CD16 loss was more frequent and abundant. CONCLUSIONS: Our findings confirm that dysplastic features in MDS-BM may point to presence of PNH-type cells, though only few cases displayed FLAER-negative cells. We showed that identification of these cells in the granulocyte compartment of BM specimen is feasible, but-according to international guidelines-results need to be confirmed in peripheral blood.

Distinct bone marrow immunophenotypic features define the splicing factor 3B subunit 1 (SF3B1)-mutant myelodysplastic syndromes subtype.

Splicing factor 3B subunit 1 (SF3B1) mutations define a distinct myelodysplastic syndromes (MDS) patient group with a relatively favourable disease course and high response rates to luspatercept. Few data are available on bone marrow phenotype beyond ring sideroblasts in this subgroup of patients with MDS. In the present study, we identified immunophenotypic erythroid, myelomonocyte and progenitor features associated with SF3B1 mutations. In addition, we illustrate that SF3B1-mutation type is associated with distinct immunophenotypic features, and show the impact of co-occurrence of a SF3B1 mutation and a deletion of chromosome 5q on bone marrow immunophenotype. These genotype-phenotype associations and phenotypic subtypes within SF3B1-MDS provide leads that may further refine prognostication and therapeutic strategies for this particular MDS subgroup.

Genomic array as compared to karyotyping in myelodysplastic syndromes in a prospective clinical trial.

Karyotyping is considered as the gold standard in the genetic subclassification of myelodysplastic syndrome (MDS). Oligo/SNP-based genomic array profiling is a high-resolution tool that also enables genome wide analysis. We compared karyotyping with oligo/SNP-based array profiling in 104 MDS patients from the HOVON-89 study. Oligo/SNP-array identified all cytogenetically defined genomic lesions, except for subclones in two cases and balanced translocations in three cases. Conversely, oligo/SNP-based genomic array profiling had a higher success rate, showing 55 abnormal cases, while an abnormal karyotype was found in only 35 patients. In nine patients whose karyotyping was unsuccessful because of insufficient metaphases or failure, oligo/SNP-based array analysis was successful. Based on cytogenetic visible abnormalities as identified by oligo/SNP-based genomic array prognostic scores based on IPSS/-R were assigned. These prognostic scores were identical to the IPSS/-R scores as obtained with karyotyping in 95%-96% of the patients. In addition to the detection of cytogenetically defined lesions, oligo/SNP-based genomic profiling identified focal copy number abnormalities or regions of copy neutral loss of heterozygosity that were out of the scope of karyotyping and fluorescence in situ hybridization. Of interest, in 26 patients we demonstrated such cytogenetic invisible abnormalities. These abnormalities often involved regions that are recurrently affected in hematological malignancies, and may therefore be of clinical relevance. Our findings indicate that oligo/SNP-based genomic array can be used to identify the vast majority of recurrent cytogenetic abnormalities in MDS. Furthermore, oligo/SNP-based array profiling yields additional genetic abnormalities that may be of clinical importance.

Implementation of erythroid lineage analysis by flow cytometry in diagnostic models for myelodysplastic syndromes.

Flow cytometric analysis is a recommended tool in the diagnosis of myelodysplastic syndromes. Current flow cytometric approaches evaluate the (im)mature myelo-/monocytic lineage with a median sensitivity and specificity of ~71% and ~93%, respectively. We hypothesized that the addition of erythroid lineage analysis could increase the sensitivity of flow cytometry. Hereto, we validated the analysis of erythroid lineage parameters recommended by the International/European LeukemiaNet Working Group for Flow Cytometry in Myelodysplastic Syndromes, and incorporated this evaluation in currently applied flow cytometric models. One hundred and sixty-seven bone marrow aspirates were analyzed; 106 patients with myelodysplastic syndromes, and 61 cytopenic controls. There was a strong correlation between presence of erythroid aberrancies assessed by flow cytometry and the diagnosis of myelodysplastic syndromes when validating the previously described erythroid evaluation. Furthermore, addition of erythroid aberrancies to two different flow cytometric models led to an increased sensitivity in detecting myelodysplastic syndromes: from 74% to 86% for the addition to the diagnostic score designed by Ogata and colleagues, and from 69% to 80% for the addition to the integrated flow cytometric score for myelodysplastic syndromes, designed by our group. In both models the specificity was unaffected. The high sensitivity and specificity of flow cytometry in the detection of myelodysplastic syndromes illustrates the important value of flow cytometry in a standardized diagnostic approach. The trial is registered at www.trialregister.nl as NTR1825; EudraCT n.: 2008-002195-10.