Integrating Molecular Alterations with Immunophenotype and Clinical Characteristics in Myelodysplastic Syndromes: A Single-Center Study

Abstract

Continuous development of molecular and immunophenotypic techniques enables more precise diagnoses and more accurate assessment of prognosis in myelodysplastic syndromes (MDS). However, the relationship between genetic alterations and immunophenotype remains very poorly understood. The analysis included 30 patients diagnosed at a tertiary center who were eligible for azacitidine treatment. Next-generation sequencing (NGS) was performed at the start of the study to assess the mutation status of 40 genes associated with MDS pathogenesis. In addition, multiparametric flow cytometry (MFC) was performed to assess the ELN score (Ogata score) and, additionally, to detect an abnormal CD11b/HLA-DR and CD11b/CD13 expression pattern. In the studied patient population, higher ELN score results were found in patients with mutations in epigenetic modifiers and pathogenic mutations of the tumor suppressor genes. Signal pathway mutations were associated with lower platelet counts at diagnosis. The results of this study indicate a correlation between molecular abnormalities and deviations in cell immunophenotype. Investigating this correlation may, in the future, allow the development of new scales that allow a more sensitive and specific diagnosis of MDS and a more precise prediction of its course.

Keywords: Flow Cytometry; MDS; MFC; NGS; Ogata score.

Figure 1

Values of individual parameters in the ELN 2012 score in each patient in the study group (axis X). (A) Percentage of B progenitors and myeloblasts. (B) Number of B lymphocytes. (C) Number of myeloblasts. (D) CD45 MFI on lymphocytes and myeloblasts. (E) Side Scatter Count (SSC) of lymphocytes and granulocytes.

Figure 1

Values of individual parameters in the ELN 2012 score in each patient in the study group (axis X). (A) Percentage of B progenitors and myeloblasts. (B) Number of B lymphocytes. (C) Number of myeloblasts. (D) CD45 MFI on lymphocytes and myeloblasts. (E) Side Scatter Count (SSC) of lymphocytes and granulocytes.

Figure 1

Values of individual parameters in the ELN 2012 score in each patient in the study group (axis X). (A) Percentage of B progenitors and myeloblasts. (B) Number of B lymphocytes. (C) Number of myeloblasts. (D) CD45 MFI on lymphocytes and myeloblasts. (E) Side Scatter Count (SSC) of lymphocytes and granulocytes.

Figure 2

Eight-color flow cytometric analysis. (A–G,I) bone marrow of a patient with MDS—Ogata score 3 points plus an abnormal neutrophil maturation pathway. (H,J) Bone marrow of a patient with thrombocytopenia of other origin than MDS—Ogata score 0 points, comparison of neutrophil maturation with a patient with MDS. The gating strategy: (A)—discrimination of doublets (FSC-A vs. FSC-H)- singlets constitute 95% of all bone marrow cells; (B)—discrimination of cells debris and gating cells SSC low (FSC-A vs. SSC-A)—SSC low cells constitute 52% of bone marrow cells, after excluding debris and doublets; (C)—gating myeloblasts CD117+ and mast cells for quality control; (D)—gating B-cell progenitors (CD10 vs. SSC-A)—percentage among all bone marrow cells; (E)—gating blast cells CD34+ (CD34 vs. SSC-A)—percentage among all bone marrow cells CD45+; (F)—gating of the bone marrow populations with particular emphasis on CD45 positive cells (CD45 vs. SSC-A): blue—neutrophils, red—blast cells, purple—monocytes, pink—B progenitors, green—lymphocytes, orange—erythroblasts—percentages of the presented populations are described among all bone marrow cells; (G)—neutrophils with abnormal maturation pathway (CD11b vs. CD13); (H)—neutrophils with normal maturation pathway (CD11b vs. CD13); (I)—neutrophils with abnormal maturation pathway (CD11b vs HLA-DR); (K)—comparison of neutrophil percentages in specific gates marked on (G,H); (L)—comparison of neutrophil percentages in specific gates marked on (I,J), #Events—Number of events.

Figure 2

Eight-color flow cytometric analysis. (A–G,I) bone marrow of a patient with MDS—Ogata score 3 points plus an abnormal neutrophil maturation pathway. (H,J) Bone marrow of a patient with thrombocytopenia of other origin than MDS—Ogata score 0 points, comparison of neutrophil maturation with a patient with MDS. The gating strategy: (A)—discrimination of doublets (FSC-A vs. FSC-H)- singlets constitute 95% of all bone marrow cells; (B)—discrimination of cells debris and gating cells SSC low (FSC-A vs. SSC-A)—SSC low cells constitute 52% of bone marrow cells, after excluding debris and doublets; (C)—gating myeloblasts CD117+ and mast cells for quality control; (D)—gating B-cell progenitors (CD10 vs. SSC-A)—percentage among all bone marrow cells; (E)—gating blast cells CD34+ (CD34 vs. SSC-A)—percentage among all bone marrow cells CD45+; (F)—gating of the bone marrow populations with particular emphasis on CD45 positive cells (CD45 vs. SSC-A): blue—neutrophils, red—blast cells, purple—monocytes, pink—B progenitors, green—lymphocytes, orange—erythroblasts—percentages of the presented populations are described among all bone marrow cells; (G)—neutrophils with abnormal maturation pathway (CD11b vs. CD13); (H)—neutrophils with normal maturation pathway (CD11b vs. CD13); (I)—neutrophils with abnormal maturation pathway (CD11b vs HLA-DR); (K)—comparison of neutrophil percentages in specific gates marked on (G,H); (L)—comparison of neutrophil percentages in specific gates marked on (I,J), #Events—Number of events.