Maria Luz Sanchez

The immunophenotype of different immature, myeloid and B-cell lineage-committed CD34+ hematopoietic cells allows discrimination between normal/reactive and myelodysplastic syndrome precursors

Occurrence of phenotypic abnormalities in CD34+ hematopoietic progenitor and precursor cells (HPC) and their major B-cell and nonlymphoid compartments has been frequently reported in myelodysplastic syndromes (MDS). Here, we analyze for the first time the numerical and phenotypic abnormalities of different maturation-associated subsets of bone marrow (BM) CD34+ HPC from 50 newly diagnosed MDS patients in comparison to normal/reactive BM (n=29). Our results confirm the existence of heterogeneously altered phenotypes among CD34+ HPC from MDS and indicate that such variability depends both on the relative distribution of the different subsets of CD34+ HPC committed into the different myeloid and B-lymphoid compartments, and their immunophenotype (for example, higher reactivity for CD117 and CD13 and lower expression of CyMPO, CD64 and CD65 on CD34+ immature and neutrophil precursors), a clear association existing between the accumulation of CD34+ HPC and that of immature CD34+ HPC. Interestingly, expansion of erythroid- and neutrophil-lineage CD34+ cells is detected in low-grade MDS at the expense of CD34+ plasmacytoid dendritic cell and B-cell precursors, while expansion of immature CD34+ precursors occurs in high-grade MDS. On the basis of the number and severity of the phenotypic abnormalities detected, a scoring system is proposed that efficiently discriminates between normal/reactive and MDS CD34+ HPC, the mean score significantly increasing from low- to high-grade MDS.

Normal patterns of expression of glycosylphosphatidylinositol-anchored proteins on different subsets of peripheral blood cells: a frame of reference for the diagnosis of paroxysmal nocturnal hemoglobinuria.

Evaluation of the expression of glycosylphosphatidylinositol‐anchored membrane proteins (GPI‐AP) is currently used for the diagnosis of paroxysmal nocturnal hemoglobinuria (PNH). In this study, we analyzed the amount of expression of a wide variety of GPI‐AP in different subsets of hematopoietic cells present in normal peripheral blood (PB), to establish their normal patterns of expression and provide a frame of reference for the definition of the best combination of GPI‐AP and PB cell subsets to be applied in the diagnosis and monitoring of PNH.

Evaluation of the WHO criteria for the classification of patients with mastocytosis

Diagnosis and classification of mastocytosis is currently based on the World Health Organization (WHO) criteria. Here, we evaluate the utility of the WHO criteria for the diagnosis and classification of a large series of mastocytosis patients (n=133), and propose a new algorithm that could be routinely applied for refined diagnosis and classification of the disease. Our results confirm the utility of the WHO criteria and provide evidence for the need of additional information for (1) a more precise diagnosis of mastocytosis, (2) specific identification of new forms of the disease, (3) the differential diagnosis between cutaneous mastocytosis vs systemic mastocytosis, and (4) improved distinction between indolent systemic mastocytosis and aggressive systemic mastocytosis. Based on our results, a new algorithm is proposed for a better diagnostic definition and prognostic classification of mastocytosis, as confirmed prospectively in an independent validation series of 117 mastocytosis patients.

Quantitative Analysis of the Expression of Glycosylphosphatidylinositol-Anchored Proteins During the Maturation of Different Hematopoietic Cell Compartments of Normal Bone Marrow

Glycosylphosphatidylinositol‐anchored proteins (GPI‐AP) are a heterogeneous group of proteins deficiently expressed in patients with paroxysmal nocturnal hemoglobinuria. Up till now, no study has been reported in which the exact patterns of expression of a large number of GPI‐AP are quantitatively evaluated in normal bone marrow (BM) cells classified according to their lineage and maturation stage.

Bone marrow cells from myelodysplastic syndromes show altered immunophenotypic profiles that may contribute to the diagnosis and prognostic stratification of the disease: A pilot study on a series of 56 patients

A heterogeneous spectrum of immunophenotypic abnormalities have been reported in myelodysplastic syndromes (MDS). However, most studies are restricted to the analysis of CD34+ cells and/or other major subsets of CD34− cells, frequently not exploring the diagnostic and prognostic impact of immunophenotyping.

Impact of trisomy 12, del(13q), del(17p), and del(11q) on the immunophenotype, DNA ploidy status, and proliferative rate of leukemic B‐cells in chronic lymphocytic leukemia

B‐cell chronic lymphocytic leukemia (B‐CLL) is a well‐defined clinical entity with heterogeneous molecular and cytogenetic features. Here, we analyze the impact of trisomy 12, del(13q), del(17p), and del(11q) as determined by interphase fluorescence in situ hybridization analysis of purified neoplastic B‐CLL cells on their immunophenotype, DNA ploidy status and proliferative rate.

Cytogenetic profiles in multiple myeloma and monoclonal gammopathy of undetermined significance: a study in highly purified aberrant plasma cells

Cytogenetic studies in clonal plasma cell disorders have mainly been done in whole bone marrow or CD138+ microbead-enriched plasma cells and suggest that recurrent immunoglobulin heavy chain translocations - e.g. t(4;14) -are primary oncogenetic events. The aim of this study was to determine cytogenetic patterns of highly purified aberrant plasma cells (median purity ≥98%) in different clonal plasma cell disorders. We analyzed aberrant plasma cells from 208 patients with multiple myeloma (n=148) and monoclonal gammopathy of undetermined significance (n=60) for the presence of del(13q14), del(17p13) and t(14q32) using multicolor interphase fluorescence in situ hybridization. Additionally, immunoglobulin heavy chain gene arrangements were analyzed and complementarity determining region 3 was sequenced in a subset of patients and combined multicolor interphase fluorescence in situ hybridization/immunofluorescent protein staining analyses were performed in selected cases to confirm clonality and cytogenetic findings. At diagnosis, 96% of cases with multiple myeloma versus 77% of monoclonal gammopathy of undetermined significance cases showed at least one cytogenetic alteration and/or hyperdiploidy. The cytogenetic heterogeneity of individual cases reflected coexistence of cytogenetically-defined aberrant plasma cell clones, and led to the assumption that karyotypic alterations were acquired stepwise. Cases of multiple myeloma and monoclonal gammopathy of undetermined significance frequently showed different but related cytogenetic profiles when other cytogenetic alterations such as deletions/gains of the immunoglobulin heavy chain or the fibroblast growth factor receptor 3 were additionally considered. Interestingly, in 24% of multiple myeloma versus 62% of monoclonal gammopathy of undetermined significance patients with an immunoglobulin heavy chain translocation, aberrant plasma cells with and without t(14q32) coexisted in the same patient. Our data suggest that recurrent immunoglobulin heavy chain translocations might be absent in the primordial plasma cell clone in a significant proportion of patients with clonal plasma cell disorders carrying these cytogenetic alterations.

Impact of BCR/ABL gene expression on the proliferative rate of different subpopulations of haematopoietic cells in chronic myeloid leukaemia

Despite the effects of BCR ABL on cell proliferation, no study has compared the proliferative rate of different haematopoietic cell compartments from chronic myeloid leukaemia (CML) with those of normal bone marrow (NBM). We comparatively analysed the cell cycle distribution and BCR/ABL expression in different compartments of BM cells from 15 CML and 11 NBM. Overall, our results showed similar proliferative indices in CML patients and NBM. However, CD34+ myeloid precursors from CML patients displayed an increased proportion of S + G2/M‐phase cells (P = 0·04), while no significant differences were found between CML and NBM for other BM cell subsets analysed. In BM cells separated by fluorescence‐activated cell sorting, decreasing levels of BCR/ABL mRNA were found from CD34+/CD38+ myeloid precursors to myeloblasts; BCR/ABL expression increased afterwards with a peak at the myelocyte/metamyelocyte stage, decreasing in the more mature band/neutrophil compartment. Unexpectedly, BCR/ABL gene expression showed an inverse correlation with the proportion of S + G2/M‐phase cells (R = −0·33; P = 0·04). These results suggest that in CML, BCR/ABL expression is associated with an increased proliferation of CD34+ myeloid haematopoietic progenitor cells but not of other more mature myeloid precursors, as confirmed by the observation of an inverse correlation between the amount of BCR/ABL transcripts and the proportion of S + G2/M‐phase cells.

Detailed immunophenotypic characterization of different major and minor subsets of peripheral blood cells in patients with paroxysmal nocturnal hemoglobinuria.

BACKGROUND: Paroxysmal nocturnal hemoglobinuria (PNH) is characterized by a deficient expression of glycosylphosphatidylinositol‐anchored proteins (GPI‐APs), due to somatic mutations of the phosphatidylinositolglycan complementation Class A (PIG‐A) gene.

Immunophenotypical, morphologic, and functional characterization of maturation-associated plasmacytoid dendritic cell subsets in normal adult human bone marrow

BACKGROUND: Information about maturation of plasmacytoid dendritic cell precursors (pre‐pDCs) in normal bone marrow (BM) remains limited.