Susana Barrena

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.

Aberrant expression of tetraspanin molecules in B-cell chronic lymphoproliferative disorders and its correlation with normal B-cell maturation

Tetraspanin proteins form signaling complexes between them and with other membrane proteins and modulate cell adhesion and migration properties. The surface expression of several tetraspanin antigens (CD9, CD37, CD53, CD63, and CD81), and their interacting proteins (CD19, CD21, and HLA-DR) were analyzed during normal B-cell maturation and compared to a group of 67 B-cell neoplasias. Three patterns of tetraspanin expression were identified in normal B cells. The first corresponded to bone marrow CD10+ B-cell precursors (BCP) which showed high expression of CD81 and CD9, low reactivity for CD53 and negativity for CD37. CD10− B-lymphocytes showed downregulation of CD9/CD81 and upregulation of CD53/CD37. Plasma cells showed re-expressed CD9 and downregulated CD37. Hierarchical clustering analysis of flow cytometry immunophenotypic data showed a good correlation between the tumor differentiation stage and the pattern of tetraspanin expression, with all analyzed individual samples classified into three major groups, independently of their normal or neoplastic origin. Despite this, neoplastic B-cells frequently showed aberrantly high/low expression of the different markers analyzed. Interestingly, in B-cell chronic lymphocytic leukemia, abnormal expression of CD53 and CD9 were associated with different patterns of disease infiltration, which would support the role of these molecules on modulating adhesion and migration of neoplastic B cells.

Automated pattern-guided principal component analysis vs expert-based immunophenotypic classification of B-cell chronic lymphoproliferative disorders: a step forward in the standardization of clinical immunophenotyping

Immunophenotypic characterization of B-cell chronic lymphoproliferative disorders (B-CLPD) is becoming increasingly complex due to usage of progressively larger panels of reagents and a high number of World Health Organization (WHO) entities. Typically, data analysis is performed separately for each stained aliquot of a sample; subsequently, an expert interprets the overall immunophenotypic profile (IP) of neoplastic B-cells and assigns it to specific diagnostic categories. We constructed a principal component analysis (PCA)-based tool to guide immunophenotypic classification of B-CLPD. Three reference groups of immunophenotypic data files—B-cell chronic lymphocytic leukemias (B-CLL; n=10), mantle cell (MCL; n=10) and follicular lymphomas (FL; n=10)—were built. Subsequently, each of the 175 cases studied was evaluated and assigned to either one of the three reference groups or to none of them (other B-CLPD). Most cases (89%) were correctly assigned to their corresponding WHO diagnostic group with overall positive and negative predictive values of 89 and 96%, respectively. The efficiency of the PCA-based approach was particularly high among typical B-CLL, MCL and FL vs other B-CLPD cases. In summary, PCA-guided immunophenotypic classification of B-CLPD is a promising tool for standardized interpretation of tumor IP, their classification into well-defined entities and comprehensive evaluation of antibody panels.

Generation of flow cytometry data files with a potentially infinite number of dimensions.

Immunophenotypic characterization of B‐cell chronic lymphoproliferative disorders (B‐CLPD) is associated with the use of increasingly larger panels of multiple combinations of 3 to ≥6 monoclonal antibodies (Mab), data analysis being separately performed for each of the different stained sample aliquots. Here, we describe and validate an automated method for calculation of flow cytometric data from several multicolor stainings of the same cell sample—i.e., the merging of data from different aliquots stained with partially overlapping combinations of Mab reagents (focusing on ≥1 cell populations)—into one data file as if it concerned a single “super” multicolor staining. Evaluation of the performance of the method described was done in a group of 60 B‐CLPD studied at diagnosis with 18 different reagents in a panel containing six different 3‐ and 4‐color stainings, which systematically contained CD19 for the identification of B‐cells. Our results show a high degree of correlation and agreement between originally measured and calculated data about cell surface stainings, providing a basis for the use of this approach for the generation of flow cytometric data files containing information about a virtually infinite number of stainings for each individual cellular event measured in a sample, using a limited number of fluorochrome stainings.

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.

A probabilistic approach for the evaluation of minimal residual disease by multiparameter flow cytometry in leukemic B-cell chronic lymphoproliferative disorders.

Multiparameter flow cytometry has become an essential tool for monitoring response to therapy in hematological malignancies, including B‐cell chronic lymphoproliferative disorders (B‐CLPD). However, depending on the expertise of the operator minimal residual disease (MRD) can be misidentified, given that data analysis is based on the definition of expert‐based bidimensional plots, where an operator selects the subpopulations of interest. Here, we propose and evaluate a probabilistic approach based on pattern classification tools and the Bayes theorem, for automated analysis of flow cytometry data from a group of 50 B‐CLPD versus normal peripheral blood B‐cells under MRD conditions, with the aim of reducing operator‐associated subjectivity. The proposed approach provided a tool for MRD detection in B‐CLPD by flow cytometry with a sensitivity of ≤8 × 10−5 (median of ≤2 × 10−7). Furthermore, in 86% of B‐CLPD cases tested, no events corresponding to normal B‐cells were wrongly identified as belonging to the neoplastic B‐cell population at a level of ≤10−7. Thus, this approach based on the search for minimal numbers of neoplastic B‐cells similar to those detected at diagnosis could potentially be applied with both a high sensitivity and specificity to investigate for the presence of MRD in virtually all B‐CLPD. Further studies evaluating its efficiency in larger series of patients, where reactive conditions and non‐neoplastic disorders are also included, are required to confirm these results.

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.

Quantitative analysis of bcl-2 expression in normal and leukemic human B-cell differentiation

Lack of apoptosis has been linked to prolonged survival of malignant B cells expressing bcl-2. The aim of the present study was to analyze the amount of bcl-2 protein expressed along normal human B-cell maturation and to establish the frequency of aberrant bcl-2 expression in B-cell malignancies. In normal bone marrow (n=11), bcl-2 expression obtained by quantitative multiparametric flow cytometry was highly variable: very low in both CD34+ and CD34− B-cell precursors, high in mature B-lymphocytes and very high in plasma cells. Bcl-2 expression of mature B-lymphocytes from peripheral blood (n=10), spleen (n=8) and lymph node (n=5) was significantly higher (P<0.02) in CD23− as compared to CD23+ B cells, independent of the type of tissue analyzed. Upon comparison with normal human B-cell maturation, bcl-2 expression in neoplastic B cells from 144 patients was found to be aberrant in 66% of the cases, usually corresponding to bcl-2 overexpression (63%). Follicular lymphoma (FL) carrying t(14;18) and MALT lymphoma were the only diagnostic groups constantly showing overexpression of bcl-2. Bcl-2 overexpression was also frequently found in precursor B-acute lymphoblastic leukemia (84%), typical (77%) and atypical (75%) B-cell chronic lymphocytic leukemia, prolymphocytic leukemia (two of three cases), mantle cell lymphoma (55%), but not in t(14;18)− FL, splenic marginal zone lymphoma, Burkitt lymphoma and multiple myeloma.

Flow cytometry immunophenotyping of fine-needle aspiration specimens: utility in the diagnosis and classification of non-Hodgkin lymphomas

Barrena S, Almeida J, García‐Macias M D C, López A, Rasillo A, Sayagués J M, Rivas R A, Gutiérrez M L, Ciudad J, Flores T, Balanzategui A, Caballero M D & Orfao A
(2011) Histopathology 58, 906–918
Flow cytometry immunophenotyping of fine‐needle aspiration specimens: utility in the diagnosis and classification of non‐Hodgkin lymphomas

CD34+ Cells from Acute Myeloid Leukemia, Myelodysplastic Syndromes, and Normal Bone Marrow Display Different Apoptosis and Drug Resistance–Associated Phenotypes

Myelodysplastic syndromes and acute myeloid leukemia (AML) are heterogeneous disorders in which conflicting results in apoptosis and multidrug resistance (MDR) have been reported. We have evaluated by multiparameter flow cytometry the expression of apoptosis- (APO2.7, bcl-2, and bax) and MDR-related proteins [P-glycoprotein (P-gp), multidrug resistance protein (MRP), and lung resistance protein (LRP)] specifically on bone marrow (BM) CD34+ cells, and their major CD32−/dim and CD32+ subsets, in de novo AML (n = 90), high-risk myelodysplastic syndrome (n = 9), and low-risk myelodysplastic syndrome (n = 21) patients at diagnosis, and compared with normal BM CD34+ cells (n = 6). CD34+ myeloid cells from AML and high-risk myelodysplastic syndrome patients displayed higher expression of bcl-2 (P < 0.0001) and lower reactivity for APO2.7 (P = 0.002) compared with low-risk myelodysplastic syndrome and normal controls. Similar results applied to the two predefined CD34+ myeloid cell subsets. No significant differences were found in the expression of P-gp, MRP, and LRP between low-risk myelodysplastic syndrome patients and normal BM, but decreased expression of MRP (P < 0.03) in AML and high-risk myelodysplastic syndromes and P-gp (P = 0.008) in high-risk myelodysplastic syndromes were detected. Hierarchical clustering analysis showed that low-risk myelodysplastic syndrome patients were clustered next to normal BM samples, whereas high-risk myelodysplastic syndromes were clustered together and mixed with the de novo AML patients. In summary, increased resistance to chemotherapy of CD34+ cells from both AML and high-risk myelodysplastic syndromes would be explained more appropriately in terms of an increased antiapoptotic phenotype rather than a MDR phenotype. In low-risk myelodysplastic syndromes abnormally high apoptotic rates would be restricted to the CD34− cell compartments.