Er van Wering

Flow cytometric analysis of normal B cell differentiation: a frame of reference for the detection of minimal residual disease in precursor-B-ALL.

During the last two decades, major progress has been made in the technology of flow cytometry and in the availability of a large series of monoclonal antibodies against surface membrane and intracellular antigens. Flow cytometric immunophenotyping has become a diagnostic tool for the analysis of normal and malignant leukocytes and it has proven to be a reliable approach for the investigation of minimal residual disease (MRD) in leukemia patients during and after treatment. In order to standardize the flow cytometric detection of MRD in acute leukemia, a BIOMED-1 Concerted Action was initiated with the participation of six laboratories in five different European countries. This European co-operative study included the immunophenotypic characterization and enumeration of different precursor and mature B cell subpopulations in normal bone marrow (BM). The phenotypic profiles in normal B cell differentiation may form a frame of reference for the identification of aberrant phenotypes of precursor-B cell acute lymphoblastic leukemias (precursor-B-ALL) and may therefore be helpful in MRD detection. Thirty-eight normal BM samples were anal- yzed with five different pre-selected monoclonal antibody combinations: CD10/CD20/CD19, CD34/CD38/CD19, CD34/ CD22/CD19, CD19/CD34/CD45 and TdT/CD10/CD19. Two CD19− immature subpopulations which coexpressed B cell-associated antigens were identified: CD34+/CD22+/CD19− and TdT+/CD10+/CD19−, which represented 0.11u2009±u20090.09% and 0.04u2009±u20090.05% of the total BM nucleated cells, respectively. These immunophenotypes may correspond to the earliest stages of B cell differentiation. In addition to these minor subpopulations, three major CD19+ B cell subpop- ulations were identified, representing three consecutive maturation stages; CD19dim/CD34+/TdT+/CD10bright/CD22dim/ CD45dim/CD38bright/CD20− (subpopulation 1), CD19+/CD34−/ TdT−/CD10+/CD22dim/CD45+/CD38bright/CD20dim (subpopulation 2) and CD19+/CD34−/TdT−/CD10−/CD22bright/CD45bright/CD38dim/ CD20bright (subpopulation 3). The relative sizes of subpopulations 1 and 2 were found to be age related: at the age of 15 years, the phenotypic precursor-B cell profile in BM changed from the childhood x91immature profile (large subpopulations 1 and 2/small subpopulation 3) to the adult x91mature profile (small subpopulation 1 and 2/large subpopulation 3). When the immunophenotypically defined precursor-B cell subpopulations from normal BM samples are projected in fluorescence dot-plots, templates for the normal B cell differentiation pathways can be defined and so-called x91empty spaces where no cell populations are located become evident. This allows discrimination between normal and malignant precursor-B cells and can therefore be used for MRD detection.

BIOMED-I concerted action report: flow cytometric immunophenotyping of precursor B-ALL with standardized triple-stainings. BIOMED-1 Concerted Action Investigation of Minimal Residual Disease in Acute Leukemia: International Standardization and Clinical Evaluation.

The flow cytometric detection of minimal residual disease (MRD) in precursor-B-acute lymphoblastic leukemias (precursor-B-ALL) mainly relies on the identification of minor leukemic cell populations that can be discriminated from their normal counterparts on the basis of phenotypic aberrancies observed at diagnosis. This technique is not very complex, but discordancies are frequently observed between laboratories, due to the lack of standardized methodological procedures and technical conditions. To develop standardized flow cytometric techniques for MRD detection, a European BIOMED-1 Concerted Action was initiated with the participation of laboratories from six different countries. The goal of this concerted action was to define aberrant phenotypic profiles in a series of 264 consecutive de novo precursor-B-ALL cases, systematically studied with one to five triple-labelings (TdT/CD10/CD19, CD10/CD20/CD19, CD34/CD38/CD19, CD34/CD22/CD19 and CD19/CD34/CD45) using common flow cytometric protocols in all participating laboratories. The use of four or five triple-stainings allowed the identification of aberrant phenotypes in virtually all cases tested (127 out of 130, 98%). These phenotypic aberrancies could be identified in at least two and often three triple-labelings per case. When the analysis was based on two or three triple-stainings, lower incidences of aberrancies were identified (75% and 81% of cases, respectively) that could be detected in one and sometimes two triple-stainings per case. The most informative triple staining was the TdT/CD10/CD19 combination, which enabled the identification of aberrancies in 78% of cases. The frequencies of phenotypic aberrations detected with the other four triple-stainings were 64% for CD10/CD20/CD19, 56% for CD34/CD38/CD19, 46% for CD34/CD22/CD19, and 22% for CD19/CD34/CD45. In addition, cross-lineage antigen expression was detected in 45% of cases, mainly coexpression of the myeloid antigens CD13 and/or CD33 (40%). Parallel flow cytometric studies in different laboratories finally resulted in highly concordant results (>90%) for all five antibody combinations, indicating the high reproducibility of our approach. In conclusion, the technique presented here with triple-labelings forms an excellent basis for standardized flow cytometric MRD studies in multicenter international treatment protocols for precursor-B-ALL patients.

BIOMED-1 Concerted Action report : Flow cytometric characterization of CD7+ cell subsets in normal bone marrow as a basis for the diagnosis and follow-up of T cell acute lymphoblastic leukemia (T-ALL)

The European BIOMED-1 Concerted Action was initiated in 1994 to improve and standardize the flow cytometric detection of minimal residual disease (MRD) in acute leukemia (AL). Three different protocols were defined to identify the normal subsets of B, T and myeloid cells in bone marrow (BM), and were applied to the different types of AL in order to study aberrant immunophenotypes. Using sensitive acquisition methods (‘live gate’) T cell subsets in normal BM could be identified with five triple-stains: CD7/CD5/CD3, CD7/CD4/CD8, CD7/CD2/CD3, CD7/CD38/CD34 and TdT/CD7/surface or cytoplasmic (cy)CD3 (antibodies conjugated with FITC/PE/PECy5 or PerCP, respectively). The identification of T cell subsets in BM allowed definition of ‘empty spaces’ (ie areas of flow cytometric plots where normally no cells are found). All studied T-ALL cases (n = 65) were located in ‘empty spaces’ and could be discriminated from normal T cells. The most informative triple staining was TdT/CD7/cyCD3, which was aberrant in 91% of T-ALL cases. In most cases, two or more aberrant patterns were found. Apparently the immunophenotypes of T-ALL differ significantly from normal BM T cells. This is mostly caused by their thymocytic origin, but also the neoplastic transformation might have affected antigen expression patterns. Application of the five proposed marker combinations in T-ALL contributes to standardized detection of MRD, since cells persistent or reappearing in the ‘empty spaces’ can be easily identified in follow-up BM samples during and after treatment.

Differences in Immunoglobulin and T-Cell Receptor Gene Rearrangement Patterns in Acute Lymphoblastic Leukemia Between Diagnosis and Relapse

Rearrangements of immunoglobulin (Ig) and T-cell receptor (TcR) genes can be used to establish clonality in lymphoproliferative diseases [1–3]. Rearrangements and/or deletions of Ig heavy chain (IgH) genes occur in about 98% of precursor-B-acute lymphoblastic leukemia (ALL) and in 10 to 15% of T-ALL, whereas rearrangements and/or deletions of Ig light chain (IgL) genes [Ig kappa chain (Igκ) or Ig lambda chain (Igλ)] are reported to occur in 5 to 50% of precursor-B-ALL and in 0% of T-ALL [4–7]. Rearrangements and/or deletions of TcR-β, TcR-γ, and TcR-δ genes were found in 33%, 55%,and 80% of precursor-B-ALL, and in 89%, 91%, and 96% of T-ALL, respectively [4,5].

Molecular Biology of Acute Lymphoblastic Leukemia: Implications for Detection of Minimal Residual Disease

Acute lymphoblastic leukemias (ALL) are characterized by high frequencies of clonal chromosome aberrations (ploidy aberrations and translocations) as well as by clonal rearrangements of immunoglobulin (Ig) and T-cell receptor (TcR) genes. These two types of clonal molecular characteristics can be used as leukemia-specific markers for detection of minimal residual disease (MRD) by use of polymerase chain reaction (PCR) technology.