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Multiparameter Phenotype Mapping of Normal and Post-Chemotherapy B Leukemia (1997) 11, 1266–1273 1997 Stockton Press All rights reserved 0887-6924/97 $12.00 Multiparameter phenotype mapping of normal and post-chemotherapy B lymphopoiesis in pediatric bone marrow MN Dworzak, G Fritsch, C Fleischer, D Printz, G Fro¨schl, P Buchinger, G Mann and H Gadner Children’s Cancer Research Institute, St Anna Kinderspital, Kinderspitalgasse 6, A-1090 Vienna, Austria We studied the differentiation profiles of B cell precursors Materials and methods (BCP) in normal and post-chemotherapy pediatric bone marrow (BM) using multiparameter flow cytometry. The goal of our study was to draw a comprehensive phenotypic map of the Sample description three major maturational BCP stages in BM. By correlating lin- eage-associated markers, CD45RA, and several adhesion mol- Pediatric bone marrow (BM) samples (n = 44) were obtained ecules, the stage-specific patterns were found to differ in cer- from children undergoing diagnostic BM aspiration for the fol- tain details from previously published concepts. Among the earliest BCP, a subset of CD34+CD10lo precursors was repeat- lowing diagnoses in the absence of BM involvement at the edly observed in addition to the well characterized time of immunologic investigation: suspicion of storage dis- CD34+CD10hiCD19+ majority of cells. Only two-thirds of these ease (n = 1), bacterial lymphadenitis (n = 1), neuroblastoma CD34+CD10lo cells expressed CD19. However, uniformity of (n = 1), severe aplastic anemia in remission (n = 2), single sys- phenotypic features, absence of T lineage markers, and the tem Langerhans cell histiocytosis (n = 1), idiopathic throm- regeneration kinetics after chemotherapy suggest the B lineage = + lo bocytopenic purpura (n 1), early stage (localized) non- affiliation of the CD34 CD10 precursors in general. In the more = = mature BCP, expression of CD10, CD20, cytoplasmic and sur- Hodgkin lymphoma (T-NHL: n 2, B-NHL: n 1) as well as face m chains (cm and sm) was observed to overlap more than acute leukemia in follow-up with confirmed complete clinical previously recognized. We found that CD20 and cm appear remission at the time of immunologic investigation (B lineage early during B cell ontogeny (already on CD34+ BCP), and that ALL: n = 32, T-ALL: n = 1, AML: n = 1). The median age of the CD10 is lost late, following the onset of sm expression. Differ- 2 patients at BM aspiration was 512 years (range: 5 months to ences between normal and post-chemotherapy BM specimens 7 1612 years). Among the leukemia/lymphoma patients after regarding the phenotypic appearance of BCP were exclusively = due to differences in the subset composition, as post-chemo- chemotherapy (n 35), the median time from remission to BM 3 therapy samples showed a preponderance of immature stages. sampling was 11 months (range: 2 months to 412 years). The Our observations may build a framework for comparing leu- five patients with the shortest time from remission to BM aspir- kemic cells with their normal counterparts to define possible ation (1 or 2 months) have subsequently stayed event free until leukemia-associated aberrations useful for residual disease the last evaluation, 10 months to 1 7 years later. Only two studies. 12 Keywords: B cell precursor; lymphopoiesis; bone marrow; pheno- patients have relapsed after the immunologic evaluation type; flow cytometry (AML) M2 1 month later, C-ALL 4 months later). The median observation time from BM aspiration to final evaluation was 2 6 112 years (range: 1 month to 212 years). The two patients with the shortest follow-up (1 and 2 months) have been in con- Introduction 3 firmed remission for 312 years and 4 months, respectively. All leukemia/lymphoma patients described in this paper were The mainstays in defining the phenotypic patterns of B cell treated according to BFM (Berlin–Frankfurt–Munster) treat- differentiation have already been documented during the last ¨ ment protocols. In brief, for ALL a multi-agent induction ther- two decades.1–4 In brief, the CD19 antigen is expressed apy administered in three cycles over the first six months was throughout all stages of B cell maturation,1,5 while CD10 is highly expressed in the most immature (CD34+) B cell precur- followed by a low-dose maintenance therapy until completion sors (BCP),3 and is lost at a later stage concomitantly with gain of the second therapy year. All data concerning these patients of surface expression of CD20, IgM and CD22.2 More were obtained from the Austrian study center of the recently, this classical model of B cell differentiation was chal- international BFM study group. lenged, as new antigenic patterns,6–8 new insights into the sequence of antigen acquisition in B cell ontogeny,9 and tissue-related differences in the coordinate patterns of B Antibodies lymphoid antigen expression were documented.10,11 In the present study, our aim was comprehensively to rede- Fluorescein isothiocyanate (FITC)-, and phycoerythrin (PE)- fine the phenotypic patterns of the early stages of normal B labeled, or unconjugated pure monoclonal antibodies cell differentiation in postnatal bone marrow by multi- (MoAbs) were used: CD7 (DK24-FITC), CD10 (SS2/36-PE), parameter flow cytometry. By comparing normal and post- CD11a (MHM24 pure), CD19 (HD37-FITC, pure), CD20 (B- chemotherapy bone marrow, we focused on sample-inherent Ly1-FITC), CD44 (DF1485 pure) and CD45 (T29/33-FITC) differences in BCP phenotype and subset composition. Our were all from Dako (Glostrup, Denmark); CD34 (HPCA2- approach could be of particular interest for the development FITC, -PE) fom Becton Dickinson (BD; Sunnyvale, CA, USA); of techniques for immunologic residual disease investigations CD7 (3A1-RD1-PE) and CD45RA (2H4-RD1-PE, pure) from in B lineage acute lymphoblastic leukemia (ALL). Coulter Immunology (Krefeld, Germany); CD49d (HP2/1 pure), and anti-human TdT (H Tdt-1, 3, 4-FITC) from Immuno- tech (Marseille, France); CD49e (P1D6 pure) from Oncogene Correspondence: MN Dworzak Sciences (Cambridge, MA, USA); CD99 (culture supernatant Received 20 September 1996; accepted 10 April 1997 of the hybrid cell line 12E7) from R Levy (Stanford University, Phenotypic map of B lymphopoiesis in pediatric BM MN Dworzak et al 9 1267 CA, USA). FITC-conjugated F(ab )2 fragments of polyclonal Results rabbit anti-human m-heavy-chain antibodies were from Dako. Definition and quantification of BCP stages Immunofluorescence staining procedure The BCP content of the BM specimens was estimated by stain- ing with CD19. A median of 16% of MNC was CD19+ BCP Mononuclear cells (MNC) were isolated from the BM speci- = mens by density gradient centrifugation and washed twice in (range 2.9–47.6%, n 32). For a further subdivision, CD19 RPMI 1640 medium containing 2% fetal calf serum (FCS; both was correlated with CD45RA, which has been shown from Gibco, Paisley, UK) prior to immunofluorescence labe- (together with CD45) to define three major stages of BCP due 12 ling. Staining protocols employed three-color investigations in to differences in antigen density. CD99 was added especially for the definition of the most immature BCP (stage all experiments. All antibodies were used at concentrations + +++ × 5 1, CD45RA CD99 ; Figure 1, black) as reported pre- titrated for optimal staining. In brief, 1 to 2 10 MNC in 13,14 conventional analyses as well as at least 5 × 105 cells per viously. The next developmental stage (BCP 2, blue) was CD45RA++CD99+/++, and the most mature stage (BCP 3, red) analysis in gating experiments, were first incubated with the +++ + ++ 9 CD45RA CD99( )/ (see Figure 1). According to this subdiv- unconjugated MoAbs and then with biotinylated rabbit F(ab )2 anti-mouse Ig antibodies (from Dako). To exclude non-specific ision, any difference in the marker profiles of BCP from differ- staining via unsaturated binding sites of the second step ent BM specimens could be attributed exclusively to differ- reagent, this was followed by incubation with unlabeled irrel- ences in the subset composition. We found that specimens of ALL patients in remission and regenerating after induction evant murine IgG1 (MOPC21; Sigma, St Louis, MO, USA). Without washing, the directly labeled CD MoAbs were then therapy, were reduced in their total B cell content, but had a added simultaneously and together with streptavidin-R670 considerable relative expansion of stage 1 BCP (Table 1). (Gibco). Murine irrelevant isotype-matched fluorescent Patients at the end of low-dose maintenance chemotherapy as 9 well as the non-leukemic probands (without any prior MoAbs as well as irrelevant F(ab )2 fragments of rabbit Ig-FITC (all from Dako) were always included in the staining protocols chemotherapy) showed a preponderance of more mature as negative controls. Incubations were performed at 4°Cin stages. Due to the relatively small number of normal BM the dark over 30 min and were followed by washing steps donors, age-related differences were not assessed. using PBS (0.13 mol/l NaCl, 7 mmol/l Na2HPO4, 3 mmol/l NaH2PO4) with 2% FCS. Cellular permeabilization for the investigation of TdT and cytoplasmic m-chain expression was Phenotypic analysis of stage 1 BCP cells performed using a commercially available formaldehyde- 3 based erythrocyte lysing solution (BD) supplemented with Tri- The earliest B cells in the BM express CD10 and CD34. In ton-X (0.015%). The concentration of the latter had been accordance, these markers were found on the corresponding titrated for optimal permeabilization together with a good BCP subsets in our analysis (stage 1). A majority of stage 1 preservation of scattering and fluorescence properties. The cells typically displayed high levels of CD10 positivity (stage permeabilization step was always done after labeling of sur- 1B; Figure 2, green subset). However, precursor cells (11.5% = face antigens. Prior to flow cytometry, cell suspensions were median of total BCP 1; range 0–41%, n 19) with consider- passed through a 30 mm mylon mesh (Swiss Silk Bolting Mfg, ably lower expression of CD10 were also observed (Figure 2, lo + Zurich, Switzerland).
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