Studies on B Cell Precursors

Fine characterization of childhood and adult acute lymphoblastic leukemia (ALL) by a proB and preB surrogate light chain-specific mAb and a proposal for a new B cell ALL classification

Abstract

The expression of the surrogate light chain (ΨL) – made of the λ-like (or λ5) and the VpreB proteins – is a B cell-specific maturation marker. Using an anti-human VpreB mAb (4G7), we recently identified in human normal bone marrows, proB and preB cells that express the ΨH–ΨL proB (proBCR) and the μ-ΨL preB (preBCR) receptors, respectively. In the present study, FACS and biochemical analysis confirm the broad proB and preB reactivity of the 4G7 mAb that contrasts with the narrow specificity of other available anti-ΨL reagents for preB cells. This mAb was used to explore intracytoplasmic and cell surface expression of the VpreB protein on a series of 92 precursor B cell ALLs (from 40 child and 52 adult patients), in combination with 24 other mAbs. The major result concerns the identification within proB (or BI) and common (or BII) ALLs, of proBCR and proBCR+ ALLs that express the VpreB in the cytoplasm or at the cell surface, respectively. The percentage of ALLs within these two VpreB sub-groups differ considerably between the ALL origin. In the pediatric series, ALLs present in the majority a proBCR+ phenotype whereas we observed a proBCR phenotype for adult ALLs. Based on VpreB expression, and in combination with other published data, we propose a refined classification for precursor B cell ALLs.

Introduction

In adult bone marrow, the intermediates of normal B cell differentiation, proB, preB, immature B and mature B cells are well characterized by cell surface marker expression, morphological criteria and by the status of Ig gene rearrangements. The differentiation processes are strictly coordinated and at least two ‘quality control’ checkpoints exist, one dependent on the preB receptor (preBCR) at the transition from large to small preB cells1 and the second due to the B cell receptor (BCR), at the immature B cell stage.2 PreB and B cells are submitted to positive selection for survival, whereas in immature B cells receptor editing and negative selection occurs to eliminate autoreactive B cells.3

PreB cells (CD34 CD10+ CD19++ TdT) express a small amount of the μ chain at the cell surface in association with the surrogate light chain (ΨL) and the Igα-Igβ transducing module to form the so-called preBCR. The ΨL is composed of two polypeptides encoded by λ-like45 (or λ5 in mice6) and VpreB78 genes, related to the Cλ and Vλ Ig light chain domains, respectively. It is now well established that the preBCR is required for: (1) the transition from large to small preB; (2) the repertoire selection of preB cells;91011 (3) the amplification of this compartment; and (4) the control of allelic exclusion at the H chain locus.12 A signal transduction activity of this receptor, that is expressed at a very low level at the surface of preB cells, has been clearly demonstrated.1314

ProB cells (CD34+ CD10+ CD19+ TdT+) also produce ΨL but discrepancies exist in the literature about the cell surface expression of the ΨL in such cells.1516171819 Recently, by using a new series of anti-human VpreB mAbs, we definitively established the existence of surface ΨL chain in sub-populations of both proB and preB cells and we identified the nature of the cell surface ΨH–ΨL proB cell complex (proBCR).20 Moreover, by comparison with anti-surrogate light chain mAbs, SLC1/SLC21516 from Cooper's group, we showed that discrepancies regarding ΨL cell surface expression at the proB cell stage, were due to subtle differences in mAb specificity.20

B acute lymphoblastic leukemias (ALLs) represent 75–80% and 85–90% of adult and childhood ALLs, respectively. Their diagnosis is based on morphological, cytochemical and immunological features. The French–American–British (FAB) group2122 established a morphological classification and identified three subgroups, L1, L2 and L3; most ALLs are of subgroup L1. Immunophenotypic classification of B ALLs uses a panel of mAbs that identify the maturation stage of leukemic cells. The European Group for the Immunological Characterization of Leukemias (EGIL) classifies B ALLs (that express CD19 and/or CD79a and/or CD22, but at least two of these markers) in four subgroups according to CD10, μ and κ/λ Ig expression:23 (1) BI ALLs (10% and 5% of adult and childhood ALLs, respectively), that correspond to proB ALLs, are CD19+ and CD10; (2) BII ALLs (the major immunological subtype in both adult and childhood ALLs) also referred to as ‘common ALLs’, co-express CD19 and CD10; (3) BIII ALLs (10% and 15% of adult and childhood ALLs, respectively) that correspond to preB ALLs, are characterized by the expression of cytoplasmic μ chains and either do or do not express CD10; and (4) BIV ALLs (4% of adult and 3% of childhood ALLs) or mature B ALLs in which there is expression of surface IgM or cytoplasmic κ or λ Ig chains, with or without co-expression of CD10.

Karasuyama's group recently described three new anti-ΨL mAbs (against λ-like, VpreB or the μ-ΨL preBCR) that were used to characterize childhood ALLs.24 Different preB ALLs can be distinguished based on the expression of the cell surface preBCR, leading to a new ALL classification composed of five subgroups: ProBI, ProBII, PreB preBCR, preB preBCR+ and mature B ALLs. In the present paper, using the 4G7 anti-VpreB mAb that recognizes the preBCR but also the proBCR,20 we have explored ΨL expression in both childhood and adult B ALLs in order to improve the diagnosis of precursor B cell ALLs. Our data allowed us to split proB (or BI) and common (or BII) ALLs, ie the major immunological subtype of ALLs, into two new subgroups based on the cell surface expression of the proBCR. In combination with data from Karasuyama's group, a new refined B ALL classification is proposed.

Materials and methods

Flow cytometric analysis of cell lines

RS4.1125 and JEA217 are μ proB cell lines. NALM626 and Namalwa (NWA) are μ+ preB and a μ++ mature B cell lines, respectively. Cells were maintained at 37°C and 7% CO2 in RPMI medium supplemented with penicillin, streptomycin, 10% FCS, 2 mM L-glutamine and 1 mM sodium pyruvate. The JEA2 proB cell line was cultivated for 4 days in RPMI medium containing 20 ng/ml of recombinant human IL-7 (Immugenex Corporation, Los Angeles, CA, USA).

For direct staining of cell lines, 106 cells were incubated at 4°C for 20 min in 70 μl of PBS, 0.2% BSA, 0.1% sodium azide and 10% normal mouse serum containing 0.25 μg of PE- labeled anti-VpreB 4G7, as previously described.20 PE-labeled IgG1 (Immunotech, Marseille, France) served as isotype-matched control mAb. For indirect staining with HSL11 (anti-λ-like), HSL96 (anti-VpreB) and HSL2 (anti-preBCR)24 we used 3, 6.25 and 25 μg/ml of mAbs, respectively, in the same incubation conditions. Anti-HEL IgG1 (kindly provided by P Machy, CIML, Marseille, France) served as isotype-matched control mAb. Staining was revealed with a PE-labeled goat F(ab′)2 anti-mouse IgG+IgM (H+L) (Jackson Immunoresearch, West Grove, PA, USA). Direct staining of cell surface μ chain was achieved using FITC-labeled F(ab′)2 polyclonal rabbit anti-human IgM and compared with a FITC-conjugated F(ab′)2 rabbit negative control (Dakopatts, Glostrup, Denmark). Stained cells were analyzed using a Becton Dickinson (Immunocytometry Systems, CA, USA) FACScan device.

Cell surface biotinylation and immunoprecipitation

Viable cells (50 × 106) in 2 ml ice-cold PBS were incubated with 1 mg of Sulfo-NHS-LC-Biotin (Pierce, Rockford, IL, USA), 30 min at 49°C. After five washes with ice-cold PBS, labeled cells were lysed with 1 ml of NP-40 lysis buffer (1% NP-40, 150 mM NaCl, 20 mM Tris pH8, 1 mM PMSF, pepstatin, leupeptin, antipain and iodoacetamide). After three successive incubations with 100 μl of protein A-Sepharose saturated with 4% nonfat milk, cell lysates were incubated for 6 h at 4°C with the indicated antibodies (10 μg) preadsorbed overnight on protein G Sepharose (50 μl). Immunoprecipitates were washed, suspended in 70 μl of reducing Laemmli sample buffer, boiled for 10 min, and subjected to SDS-PAGE (5–15%) in reducing conditions. The proteins were transferred to Immobilon-P membranes (Millipore Corporation, Bedford, MA, USA). After blocking with 5% BSA in PBST (PBS, 0.1% Tween 20) for 1 h, membranes were revealed by incubation with a 1:5000 dilution of streptavidin-HRPO (Amersham, Buckingham, UK).

Immunoprecipitations were performed using mouse anti-IgM mAb (Immunotech, Marseille, France), anti-VpreB (4G7) and anti-HEL IgG1 as an irrelevant control. In Western blot experiments, the VpreB was detected by the anti-VpreB 4G7 mAb followed by HRPO-conjugated goat anti-mouse IgG (Sigma, Saint Quentin Fallavier, France), as previously described.2027

Patient samples

The diagnosis of ALLs was based on conventional morphological and cytochemical studies on blood or bone marrow samples, according to the French–American–British (FAB) criteria.2122 This study was mainly performed retrospectively on 92 ALLs selected on the availability of cellular material, but also includes fresh consecutive ALLs. We analyzed 40 childhood ALLs, 19 females/21 males, median age of 8 years (1 to 16), median WBC of 47.7 × 109/L (1.98–800 × 109/l). They were all enrolled in the current FRALLE 93 therapeutic protocol.28 We explored 52 adult ALLs, 23 females/29 males, median age of 45 years (18 to 83), median WBC of 76 × 109/l (2–816 × 109/l). They were all enrolled in the current LALA94 therapeutic protocol,29 except for patient No. 25 owing to his advanced age.

Immunophenotyping analysis of patient samples

Immunological features were studied by cytofluorometry using direct immunofluorescence as previously described with a panel of 24 mAbs.30 Immunophenotyping was performed on bone marrow samples after Ficoll–Hypaque gradient or on total peripheral blood after red blood cells lysis when an individual's blast cell count was greater than 50 × 109/l.

FITC or PE-conjugated mAbs recognizing MHC class I, class II, CD34, lymphoid T antigens CD1a, CD2, CD3, CD4, CD5, CD7, CD8, lymphoid B antigens CD10, CD19, CD20, CD22, CD24, κ, λ, μ chains and myeloid antigens CD13, CD33, CD14, CD15, MPO provided by Immunotech and Dako (Trappes, France) were used. Surface and cytoplasmic VpreB chain expression (sVpreB and cVpreB, respectively) were evaluated with PE-labeled anti-VpreB (4G7) mAb using 0.25 μg/5 × 105 cells. Intracytoplasmic VpreB staining was performed in sVpreB-negative ALLs after permeabilization with IntraPrep Permeabilization Reagent (Immunotech). PE-labeled IgG1 (Immunotech) served as isotype-matched control mAb.

Fluorescent cells were analyzed on a FACScan (Becton Dickinson, Immunocytometry Systems, CA, USA) cytometer for adult samples and on an EPICS XL (Coultronics, Miami, FL, USA) cytometer for children's samples. A minimum of 5000 cells was gated on forward and side scatter parameters and at least 80% of blast cells were analyzed on each sample. For recent cases, blast cells were gated according to CD45 expression. A percentage of labeled cells above 5% was considered as positive. In all cases, negative controls included mouse irrelevant IgG1 or IgG2a mAbs, directly conjugated to FITC or PE.

RT-PCR analysis of fusion transcripts

ALLs samples were analyzed for genetic alterations by systematic karyotyping and RT-PCR screening. A search for five fusion transcripts was performed by RT-PCR: MLL-AF4,31 TEL-AML1,32 E2A-PBX133 and BCR-ABL for both minor (m) and major (M) breakpoint translocations.34 From January 1998 onwards, we used the primer combinations designed during the BIOMED 1 concerted action.35 RNA isolation, reverse transcription and polymerase chain reaction conditions were performed as previously described.36 TEL-AML1 positivity was always confirmed on two different experiments with two different samples and/or by fluorescence in situ hybridization (FISH) analysis. For MLL-AF4, E2A-PBX1 and BCR-ABL positive patients, there was a complete concordance with the karyotype.

Statistics

For statistical evaluation, the Student's t-test was used. A P value <0.05 was considered significant.

Results

Comparison of the fine specificity of anti-surrogate light chain mAbs

We recently reported that the 4G7 anti-human VpreB mAb was able to detect cell surface expression of the ΨL on both proB and preB cell lines and on the corresponding cells in normal bone marrow.20 Biochemical analysis performed with this mAb reveals both the ΨH–ΨL proB cell complex (proBCR) and the μ-ΨL preB receptor (preBCR).20 Tsuganezawa et al24 have also produced anti-human ΨL reagents by immunizing mouse against a preBCR (mouse μ and human ΨL hybrid). HSL11 and HSL96 react with λ-like (or λ5) and VpreB, respectively, whereas HSL2 does not bind to individual components but does bind to the completely assembled preBCR. All three mAbs detected preBCR but not proBCR surface expression. As these reagents were tested in a series of proB cell lines different from ours, in particular those that we have found to be positive with the 4G7 anti-VpreB mAb, we first compared the fine specificity of HSL96, HSL2 and HSL11 to that of 4G7 on different early cell lines. As previously reported independently by the two groups,2024 the four mAbs allowed the detection of the expression of the ΨL on NALM6, a typical μ-positive preB cell line, but not on the RS4.11, a μ-negative proB cell line (Figure 1). We did not observe any labeling by the HSL series on the JEA2 proB cell line even when these cells were cultivated for 4 days in the presence of IL7, a treatment that considerably enhances ΨL chain surface expression, as detected by the 4G7 mAb. These results are in agreement with the preB (μ-ΨL) conformational nature of the epitope recognized by the HSL2 mAb.24 For the HSL96 and HSL11, although these mAbs immunoprecipitated free VpreB or λ-like proteins, respectively24 it seems that their respective epitopes are lost in a proB cell configuration, ie when the ΨL is associated with the ΨH chain complex. This point was further analyzed by using a biochemical approach.

Figure 1
figure1

 Surface FACS analysis of human cell lines using anti-surrogate light chain and anti-μ mAbs. ProB (RS4.11, JEA2), preB (NALM6) and B (NAMALWA, NWA) cell lines were analyzed using anti-VpreB (4G7, HSL96), anti-λ-like or λ5 (HSL11), anti-preBCR (HSL2) and anti-μ mAbs. The 4G7 mAb was PE-labeled whereas HSL96, 11 and 224 were indirectly revealed using a PE-conjugated goat F(ab′)2 anti-mouse IgG+IgM (H+L). Staining profiles with these mAbs (shaded figures) and staining profiles with isotype-matched control mAbs (open figures) were overlaid in the histograms. JEA2 cells were also cultured with 20 ng/ml of IL-7 for 4 days before being analyzed (row 3). The 4G7 anti-VpreB labeling is completely inhibited by 2 μg/ml of the relevant COOH-terminal VpreB peptide.20

Biotinylated cell surface proteins from JEA2 and NALM6 cell lines were immunoprecipitated using anti-ΨL, anti-μ and irrelevant γ mAbs, submitted to SDS-PAGE, transferred to a membrane filter and revealed by streptavidin-HRPO (Figure 2). As previously reported,20 the 16 kDa VpreB protein (faintly labeled but clearly visible following immunoblotting), the λ-like component at 20 kDa, a strongly biotinylated p105 and a faintly labeled p130 protein were immunoprecipitated from the JEA2 cell line, using the anti-VpreB (4G7) mAb. By contrast, under the same conditions the three HSL mAbs did not reveal either the p105 or the p130 protein on JEA2 cells. Using the anti-λ-like (HSL11) and the anti-VpreB (HSL96) mAbs, trace amounts of the ΨL components were detected and this was also true, after long exposure, for the ‘conformational preB’ HSL2 mAb. These data suggest either that the ΨL may be expressed alone at the cell surface of the proB cell line (but in so low a concentration as to not be detectable by FACS analysis) or that some internal labeling may have occurred. Using the NALM6 preB cell line, the four anti-ΨL reagents recognized the cell surface preBCR since μ, λ-like and VpreB proteins were detectable (Figure 2).

Figure 2
figure2

 Biochemical characterization of cell surface proBCR and preBCR complexes by using anti-surrogate light chain and anti-μ mAbs. Upper panel: the proB (JEA2) and preB (NALM6) cells lines were surface labeled with biotin and lysed with 1% NP-40 lysis buffer. Lysates (5 × 107 cells) were incubated with either IgG1 control, anti-VpreB (4G7, HSL96), anti-λ-like or λ5 (HSL11), anti-preBCR (HSL2) or anti-μ mAbs. Immunoprecipitates were submitted to a gradient SDS-PAGE (5–15%) under reducing conditions and transferred on to Immobilon P membrane. Cell surface biotinylated proteins were detected by streptavidin-peroxidase substrate. Lower panel: the membrane was stripped and then incubated with the anti-VpreB 4G7 mAb and revealed by a peroxidase-conjugated goat anti-mouse IgG.

These data are in agreement with the immunofluorescence analysis and confirm the preB cell specificity of the HSL reagents. Even though these mAbs are able to detect the ΨL chain in the proB cell line, they are totally unable to detect the ΨH(p105/p130)-ΨL association. In contrast, the 4G7 mAb detects both cell surface expression of μ-ΨL preB and ΨH-ΨL proB cell complexes. Owing to these properties, the 4G7 mAb was used to analyze a panel of B cell ALLs, with a special focus on precursor B cell ALLs.

Analysis of ΨL expression in adult and childhood precursor B cell ALLs

We performed FACS analysis on a series of 92 ALLs from both childhood (40 samples) and adult (52 samples) origin that had been first assigned to the B cell lineage by conventional phenotypic analysis. This latter characterization was achieved using a panel of 24 mAbs, including the anti-VpreB 4G7 mAb.2030 A genetic alteration analysis, that included cytogenetic and PCR detection of MLL-AF4, TEL-AML1, BCR-ABL and E2A-PBX1 fusion transcripts, was also performed.31323334

The percentage of positive cells by FACS staining using selected mAbs and the presence of fusion transcripts are reported in Table 1A, B and C. The results are presented for proB (CD19+ CD10), common (CD19+ CD10) and preB (CD19+ CD10+/−) ALLs in Table 1A, B and C, respectively. For each ALL subgroup the classification was based on VpreB expression (ie no VpreB expression, cytoplasmic and surface VpreB expression).

Table 1 Table 1A Phenotyping of proB or BI (CD19+ CD10μ) ALLs
Table 2 Table 1B Phenotyping of common or BII (CD19+ CD10+μ) ALLs
Table 3 Table 1C Phenotyping of preB or BIII (CD19+ CD10+/− μ+) ALLs

As shown in Table 1A, cells derived from proB ALL patients (three children and six adults) express CD34, CD19 and CD22 (except for No. 2 and No. 4 that are CD34 and CD22, respectively), but neither CD10, CD20 (with the exception of Nos. 1 and 6) nor the μ chain. With regard to VpreB expression, with one exception (No. 9) this marker was expressed only intracellularly. Of the five fusion transcripts tested only the MLL-AF4 was detected in this group, for three ALL samples.

Common ALLs (Table 1B; 25 children and 39 adults) was the largest B ALL group analyzed (70% of tested samples). They all expressed CD19 and CD10 in the absence of the μ chain. The CD34 and CD22 markers were generally present. In a significant proportion of cases the presence of the CD20 marker was observed, indicating the presence of more mature precursors cells. Differences existed within the ALL origin on the basis of myeloid markers, since 28% (7/25) of childhood and 44% (17/39) of adult-derived ALLs express the CD13 and/or the CD33 markers. Moreover, in adults, the labeling intensity of these markers is generally higher.

Regarding VpreB expression, these common ALLs can be subdivided into three subgroups (Table 1B): (1) ALLs Nos 1 to 6 are totally VpreB negative; (2) ALLs Nos 7 to 40 are cytoplasmic VpreB positive; and (3) ALLs Nos 41 to 64 express VpreB into the cytoplasm and at the cell surface. VpreB FACS staining profiles for representative ALL subgroups are presented in Figure 3.

Figure 3
figure3

 Surface or intracytoplasmic FACS analysis of selected common (or BII) human ALLs, using anti-CD19, anti-VpreB and anti-μ mAbs. In a (patient No. 5), b (patient No. 32) and c (patient No. 57), a representative ALL sample with no VpreB expression, intracytoplasmic and both intracytoplasmic + surface VpreB expression, respectively, is shown. The anti-CD19 and anti-μ mAbs were FITC-labeled whereas the anti-VpreB (4G7) was PE-labeled. Intracytoplasmic μ and VpreB staining was performed after permeabilisation with IntraPrep Permeabilisation Reagent. Staining profiles with these mAbs (shaded figures) and staining profiles with isotype-matched control mAbs (open figures) were overlaid in the histograms.

When we consider either the adult or the childhood series, the percentage of ALLs that express cytoplasmic or cytoplasmic + surface VpreB varied considerably (Tables 1B and 2): 67% of adult ALLs (compared to 32% in children) expressed the VpreB only in the cytoplasm and 60% of childhood ALLs (compared to 23% in adults) expressed the VpreB at the cell surface. Moreover, we noted that the origin of the genetic alteration does not seem to influence the expression of the VpreB epitope (Table 2). In the adult series, either BCR-ABL-positive (61%) or -negative (72%) ALLs expressed cytoplasmic VpreB, and in childhood either TEL-AML1-positive (63%) or -negative (56%) ALLs were characterized by VpreB cell surface expression.

Table 4  VpreB expression in common or BII (CD19+ CD10+) ALLs

Compared to the physiological situation, these data suggest that common ALLs represent homogeneous groups of mature sVpreB+ μ proB cells in children and more immature cVpreB+ μ proB cells in adults. Moreover, the immature proB phenotype in adults correlates with the expression of myeloid markers.

ALLs in Table 1C (12 children and seven adults) express a preB phenotype, since they are all CD19 and cytoplasmic μ chain positive. Except for two cases (Nos 4 and 11), they also expressed CD10 and were generally positive for CD34 and CD20. For this preB ALL group, with diverse genetic alterations, we observed only two subgroups based on VpreB expression: six ALLs were characterized by the uniquely intracytoplasmic VpreB expression and 13 ALLs by the presence of VpreB at the cell surface. In contrast to common ALLs, we did not observe a preferential expression of surface VpreB, in children. Therefore, just as in normal B cell differentiation, these data suggest that preB ALLs either does or does not express the μ-ΨL preBCR at the cell surface.

Discussion

Since the discovery of the ΨL complex, attempts have been made to obtain a detailed characterization of ALLs. The first reports used a combination of Ig gene status and λ-like/VpreB gene expression,58 but recently the fine characterization of anti-ΨL mAbs has allowed a precise sub-classification of precursor B cell ALLs. In the case of childhood preB ALLs, the leukemias can be subdivided into cell surface preBCR and preBCR+ subtypes.24 As we also recently characterized a new series of anti-human VpreB mAbs,2027 among which 4G7 identified surface ΨL expression on normal proB and preB cells, we decided to analyze the VpreB expression on a large panel of both childhood and adult precursor B cell ALLs.

We first compared the fine mAb specificities of 4G7 with that of the 3 HSL mAbs from Karasuyama's group on the same proB and preB cell line.24 By FACS (Figure 1) and biochemical (Figure 2) analysis we have definitively established that the 4G7 mAb has a uniquely broad specificity in that it detects both the ΨH-ΨL proBCR and the μ-ΨL preBCR at the cell surface of proB and preB cell lines, respectively. By contrast, the three HSL mAbs display preBCR specificity, as is the case for the SLC 1/21516 and VpreB 8/937 mAbs obtained from Cooper's group.20 Currently, only one other anti-VpreB mAb (No. 688) has been reported to be capable of detecting ΨL cell surface expression on human proB cell lines,18 but this mAb is of the μ isotype and does not allow a clear identification of the cell surface proBCR, in contrast to the γ1κ 4G7 mAb.20

Precursor B cell ALLs from 40 children and 52 adults were systematically analyzed by FACS using the 4G7 mAb, in combination with selected mAbs that allow an unambiguous B cell classification. The results are summarized in Table 3 (top) for the ProB (or BI), common (or BII) and preB (or BIII) subgroups. First, we observed that the percentage of ALLs within each group corresponds to that already reported in the literature,23 common ALLs being the largest subgroup in both children and adults. In regard to VpreB expression and the corresponding subgroup classification, the results provoke several comments. For proB ALLs, the large majority expresses the VpreB only in the cytoplasm, a situation expected for an early B cell phenotype. For common ALLs, there is a good correlation between the expression of CD10 (and to a lesser degree CD20) and the presence of more mature, surface VpreB-positive, ALLs. As mentioned in Table 2, we observed a dichotomy between childhood and adult ALLs: the majority of the former expresses the cell surface proBCR and thus presents a more mature phenotype than the latter. Moreover, we noted that these results are not linked to the origin of the genetic alteration, ie TEL-AML1 in children and BCR-ABL in adults. In this common ALL subgroup the presence of six VpreB-negative ALLs is puzzling (Table 3). We cannot exclude the possibility that the non-expression of the VpreB epitope is fortuitous and that the λ-like chain may be present. The fact that 5% of leukemic B ALLs neither express λ-like nor VpreB,24 however, may also indicate the existence of very early B cell precursors that express CD19 and CD10 before surrogate light chain components (see below). For cμ+ preB ALLs both intracytoplasmic and surface VpreB-positive leukemias are detected supporting the existence of surface preBCR and surface preBCR+ cells, as Karasuyama's group reported.24 At this stage we do not know if preBCR cells are more immature or more differentiated than preBCR+ cells. It is possible that cell surface expression of the preBCR has failed in preBCR cells due to structural constraints on the interactions between μ and ΨL, and these cells in a physiological situation would not be selected for further differentiation.9101138 However, we cannot exclude the possibility that these cells have been selected and correspond to intermediates between preBCR+ and immature B cells, in which the expression of the ΨL chain is down-regulated and light chain gene rearrangements initiated. Co-transfection of ΨL-positive proB cells with the corresponding μ vectors and search for cell surface expression of the preBCR,3839 would allow one to distinguish between these two possibilities.

Table 5  Summary of VpreB expression in human precursor B cell ALLs

Since expression of the ΨL chain is more relevant to physiological B cell differentiation than that of CD10, it is tempting to consider that CD19+ CD10+cVpreB common ALLs have an earlier origin than CD19+ CD10cVpreB+ proB ALLs, as outlined above. On this basis, we would like to propose a refined classification of precursor B cell ALLs based on the expression of the VpreB protein (Table 3, bottom): (1) a subgroup characterized by expression of CD19+, with or without CD10, but surrogate light chain negative, designated proBI ALLs. This subgroup represents 5% and 7.5% of childhood and adult precursor B cell ALLs, respectively; (2) CD19+ VpreB+ ALLs, irrespective of CD10 expression, would be split into early proBII proBCR and late proBII proBCR+, that do not and do express the cell surface proB cell complex, respectively. When all proBII ALLs are considered, proBCR+ expression in children (37.5%), is significantly higher (P = 0.0009) than in adults (19%); (3) CD19++ ALLs would also be split, as already proposed by Tsuganezawa et al,24 into preB preBCR and preB preBCR+ ALLs, that do not or do express the preBCR at the cell surface, respectively, without a clear assignment of the precise differentiation stage, as discussed above. In both studies regarding childhood ALLs, the same percentage for these two sub-populations was observed, ie approximately 10% and 20% of preBCR and preBCR+ ALLs, respectively.

In conclusion, we demonstrate that FACS analysis with a limited number of mAbs, including the 4G7 anti-VpreB reagent, is an easy process for the diagnosis of B leukemias. Moreover, the major proBII ALL group in both adults (87%) and children (65%), has been characterized in detail and shown to contain clearly different subgroups. We were able to demonstrate that the majority of adult and childhood precursor B cell ALLs pertains to the immature proBCR and the mature proBCR+ subgroups, respectively. It remains to be established if the clear identification of proBII proBCR and proBCR+ would aid the prognosis of B ALLs. A further and larger prospective study, with the analysis of the clinical outcome in a multicentric protocol, is needed to conclude an eventual impact of the immunological presentation among the factors of prognosis, previously described.

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Acknowledgements

We thank D Blaise, R Bouhabdalla and G Michel for referring patients and M-J Mozziconacci, M Lafage-Pochitaloff and A-M Vagner-Capodano for the ALL karyotyping. For excellent technical assistance we are indebted to Y Toiron (RT-PCR analysis) and to F Baudoin, D Jayme and N Geoffroy (immunophenotyping). We also thank V-J Bardou for the statistical analysis, M Fougereau and J Ewbank for critical reading of the manuscript. Anti-human surrogate light chain mAbs (HSL96, 11 and 2) were kindly provided by H Karasuyama. This work was supported by CNRS (Centre National de la Recherche Scientifique), INSERM (Institut National de la Santé et de la Recherche Médicale), ARC (Association de Recherche contre le Cancer, grant No 6345), La Ligue contre le Cancer (Comité des Bouches du Rhône No. 6027–01 and No. 9592), La Ligue Nationale contre le Cancer and La Fondation de France (Comité Leucémie, No. 99002024).

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Correspondence to C Schiff.

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Lemmers, B., Arnoulet, C., Fossat, C. et al. Fine characterization of childhood and adult acute lymphoblastic leukemia (ALL) by a proB and preB surrogate light chain-specific mAb and a proposal for a new B cell ALL classification. Leukemia 14, 2103–2111 (2000). https://doi.org/10.1038/sj.leu.2401932

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Keywords

  • VpreB
  • proBCR
  • preBCR
  • TEL-AML1
  • BCR-ABL
  • leukemia classification

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