(2004) 18, 1997–2001 & 2004 Nature Publishing Group All rights reserved 0887-6924/04 $30.00 www.nature.com/leu IgH DJ rearrangements within T-ALL correlate with cCD79a expression, an immature/TCRcd phenotype and absence of IL7Ra/CD127 expression

V Asnafi1,4, K Beldjord1,2, R Garand3, C Millien1,4, M Bahloul1, P LeTutour1, L Douay2, F Valensi1,4 and E Macintyre1,4

1Biological Hematology, Assistance Publique, Hopitaux de Paris (AP-HP), Necker-Enfants-Malades, France; 2Trousseau, Paris, France; 3CHU Nantes, France; and 4INSERM EMIU210 and Universite´ Paris V, France

cCD79a and IgH VDJ/DJ rearrangements are considered to be B lymphoid restricted is not clear. CD127 (IL7Ra) interaction has relatively specific for B lymphoid precursors. We looked for been reported to be a nonredundant cytokine for murine B and both in cCD3 þ , CD7 þ , CD19- T-ALLs classified by TCR status T lymphopoiesis but its role in human B lymphoid development into ab or cd/immature (IM) lineages, with individualization of 1,14 HOX11L2 þ T-ALLs since they represent an intermediate ab/cd is controversial. An early CD34 þ , CD19À, CD10À, category. cCD79a was expressed at low levels in 47% of T-ALL cCD79a þ , CD127 þ cord blood B progenitor has recently and was most frequent in IMc T-ALLs. IgH rearrangements were been shown to demonstrate IgH DJ rearrangement and macro- common in cd/IM (45%) and HOX11L2 þ (35%) T-ALLs com- phage, natural killer (NK), B and T lymphoid potential.15 IgH DJ pared to HOX11L2-negative cases (3%; Po0.001). CD127 (IL7Ra) rearrangements have also been reported in normal immature expression was also more common in the cd/IM lineage but its murine,16 but not human,12 thymocytes and in 22% of pediatric expression was virtually mutually exclusive of IgH rearrange- 17 ment. Low-level cCD79a expression alone should therefore not T-ALLs, particularly in TCRgd þ cases. We have recently be interpreted as evidence of B lineage affiliation in immature described a TCR-based classification which allows separation of . cd/IM lineage T-ALLs potentially include two distinct T-ALLs into ab-lineage (TCRab/cTCRb expressing and/or TCRb categories: predominantly IgH þ , cCD79a þ , CD127- cases VDJ rearranged T-ALLs) and gd lineage/immature (gd/IM) which retain cd and B lymphoid potential and IgHÀ, cCD79aÀ, leukemias (TCRgd expressing or absence of complete TCRb CD127 þ cases with restricted T lineage potential. 18 Leukemia (2004) 18, 1997–2001. doi:10.1038/sj.leu.2403531 rearrangement). IM T-ALLs can be divided into those which Published online 14 October 2004 have undergone TCRd (IMd) or TCRg (IMg) rearrangement and Keywords: T-ALL; CD79a; CD127/IL7Ra; IgH rearrangements; IM0 T-ALLs, with all TCR in a germline configuration. lymphopoiesis IMd/g T-ALLs clearly include gd precursors,19 but they poten- tially also include leukemias with wider lymphoid potential. We therefore collectively refer to TCRgd þ and IMd/g T-ALLs as Introduction belonging to a gd/IM lineage. HOX11L2 expression in T-ALL is associated with a maturation arrest intermediate to the ab and gd The lymphoid (BCR) complex includes a lineages20 and, as such have been individualized in the present membrane-bound Ig and a transmembrane signal transducing study. In an attempt to further analyze lineage affiliation and to heterodimer, CD79, the B-cell equivalent of CD3 (reviewed in assess the diagnostic significance of CD79a, we compared LeBien1). CD79 comprises two polypeptides; CD79a (Iga) and cCD79a and CD127 expression with IgH DJ and VDJ CD79b (Igb). While CD79b appears to be expressed as a single rearrangement in T-ALLs defined by their ab vs gd/IM lineage isoform, CD79a is expressed from two alternatively spliced and their HOX11L2 þ expression. transcripts, with the smaller one representing an immature form, whose is predicted not to form disulfide-linked dimers with CD79b but to maintain the transmembrane and cytoplas- 2 Materials and methods mic portions, thus permitting immunological detection. Cyto- plasmic (c)CD79a is expressed prior to CD19 during B 3 Diagnostic peripheral blood or bone marrow samples from 149 lymphoid development and is expressed by virtually all B-cell 4,5 T-ALLs were analyzed with informed consent. Their clinical, precursor ALLs (BCP-ALL). It is considered to represent a B 6 immunological and genotypic characteristics and RAG1 expres- lineage restricted marker, although weak cCD79a expression sion have been described.18–20 Inclusion criteria for T-ALLs has been detected by or flow cytometry 7–10 included availability of material for monocentrique cCD79a in 10–40% of a limited number of T-ALLs. Incomplete staining, c/surface CD3 þ and CD7 positivity and CD19 and immunoglobulin (Ig) DJ rearrangements first occur in CD34 þ , myeloperoxidase (MPO) negativity. Controls included 35 AMLs CD19À, CD10 þ , cCD79a þ early B cells, whereas VDJ and 17 BCP-ALLs, defined by EGIL and FAB criteria, and 2 rearrangements commence in CD19 þ , pro-B cells, just prior 11–13 neonatal thymi. IL7Ra staining was performed using the SB/14- to appearance of cytoplasmic Igm and pre-BCR expression. PE anti-CD127 monoclonal (Becton Dickinson, Whether the earliest IgH DJ rearranged, CD79a þ precursors are Biosciences). cCD79a (HM57-phycoerythrin, DAKO, Glostrup Denmark) was detected by direct labeling of permeabilized cells Correspondence: E Macintyre, Laboratoire d’He´matologie, Tour Pasteur, Hoˆpital Necker, 149-161, rue de Se`vres, 75743 Paris cedex (Harla Sera-Lab, England), and quantitated as a Ratio of mean 21 15, France; Fax: þ 33 1 44 38 17 45; E-mail: elizabeth.macintyre@ Fluorescence Intensity (RFI). The mean cCD79a fluorescent nck.ap-hop-paris.fr intensity of the blast population, defined by CD45/SSC (rarely Financial support: This work was supported by the fondation contre la SSC/FSC) gating and exclusion of residual normal B leuce´mie de la Fondation de France, l’Association de la Recherche sur by double staining with CD7 or cCD3, was divided by the mean le Cancer (ARC), the League Contre le Cancer, the Direction de fluorescent intensity of the isotype control. IgH VDJ rearrange- Recherche Clinique de l’Assistance Publique-Hoˆpitaux de Paris (PHRC 97-106) and the Biomed-2 BMH4-CT98-3936 concerted action. ments were detected by multiplex, multifluorescent PCR, as 22 Received 11 May 2004; accepted 19 July 2004; Published online described. IgH DJ rearrangements were analyzed by two 14 October 2004 multiplex PCR with DH1-6 or DH7 primers developed within IgH DJ rearrangements within T-ALL V Asnafi et al 1998 the BIOMED-2 Concerted Action, ‘PCR-based clonality studies nonfunctional isoforms, we evaluated which form of CD79a for early diagnosis of lymphoproliferative disorders (PL96- transcripts predominated in T-cell lines, in order to avoid RT- 3936).23 PCR amplification of normal residual B lymphocytes in T-ALL CD79a and CD79b amplification were performed by RT-PCR. samples. As shown in Figure 2, both CD79a and CD79b were Briefly, 100 ng cDNA was amplified for 35 cycles (941C30s,551C amplified from peripheral blood lymphocytes and B-cell lines, 30 s and 681C 1 min) in the presence of 0.2 mM of each primer, 2 mM neither from the K562 erythroid/myeloid cell line, and only MgCl2 and 1 U Taq polymerase in a 20 ml final volume. Primers CD79a from thymus and T-cell lines. Full-length transcripts CD79a sense: TCAAACTAACCAACCCACTGGGAG, CD79a predominated in all T-cell lines, with no obvious difference in reverse: GTGAGCTGAGACACTGGAGC, CD79b sense: GTCGGG the proportion of shorter transcripts between T- and B-cell lines. GACAGAGCGGTGAC, CD79b reverse: GCAGCTCC GAAGCA It therefore appears that the CD79a expressed by T-ALLs is GTCACTG and were provided by C Schiff, Centre d’Immunologie potentially functional. Marseille-Luminy. RAG1 quantification was performed by RQ-PCR and ex- pressed relative to logarithmic dilutions of the HBP-ALL T-cell IgH rearrangements correlates with cCD79a expression line after normalization by the ABL housekeeping .18 and is virtually restricted to a gd/IM phenotype

IgH rearrangements were seen in 31/141 (22%) T-ALLs. DJ Results rearrangement was seen in 28 cases (one case was also IgH VDJ þ ), VDJ in four. As described,17 there was a high frequency 0 cCD79a staining in B, T and myeloid acute leukemia of 3 DH segment usage, since 10/31 rearranged alleles used

All BCP-ALLs other than one pro-B case showed strong cCD79a Table 1 cCD79a and CD127/IL7Ra expression in T-ALLs classified expression, defined by an RFI410 (Figure 1a: mean RFI 29.7; by TCR status range 5.2–71) and all FAB M1–5 AML were negative defined by RFIo2 (mean RFI 1.2; 0.5–1.7). T-ALLs showed a continuous cCD79aa DH-JH/IgHb CD127* spectrum of low-level positivity (mean 2.9, 0.7–39). Similar levels of expression were seen in two unsorted, neonatal thymi. IM0 3/7 (43%) 0/8 (0%) 1/5 (20%) lineage Overall, 67/142 47% demonstrated an RFI42 and were gd IMd 3/9 (33%) 3/9 (33%) 3/7 (43%) considered cCD79a positive. In all, 13 T-ALLs demonstrated IMg 15/18 (83%) 10/16 (63%) 4/9 (44%) intermediate cCD79a levels (RFI 5–10) and one, high-level TCRgd 8/24 (33%) 9c/24 (38%) 9/18 (50%) expression. Given the homogeneous cCD79a positivity in T-ALL HOX11L2+ 9/19 (47%) 7/20 (35%) 2/10 (20%) (Figure 1b), RFI allows more objective assessment of the ablineage 29/65 (45%) 2/64 (3%) 13/35 (37%) intensity of expression than percentage positivity and avoids Total ¼ 149 67/142 (47%) 31/141 (22%) 32/84 (38%) the erroneous impression of positivity in a subpopulation. acCD79a expression was considered positive if RFI 42 and CD127 cCD79a expression was most common in IMg T-ALLs in positive if greater than 20%. There was no difference in the incidence comparison with the other categories: 15/18 (83%) vs 52/124 of cCD79a or CD127 expression in pediatric and adult T-ALLs (data (42%), P ¼ 0.001 (Table 1). Intermediate RFI levels were also not shown). bIgH DJ were found in 28 T-ALLs, one of which also demonstrated VDJ more common in this category (5/18; 28% compared to 9/124; rearrangement. VDJ were identified in three HOX11L2+ ab-lineage and 7%, P ¼ 0.006). one TCRgd T-ALL. IgH DJ were found in 14/50 (28%) CD127- T-ALLs In order to determine whether the CD79a protein detected in compared to only 2/30 (7%) CD127+ cases (P ¼ 0.02). T-ALLs corresponded to the alternatively spliced, shorter, cIncludes three HOX11L2+ TCRgd T-ALLs.

Figure 1 cCD79a relative fluorescent intensity (RFI) expression in acute leukemia. (a) RFI by diagnostic category, with T-ALLs classified by TCR status. Cases were considered low-level positive if RFI 2–5 and intermediate if RFI 5–10. Controls included two unsorted neonatal thymi, 35 AML, including seven FAB M1, eight M2, three M3, four M4 and 13 M5, and 17 BCP-ALL. (b) Representative histograms of intermediate (UPN269, IMg) and low level (UPN160; IMg) cCD79a positivity in T-ALL. The minor population of strongly positive cells for UPN 160 and 1972 correspond to B lymphocytes. UPN ¼ unique patient number.

Leukemia IgH DJ rearrangements within T-ALL V Asnafi et al 1999 DH7, in keeping with primitive rearrangements. IgH rearrange- low-level RAG1 expression, whereas TCRd Jd1, mature TCRg and ment was virtually restricted to the gd/IM cases (22/49; 45%) and complete TCRb VDJ are only found in T-ALLs with levels above HOX11L2 þ cases (7/20; 35%) and was extremely rare in all 1,E-02.18 The present data are in keeping with a requirement for other ab-lineage cases (2/64; 3%, Po0.001). As with cCD79a, higher RAG1 levels for IgH VDJ compared to DJ rearrangement. IgH rearrangement was most common in IMg T-ALLs. It was more common in cCD79a þ compared to cCD79aÀ cases, both within the overall population (20/63, 31% vs 10/70, 14%, IL7Ra/CD127 expression define two subgroups of respectively; P ¼ 0.02) and within the gd/IM subgroup (15/25, T-ALLs 60% vs 6/23, 25%; P ¼ 0.02). It is noteworthy that no Ig rearrangements were detected in the ab-lineage T-ALL with high Since IL7 plays an early role in lymphoid lineage specification, level CD79a expression (RFI 39). These data demonstrate that we looked for CD127 expression in 84 T-ALLs. Totally, 32 (38%) IgH rearrangement is virtually restricted to the gd/IM and were positive (Table 1). Once again the highest incidence was HOX11L2 þ subgroups of T-ALLs and correlates with cCD79a seen in the gd/IM lineage, in keeping with the fact that IL7 is expression. important for gd development.14 CD127 expression was rare in HOX11L2 þ ab-lineage T-ALLs. Strikingly, IgH rearrangements and CD127 expression were mutually exclusive, particularly IgH DJ rearrangement occur in low-level RAG1 within the gd/IM lineage, when Ig rearrangements were seen in expression within gd/ IM T-ALLs 11/17 (65%) CD127 negative compared to only 2/15 (13%) CD127 positive cases; P ¼ 0.003. This is in keeping with IL7- As shown in Figure 3, all four IgH VDJ rearranged T-ALLs mediated orientation towards a T/NK/dendritic-macrophage demonstrated high-level RAG1 expression, as did 6/7 HOX11L2 precursor, as recently described,15 rather than towards the and both ab-lineage cases with DJ rearrangement. In contrast, B-cell lineage. Among gd/IM T-ALLs, only 2/14 cCD79a þ /IgH IgH DJ rearrangement in gd/IM cases occurred in the presence D-J þ cases were CD127 þ compared to seven of the 11 of much lower RAG1 levels, when IgH VDJ rearrangement cCD79aÀ/IgH D-J- cases (P ¼ 0.02). was not seen. We have previously shown that partial TCRd and These data would suggest that IMd/g T-ALLs include early TCRg rearrangements occur in the presence of relatively two distinct categories: predominantly Ig þ , cCD79a þ ,

Figure 2 RT-PCR amplification of full length and smaller, alternatively spliced transcripts of CD79a and CD79b from B T and myeloid cell lines, as indicated/transcript lengths are shown as base pairs on the left.

Figure 3 RQ-PCR quantification of RAG1 transcript level from IgH DJ and VDJ-positive and -negative T-ALLs. Levels of expression are expressed on a logarithmic scale, relative to the HPB-ALL cell line, classed as 1.

Leukemia IgH DJ rearrangements within T-ALL V Asnafi et al 2000 CD127À cases, which retain gd and B lymphoid potential, and ab lymphopoiesis is also established but its role in human B IgÀ, cCD79aÀ, CD127 þ cases which have T rather than B lymphopoiesis is less clear. Several lines of evidence suggest lineage potential. important differences between murine and human B lympho- poiesis. Severe combined immuno-deficiency patients with IL7Ra mutations lack T lymphopoiesis but demonstrate normal Discussion B-cell development25 and human B-cell development in vitro does not require IL7.26 We have recently demonstrated that Flow cytometric analysis confirmed immunohistochemical T-ALLs reproduce, at least in part, normal thymic populations.18 evidence of cCD79a expression in approximately 50% of The identification of independent IgH þ , cCD79a þ , CD127À T-ALLs, demonstrating that low-level positivity in acute leuke- and IgÀ, cCD79aÀ, CD127 þ T-ALLs within the IMd/g category mia should not be taken to indicate a B lineage restricted nature. suggests that IL7Ra expression within an immature CD7 þ , Expressing results as RFI allows distinction of BCP-ALL with cCD3 þ CD4/8 double-negative precursor identifies early T-cell high-level expression and T lineage cases with low-level lineage commitment whereas its absence, particularly in the expression, with little overlap. Our data indicate that semi- presence of CD79a expression and/or IgH DJ rearrangement quantification of cCD79a expression is preferable to histological indentifies a precursor with B and gd lineage potential. Whether assessment, when expression levels are difficult to evaluate. the former population demonstrates ab-lineage rather than gd- cCD79a is one of the most discriminating markers within the lineage potential is not clear. The data presented here will allow EGIL classification of acute leukemia,6 with a value of 2 for selection of normal thymic precursors in which the preferential B lineage affiliation. We suggest that such a value should be development potential suggested above can be tested. restricted to cases with high level (RFI45–10) cCD79a We have shown that HOX11L2 þ T-ALLs are associated with expression. an intermediate stage of maturation arrest between the ab and gd The significance of low-level cCD79a expression in T-ALLs is lineages.20 These T-ALLs frequently demonstrate IgH DJ not clear. Both cCD79a expression and IgH DJ rearrangement rearrangement but differ from gd-lineage, IgH DJ þ T-ALLs by precede CD19 expression in pro-B cells,3,11,12 but the demon- their relatively high RAG1 levels and by less-frequent CD127 stration that a CD34 þ , CD19À, CD10À, CD79a þ , CD127 þ , expression. Both may be explained by relative maturity, since IgH DJ rearranged cord blood B progenitor has macrophage, the majority of HOX11L2 þ T-ALLs are arrested at the IMb, NK, B and T lymphoid potential15 suggested that it is not yet B pre-ab or pTa þ /TCRgd þ stages. It is noteworthy that IgH DJ lymphoid restricted. We show that both CD79a expression and, rearrangements were not seen in HOX11 þ T-ALLs (data not particularly, IgH DJ rearrangement are preferentially associated shown), which demonstrate IMb and pre-ab but not TCRgd þ with TCRgd expressing T-ALLs and their precursors, IMd/g stages of maturation arrest. Given the high level of homology T-ALLs. It is possible that cross-lineage IgH rearrangements in between HOX11 and HOX11L2, comparison of the conse- T-ALL reflect transformation of a CD34 þ /CD19À precursor, quences of their expression on Ig/TCR accessibility is which has initiated IgH DJ rearrangement and retains B and gd likely to improve our understanding of normal and pathological rather than ab-lineage potential. We believe that IgH DJ lymphoid development. rearrangements and cCD79a expression in IMd/g T-ALLs are not simply a reflection of immaturity, since if this were the case, the incidence would be at least as high in IMd as IMg and potentially even higher in IM0 T-ALL, which was not the case. References These data are suggestive of a continuous spectrum of lymphoid cells, with the gd-lineage being intermediate between the ab and 1 LeBien TW. Fates of human B-cell precursors. Blood 2000; 96: B lymphoid lineages. 9–23. An alternative explanation could be aberrant postoncogenic 2 Benlagha K, Guglielmi P, Cooper MD, Lassoued K. Modifications events resulting from ongoing RAG activity within transformed of Igalpha and Igbeta expression as a function of B lineage precursors. We have shown that RAG1 activity was 10–100-fold differentiation. J Biol Chem 1999; 274: 19389–19396. 3 Dworzak MN, Fritsch G, Froschl G, Printz D, Gadner H. Four- higher within ab-lineage T-ALLs than IM and the majority of color flow cytometric investigation of terminal deoxynucleotidyl 18 TCRgd T-ALLs. Virtual absence of IgH rearrangements in ab- transferase-positive lymphoid precursors in pediatric bone mar- lineage T-ALLs is against postoncogenic events due to ongoing row: CD79a expression precedes CD19 in early B-cell ontogeny. RAG-mediated recombinase activity. The fact that the majority Blood 1998; 92: 3203–3209. of IgH DJ þ T-ALLs demonstrated low-level RAG1 transcripts 4 Buccheri V, Mihaljevic B, Matutes E, Dyer MJ, Mason DY, suggests either that a prior wave of RAG1 expression led to their Catovsky D. mb-1: a new marker for B-lineage lymphoblastic leukemia. Blood 1993; 82: 853–857. rearrangement or that, as for initial TCRd and TCRg rearrange- 5 Mason DY, Cordell JL, Brown MH, Borst J, Jones M, Pulford K et al. 18 ments, low-level RAG1 expression suffices for IgH DJ (but not CD79a: a novel marker for B-cell in routinely processed VDJ) rearrangement. This does not explain the virtual absence of tissue samples. Blood 1995; 86: 1453–1459. IgH DJ rearrangement in ab-lineage precursor T-ALLs, suggest- 6 Bene MC, Castoldi G, Knapp W, Ludwig WD, Matutes E, Orfao A ing that IgH locus accessibility differs in ab and gd lineage et al. Proposals for the immunological classification of acute precursors. leukemias. European Group for the Immunological Characteriza- tion of Leukemias (EGIL). Leukemia 1995; 9: 1783–1786. Approximately one-third of T-ALLs expressed CD127, the 7 Hashimoto M, Yamashita Y, Mori N. Immunohistochemical IL7Ra. As for IgH DJ rearrangement and CD79a expression, the detection of CD79a expression in precursor lymphoblastic highest incidence of expression was found in the gd-lineage /leukaemias. J Pathol 2002; 197: 341–347. cases. This is in keeping with the known role of IL7 in TCRgd 8 Finolezzi E, Corral L, Martelli MP, De Propris MS, Falini B, Cimino development.24 More strikingly, it was possible to divide IMd/g G. Relevance of CD79a expression for T-cell lineage attribution in À T-ALLs into predominantly Ig þ , cCD79a þ , CD127À cases CD7+/CD3 acute lymphoblastic leukemia. Haematologica 2002; 87: ELT41. and IgÀ, cCD79aÀ,CD127 þ cases. IL7 has been shown to be 9 Pilozzi E, Pulford K, Jones M, Muller-Hermelink HK, Falini B, an indispensable cytokine for both B and T murine lymphopoi- Ralfkiaer E et al. Co-expression of CD79a (JCB117) and CD3 by esis.14 Its role in human T gd and, to a lesser extent, lymphoblastic lymphoma. J Pathol 1998; 186: 140–143.

Leukemia IgH DJ rearrangements within T-ALL V Asnafi et al 2001 10 Lai R, Juco J, Lee SF, Nahirniak S, Etches WS. Flow cytometric 19 Asnafi V, Radford-Weiss I, Dastugue N, Bayle C, Leboeuf D, detection of CD79a expression in T-cell acute lymphoblastic Charrin C et al. CALM-AF10 is a common fusion transcript in leukemias. Am J Clin Pathol 2000; 113: 823–830. T-ALL and is specific to the TCRgammadelta lineage. Blood 2003; 11 Davi F, Faili A, Gritti C, Blanc C, Laurent C, Sutton L et al. Early onset 102: 1000–1006. of immunoglobulin heavy chain gene rearrangements in normal 20 Asnafi V, Beldjord K, Libura M, Villarese P, Millien C, Ballerini P human bone marrow CD34+ cells. Blood 1997; 90: 4014–4021. et al. Age-related phenotypic and oncogenic differences in T-acute 12 Bertrand III FE, Billips LG, Burrows PD, Gartland GL, Kubagawa H, lymphoblastic leukemias may reflect thymic atrophy. Blood 2004, Schroeder Jr HW. Ig D(H) gene segment transcription and March 30 [E-pub ahead of print]. rearrangement before surface expression of the pan-B-cell marker 21 Zupo S, Isnardi L, Megna M, Massara R, Malavasi F, Dono M et al. CD19 in normal human bone marrow. Blood 1997; 90: 736–744. CD38 expression distinguishes two groups of B-cell chronic 13 Lassoued K, Nunez CA, Billips L, Kubagawa H, Monteiro RC, lymphocytic leukemias with different responses to anti-IgM anti- LeBlen TW et al. Expression of surrogate chain receptors is bodies and propensity to apoptosis. Blood 1996; 88: 1365–1374. restricted to a late stage in pre-B cell differentiation. Cell 1993; 73: 22 Brumpt C, Delabesse E, Beldjord K, Davi F, Cayuela JM, Millien C 73–86. et al. The incidence of clonal T-cell receptor rearrangements in 14 Fry TJ, Mackall CL. Interleukin-7: from bench to clinic. Blood B-cell precursor acute lymphoblastic leukemia varies with age and 2002; 99: 3892–3904. genotype. Blood 2000; 96: 2254–2261. 15 Reynaud D, Lefort N, Manie E, Coulombel L, Levy Y. In vitro 23 van Dongen JJ, Langerak AW, Bruggemann M, Evans PA, Hummel identification of human pro-B cells that give rise to macrophages, M, Lavender FL et al. Design and standardization of PCR primers natural killer cells, and T cells. Blood 2003; 101: 4313–4321. and protocols for detection of clonal immunoglobulin and T-cell 16 Born W, White J, Kappler J, Marrack P. Rearrangement of IgH receptor gene recombinations in suspect lymphoproliferations: genes in normal thymocyte development. J Immunol 1988; 140: report of the BIOMED-2 Concerted Action BMH4-CT98-3936. 3228–3232. Leukemia 2003; 17: 2257–2317. 17 Szczepanski T, Pongers-Willemse MJ, Langerak AW, Harts WA, 24 Haks MC, Oosterwegel MA, Blom B, Spits HM, Kruisbeek AM. Wijkhuijs AJ, van Wering ER et al. Ig heavy chain gene Cell-fate decisions in early T cell development: regulation by rearrangements in T-cell acute lymphoblastic leukemia exhibit cytokine receptors and the pre-TCR. Semin Immunol 1999; 11: predominant DH6-19 and DH7-27 gene usage, can result in 23–37. complete V-D-J rearrangements, and are rare in T-cell receptor 25 Puel A, Ziegler SF, Buckley RH, Leonard WJ. Defective IL7R alpha beta lineage. Blood 1999; 93: 4079–4085. expression in T(À)B(+)NK(+) severe combined immunodeficiency. 18 Asnafi V, Beldjord K, Boulanger E, Comba B, Le Tutour P, Estienne Nat Genet 1998; 20: 394–397. MH et al. Analysis of TCR, pT alpha, and RAG-1 in T-acute 26 Prieyl JA, LeBien TW. Interleukin 7 independent development lymphoblastic leukemias improves understanding of early human of human B cells. Proc Natl Acad Sci USA 1996; 93: T-lymphoid lineage commitment. Blood 2003; 101: 2693–2703. 10348–10353.

Leukemia