Leukemia (1997) 11, 1364–1366  1997 Stockton Press All rights reserved 0887-6924/97 $12.00

BRIEF COMMUNICATION Germline configuration of nfkb2,c-rel and bcl3 in childhood acute lymphoblastic leukemia (ALL) S Liptay1, T Seriu2, CR Bartram2 and RM Schmid3

Departments of 1Pediatrics II and 3Internal Medicine I, University of Ulm; and 2Institute of Human Genetics, University of Heidelberg, Germany

The well-known family of NF-␬B/Rel transcription factors is a children was 6.25 years at diagnosis, ranging from 5 days to central regulator of growth, differentiation and apoptosis in 17.75 years. As shown in Table 1 the ALL samples were sub- hematopoietic cell lineages. There is increasing evidence for 11 their role in malignant transformation, especially in lym- classified by immunological criteria. Leukemic samples con- phomas. To study the possible involvement of NF-␬B/Rel genes tained more than 90% of blasts. in the development of pediatric acute lymphoblastic leukemia High molecular weight DNA was prepared from 106 bone (ALL), DNA samples from 140 patients were examined by marrow, 31 peripheral blood, and three pleural effusion cell Southern blot analysis. All samples revealed germline con- samples obtained at initial diagnosis (n = 119), first (n = 16), figuration of nfkb2,c-rel, and bcl3, indicating that structural or second relapse (n = 5). Ten micrograms of each DNA were alterations of these members of the NF-␬B/Rel family are extremely rare, if existing at all in childhood ALL. digested with EcoRI, BamHI or HindIII, separated on 0.6% Keywords: NF-␬B; NF-␬B2; c-Rel; Bcl-3; rearrangement agarose gels and transferred on to nylon membrane. For hybridization the following cDNA probes were used: a 950 bp EcoRI–AflIII fragment of NF-␬B212 containing exons 2 to 13 ␬ (exons 1a and 1b are non-coding),10,13 a1kbEcoRI–EcoRV NF- B/Rel proteins comprise an ubiquitously expressed family 7 of transcription factors which are important regulators of the fragment of c-Rel including exons 0 to 5, and a 1.4 kb full length Bcl3 c-DNA.5 Schemes of the NFKB2,8,10,13 cREL,7,14,15 immune function, acute phase response, growth and differen- 5,16 tiation of hematopoietic cells.1 More recently it became evi- and BCL3 loci are shown in Figure 1a, b and c, respect- dent that NF-␬B is also a major regulator of apoptosis.2,3 There ively. Hybridization and washing were carried out under strin- is an increasing number of reports, which suggest that NF- gent conditions. High molecular weight DNA from PB cells ␬B/Rel proteins are involved in the development of human of healthy donors were used as controls. Southern blot analy- cancer, especially hematopoietic malignancies (reviewed in sis of EcoRI digested genomic DNA of all 140 patients with Ref. 4). Structural alterations and chromosomal rearrange- different subtypes of childhood ALL showed germline con- ments of three family members, bcl3,c-rel and nfkb2, have figuration of the nfkb2,c-rel and bcl3 genes, respectively been described in hematopoietic malignancies. The bcl3 gene (Figure 2). Additional Southern blot analysis of BamHI and was cloned out of the translocation t(14;19)(q32;q13) found HindIII digested DNA confirmed these results. 5 Rearrangements in the nfkb2 gene were originally described in patients with B cell chronic lymphocytic leukemia. This 9 result in transcriptional up-regulation of bcl3. Involvement of by Neri et al by cloning the breakpoint junctions of a bcl3 in this translocation was confirmed, but is a rare abnor- t(10;14)(q24;q23) chromosomal translocation. Analysis of 6 sequences adjacent to this breakpoint led to the identification mality in chronic lymphoproliferative disorders. Alterations 9 of the c-rel oncogene was shown in human non-Hodgkin’s of lyt-10, which is identical to nfkb2. To determine whether lymphoma.7 The chromosomal location of nfkb2 was assigned structural alterations of the NFKB2 (LYT10) locus were present to chromosome 10q24, a region which is associated with in other lymphoid malignancies, the authors included 38 translocations in T cell acute lymphoblastic leukemia and B cases of non-Hodgkin’s lymphoma and 49 cases of chronic cell non-Hodgkin’s lymphoma.8 In fact, nfkb2 is involved in lymphocytic leukemia in their study. Southern blot analysis the t(10;14)(q24;q32) of B cell non-Hodgkin’s lymphoma.9 using the same restriction digests and an almost identical ␬ cDNA probe as in our study identified two additional cases While the unprocessed form of NF- B2 is localized in the 9 cytoplasm, this rearrangement leads to a truncation of the C- with rearrangements in the nfkb2 gene. Neri et al used a terminus and to a subsequent translocation of a constitutively 855 bp EcoRI–StuI fragment spanning exons 3 to 14, com- active protein into the nucleus. Similar genomic alterations pared to a 950 bp EcoRI–EcoRV probe containing the same are found in cutaneous T cell lymphomas.10 exons in our study. In the previous study, genomic DNA was This study was undertaken to determine the incidence of digested with EcoRI and BamHI; we used EcoRI, BamHI and structural alterations of the nfkb2,c-rel, and bcl3 genes in the in addition a HindIII restriction digest. Since the entire NFKB2 locus is contained in a single 18 kb EcoRI or a single 12 kb most frequent pediatric malignancy, acute lymphoblastic 9,10 leukemia (ALL). BamHI fragment, any rearrangement affecting this region After informed consent cell samples were obtained from should have been detected by the restriction digests and probe used in the present study. 140 children with ALL enrolled in the multicenter ALL trials 16 ALL/NHL-BFM 86 and AL-BFM 90 of the German Berlin– The bcl3 gene contains 9 exons, spanning 11.5 kb. Using a full length cDNA probe and genomic DNA digested with Frankfurt–Mu¨ nster (BFM) study group. The mean age of the EcoRI we detected a 15 kb and a 6 kb band spanning the entire BCL3 locus. bcl3 was identified adjacent to the break- Correspondence: S Liptay, Department of Pediatrics II, Prittwitzstra␤e points in the translocation t(14;19)(q32;q13.1) recurrently 43, 89075 Ulm, Germany found in B cell chronic lymphocytic leukemia.5 The original Received 12 March 1997; accepted 26 May 1997 breakpoint junction was found about 443 bp upstream of the Brief communication S Liptay et al 1365 Table 1 Immunophenotypes of 140 ALL samples

T-ALL (n = 19) TdT+/−, surface CD3+, CD7+, CD1− Pre T-ALL (n = 3) TdT+, cyCD3+, CD7+, CD1−, surface CD3− B-ALL (n = 3) CD19+, SIgM+(with ␬ or ␭ light-chain restriction), CD10+/− Common ALL (n = 70) TdT+, CD19+, CD10+, cylgM− Pre B-ALL (n = 27) TdT+, CD19+, cylgM+, CD10+/−, SIgM− Pre pre B-ALL (n = 18) TdT+, CD19+

Figure 1 Schematic representations of the human (a) NFKB2,8,10,13 (b) cREL7,14,15 and (c) BCL3 loci.5,16 Exons are illustrated as boxes. Restric- tion sites for EcoRI (RI), BamHI (B) and HindIII (H) are indicated. apparent major transcription initiation site resulting in an up- described, which resulted in increased expression of c-rel regulation of bcl3 transcription.5 Similar classical mRNA. Genomic DNA was digested with EcoRI and a full- t(14;19)(q32;q13) rearrangements would have been detected length v-rel probe was used.17 A similar genomic alteration in our study. However, we cannot exclude more complex would have been detected in our study. three-way rearrangements as recently reported t(7;19;14) The lack of detectable structural alterations of the nfkb2,c- (q21;q13;q32), which were only detected by a long-range rel or bcl3 genes in this study suggests that these genes play restriction mapping and would have been missed by Southern a minor role in childhood ALL despite involvement in other blot analysis with the usually available BCL3 probes.6 How- hematopoietic malignancies. ever, it is not clear whether these rearrangements affected expression levels of Bcl3. Previously, rearrangements in the REL locus were detected using genomic DNA digested with BamHI, PvuII or NdeI and a genomic probe containing most Acknowledgements of exon 4 and all of exon 5.7 The detected BamHI fragment contains exons 3, 4, 5, 6a, 6a′, 6b and 7. Since exon 6a con- This work was supported by grants from the Klinikumsvor- tains an Alu repeat14 we decided to use a cDNA probe span- stand der Universita¨t Ulm (Baustein P169) and the Deutsche ning from exon 0 to 5 to avoid cross-hybridization with other Forschungsgemeinschaft (Sonderforschungsbereich 322, Teil- Alu repeats. BamHI restriction digests in combination with this projekt A15). We gratefully acknowledge the close co-oper- cDNA probe are suitable to detect all rearrangements so far ation with the participants of the BFM-ALL trials, especially described in human malignancies. In an avian B cell lym- Professor WD Ludwig for immunophenotypic characteriz- phoma cell line an insertion in the c-rel promoter region was ation. Brief communication S Liptay et al 1366 Meeus P, Michaux JL, Scheiff JM, Noel H, Louwagnie A, Criel A, Boogaerts M, Van Orshoven A, Cassiman JJ, Van Den Berghe H. BCL3 rearrangement and t(14;19)(q32;q13) in lymphoproliferative disorders. Gene Chromosom Cancer 1996; 15: 38–47. 7 Lu D, Thompson JD, Gorski GK, Rice NR, Mayer MG, Yunis JJ. Alterations of the rel locus in human lymphoma. Oncogene 1991, 6: 1235–1241. 8 Liptay S, Schmid RM, Perkins ND, Meltzer P, Altherr MR, McPher- son JD, Wasmuth JJ, Nabel GJ. Related subunits of NF-kappa B map to two distinct loci associated with translocations in leuke- mia. Genomics 1992; 13: 287–292. 9 Neri A, Chang CC, Lombardi L, Salina M, Corradini P, Maiolo AT, Chaganti RSK, Della-Favera R. B cell lymphoma-associated chromosomal translocation involves candidate oncogene lyt-10, homologous to NF-␬B p50. Cell 1991; 67: 1075–1087. 10 Fracchiolla NS, Lombardi L, Salina M, Migliazza A, Baldini L, Berti E, Cro L, Polli E, Maiolo AT, Neri A. Structural alterations of the NF-␬B transcription factor lyt-10 in lymphoid malignancies. Figure 2 Southern blot analysis in representative ALL samples. Oncogene 1993; 8: 2839–2845. DNA of a healthy individual (lanes 1, 5, 9), a patient with pre-B ALL 11 Ludwig WD, Bartram CR, Ritter J, Raghavachar A, Hiddemann W, (lanes 2, 6, 10), T-ALL (lanes 3, 7, 11), or c-ALL (lanes 4, 8, 12) was Heil G, Harbott J, Seibt-Jung H, Teichmann JV, Riehm H. Ambigu- digested with EcoRI. Filters were hybridized with nfkb2 (lanes 1–4), ous phenotypes and genotypes in 16 children with acute leukemia c-rel (lanes 5–8), or bcl3 (lanes 9–12) probes, respectively. Sizes of as characterized by multiparameter analysis. Blood 1988; 71: lambda HindIII markers are indicated on the side of the panel (in 1518–1528. kilobases). 12 Schmid RM, Perkins ND, Duckett CS, Andrews PC, Nabel GJ. Cloning of an NF-kappa B subunit which stimulates HIV transcrip- tion in synergy with p65. Nature 1991; 352; 733–736. References 13 Liptay S, Schmid RM, Nabel EG, Nabel JG. Transcriptional regu- lation of NF-␬B2: evidence for ␬B-mediated positive and negative 1 Baeuerle PA, Henkel T. Function and activation of NF-␬B in the autoregulation. Mol Cell Biol 1994; 14: 7695–7703. immunsystem. Ann Rev Immunol 1994; 12: 141–179. 14 Brownell E, Mittereder N, Rice NR. A human rel photo-oncogene 2 Beg AA, Baltimore D. An essential role for NF-␬B in preventing cDNA containing an Alu fragment as a potential coding exon. TNF-␣-induced cell death. Science 1996; 274: 782–784. Oncogene 1989; 4: 935–942. 3 Lui ZG, Hsu H, Goeddel DV, Karin M. Dissection of TNF receptor 15 Brownell E, O’Brien SJ, Nash WG, Rice N. Genetic characteriz- 1 effector functions: JNK activation is not linked to apoptosis while ation of human c-rel sequences. Mol Cell Biol 1985; 5: 2826– NF-␬B activation prevents cell death. Cell 1996; 87: 565–576. 2831. 4 Gilmore TD, Koedood M, Piffat KA, White DW. Rel/NF-␬B/I␬B 16 McKeithan TW, Ohno H, Dickstein J, Hume E. Genomic structure proteins and cancer. Oncogene 1996; 13: 1367–1378. of the candidate proto-onogene BCL3. Genomics 1994; 24: 5 Ohno H, Takimoto G, McKeithan TW. The candidate proto-onco- 120–126. gene bcl-3 is related to genes implicated in cell lineage determi- 17 Kabrun N, Bumstead N, Hayman MJ, Enrietto PJ. Characterization nation and cell cycle control. Cell 1990; 60: 991–997. of a novel promoter insertion in the c-rel locus. Mol Cell Biol 6 Michaux L, Mecucci C, Stul M, Wlodarska I, Hernandez JM, 1990; 10: 4788–4794.