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A new B-cell line showing a complex translocation (8;14;18) and BCL2 rearrangement
A New B-Cell Line Showing a Complex Translocation (8;14;18) and BCL2 Rearrangement R. B. C. van Ooteghem, E. M. E. Smit, A. Beishuizen, A. C. Lambrechts, M. v. d. Blij-Philipsen, T. J. Smilde, and A. Hagemeijer
ABSTRACT: A cell line named BOS-50 [Rotterdam suspension cell line no. 50) has been established from peripheral blood of a 69-year-old male with acute lymphoblastic leukemia (FAB type L3). Among the aberrations, cytogenetic analysis showed the presence of 14q +, 18q-, and two 8 q - marker chromosomes. With fluorescence in situ hybridization [FISH) we characterized the chromosomal translocations, t(8;14) and t(14;18), in which the same chromosome 14 is involved. PCB analysis demonstrated the presence of an IGH-BCL2 rearrangement with a breakpoint in the minor cluster region (mcr) confirming the t[14;18) characteristic for follicular lymphoma. Additional studies showed high expression of BCL2 protein, an early B-cell immunophenotype, and an unusual pattern of IGH gene rearrangement.
INTRODUCTION B-ALL represents about 5% of all ALL cases and shows a particular morphology of blasts reminiscent of lymphoblasts in Burkitt lymphoma with vacuolated cytoplasm (FAB-L3). Nonrandom chromosome abnormalities are consistently found in Burkitt lymphoma; these include t(8;14)(q24;q32) or one of the variants, t(2;8)(p12;q24) and t(8;22)(q24;q11) [1-3]. In the case of t(8;14), Dalla-Favera et al. [4] have shown that the MYC oncogene, which is located on band 8q24, translocates to the IGH locus on chromosome 14. On the contrary, in both t(2;8) and t(8;22) MYC remains on the rearranged chromosome 8 and the kappa or lambda immunoglobulin light-chain constant regions, respectively, translocate to chromosome 8 [5]. In all cases MYC comes into the neighborhood of an immunoglobulin enhancer. Under the influence of this enhancer MYC is deregulated and transcribed constitutively at an elevated level. The product of the MYC oncogene is a nuclear protein capable of binding DNA. While the function of the MYC gene product is not yet clear, it seems likely that it is involved in the control of cell proliferation [6]. Another translocation, t(14;18)(q32;q21), involving the IGH locus on 14q32 and the BCL2 locus on 18q21, is found in follicular lymphoma [7], but may be observed in other B-cell
From the Departments of Cell Biology and Genetics (R. B. C. v. 0., E. M. E. S., A. H.), and Immunology (A. B.), Erasmus University, Rotterdam; Dr. Daniel Den Hoed Cancer Center (A. C. L.), Rotterdam; Department of Clinical Genetics (M. v. d. B.-P.), Veldhoven; Groot Ziekengasthuis (T. ]. S.), 's-Hertogenbosch, The Netherlands. Address reprint requests to: Anne Hagemeijer, Department of Cell Biology and Genetics, Erasmus University, P.O. Box 1738, 3000 DR Rotterdam, The Netherlands. Received July 2Z 1993; accepted October 25, 1993.
malignancies as well. The BCL2 gene is translocated to 14q32 into the IGH gene and is consequently deregulated and transcribed at a high rate. The coding regions of the BCL2 gene are left intact in all t(14;18) translocations so that the BCL2IGH transcript produced encodes a normal BCL2 protein. BCL2 activation prevents the cells from undergoing programmed cell death (apoptosis), a phenomenon corresponding to the low-grade malignancy oft_he follicular lymphoma [8]. Recently, a few cases of highly malignant leukemia and/or lymphoma carrying both types of rearrangements, i.e., t[8 ;14) and t(14;18), have been reported. These two translocations usually involve both chromosomes 14 [9-17]. However, in a lymphoblastoid cell line a three-way translocation between chromosomes 8, 14, and 18 is described by Kiem et al. [18]. We report here on the establishment and characterization of a new cell line with a complex t(8;14;18), a Burkitt-like morphology of blast cells, early B-cell immunophenotype, and BCL2 rearrangement studied by PCR. Fluorescence in situ hybridization (FISH) with chromosome-specific probes and IGH gene rearrangement studies were applied to analyze this complex translocation. CASE REPORT
A 69-year-old man presented with complaints of fatigue, loss of appetite, weight loss, and night sweats. He had a 10-year history of diabetes and hypertension, the former treated with glibenclamide and metformin. Physical examination showed pallor. He had a slightly enlarged liver, no lymphadenopathy or splenomegaly. Laboratory investigations showed a high ESR, Fib 6.1 mmol/L, platelets 63 x 10E9/L, leukocytes 4.1 x 10E9/L with 1% eosinophils, 47% granulocytes, 2 % monocytes, 5% myelocytes, 2% metamyelocytes, 10% band nan87
© 1994 Elsevier Science Inc. 655 A v e n u e of the Americas, N e w York, NY 10010
Cancer Genet Cytogenet 7 4 : 8 7 - 9 4 (1994)
0165-4608/94/$07.00
88 trophils, 33 % lymphocytes. There were no signs of hemolysis or intravascular coagulation. LDH (4152 U/L) and glucose (12.5 retool/L) were elevated. Virus serology for EBV, HSV I and 2, hepatitis B, and HIV was negative. A bone marrow aspirate contained 90% large blast-like cells with an abundant chromatin in the nucleus and two to three nucleoli. The basophilic cytoplasm revealed multiple vacuoles (Burkittlike lymphoblasts). These blasts did not stain with Sudan black, PAS, acid phosphatase, and nonspecific esterase. Immunophenotypic evaluation revealed a monoclonal B-cell population, expressing CD20 and CD19 with an IgM kappa membrane expression. These cells were negative for CD5, CD10 (CALLA), intracytoplasmatic immunoglobulins, and terminal deoxynucleotidyl-transferase (TdT). Thus, morphology and immunologic phenotype identified an ALL L3 subtype (Burkitt-like). Treatment with daunomycin, cyclophosphamide, vincristine, prednisone, and intrathecal methotrexate resulted in a complete remission. Consolidation therapy with asparaginase was given, but 2 months later the leukemia relapsed. The patient refused further treatment and died at home 1 month later. MATERIALS AND METHODS
Cell Line Peripheral blood, which contained less than 2 % of B-lymphocytes, was obtained at the time of relapse. Enriched leukocyte suspension was obtained by sedimentation in high molecular weight dextran sulphate and put into culture at high cell density in RPMI 1640, supplemented with 10% fetal calf serum, glutamine, and antibiotics. The cells were kept in a humidified incubator with a pCO2 of 5% in air and checked regularly for growth. Cell debris was removed cautiously and the medium was only partially renewed once a week. After 4 weeks, clumps of growing cells were visible and I month later the line was considered to be established, as it could be split I to 2 regularly, harvested, and characterized. As a control for the hybridization experiment we used a cell line named 1301 carrying the t(8;14), and SUDHL-4, another cell line carrying the classical t(14;18) [19]. Cytogenetics Cytogenetics was performed on a 24-hour culture of bone marrow cells at diagnosis and at several occasions on the cell line after establishment in vitro, according to standard procedures. The chromosomes were identified with GTG banding at diagnosis and with RFA, QFQ, and GTG banding on the cell line. Fluorescent in situ hybridization (FISH} Probes: The probes were D8Z2, recognizing highly repetitive alphoid sequences in the centromeric region of chromosome 8 [20], and the chromosome-specific plasmid libraries pBS-8 and pBS-18, a generous gift from J. Gray (University of California) [21]. Cos-1.16, a cosmid probe recognizing the constant region of the IGH locus on chromosome 14, was given by G. Grosveld and a 5.7-kb genomic probe for the MYC oncogene [22] was also used.
R . B . C . van Ooteghem et al. Methods: The DNA probes were labeled with biotin-16-dUTP or digoxygenin-ll-dUDP by standard nick translation, precipitated, and resnspended in hybridization mixture (50% formamide, lO% dextmn sulphate in 2 x SSCP) with salmon sperm DNA (0.5 ~gr/~l) and Cot-1 DNA (0.5 ~gr/~l) when competitive hybridization was necessary. The probe concentration was as follows: 0.5 ngr/~l for centromeric probe, 7.5 ngr/~l for cosmids and for chromosome-specific libraries. After probe denaturation (4 minutes at 72°C), the repeat sequences were allowed to preanneal with Cot-1 DNA for I hour at 37°C, in the case of cosmids or libraries. Metaphase spreads on glass were pretreated with RNAse and a low concentration of pepsin, posffixed, and denatured at 72°C for 2.5 minutes in 70% formamide. Probes were hybridized overnight at 37°C in a humidified box. After washing, the hybridization sites were visualized by immunochemistry using two layers of FITC or TRITC and observed in fluorescence. Chromosome identification was obtained by DAPI banding or co-hybridization with a known probe. Details of the techniques were slightly modified according to probe characteristics [23, 24]. For each probe tested at least 20 metaphases were analyzed by FISH.
DNA Analysis of BCL2 and IGH Gene Rearrangement High molecular weight DNA was extracted from ROS-50. For PCR analysis of t[14;18) two primers were defined: a sense MC12 primer (5' TTGATGGCCTTTGCTGAGA3') and an antisense JH consensus primer (5' TGAGGAGACGGTGACC 3') [25). One microgram of DNA in a reaction volume of 50 ~tl was subjected to 30 cycles of PCR amplification [26]. Amplified products were analyzed by Southern blotting and hybridized with the pFL-2 probe specific for the minor cluster region of chromosome 18 [gift from M. L. Cleary [27]). The configuration of the IGH genes was analyzed in Bgl/II digests, as well as in BamHI/HindIII double digests with a 32p random oligonucleotide-labeled JH probe [28]. The JH probe [IGHJ6) is a 1.0-kb probe, which recognizes sequences that are just 3' of the JH gene segments [Beishuizen et al., Leukemia 1993; 7:2045-2053). lmmunophenotype and BLC2 Protein Expression The cells from cell line ROS-50 were extensively phenotyped using a panel of monoclonal antibodies exactly as reported for diagnosis of leukemia and lymphoma [29]. Expression of the BCL2 protein was analyzed on cytocentrifugated cell preparations, both unfixed and fixed in acetone for 10 minutes. In an indirect immunofluorescence assay, anti-BCL2 antibody (100-a, gift of D. Y. Mason, Oxford, U.K.) was used, with an FITC-conjugated goat anti-mouse immunoglobulin antibody (Nordic batch no. 3815) in the second step [30]. RESULTS Cell Line and Immunophenotyping The cell line ROS-50 grew spontaneously in vitro out of a blood cell suspension containing less than 2 % of B-lymphocytes. The patient at that time suffered from a bone marrow relapse of a previously treated B-ALL, FAB type L3. Immunologic marker analysis of the ROS-50 cells showed
t ( 8 ; 1 4 ; 1 8 ) i n a B-cell L i n e
Figure 1
89
R-banded karyotype of the ROS-50 cell line. Asterisks indicate the structurally abnormal chromosomes.
a m o n o c l o n a l B-cell p o p u l a t i o n p o s i t i v e for S m K , S m ~ , a n d p a r t i a l l y for SinS. T h e m a j o r i t y of cells (95-100%) w e r e positive for t h e B-cell m a r k e r s CD10, CD19, CD20, CD24, CD3Z FMC7, a n d HLA-DR. Part of t h e c e l l s e x p r e s s e d CD21, t h e trans§errin r e c e p t o r CD71, a n d t h e p r e c u r s o r cell m a r k e r CD34 (50%, weak). T h e ROS-50 c e l l s w e r e n e g a t i v e for TdT. T h e c o m b i n a t i o n of CD34 a n d a m a t u r e B-cell m a r k e r (FMC7) is u n u s u a l . CD10 p o s i t i v i t y c a n i n d i c a t e a p r e c u r s o r B - p h e n o t y p e or a f o l l i c u l a r B-cell p h e n o t y p e . T h e o r i g i n a l l e u k e m i a
Table 1
w a s CD10 n e g a t i v e b u t t h e r e l a p s e - d e r i v e d cell l i n e w a s CD10 + (95% of t h e cells). T h i s c o u l d i n d i c a t e c l o n a l evolut i o n or c l o n a l s e l e c t i o n . Cytogenetics At d i a g n o s i s , 14 b o n e m a r r o w m e t a p h a s e s w e r e a n a l y z e d ; a m o n g t h e m , t h r e e s h o w e d a 46,XY k a r y o t y p e a n d 11 w e r e a b n o r m a l . T h e 8q24 b a n d o n o n e c h r o m o s o m e 8 was s l i g h t l y s h o r t e r a n d s t a i n e d a b n o r m a l l y , o n e c h r o m o s o m e 14 w a s too
C h r o m o s o m a l hybridization signals of various probes on metaphases from ROS-50, 1301, and S U D H L - 4 cell lines Number of positive signals in 20 metaphases
Cell line translocation ROS-50 t(8;14;18)
1301 t(8;14)
SUDHL-4 t(14;18)
Chromosome Probe
Locus
8
der(8)
der(8)
14
14q +
18
18q -
D8Z2 MYC IGH pBS18 IGH MYC DSZ2 IGH
Centromere 8 8q24 14q32 Paint 18 14q32 8q24 Centromere 8 14q32
60 a 19 0 0 0 17 40 a 0
60 a 0 17 19 0 0 40 ° -
60 a 0 18 20 -
0 0 13 0 18 0 0 11 (weak)
0 10 0 0 19 12 0 0
0 0 0 17 0 0 0 0
0 0 6 (weak) 19 -
a Co-hybridizationof probe DSZ2 with Myc, IGH, and pBS 18:20 metaphases scoredin each experiment. b _, Chromosome not presentin the cellline.
19
18q
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20
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iiiiiiiiiii 8q24-qter
14 der(14)
18 der(18)
Figure 3 Schematic representation of the chromosome pairs 8, 14, and 18 of ROS-50 cells. Shown are the sites of break and the nature of exchanged chromosomal segments, as indicated by FISH studies.
long, a n d one c h r o m o s o m e 18 was too short. All the other 43 chromosomes appeared normal. No cytogenetic study was done at relapse. The ROS-50 cell line showed a rather homogeneous karyotype: 47,XY,der(8), + der(8),add (14)(q32), der(7;17)(q10;q10),del(18)(q21), as shown in Figure 1. This was interpreted as a t(8;14;18)(q24;q32;q22), w i t h d u p l i c a t i o n of der(8), trisomy for 7(t, and m o n o s o m y 17p. A m i n o r i t y (16%) of cells showed additional aberrations such as only one der(8) and trisomy 11. The karyotype of the cell line 1301 was 47,XY, ÷Y,t(8;14) (q24;q32),der(14)t(8;14)t(14;19)(p12;p11 or q11),der(19) t(14;19). At the time of study the karyotype of SUDHL-4 was hyperd i p l o i d a n d showed more abnormalities than previously reported [19] i.e., structural rearrangements of chromosomes 2, 3, 8, 12, 13, trisomy 7, and a classical t(14;18)(q32;q21) with two copies of der(18).
FISH Studies Results obtained by hybridization of the various probes on
metaphases from the ROS-50, 1301, and SUDHL-4 cell lines are s u m m a r i z e d in Table 1 and shown in Figure 2. The D8Z2 probe revealed three clear centromeric spots on metaphases and interphase cells from the ROS-50 cell line, indicating the presence of a normal chromosome 8 and two der(8) (Fig. 2). In most experiments D8Z2 was co-hybridized with another probe in order to identify easily the chromosome 8 and der(8). The probe for the MYC oncogene is relatively small and p r o d u c e d faint but clear signals on the normal 8q24 and on the 14q ÷ in ROS-50 cells. The two der(8) d i d not hybridize to the MYC probe (Fig. 2A). This was indicative of a translocation (8;14) displacing MYC to the der(14). On the cell line 1301 the MYC probe also hybridized to the normal 8 and the 14q ÷ (not shown). Using a cosmid probe for IGH, clear signals were obtained on the normal chromosome 14 in all three cell lines, on the two der(8) in ROS-50 (Fig. 2C), on the 14q ÷ in line 1301, and at the two der(18) in SUDHL-4. Furthermore, weak signals were seen in half of the metaphases on the 18q- in ROS-50 and on both 15q14 regions in all lines. The latter is a known
Figure 2 Hybridization results on metaphase spreads from ROS-50 cells (A,C,E) and corresponding DAPI staining (B,D,F}. (A) Double hybridization with MYC and D8Z2 probes. The DSZ2 probe gives a bright hybridization signal on the centromere of three chromosomes. MYC signals (arrowhead) are visible on the normal chromosome 8 and on the der(14) chromosome. (C) Double hybridization with IGH and DSZ2 probes. Arrowheads indicate IGH signals on the normal chromosome 14 and the two der(8) chromosomes; the asterisk indicates IGH signal on the der(18) chromosome. Secondary sites are visible on the proximal part of 15q. (E) Double hybridization with pBS-18 and D8Z2 probes. Arrowheads indicate part of chromosome 18 on the two der(8) chromosomes. In addition, paints of two chromosomes 18 are clearly visible.
92
R . B . C . van Ooteghem et al.
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-G Figure 5 PCR analysis of ROS-50 DNA: 1/20 of the amplified product electrophoresed and hybridized with pFL-2 a mcr-specific probe. The marker lane contains 50 ngr of Phix DNA digested with HaeIII and shows bands of 1353, 603, and 201 bp.
2.3
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Bglll, JH probe
BamHI/Hindlll, JH probe
Figure 4 Southern blot analysis of Bgl/II and BamHI/HindI~ double digests of a control cell line DNA with germ line IGH genes and DNA from the ROS-50 cell line, probed with a JH probe (IGHJ6). In ROS-50 DNA, no germ line band was detected but three rearranged IGH bands are visible.
secondary hybridization site of this probe. This indicated involvement of the IGH gene in the chromosome 14 rearrangemerits with various breakpoints around or within the region of the gene identified by our cosmid probe, i.e., JH to C~. The pBS-18 library painted four chromosomes in ROS50, i.e., 18, 18q - , and the distal part of both der(8) (Fig. 2E). This showed the composite nature of the der(8) that contains sequences of the IGH gene derived from chromosome 14 and part of chromosome 18 distal to 18q21 (Fig. 3).
DNA Analysis of IGH and BCL2 Rearrangements Southern blot analysis of IGH gene rearrangements with the JH probe revealed in both Bg/II and BamHI/HindIII digestions three rearranged IGH gene bands with different density (one band with a larger density compared to the other two bands) (Fig. 4). From these data it is not possible to decide which chromosome is actually coding. The heavier band was interpreted as representing sequences carried by the duplicated der(8), while the two other rearranged bands of
comparable density could be attributed to the normal chromosome 14 and to the t(14;18). PCR analysis of ROS-50 DNA revealed a breakpoint in the mcr region of chromosome 18. The fragment size of the amplified product was visualized by ethidium bromide staining and hybridization with pFL-2 and estimated to be about 550 base pairs (bp) (Fig. 5). This demonstrated the presence of a IGH-BCL2 rearrangement and confirmed the presence of a t(14;18). Furthermore, the immunofluorescent assay using an anti-BCL2 antibody showed a bright fluorescence in the cytoplasm of all cells, both on unfixed and fixed slides, clearly demonstrating a high expression of BCL2 protein. DISCUSSION Cytogenetic and FISH analysis of the ROS-50 cell line showed a complex chromosomal rearrangement that can be interpreted as a combination of two nonrandom translocations, i.e., t(14;18)(q32;q21) and t(8;14)(q24;q32), both involving the same chromosome 14. This implies involvement of the BCL2 gene on chromosome 18 and of the MYC oncogene on chromosome 8. Indeed, a breakpoint in the mcr of the BCL2 gene was shown by PCR analysis, high expression of the BCL2 protein was demonstrated, and translocation of MYC from chromosome 8 to 14q+ was shown by FISH. Furthermore, FISH turned out to be a powerful tool to analyze this double translocation involving the same chromosome 14. The Cos-1.16 probe is not rigorously mapped on the IGH sequences but is known to encompass the switch I~ and C~ and to extend 5' into the J region (Fig. 6). If we keep in mind the 3'-'5' orientation of the IGH gene on chromosome 14, the FISH results suggest the following scenario:
t(8;14;18) in a B-cell L i n e
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tt(14;18) tt(8;14) F i g u r e 6 Schematic representation of the molecular organization of the IGH gene on chromosome 14. The solid bar indicates the probes used for FISH analysis, arrows indicate the putative breakpoints. The exact boundaries of the probe are not known.
T h e t r a n s l o c a t i o n (14;18) o c c u r r e d at first, p o s s i b l y w i t h a b r e a k p o i n t in the J r e g i o n of the IGH gene, t h u s distal o n c h r o m o s o m e 14. T h i s g e n e r a t e d a der(18) that o n l y w e a k l y h y b r i d i z e s w i t h o u r IGH p r o b e a n d a der(14) s h o w i n g positive hybridization for the IGH probe. In the subsequent t(8;14), t h e s a m e c h r o m o s o m e der(14) was b r o k e n in the IGH r e g i o n p r o x i m a l to o u r probe. S u c h event e x p l a i n s t h e IGH-positive signal as w e l l as the c h r o m o s o m e 18 p a i n t i n g o n t h e der(8) a n d the loss of IGH signal o n the der(14). T h i s is s c h e m a t i z e d in Figures 3 and 6. S o u t h e r n blot analysis s h o w e d r e a r r a n g e m e n t of t h r e e n o n - g e r m l i n e b a n d s (Fig. 4). T h i s was also s e e n w i t h o t h e r JH probes (data not s h o w n ) a n d c a n be the c o n s e q u e n c e of the c o m p l e x t r a n s l o c a t i o n w i t h b r e a k in the JI-I-C~ area, as tentatively d e p i c t e d in F i g u r e 6. Very r e c e n t l y Z e l e n e t z et al. [31] r e p o r t e d o n t h e f r e q u e n t o c c u r r e n c e of interstitial d e l e t i o n of der(14) in t(14;18) carr y i n g l y m p h o m a . In ROS-50 cell line, instead of a d e l e t i o n ,
a translocation w i t h 8q24 r e s e m b l i n g the class switch recomb i n a t i o n event c o u l d have h a p p e n e d . F u r t h e r investigations are n e e d e d to d i s c l o s e t h e m o l e c u l a r structure of t h e rearr a n g e d c h r o m o s o m a l regions. T h e a s s o c i a t i o n of t(8;14) a n d t(14;18) is rare, as r e v i e w e d by Brito-Babapulle et al. [9]. In Table 2 we s u m m a r i z e the data of reported cases w i t h t(14;18) and t(8;14), excluding variants. Five cases p r e s e n t e d w i t h a history of f o l l i c u l a r l y m p h o m a p r e c e d i n g l y m p h o b l a s t i c l e u k e m i a , w h i l e in seven other cases [ i n c l u d i n g ours) acute l e u k e m i a was the presenting diagnosis. T h e FAB classification was L2 or L3 and in s o m e investigated cases the blast cells were TdT-positive. Our case p r e s e n t e d as acute l e u k e m i a of t h e FAB-L3 type, w i t h Burkitt-like l y m p h o b l a s t s , a n d o n l y t r a n s i e n t r e s p o n s e to therapy. T h i s is v e r y m u c h in l i n e w i t h o t h e r m o r p h o l o g i cally s i m i l a r cases that carry o n l y a t(8;14) a n d suggests that i n d e e d d e r e g u l a t i o n of MYC has taken place. As described above, we have cytogenetic e v i d e n c e that the
Table 2 Cases of l y m p h o b l a s t i c l e u k e m i a or l y m p h o m a w i t h t(14;18)(q32;q21) a n d an a d d i t i o n a l t(8;14)(q24;32) Cell line
Patient Reference sex { a g e }
SU-DUL5
[18]
KHM-2B Ros 50 DS 380 NA
[13] This study b [14] [10]
NA NA
[11] [9]
NA
[16] [171
NA
NA NA NA
[15]
Diagnosis
Additional translocation Tdt
BCL-2
c-MYC BCL-2 c-MYC
(DNA}
(RNA/protein}
M (30) Lymphoblastic lymphoma M (62) B-ALL L3 M {69) B-ALL L3
t(8; 14)a
ND
lUG
lUG
t(8;14} t(8;14) a
ND -
lUG R
luG ND
F (70) Malignant lymphoma M (15) C-ALL L2 F (50) Diffuse large-cell lymphoma/pre-B-ALL M (21) C-ALL L2 F (73) B-ALL L3
t(6; 14) t(6;14) t(8;14)
+ +
R R luG
ND G/G luG
- /ND ND
t(8; 14) t(8;14)
+ -
ND luG
M (36) ALL L3 M (62) Immature ALL M (64) Immunoblastic lymphoma F (29) ALL-L2 M (44) Follicular lymphoma with interfollicular lymphoblastic lymphoma
t(6;14) t(8;14) t(8;14}
ND -
ND BCL-2-JH fragment luG ND ND
t(8; 14) t(8; 14)
+
ND IUG
+
- /-
+/ND
Survival Previous follicular (months} lymphoma NA
NA
3 5
No No
+/ND ND ND
6 5 2
No No Yes
ND ND
ND ND
3 0.25
No Yes c
lUG ND ND
ND ND ND
ND ND ND
3 3 4
No No Yes
ND IUG
ND ND
ND ND
29 8
Yes No
+/ND +/ND ND/+ ND
Abbreviations: ND, not done; R, rearranged; G, germline; NA, not available; survival after diagnosis. a In these cases the same chromosome 14 was involved in the t(8;14) and in the t(14;18). b A.C. Lambrechts et al., personal communication (manuscript in preparation). c Small cleaved lymphocytes in paratrabecular areas indicative of previously undetected follicular lymphoma.
94 t(14;18) p r e c e d e d the t(8;14) but t h e t i m e lapse b e t w e e n t h e two events is u n k n o w n . Also, there w e r e no obvious c l i n i c a l c h a n g e s p r e c e d i n g the onset of f u l l - b l o w n l e u k e m i a . T h e t(14;18) in follicular l y m p h o m a results in o v e r e x p r e s s i o n of the BCL2 oncogene. T h i s was also the case in the ROS-50 cell line. In the single-cell line (SU-DUL5) r e p o r t e d w i t h t(14;18) a n d t(8;14) i n v o l v i n g the s a m e c h r o m o s o m e 14 [18], BCL2 was not expressed and s e q u e n c i n g of the der(8) showed translocation of BCL2 o n the der(8) b u t no e v i d e n c e for chrom o s o m e 14 translocation. In our case, part of the IGH is translocated to t h e der(8) together w i t h 18q21-qter, t h u s p o s s i b l y m a i n t a i n i n g BCL2 u n d e r the control of the IGH enhancer. The authors thank E.J. Harthoorn-Lasthuizen, M.D. for referring this patient, A. Geurts van Kessel, Ph.D., for a first appraisal of cytogenetic data and R. Boucke for typing the manuscript. Part of the work was supported by the Netherlands Cancer Foundation.
REFERENCES 1. Zech L, Haglund U, Nilsson K, Klein G (1976}: Characteristic chromosomal abnormalities in biopsies and lymphoid-cell lines from patients with Burkitt and non-Burkitt lymphomas. Int J Cancer 17:47-56. 2. Berger R, Bernheim A, Weh HJ, Flandrin G, Daniel MT, Brouet JT, Colbert N (1979): A new translocation in Burkitt's tumor ceils. Hum Genet 53:111-112. 3. Van Den Berghe H, Parloir H, Gosseye S, Englebienne V, Coruu G, Sokal G (1979): Variant translocation in Burkitt lymphoma. Cancer Genet Cytogenet 1:9-14. 4. Dalla-Favera R, Bregni M, Erikson J, Patterson D, Gallo RC, Croce CM (1982): Human c-myc onc gene is located in the region of chromosome 8 that is translocated in Burkitt lymphoma cells. Proc Natl Acad Sci USA 79:7824-7827. 5. Croce CM, Thierfelder W, Erikson J, Nishikura K, Finan J, Lenoir G, Newel PC (1983): Transcriptional activation of an unrearranged and untmnslocated c-myc oncogene by translocation of C~, locus in Burkitt lymphoma. Proc Natl Acad Sci USA 80:6922-6926. 6. Croce CM, Nowell PC (1985): Molecular basis of human B-cell neoplasia. Blood 65:1-7. 7. Cotter FE (1990): The role of the bcl-2 gene in lymphoma. Br J Haematol 75:449-453. 8. Hockenbery D, Nunez G, Milliman C, Schreiber RD, Korsmeijer SJ (1990): bcl-2 is an inner mitochondrial membrane protein that blocks programmed cell death. Nature 348:334-336. 9. Brito-Babapulle V, Crawford A, Khokhar T, Laffan M, Matutes E, Fairhead S, Catovsky D (1991): Translocations t(14;18) and t(8;14) with rearranged bcl-2 and c-myc in a case presenting as B-ALL (L3). Leukemia 5:83-87. 10. Gauwerky CE, Hoxie J, Nowell PC, Croce CM (1988): Pre-B cell leukemia with a t(8;14) and a t(14;18) translocation is preceded by follicular lymphoma. Oncogene 2:431-435. 11. Mufti GJ, Hamblin TJ, Oscier DG, Johnson S (1983): Common ALL with pre-B cell features showing (8;14) and (14;18) chromosome translocations. Blood 62;1142-1146. 12. Koduru PRK, Offit K (1991): Molecular structure of double reciprocal translocations: significance in B-cell lymphomagenesis. Oncogene 6:145-148. 13. Matsuzaki H, Hate H, Asou N, Suzushima H, Akahoshi Y, Yoshida M, Nagakura S, Ishii T, Sanada I, Takatsuki K (1990): Establishment and characterization of acute B-cell lymphocytic leukemia cell line showing (8;14) and (14;18) chromosome translocation. Acta Haematol 84:156-161. 14. Pegoraro L, Palumbo A, Erikson J, Falda M, Giovanazzo B, Emanuel BS, Revere G, Nowell PC, Croce CM (1984): A 14;18 and an 8;14 chromosome translocation in a cell line derived
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from an acute B-cell leukemia. Proc Natl Acad Sci USA 81:7166-7170. de Jong D, Voetdijk BMH, Beverstock GC, van Ommen GJB, Willemze R, Kluin PH (1988): Activation of the c-myc oncogene in a precursor B-cell blast crisis of follicular lymphoma, presenting as composite lymphoma. N Engl J Med 318:1373-1378. Kramer MHH, Raghoebier S, Beverstock GC, de Jong D, Kluin PM, Kluin-Nelemans JC (1991): De nero acute B-cell leukemia with translocation t(14;18): an entity with a poor prognosis. Leukemia 5:473-478. Thangavelu M, Olopade O, Beckman E, Vardiman JW, Larson RA, McKeithan TW, Le Beau MM, Rowley JD (1990): Clinical, morphologic, and cytogenetic characteristics of patients with lymphoid malignancies characterized by both t(14;18)(q32;q21) and t(8;14)(q24;q32) or t(8;22)(q24;q11). Genes Chrom Cancer 2:147-158. Kiem HP, Nourse J, Saltman DL, Blume KG, Cleary ML (1990): Concurrent activation of c-myc and inactivation of Bcl-2 by chromosomal translocation in a lymphoblastic cell line. Oncogene 5:1815-1819. Kaiser-McCaw Hecht B, Epstein AL, Berger CS, Kaplan HS, Hecht F (1985): Histocytic lymphoma cell lines and cytogenetic studies. Cancer Genet Cytogenet 14:205-218. Donlon TA, Bruns GA, Latt SA, Mulholland J, Wijman AR (1987): A chromosome 8 enriched alphoYd repeat. Cytogenet Cell Genet 46:607. Collins C, Kuo WL, Seagraves R, Fuscoe J, Pinkel D, Gray JW (1991): Construction and characterization of plasmid libraries enriched in sequences from single human chromosomes. Genomics 11:997-1008. Stehelin D, Saule S, Roussel M (1979): Three new types of viral oncogenes in defective avian leukemia viruses I. Specific nucleotide sequences of cellular origin correlate with specific transformation. Cold Spring Harbor Symposia on Quantitative Biology, 1215-1223. Pinkel D, Landegent J, Collins C (1988): Fluorescence in situ hybridization with human chromosome-specific libraries: Detection of trisomy 21 and translocations of chromosome 4. Proc Natl Acad Sci USA 85:9138-9142. Arnoldus EPJ, Wiegant J, Noordermeer IA, Wessels JW, Beverstock GC, Grosveld GC, van der Ploeg M, Raap AK (1990): Detection of the Philadelphia chromosome on interphase nuclei. Cytogenet Cell Genet 54:108-111. Ngan B-O, Nourse J, Cleary ML (1989): Detection of chromosomal translocation t(14;18) within the minor cluster region of bcl-2 by polymerase chain reaction and direct genomic sequencing of the enzymatically amplified DNA in follicular lymphomas. Blood 73:1759-1762. Lambrechts AC, de Ruiter PE, Dorssers LCJ, van 't Veer MB (1992): Detection of residual disease in translocation (14;18) positive non-Hodgkin's lymphoma using the polymemse chain reaction: A comparison with conventional staging methods. Leukemia 6:29-34. Cleary ML, Galili N, Skier J (1986): Detection of a second t(14;18) breakpoint cluster region in human follicular lymphomas. J Exp Med 164:315-320. Van Dongen JJM, Wolvers-Tettero ILM (1991): Analysis of immunoglobulin and T-cell receptor genes. Part I: Basic and Technical Aspects. Clinica Chimica Acta 198:1-92. Van Dongen JJM, Adriaansen HJ, Hooijkaas H (1988): Immunophenotyping of leukemias and non-Hodgkin lymphomas. Immunological markers and their CD codes. Neth J of Med 33:298-314. Pezella F, Tse E, Gordell J, Pulford KAF, Getter KC, Mason DY (1990): Expression of the bcl-2 oncogene protein is not specific for the t(14;18) translocation. Am J Pathol 137:225-232. Zelenetz AD, Cleary ML, Levy R (1993): A submicroscopic interstitial deletion of chromosome 14 frequently occurs adjacent to the t(14;18) translocation breakpoint in human follicular lymphoma. Genes Chrom Cancer 6:140-150.
ABSTRACT: A cell line named BOS-50 [Rotterdam suspension cell line no. 50) has been established from peripheral blood of a 69-year-old male with acute lymphoblastic leukemia (FAB type L3). Among the aberrations, cytogenetic analysis showed the presence of 14q +, 18q-, and two 8 q - marker chromosomes. With fluorescence in situ hybridization [FISH) we characterized the chromosomal translocations, t(8;14) and t(14;18), in which the same chromosome 14 is involved. PCB analysis demonstrated the presence of an IGH-BCL2 rearrangement with a breakpoint in the minor cluster region (mcr) confirming the t[14;18) characteristic for follicular lymphoma. Additional studies showed high expression of BCL2 protein, an early B-cell immunophenotype, and an unusual pattern of IGH gene rearrangement.
INTRODUCTION B-ALL represents about 5% of all ALL cases and shows a particular morphology of blasts reminiscent of lymphoblasts in Burkitt lymphoma with vacuolated cytoplasm (FAB-L3). Nonrandom chromosome abnormalities are consistently found in Burkitt lymphoma; these include t(8;14)(q24;q32) or one of the variants, t(2;8)(p12;q24) and t(8;22)(q24;q11) [1-3]. In the case of t(8;14), Dalla-Favera et al. [4] have shown that the MYC oncogene, which is located on band 8q24, translocates to the IGH locus on chromosome 14. On the contrary, in both t(2;8) and t(8;22) MYC remains on the rearranged chromosome 8 and the kappa or lambda immunoglobulin light-chain constant regions, respectively, translocate to chromosome 8 [5]. In all cases MYC comes into the neighborhood of an immunoglobulin enhancer. Under the influence of this enhancer MYC is deregulated and transcribed constitutively at an elevated level. The product of the MYC oncogene is a nuclear protein capable of binding DNA. While the function of the MYC gene product is not yet clear, it seems likely that it is involved in the control of cell proliferation [6]. Another translocation, t(14;18)(q32;q21), involving the IGH locus on 14q32 and the BCL2 locus on 18q21, is found in follicular lymphoma [7], but may be observed in other B-cell
From the Departments of Cell Biology and Genetics (R. B. C. v. 0., E. M. E. S., A. H.), and Immunology (A. B.), Erasmus University, Rotterdam; Dr. Daniel Den Hoed Cancer Center (A. C. L.), Rotterdam; Department of Clinical Genetics (M. v. d. B.-P.), Veldhoven; Groot Ziekengasthuis (T. ]. S.), 's-Hertogenbosch, The Netherlands. Address reprint requests to: Anne Hagemeijer, Department of Cell Biology and Genetics, Erasmus University, P.O. Box 1738, 3000 DR Rotterdam, The Netherlands. Received July 2Z 1993; accepted October 25, 1993.
malignancies as well. The BCL2 gene is translocated to 14q32 into the IGH gene and is consequently deregulated and transcribed at a high rate. The coding regions of the BCL2 gene are left intact in all t(14;18) translocations so that the BCL2IGH transcript produced encodes a normal BCL2 protein. BCL2 activation prevents the cells from undergoing programmed cell death (apoptosis), a phenomenon corresponding to the low-grade malignancy oft_he follicular lymphoma [8]. Recently, a few cases of highly malignant leukemia and/or lymphoma carrying both types of rearrangements, i.e., t[8 ;14) and t(14;18), have been reported. These two translocations usually involve both chromosomes 14 [9-17]. However, in a lymphoblastoid cell line a three-way translocation between chromosomes 8, 14, and 18 is described by Kiem et al. [18]. We report here on the establishment and characterization of a new cell line with a complex t(8;14;18), a Burkitt-like morphology of blast cells, early B-cell immunophenotype, and BCL2 rearrangement studied by PCR. Fluorescence in situ hybridization (FISH) with chromosome-specific probes and IGH gene rearrangement studies were applied to analyze this complex translocation. CASE REPORT
A 69-year-old man presented with complaints of fatigue, loss of appetite, weight loss, and night sweats. He had a 10-year history of diabetes and hypertension, the former treated with glibenclamide and metformin. Physical examination showed pallor. He had a slightly enlarged liver, no lymphadenopathy or splenomegaly. Laboratory investigations showed a high ESR, Fib 6.1 mmol/L, platelets 63 x 10E9/L, leukocytes 4.1 x 10E9/L with 1% eosinophils, 47% granulocytes, 2 % monocytes, 5% myelocytes, 2% metamyelocytes, 10% band nan87
© 1994 Elsevier Science Inc. 655 A v e n u e of the Americas, N e w York, NY 10010
Cancer Genet Cytogenet 7 4 : 8 7 - 9 4 (1994)
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88 trophils, 33 % lymphocytes. There were no signs of hemolysis or intravascular coagulation. LDH (4152 U/L) and glucose (12.5 retool/L) were elevated. Virus serology for EBV, HSV I and 2, hepatitis B, and HIV was negative. A bone marrow aspirate contained 90% large blast-like cells with an abundant chromatin in the nucleus and two to three nucleoli. The basophilic cytoplasm revealed multiple vacuoles (Burkittlike lymphoblasts). These blasts did not stain with Sudan black, PAS, acid phosphatase, and nonspecific esterase. Immunophenotypic evaluation revealed a monoclonal B-cell population, expressing CD20 and CD19 with an IgM kappa membrane expression. These cells were negative for CD5, CD10 (CALLA), intracytoplasmatic immunoglobulins, and terminal deoxynucleotidyl-transferase (TdT). Thus, morphology and immunologic phenotype identified an ALL L3 subtype (Burkitt-like). Treatment with daunomycin, cyclophosphamide, vincristine, prednisone, and intrathecal methotrexate resulted in a complete remission. Consolidation therapy with asparaginase was given, but 2 months later the leukemia relapsed. The patient refused further treatment and died at home 1 month later. MATERIALS AND METHODS
Cell Line Peripheral blood, which contained less than 2 % of B-lymphocytes, was obtained at the time of relapse. Enriched leukocyte suspension was obtained by sedimentation in high molecular weight dextran sulphate and put into culture at high cell density in RPMI 1640, supplemented with 10% fetal calf serum, glutamine, and antibiotics. The cells were kept in a humidified incubator with a pCO2 of 5% in air and checked regularly for growth. Cell debris was removed cautiously and the medium was only partially renewed once a week. After 4 weeks, clumps of growing cells were visible and I month later the line was considered to be established, as it could be split I to 2 regularly, harvested, and characterized. As a control for the hybridization experiment we used a cell line named 1301 carrying the t(8;14), and SUDHL-4, another cell line carrying the classical t(14;18) [19]. Cytogenetics Cytogenetics was performed on a 24-hour culture of bone marrow cells at diagnosis and at several occasions on the cell line after establishment in vitro, according to standard procedures. The chromosomes were identified with GTG banding at diagnosis and with RFA, QFQ, and GTG banding on the cell line. Fluorescent in situ hybridization (FISH} Probes: The probes were D8Z2, recognizing highly repetitive alphoid sequences in the centromeric region of chromosome 8 [20], and the chromosome-specific plasmid libraries pBS-8 and pBS-18, a generous gift from J. Gray (University of California) [21]. Cos-1.16, a cosmid probe recognizing the constant region of the IGH locus on chromosome 14, was given by G. Grosveld and a 5.7-kb genomic probe for the MYC oncogene [22] was also used.
R . B . C . van Ooteghem et al. Methods: The DNA probes were labeled with biotin-16-dUTP or digoxygenin-ll-dUDP by standard nick translation, precipitated, and resnspended in hybridization mixture (50% formamide, lO% dextmn sulphate in 2 x SSCP) with salmon sperm DNA (0.5 ~gr/~l) and Cot-1 DNA (0.5 ~gr/~l) when competitive hybridization was necessary. The probe concentration was as follows: 0.5 ngr/~l for centromeric probe, 7.5 ngr/~l for cosmids and for chromosome-specific libraries. After probe denaturation (4 minutes at 72°C), the repeat sequences were allowed to preanneal with Cot-1 DNA for I hour at 37°C, in the case of cosmids or libraries. Metaphase spreads on glass were pretreated with RNAse and a low concentration of pepsin, posffixed, and denatured at 72°C for 2.5 minutes in 70% formamide. Probes were hybridized overnight at 37°C in a humidified box. After washing, the hybridization sites were visualized by immunochemistry using two layers of FITC or TRITC and observed in fluorescence. Chromosome identification was obtained by DAPI banding or co-hybridization with a known probe. Details of the techniques were slightly modified according to probe characteristics [23, 24]. For each probe tested at least 20 metaphases were analyzed by FISH.
DNA Analysis of BCL2 and IGH Gene Rearrangement High molecular weight DNA was extracted from ROS-50. For PCR analysis of t[14;18) two primers were defined: a sense MC12 primer (5' TTGATGGCCTTTGCTGAGA3') and an antisense JH consensus primer (5' TGAGGAGACGGTGACC 3') [25). One microgram of DNA in a reaction volume of 50 ~tl was subjected to 30 cycles of PCR amplification [26]. Amplified products were analyzed by Southern blotting and hybridized with the pFL-2 probe specific for the minor cluster region of chromosome 18 [gift from M. L. Cleary [27]). The configuration of the IGH genes was analyzed in Bgl/II digests, as well as in BamHI/HindIII double digests with a 32p random oligonucleotide-labeled JH probe [28]. The JH probe [IGHJ6) is a 1.0-kb probe, which recognizes sequences that are just 3' of the JH gene segments [Beishuizen et al., Leukemia 1993; 7:2045-2053). lmmunophenotype and BLC2 Protein Expression The cells from cell line ROS-50 were extensively phenotyped using a panel of monoclonal antibodies exactly as reported for diagnosis of leukemia and lymphoma [29]. Expression of the BCL2 protein was analyzed on cytocentrifugated cell preparations, both unfixed and fixed in acetone for 10 minutes. In an indirect immunofluorescence assay, anti-BCL2 antibody (100-a, gift of D. Y. Mason, Oxford, U.K.) was used, with an FITC-conjugated goat anti-mouse immunoglobulin antibody (Nordic batch no. 3815) in the second step [30]. RESULTS Cell Line and Immunophenotyping The cell line ROS-50 grew spontaneously in vitro out of a blood cell suspension containing less than 2 % of B-lymphocytes. The patient at that time suffered from a bone marrow relapse of a previously treated B-ALL, FAB type L3. Immunologic marker analysis of the ROS-50 cells showed
t ( 8 ; 1 4 ; 1 8 ) i n a B-cell L i n e
Figure 1
89
R-banded karyotype of the ROS-50 cell line. Asterisks indicate the structurally abnormal chromosomes.
a m o n o c l o n a l B-cell p o p u l a t i o n p o s i t i v e for S m K , S m ~ , a n d p a r t i a l l y for SinS. T h e m a j o r i t y of cells (95-100%) w e r e positive for t h e B-cell m a r k e r s CD10, CD19, CD20, CD24, CD3Z FMC7, a n d HLA-DR. Part of t h e c e l l s e x p r e s s e d CD21, t h e trans§errin r e c e p t o r CD71, a n d t h e p r e c u r s o r cell m a r k e r CD34 (50%, weak). T h e ROS-50 c e l l s w e r e n e g a t i v e for TdT. T h e c o m b i n a t i o n of CD34 a n d a m a t u r e B-cell m a r k e r (FMC7) is u n u s u a l . CD10 p o s i t i v i t y c a n i n d i c a t e a p r e c u r s o r B - p h e n o t y p e or a f o l l i c u l a r B-cell p h e n o t y p e . T h e o r i g i n a l l e u k e m i a
Table 1
w a s CD10 n e g a t i v e b u t t h e r e l a p s e - d e r i v e d cell l i n e w a s CD10 + (95% of t h e cells). T h i s c o u l d i n d i c a t e c l o n a l evolut i o n or c l o n a l s e l e c t i o n . Cytogenetics At d i a g n o s i s , 14 b o n e m a r r o w m e t a p h a s e s w e r e a n a l y z e d ; a m o n g t h e m , t h r e e s h o w e d a 46,XY k a r y o t y p e a n d 11 w e r e a b n o r m a l . T h e 8q24 b a n d o n o n e c h r o m o s o m e 8 was s l i g h t l y s h o r t e r a n d s t a i n e d a b n o r m a l l y , o n e c h r o m o s o m e 14 w a s too
C h r o m o s o m a l hybridization signals of various probes on metaphases from ROS-50, 1301, and S U D H L - 4 cell lines Number of positive signals in 20 metaphases
Cell line translocation ROS-50 t(8;14;18)
1301 t(8;14)
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Figure 3 Schematic representation of the chromosome pairs 8, 14, and 18 of ROS-50 cells. Shown are the sites of break and the nature of exchanged chromosomal segments, as indicated by FISH studies.
long, a n d one c h r o m o s o m e 18 was too short. All the other 43 chromosomes appeared normal. No cytogenetic study was done at relapse. The ROS-50 cell line showed a rather homogeneous karyotype: 47,XY,der(8), + der(8),add (14)(q32), der(7;17)(q10;q10),del(18)(q21), as shown in Figure 1. This was interpreted as a t(8;14;18)(q24;q32;q22), w i t h d u p l i c a t i o n of der(8), trisomy for 7(t, and m o n o s o m y 17p. A m i n o r i t y (16%) of cells showed additional aberrations such as only one der(8) and trisomy 11. The karyotype of the cell line 1301 was 47,XY, ÷Y,t(8;14) (q24;q32),der(14)t(8;14)t(14;19)(p12;p11 or q11),der(19) t(14;19). At the time of study the karyotype of SUDHL-4 was hyperd i p l o i d a n d showed more abnormalities than previously reported [19] i.e., structural rearrangements of chromosomes 2, 3, 8, 12, 13, trisomy 7, and a classical t(14;18)(q32;q21) with two copies of der(18).
FISH Studies Results obtained by hybridization of the various probes on
metaphases from the ROS-50, 1301, and SUDHL-4 cell lines are s u m m a r i z e d in Table 1 and shown in Figure 2. The D8Z2 probe revealed three clear centromeric spots on metaphases and interphase cells from the ROS-50 cell line, indicating the presence of a normal chromosome 8 and two der(8) (Fig. 2). In most experiments D8Z2 was co-hybridized with another probe in order to identify easily the chromosome 8 and der(8). The probe for the MYC oncogene is relatively small and p r o d u c e d faint but clear signals on the normal 8q24 and on the 14q ÷ in ROS-50 cells. The two der(8) d i d not hybridize to the MYC probe (Fig. 2A). This was indicative of a translocation (8;14) displacing MYC to the der(14). On the cell line 1301 the MYC probe also hybridized to the normal 8 and the 14q ÷ (not shown). Using a cosmid probe for IGH, clear signals were obtained on the normal chromosome 14 in all three cell lines, on the two der(8) in ROS-50 (Fig. 2C), on the 14q ÷ in line 1301, and at the two der(18) in SUDHL-4. Furthermore, weak signals were seen in half of the metaphases on the 18q- in ROS-50 and on both 15q14 regions in all lines. The latter is a known
Figure 2 Hybridization results on metaphase spreads from ROS-50 cells (A,C,E) and corresponding DAPI staining (B,D,F}. (A) Double hybridization with MYC and D8Z2 probes. The DSZ2 probe gives a bright hybridization signal on the centromere of three chromosomes. MYC signals (arrowhead) are visible on the normal chromosome 8 and on the der(14) chromosome. (C) Double hybridization with IGH and DSZ2 probes. Arrowheads indicate IGH signals on the normal chromosome 14 and the two der(8) chromosomes; the asterisk indicates IGH signal on the der(18) chromosome. Secondary sites are visible on the proximal part of 15q. (E) Double hybridization with pBS-18 and D8Z2 probes. Arrowheads indicate part of chromosome 18 on the two der(8) chromosomes. In addition, paints of two chromosomes 18 are clearly visible.
92
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Figure 4 Southern blot analysis of Bgl/II and BamHI/HindI~ double digests of a control cell line DNA with germ line IGH genes and DNA from the ROS-50 cell line, probed with a JH probe (IGHJ6). In ROS-50 DNA, no germ line band was detected but three rearranged IGH bands are visible.
secondary hybridization site of this probe. This indicated involvement of the IGH gene in the chromosome 14 rearrangemerits with various breakpoints around or within the region of the gene identified by our cosmid probe, i.e., JH to C~. The pBS-18 library painted four chromosomes in ROS50, i.e., 18, 18q - , and the distal part of both der(8) (Fig. 2E). This showed the composite nature of the der(8) that contains sequences of the IGH gene derived from chromosome 14 and part of chromosome 18 distal to 18q21 (Fig. 3).
DNA Analysis of IGH and BCL2 Rearrangements Southern blot analysis of IGH gene rearrangements with the JH probe revealed in both Bg/II and BamHI/HindIII digestions three rearranged IGH gene bands with different density (one band with a larger density compared to the other two bands) (Fig. 4). From these data it is not possible to decide which chromosome is actually coding. The heavier band was interpreted as representing sequences carried by the duplicated der(8), while the two other rearranged bands of
comparable density could be attributed to the normal chromosome 14 and to the t(14;18). PCR analysis of ROS-50 DNA revealed a breakpoint in the mcr region of chromosome 18. The fragment size of the amplified product was visualized by ethidium bromide staining and hybridization with pFL-2 and estimated to be about 550 base pairs (bp) (Fig. 5). This demonstrated the presence of a IGH-BCL2 rearrangement and confirmed the presence of a t(14;18). Furthermore, the immunofluorescent assay using an anti-BCL2 antibody showed a bright fluorescence in the cytoplasm of all cells, both on unfixed and fixed slides, clearly demonstrating a high expression of BCL2 protein. DISCUSSION Cytogenetic and FISH analysis of the ROS-50 cell line showed a complex chromosomal rearrangement that can be interpreted as a combination of two nonrandom translocations, i.e., t(14;18)(q32;q21) and t(8;14)(q24;q32), both involving the same chromosome 14. This implies involvement of the BCL2 gene on chromosome 18 and of the MYC oncogene on chromosome 8. Indeed, a breakpoint in the mcr of the BCL2 gene was shown by PCR analysis, high expression of the BCL2 protein was demonstrated, and translocation of MYC from chromosome 8 to 14q+ was shown by FISH. Furthermore, FISH turned out to be a powerful tool to analyze this double translocation involving the same chromosome 14. The Cos-1.16 probe is not rigorously mapped on the IGH sequences but is known to encompass the switch I~ and C~ and to extend 5' into the J region (Fig. 6). If we keep in mind the 3'-'5' orientation of the IGH gene on chromosome 14, the FISH results suggest the following scenario:
t(8;14;18) in a B-cell L i n e
93
Vl -- V200
Telomere 5 ' ~ i ~ ~
D1 - D12
~
¢
~
~
J1 - J6
~
,
C Region Genes
"
p
6
T3 ')'1(:/1 ")"2 "Y4 ~
/3' Centromere
a
tt(14;18) tt(8;14) F i g u r e 6 Schematic representation of the molecular organization of the IGH gene on chromosome 14. The solid bar indicates the probes used for FISH analysis, arrows indicate the putative breakpoints. The exact boundaries of the probe are not known.
T h e t r a n s l o c a t i o n (14;18) o c c u r r e d at first, p o s s i b l y w i t h a b r e a k p o i n t in the J r e g i o n of the IGH gene, t h u s distal o n c h r o m o s o m e 14. T h i s g e n e r a t e d a der(18) that o n l y w e a k l y h y b r i d i z e s w i t h o u r IGH p r o b e a n d a der(14) s h o w i n g positive hybridization for the IGH probe. In the subsequent t(8;14), t h e s a m e c h r o m o s o m e der(14) was b r o k e n in the IGH r e g i o n p r o x i m a l to o u r probe. S u c h event e x p l a i n s t h e IGH-positive signal as w e l l as the c h r o m o s o m e 18 p a i n t i n g o n t h e der(8) a n d the loss of IGH signal o n the der(14). T h i s is s c h e m a t i z e d in Figures 3 and 6. S o u t h e r n blot analysis s h o w e d r e a r r a n g e m e n t of t h r e e n o n - g e r m l i n e b a n d s (Fig. 4). T h i s was also s e e n w i t h o t h e r JH probes (data not s h o w n ) a n d c a n be the c o n s e q u e n c e of the c o m p l e x t r a n s l o c a t i o n w i t h b r e a k in the JI-I-C~ area, as tentatively d e p i c t e d in F i g u r e 6. Very r e c e n t l y Z e l e n e t z et al. [31] r e p o r t e d o n t h e f r e q u e n t o c c u r r e n c e of interstitial d e l e t i o n of der(14) in t(14;18) carr y i n g l y m p h o m a . In ROS-50 cell line, instead of a d e l e t i o n ,
a translocation w i t h 8q24 r e s e m b l i n g the class switch recomb i n a t i o n event c o u l d have h a p p e n e d . F u r t h e r investigations are n e e d e d to d i s c l o s e t h e m o l e c u l a r structure of t h e rearr a n g e d c h r o m o s o m a l regions. T h e a s s o c i a t i o n of t(8;14) a n d t(14;18) is rare, as r e v i e w e d by Brito-Babapulle et al. [9]. In Table 2 we s u m m a r i z e the data of reported cases w i t h t(14;18) and t(8;14), excluding variants. Five cases p r e s e n t e d w i t h a history of f o l l i c u l a r l y m p h o m a p r e c e d i n g l y m p h o b l a s t i c l e u k e m i a , w h i l e in seven other cases [ i n c l u d i n g ours) acute l e u k e m i a was the presenting diagnosis. T h e FAB classification was L2 or L3 and in s o m e investigated cases the blast cells were TdT-positive. Our case p r e s e n t e d as acute l e u k e m i a of t h e FAB-L3 type, w i t h Burkitt-like l y m p h o b l a s t s , a n d o n l y t r a n s i e n t r e s p o n s e to therapy. T h i s is v e r y m u c h in l i n e w i t h o t h e r m o r p h o l o g i cally s i m i l a r cases that carry o n l y a t(8;14) a n d suggests that i n d e e d d e r e g u l a t i o n of MYC has taken place. As described above, we have cytogenetic e v i d e n c e that the
Table 2 Cases of l y m p h o b l a s t i c l e u k e m i a or l y m p h o m a w i t h t(14;18)(q32;q21) a n d an a d d i t i o n a l t(8;14)(q24;32) Cell line
Patient Reference sex { a g e }
SU-DUL5
[18]
KHM-2B Ros 50 DS 380 NA
[13] This study b [14] [10]
NA NA
[11] [9]
NA
[16] [171
NA
NA NA NA
[15]
Diagnosis
Additional translocation Tdt
BCL-2
c-MYC BCL-2 c-MYC
(DNA}
(RNA/protein}
M (30) Lymphoblastic lymphoma M (62) B-ALL L3 M {69) B-ALL L3
t(8; 14)a
ND
lUG
lUG
t(8;14} t(8;14) a
ND -
lUG R
luG ND
F (70) Malignant lymphoma M (15) C-ALL L2 F (50) Diffuse large-cell lymphoma/pre-B-ALL M (21) C-ALL L2 F (73) B-ALL L3
t(6; 14) t(6;14) t(8;14)
+ +
R R luG
ND G/G luG
- /ND ND
t(8; 14) t(8;14)
+ -
ND luG
M (36) ALL L3 M (62) Immature ALL M (64) Immunoblastic lymphoma F (29) ALL-L2 M (44) Follicular lymphoma with interfollicular lymphoblastic lymphoma
t(6;14) t(8;14) t(8;14}
ND -
ND BCL-2-JH fragment luG ND ND
t(8; 14) t(8; 14)
+
ND IUG
+
- /-
+/ND
Survival Previous follicular (months} lymphoma NA
NA
3 5
No No
+/ND ND ND
6 5 2
No No Yes
ND ND
ND ND
3 0.25
No Yes c
lUG ND ND
ND ND ND
ND ND ND
3 3 4
No No Yes
ND IUG
ND ND
ND ND
29 8
Yes No
+/ND +/ND ND/+ ND
Abbreviations: ND, not done; R, rearranged; G, germline; NA, not available; survival after diagnosis. a In these cases the same chromosome 14 was involved in the t(8;14) and in the t(14;18). b A.C. Lambrechts et al., personal communication (manuscript in preparation). c Small cleaved lymphocytes in paratrabecular areas indicative of previously undetected follicular lymphoma.
94 t(14;18) p r e c e d e d the t(8;14) but t h e t i m e lapse b e t w e e n t h e two events is u n k n o w n . Also, there w e r e no obvious c l i n i c a l c h a n g e s p r e c e d i n g the onset of f u l l - b l o w n l e u k e m i a . T h e t(14;18) in follicular l y m p h o m a results in o v e r e x p r e s s i o n of the BCL2 oncogene. T h i s was also the case in the ROS-50 cell line. In the single-cell line (SU-DUL5) r e p o r t e d w i t h t(14;18) a n d t(8;14) i n v o l v i n g the s a m e c h r o m o s o m e 14 [18], BCL2 was not expressed and s e q u e n c i n g of the der(8) showed translocation of BCL2 o n the der(8) b u t no e v i d e n c e for chrom o s o m e 14 translocation. In our case, part of the IGH is translocated to t h e der(8) together w i t h 18q21-qter, t h u s p o s s i b l y m a i n t a i n i n g BCL2 u n d e r the control of the IGH enhancer. The authors thank E.J. Harthoorn-Lasthuizen, M.D. for referring this patient, A. Geurts van Kessel, Ph.D., for a first appraisal of cytogenetic data and R. Boucke for typing the manuscript. Part of the work was supported by the Netherlands Cancer Foundation.
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