A unique 6-cell non-Hodgkin's lymphoma (NHL) cell line. (Karpas 422). bearing both t(14;18) and t(4;ll) chromo- somal translocations as well as several other ...
A New Human B-Cell Non-Hodgkin's Lymphoma Cell Line (Karpas 422) Exhibiting Both t( 14;18) and t ( 4 ; l l ) Chromosomal Translocations By Martin J.S. Dyer, Patricia Fischer, Elisabeth Nacheva, Wayne Labastide, and Abraham Karpas A unique 6-cell non-Hodgkin's lymphoma (NHL) cell line (Karpas 422). bearing both t(14;18) and t ( 4 ; l l ) chromosomal translocations as well as several other chromosomal abnormalities, has been established from the pleural effusion of a patient with chemotherapy-resistant NHL. This cell line has the same karyotypic features as malignant cells from the patient. The major cell clone is characterized chromosomallyby 46,XX t(2;10)(p23;q22.1), t(4;ll l(q21.3; q23.11, t(14;18)(q32.1;q21.3), t(4;16)(q21.3;pl3.1). Both phenotypically and genotypically, the cell line has features of a mature 6-cell neoplasm with no evidence for commit-
ment to other lineages. Rearrangements of the C-ETS-1 oncogene and N-CAM-1 and CD3 genes that map to 11q23 were not detected by conventional Southern analysis. BCL-2 was rearranged within the major breakpoint cluster. The K422 cell line has a unique karyotype; this is the first occasion that the t ( 4 ; l l ) translocation has been described in a t(14;18) lymphoma. The cell line will be of value in determining the molecular nature of the t ( 4 ; l l ) translocation. 0 1990 by The American S o c i e t y of Hematology.
T
NHL was made and she was transferred to Addenbrooke's Hospital (Cambridge, UK) for treatment. Initially she received intravenous (IV) cyclophosphamide, hydroxydaunorubicin,vincristine, and oral prednisolone. Repeat CT scan after three courses showed only partial regression of the mass and therapy was changed to IV ifosfamide, etoposide, and methotrexate with folinic acid rescue (IMVP16). The mass remained unchanged after three courses of this protocol and she commenced oral chlorambucil 10 mg daily as a palliative measure. By June 1987 she developed a very large palpable abdominal mass and large bilateral pleural effusions. Drainage of the effusions was performed; they contained large numbers of lymphoid cells having irregular nuclei with a single nucleolus and abundant cytoplasm.Numerous abnormal mitoses were present. The cell line was obtained from this sample (see below). Further IV chemotherapy was given but the patient died 10 days later with septicemia,left ventricular failure, and renal failure. Postmortem examination showed a 6-cm thick retroperitoneal mass encasing retroperitoneal organs and extending into the pelvic cavity and through the diaphragm to the apex of the heart. There was considerablemediastinal lymphadenopathy.
HE ASSOCIATION between cytogenetic abnormalities and the phenotype of hematologic malignancies is
well-established; a paradigm is the frequent occurrence of the t( 14;18) in B-cell lineage neoplasms.'s2This translocation brings together the immunoglobulin (Ig) heavy chain locus on chromosome 14 with the BCL-2 protooncogene sequences on chromosome 18,3.4 perhaps as the result of a mistake .~ lymphomas during the Ig D-J recombination p r o c e ~ sMany with a t( 14;18) translocation pursue an indolent clinical course. However, acquisition of further cytogenetic change and, particularly, further chromosomal translocations involving the C-MYC oncogene on chromosome 8 may result both in a change of phenotype and more aggressive behavior of the ne~plasm.~.~ Similarly, the t(4;l l)(q21;q23.1) has been found primarily in acute leukemias of early infancy where it has been associated with a poor response to conventional therapy.'.'' Most leukemias with this translocation appear to have made some commitment to the B-cell lineage and many have the phenotype of B-cell precursors, but evidence for commitment to myeloid, monocytoid, and T-cell lineage may also be found."-'5 Recently there has been a case report of a t ( 4 ; l l ) occurring in a patient with a diffuse, large-cell nonHodgkin's lymphoma (NHL).I6 We document the occurrence of a t ( 4 ; l l ) translocation in a case of N H L bearing a t(14;18) and describe the establishment of a cell line of mature B-cell phenotype and genotype which has the same cytogenetic changes. This cell line will be of value in determining the molecular nature of the t(4;ll) and other chromosomal translocations. CASE HISTORY
A 72-year-old woman presented in May 1986 with a 5-week history of epigastric discomfort, nausea, and vomiting. She had idiopathic lymphedema of both legs since adolescence but no other significant illnesses. She was on no medication. Computed tomography (CT) scan showed a large retroperitoneal mass and a small right pleural effusion but no lymphadenopathyand no infiltration of liver or spleen. Bone marrow aspirates and trephines taken at diagnosis and throughout her illness were normal. Biopsy of the retroperitoneal mass was performed at laparotomy. Histology showed medium-sized lymphocytes with single nucleoli and some cytoplasmic vacuolation. Mitoses were infrequent. No immunophenotypic or genotypic data are available from this specimen. A diagnosis of intra-abdominal
Blood, Vol75, No 3 (February 11, 1990: pp 709-7 14
RESULTS
Cell culture and cytology of K422 cell line. Cells from the pleural fluid were cultured in R P M I 1640 medium containing 20% fetal calf serum (FCS), penicillin 100 U/mL, and streptomycin 50 pg/mL and incubated a t 37OC
From the Department of Haematology, University of Cambridge, UK. Submitted July 31,1989; accepted September 20, 1989. Supported in part by grants from the Medical Research Council; the Cambridge Haematological Research Fund: St John's College, Cambridge; Research Machines, Oxford, UK, and the Kay Kendall Trust. M.J.S.D. is a Mere's student for Medical Research, St John's College, Cambridge, UK. The cell line K422 may be obtained by written request to Dr A . Karpas at the reprint address. Address reprint requests to M.J.S. Dyer, Department of Haematology. University of Cambridge. Hills Rd, Cambridge CB2 2QL.
UK. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C.section 1734 solely to indicate this fact. 0 I990 by The American Society of Hematology. 0006-4971/90/7503-0004$3.00/0
709
OYER ET AL
710
Fig cytdog~ of Kline. Wright-aai,& -rr colt tine K422. Nuclei are large and irregular with one or two nucleoli. Original magnification x 1.OOO.
in a humidified atmosphere containing 5% CO,. The cells in suspension grew rapidly forming loose clusters. After 4 weeks the concentration of FCS was lowered to 10%. Cells have been in continuous culture since August 1987. Cytologic appearances of the cell line stained with Wright's stain are shown in Fig 1. Ultrastructural appearances of K422 were unremarkable. Tests for ERNA and mycoplasma have been nega t ive. Chromosome studies. Cells were harvested by conventional methods 24 hours after the initiation of culture and at intervals of about I month thereafter. For high resolution banding, analysis was performed as described.'' Slides were S-banded with Wright's stain. The ISCN 85 nomenclature has been used to describe the abnormal chromosome markers." Twenty cells were analyzed after 24 hours in culture. Two had a normal female karyotype 46.XX. The remaining 18 cells were abnormal with four marker chromosomes: t(2: IO)(p23:q22. I), t(4:I I)(q2I .3:q23.l). t(4:16)(q21 .3:pI 3.1 ). and t(14:18)(q32.1:q21.3). Additionally. 16of these 18 cells had lost one X chromosome and were trisomic for chromosome 8. After I 2 months in culture. there were minor numerical changes with trisomies of 8. 14. 20, and 21 in Some cells. The marker chromosomes were unchanged (Fig 2). A full designation of the marker chromosomes as interpreted with H R
*
-1
1
1
6
7
t;
&4
2
3
a
i
-m4'11 r
t
4
9
.
a -
s
11
.
12
t! 13 0
19
20 Fig 2.
21
22
x -
Karyotyp. of K422 d l line. Arrowr indicate rite of oxchenge in the merkr c h m " o .
K422
LYMPHOMA CELL LINE K422
711 Table 1. Immunophenotype of K422 Cell Line
6-Cell Lineage
T-cell Lineage
Antibody
Soecificity CDlO CD19 CD20 CD37 IgM IgD IgG Ig K light chain Ig X light chain
Clone
Expression
J5/RFAL-1 RFB 19
+/-
61 WR-17
Antibody Specificity
++
+/-
++ ++
+/-
++ ++
Nonlineage
Expression
Clone
Antibody Specificity
Clone
Expression
CAMPATH-6 OKTIO
-
YTH24.5 MH54.12 MH80.103 CAMPATH- 1 YAML555
++ ++ ++ ++ ++
CD 1 CD2
NA 1/34 M H 6 16
-
CD250L2-R) CD38
CD3 CD4 CD6 CD7 CD8
CAMPATH-3 OKT4 CAMPATH-4 CAMPATH-2 CAMPATH-8
-
CD45
-
-
CD45R CDw52 HLADR
-
lmmunophenotyping was performed as d e s ~ r i b e d . ' ~ Background .~~ staining was less than 1.5%. Characterizationof the antibodies used in this study has been performed at the International Workshops on Leukocyte Differentiation antigen^.^',^^ Anti-human Ig reagents were obtained from Serotec, Oxford, UK. No staining was obtained with CD14 (monocytic lineage) and CD33 (myeloid lineage) monoclonal antibodies. The strong CDw52 (CAMPATH-1) CD45 and CD45R expression reflects the mature 6-41 phenotype of this cell line (M.J.S. Dyer, manuscript submitted). Abbreviations: -, undetectable staining; +I-, dull positive staining; strong positive staining.
+ +,
banding analysis is as follows: mar 2/ 10:der(2)(2qter-p23:: 1Oq22.l-qter) mar 10/2:der( lo)( lOpter-q22.1::2~23-pter) mar 4/1 l:der(4)(4pter-q21.3::1 lq23.1-qter) mar 11/4: der(ll)( 1lpter-q23.1::4q21.3-qter)mar 16/4:der(16)( 16qterpl3.1::4q21.3-qter) mar 14/18:der( 14)( 14pter-q32.1:: 18q21.3-qter) mar 18/ 14:der( 18)( 18pter-q21.3::14q32.1qter). Immunophenotype of the cell line. Cells were immunophenotyped by flow cytometry on a FACS I1 (BectonDickinson, Sunnyvale, CA) flow cytometer as previously described," and on a custom-built flow cytometer.20 The phenotype of the cells was compatible with a mature B-cell NHL (Table 1). There was strong expression of CD19, CD37, and surface Ig, predominantly IgM and IgG, although 30% of cells also expressed IgD. There was weak expression of CDlO but no detectable CD5. No T-cell, monocytoid, or myeloid differentiation antigens were expressed. K422 is unusual among lymphoid cell lines in that it
expresses stably surface CAMPATH-1 (CDw52) antigen at levels comparable with those seen in normal lymphocytes (Fig 3). Although most lymphoid malignancies express abundant CDw52 antigen, surface expression in lymphoid cell lines is usually much diminished (reference 21 and Dyer and Hale, unpublished observations). Genotype analysis of the cell line. Genotypic analysis was performed as previously d e s ~ r i b e dDNA . ~ ~ probes and restriction enzymes used in this study are shown in Table 2. When probed with the Ig J H probe, three rearranged fragments were detected in a Hind111 digest (Fig 4A). One of these fragments comigrated with the rearranged BCL-2 fragment detected by the probe pFL-1 (Fig 4B). Similar comigration of rearranged J H and BCL-2 fragments was seen in both Pst-I and BamHI digests (Fig 5 ) . This indicates that the t( 14;18) translocation occurs within the major BCL-2 breakpoint region. No rearrangements were detected with probes for NNegative Control
Green Fluorescence (FITC)
Fig 3. Fluorescent profile of K422 cell line stained with CAMPATH-1 (CDw52) antibody. Viable cells were stained with antibodies as described'' and analyzed in this instance on a custom-built flow cytometer." Negative control: K422 cells stained with fluorescein-conjugated rabbit anti-rat Ig. Levels of CDw62 expression on K422 are comparable w i t h those seen on normal lymphoid cells.
p.'
Campath 1
Green Fluorescence (FITC)
DYER ET AL
712 Tabla 2. Genotypic Analysis of K422 Cell Line
Ig JH BCL-2 ma* breakpoint region BCL-2 minor breakpoint region C-MYC
Enzyme:
C76R51A
Hindlll/fsrl/Bam HI
25
pFL- 1
Hindlll/fsr I/Bam HI
26
PFL-2 CDlA
Hindlll/Bam HI
27 28
Psr-1 C-ETS- 1
€COR I
29
N-MYC
Hindlll Bam HI -sc I
30
pNb-1
31 32
NCAM-1 CD3 y. 6. e
Genotypic analysis was performed as previously described.” All probes were gel-purified DNA fragments. C-ETS-1, CD3, and NCAM-1 probes map to human chromosome 1lq23 and N-MYC maps to 2p23; however, no rearrangements were detected in conventional Southern filter analysis of K422 DNA in a range of DNA digests. K422 is germline when probed with a variety of T-cell receptor a,6.6, and y DNA probes (data not shown).
MYC, C-MYC, C-ETS-I CD3. and N-CAM-I sequences in several enzyme digests (data not shown). DISCUSSION
We describe the establishment of a 9-cell line from the pleural Ruid of a woman with a chemotherapy-resistant NHL. This cell line, Karpas 422, has the same karyotypic features as the malignant cells from the patient. The karyotype of the cell line is remarkable for having both a t(14;18) and t(4;ll). Such an association has not been previously reported; indeed there is only one report of t(4;l I ) occurring in NHL,I6 all other examples being described in acute leukemias. In this case there was no blood or bone marrow involvement throughout the course of the disease. Whether or not the t(4;l I ) and other structural chromosome changes were present from the outset is unknown; lack of responsc to various chemotherapeutic attempts suggests that this may have been so. Similar drug resistance has been seen in some but not all cases of acute leukemia with the t(4;l I).’” Although translocations of the region 1 lq23 are seen in a wide range of malignancies,” translocations of I Iq23 involving 4q21 occur predominantly in acute leukemias of the B-cell lineage.’”’? The importance of the 4q21 region is highlighted in the K422 cell line by the finding of a second translocation involving 4q21 with 16pl3.1. These observations indicate that genes in the region of 4q21 may be of importance in early B-cell development. The finding of a case of mature 9-cell malignancy with t(4;ll) does not invalidate this hypothesis; the t( l4;18) found in NHL is also thought to originate in 9-cell precursors, which then give rise to the phenotypically mature lymphoma The genes at 4q21 and 1 lq23 involved in the t(4;l 1) are unknown. A number of genes have been mapped to I lq23” and we sought rearrangements of some of these genes using conventional Southern analysis. Like others, we failed to detect any rearrangement of CD3, N-CAM-I. or C-ETS-I
23.1
>
9.4
>
Hind 111
- 10.5
6.6 > --
4.4
I
>
> 2.0 >
2.3
Probes:
lgJH
BCL-2
(PFL-l/pFL-2)
Fig 4. Demonstration of comigration of lgJH and BCL-2 rearranged fragments in K422 Hindlll-digested DNA. Left panel shows the three rearranged lgJH fragments detected in Hindlll-digested K422 DNA. Right panel shows the same filter stripped and reprobed with a mixture of both pFL-1 and pFL-2 probes; pFL-1 detects BCL-2 rearrangements within the major breakpoint clustera and pFL-2 rearrangements within a more 3‘ minor breakpoint region.” Note the comigration of the 6.4-kb lgJH rearranged fragment with a rearranged RCL-2 fragment; this was shown t o be a pFL-1 rearrangement by reprobing with the pFL-1 and pFL-2 probes individually (data not shown; see Fig 6). Solid bars denote germline fragments; arrowheads denote rearranged fragments. Sizes of fragments were determined by coelectrophoresis of X DNA digested with Hindlll.
sequences by this m e t h ~ d . “ . ~ Presently ~.’~ there is a lack of candidate genes to probe the 4q21 region. Thus, although the t(4;ll) in K422 appears cytogenetically identical to the t(4;ll) found in acute leukemias, it may be that important differences will be found once the molecular nature of these breakpoints is known. The K422 line also carries a t(2;lO) with the 2p23 breakpoint adjacent to the N-MYC locus; again, however, no rearrangements were detected on conventional Southern blots. A cell line with the t(4;ll) has been derived from a patient with acute leukemia”; the availability of this NHL cell line should further facilitate the molecular analysis of the t(4;ll) translocation, and the identification of genes on chromosome 4 implicated in 9-cell ontogeny.
LYMPHOMA CELL LINE
Samples A
B
K422
C
713
D E
A
B
C
D
A
E
B C D E
