Proc. Natl. Acad. Sci. USAVol. 87, pp. 628-632, January 1990Medical Sciences

Molecular analysis of a chromosomal translocation, t(9;14)(p13;q32),in a diffuse large-cell lymphoma cell line expressing theKi-1 antigen

(somatic cell hybrids/in situ hybridization/cloning of breakpoint/nucleotide sequencing/IGH locus)

HITOSHI OHNO*t, TAKAHISA FURUKAWA*, SHIROU FUKUHARAt, SHU QIN ZONG*, HIROSHI KAMESAKIt,THOMAS B. SHOWSt, MICHELLE M. LE BEAU§, TIMOTHY W. MCKEITHAN¶, TOSHIAKI KAWAKAMI*,AND TASUKU HONJO*Departments of *Medical Chemistry and tInternal Medicine, Kyoto University Faculty of Medicine, Kyoto 606, Japan; tDepartment of Human Genetics,Roswell Park Memorial Institute, New York State Department of Health, Buffalo, NY 14263; and §Department of Medicine, Section of Hematology/Oncology,and $Department of Pathology, University of Chicago, Chicago, IL 60637

Communicated by Janet D. Rowley, October 31, 1989 (received for review June 1, 1989)

ABSTRACT We have studied a translocation, t(9;14)(pl3;q32), in a diffuse large-cell lymphoma cell line, KIS-1,that expresses the Ki-1 (CD30) antigen. Molecular cloning ofthe immunoglobulin heavy-chain locus (IGH) of this cell linerevealed an unknown segment linked 5' to IGH. The break-point on chromosome 14 was 265 base pairs downstream fromthe 3' border of the JH6 joining gene segment. Class switchrecombination deleted most of the constant genes of IGH (CH)and juxtaposed the Ca2 gene downstream of the translocationjunction. Analysis of somatic cell hybrids and in situ chromo-somal hybridization demonstrated that the translocated seg-ment was normally located at band p13 of chromosome 9. Thechromosome 9 sequences were transcriptionally active, givingrise to transcripts of =11 kilobases. The KIS-1 cells seemed tohave a small quantity of chimeric transcripts containing bothchromosome 9 and Ca2 sequences.

A significant correlation has been observed between recur-rent chromosomal translocations involving band 14q32, andthe histologic and immunologic subtypes of human lymphoidmalignancies (1-3). For example, the t(8;14)(q24;q32) isfound in most cases of small-noncleaved-cell lymphoma, andthe t(14;18)(q32;q21) is primarily associated with follicularsmall-cleaved-cell lymphoma. The lymphoma cells charac-terized by each of these translocations show B-cell immu-nophenotypes. The biological significance of translocationsinvolving 14q32 has become apparent through the identifica-tion of an established protooncogene (4, 5) or a putativetransforming gene (6), adjacent to the site of chromosomebreaks on the reciprocal partner chromosomes.

Although some subtypes of non-Hodgkin lymphomas havewell-defined clinicopathologic features, the classification oflymphomas has remained controversial. The Southern blottechnique, usingDNA probes adjacent to chromosome trans-location breakpoints, can be an alternative to karyotypeanalysis in some cases (7) and can be used for the diagnosisof lymphoma (8). Cloning of breakpoint junctions of varioustranslocations in lymphomas will provideDNA probes for theprecise classification of lymphomas.We report the isolation and characterization of a DNA

fragment surrounding the translocation junction of a diffuselarge-cell lymphoma cell line, KIS-1, which has a newlyidentified t(9;14)(pl3;q32) (9). The short arm of chromosome9 has been translocated to a position 5' ofthe immunoglobulinheavy-chain locus (IGH) on chromosome 14, band q32, in the

KIS-1 cells. A transcriptionally active locus has been iden-tified at 9p13, flanking the breakpoint of the translocation.

MATERIALS AND METHODSKIS-1 Cell Line. The KIS-1 cell line was established from

the malignant cells of a patient with non-Hodgkin diffuselarge-cell lymphoma (9), which expressed the Ki-1 (CD30)antigen (10, 11). The karyotype of the KIS-1 cell line wascomplex. Each of 20 cells analyzed had a characteristictranslocation, t(9;14)(p13;q32); a translocation with thesebreakpoints has not been reported in other lymphoid neo-plasms. The rearranged chromosome 14 (14q+) that resultedfrom the reciprocal translocation was present in duplicate ortriplicate; the additional 14q+ homologues had chromosomalmaterial of unknown origin translocated to the short arm,resulting in a 14p+ and q+ chromosome. The normal homo-logue was absent (Fig. 1). According to the ISCN nomen-clature (12), a representative karyotype was as follows: 50,X,+ der(X)t(X;?)(p11.4;?),Y, - 1,- 14,- 17, - 22, + lp+ (HSR),t(9;14)(p13;q32.3),t( 12; 13)(q 13.1;pl 2), + der( 4)t(9;14;')(?: :14p12-*14q32.3: :9p13--9pter), + der(l4)t(9;14;?)(?::14p12-* 14q32.3::9p13--9pter),del(16)(q22), + der(17)t(17;?)(pll .2;?), + der(22)t(1 ;22)(ql 1;q13), + mar, + min.The primary lymphoma cells had a similar modal karyotypeincluding the t(9;14)(p13;q32).

Southern and Northern Blot Analyses. DNA filter hybridiza-tion was carried out according to Southern (13). RNA wasprepared from cultured cell lines (14) and hybridization wasperformed as described (14, 15). Probes used for theIGHlocuswere as follows: joining region (JH), an EcoRI-HindIII frag-ment ofH24 (16) (see Fig. 3); ,u-chain constant region (C,), anEcoRI fragment ofH24 (16); C,,. a Sac II fragment of Igyl-10(17); Ca2, an Xho 1-HindIII fragment of CH4A H Iga25 (18).The MYC probe was a 0.6-kilobase (kb) Kpn I-BamHI frag-ment containing the first exon of the gene (19).

Molecular Cloning and Nucleotide Sequencing. GenomicDNA of the KIS-1 cells was digested with Bgl II. The DNAfractions ofinterest were purified and ligated to the BamHI siteof Charon 28 phage arms. A cosmid library was constructedfrom the FLEB14-14 cell line (20). Libraries were screenedwith probes indicated in the text. DNA segments of theisolated bacteriophage and cosmid clones were subcloned intoplasmid vectors (pUC18, pUC19, and Bluescribe). The nucle-

Abbreviations: J, joining region; C, constant region; V, variableregion; D, diversity region.IThe sequences reported in this paper have been deposited in theGenBank data base (accession nos. M30453, M30454, M30455).

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A kb L \

23.1- -

9.4-_ _-6.7-4.4-

B23.1 - %-adatp9.4--o- 9 R6.7-4.4-

9 1 4

FIG. 1. Partial karyotype of chromosomes 9 and 14 from aG-banded metaphase cell from the KIS-1 cell line, illustrating thetranslocation between the short arm (p) of chromosome 9 and thelong arm (q) of chromosome 14 [t(9;14)(p13;q32.3)]. One normalchromosome 9 is on the left and one 9p- chromosome is on the right.Two chromosomes 14 are abnormal: one 14q+ chromosome on theleft and one 14p+ and q+ chromosome on the right. The 9p- and14q+ chromosomes are the result of a reciprocal translocationbetween chromosomes 9 and 14. The arrowheads indicate break-points involved in the 9;14 translocation: bands 9p13 and 14q32.3,respectively. The 14q+ chromosome is present in duplicate; one ofthese is involved in a second rearrangement and has additionalchromosomal material ofunknown origin on the short arm (14p+ andq+)[der(14)t(9;14;?)(?: :14pl2 > 14q32.3: :9pl3}>9pter)] .

otide sequences were determined by the dideoxy DNA se-quencing method using double-stranded plasmid vectors (21).Somatic Cell Hybrid Analysis. Thirty-five hybrids were

analyzed (22-24). The hybrids were characterized by kary-otype analysis, by mapped enzyme markers, and partly bymapped DNA probes (22, 24, 25). DNA from the hybrid cellpanel was digested with HindIII and analyzed by Southernblot hybridization using the isolated DNA probe.In Situ Chromosomal Hybridization. Human metaphase

cells were prepared from phytohemagglutinin-stimulated pe-ripheral blood lymphocytes. In situ hybridization was per-formed using 3H-labeled probes with a specific activity of 108dpm/,ug, as described (26). Autoradiographs were exposedfor 11 days.

RESULTSGenomic Southern Blot Hybridization. Southern blot hy-

bridization showed that the KIS-1 DNA had one rearrangedband that hybridized with the JH probe, and no germ-line JHband was observed (Fig. 2). Hybridization with the C.1 andC,,1 probes showed complete deletion ofboth copies of the C.gene and all the C. subclass genes (data not shown). Therearranged band that hybridized with the JH probe afterEcoRI or BamHI digestion was also labeled with the Ca2probe.

Cloning of the IGH Locus of the KIS-1 Cell Line. Southernanalysis of the KIS-1 DNA after Bgl II digestion showed that9.9- and 4.9-kb bands hybridized to the JH probe. Phageclones containing the 9.9- and 4.9-kb Bgl II fragments wereisolated by screening with the JH probe. The 4.9-kb Bgl II

fragment was identical with the 3' half of the 9.9-kb fragment,indicating that the 9.9-kb fragment resulted from partial BglII digestion. Restriction mapping and Southern hybridizationdemonstrated that the 9.9-kb Bgl II fragment contained therearranged JH-C,2 gene (Fig. 3). These results indicate thatthe KIS-1 cells have a single rearranged IGH locus that hasundergone class-switch recombination to the Ca2 gene, re-sulting in deletion of all of the other CH genes.

C

23.1-9.4-- _- w6.74.4-

pKIS JH CO

FIG. 2. Genomic Southern analysis of the KIS-1 cell line. Pla-cental (P) and KIS-1 DNAs (2 ,ug each) were digested with EcoRI (A),BamHI (B), or HindIl1 (C). The same filter was hybridized sequen-tially with chromosome 9-specific pKIS, JH, and C,2 probes. Arrowsindicate rearranged band of identical size. HindlIl-digested A phageDNA provided molecular size markers.

Comparison of the restriction map of the rearranged 14q+chromosome with those of the corresponding germ-line re-gions of chromosome 14 indicated that the breakpoint onchromosome 14 had occurred at a site 3' of the JH6 segment.The 5' 3.8-kb segment of the 9.9-kb Bgl II fragment did nothybridize with any VH (variable region gene ofIGH) probes.Genomic Southern analysis using the pKIS probe (Fig. 3)showed that KIS-1 DNA contained one rearranged band inaddition to the germ-line band. The rearranged band of KIS-1DNA was identical in size with the rearranged JH bands(Fig. 2).Chromosomal Mapping of the pKIS DNA. The chromo-

somal localization ofpKIS in the normal cell was determinedby Southern analysis ofa panel of human-mouse somatic cell

A JH HCHCB G12345 6/ G H1 111.111 If

J :BSJHi u

EI

HC Sg/°tB G SHCG IH S G

RS IBS BS or2

C pKIS e H*ESH GE*' S B S GG4SHE

I l

I

BS BS: BS p BS6d -- p8.5-1

BS

S pBS8.6BB

HEGGIB

BS1 kbL4--

FIG. 3. Restriction maps of the IGH locus of the KIS-1 cell lineand its germ-line counterparts. Open rectangles represent chromo-some 9 sequence (C) and solid lines represent chromosome 14sequence (A). Coding (JH, Ca,2) and switch (SE,, Sl/a2) regions areindicated by black and gray boxes, respectively. Restriction sites: E,EcoRI; B, BamHI; H, HindIII; G, Bgl II; S, Sac I; BS, BstEII; HC,HincII. EcoRI site marked by asterisk was not identified on rear-ranged chromosome 14q+ (B). Probes used for hybridization studiesare indicated (JH, pKIS, p8.5-1, and pBS8.6BB).

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Table 1. Segregation of pKIS probe with human chromosomes in HindIII-digested DNA of human-mouse cell hybrids

Human chromosomes

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 X

No. of concordant (+I+)1 1 3 3 3 2 2 0 5 2 1 4 1 3 1 3 2 1 0 4 5 0 2hybrids (-I-) 17 17 13 16 11 18 16 16 29 7 17 13 16 10 16 23 5 14 20 9 8 15 9

No. of discordant (+/-) 3 4 1 2 2 3 3 5 0 2 4 1 4 2 4 2 3 4 5 1 0 5 1hybrids (-/+) 10 13 16 14 19 12 14 14 0 23 13 17 14 20 14 7 24 16 10 21 22 13 18

% discordancy 42 49 52 46 60 43 49 54 0 74 49 51 51 63 51 26 79 57 43 63 63 55 63

Data were compiled from 35 cell hybrids involving 18 unrelated human cell lines and 3 mouse cell lines. The DNA probe pKIS was hybridizedto Southern blots containing HindIII-digested DNA from the human-mouse cell hybrids. The chromosome assignment ofpKIS was determinedby scoring the presence or absence of human bands on the blots. Concordant hybrids have either retained or lost pKIS together with a specificchromosome. Discordant hybrids either retained pKIS, but not a specific chromosome, or the reverse. The concordances are designated (+/+)or (-/-), and discordances are (+/-) or (-/+), where the first symbol denotes the presence or absence of the pKIS band and the second symboldenotes the presence or absence of the specific human chromosome. Percent discordancy indicates the degree of discordant segregation for amarker and a chromosome. A 0%o discordancy is the basis for chromosome assignment.

hybrids. The pKIS probe hybridized to a 10.4-kb HindIllfragment in human DNA, and to an 8.0-kb HindIII fragmentin mouse DNA under stringent washing conditions. Hybrid-ization to 35 cell hybrids showed that the pKIS sequencemapped to chromosome 9. All other chromosomes segre-gated discordantly (Table 1).To localize the pKIS DNA by an independent method,

three pKIS probes (Fig. 3) were also hybridized to normalmetaphase chromosomes. In each case, we observed specificlabeling of chromosome 9 only; the largest number of grainswas at 9p13 (Fig. 4).These data indicate that the phage clones we have isolated

contain the breakpoint junction of the t(9;14)(p13;q32). Theshort arm of chromosome 9 distal to 9p13 was translocated tothe 5' side of IGH, deleting all the VH, D (diversity), and JHsegments on the 14q+ chromosome. This nonproductiverearrangement ofIGH and loss of the normal chromosome 14are presumably responsible for the absence of immunoglob-ulin expression on the KIS-1 cell surface (9).

Nucleotide Sequence Analysis of the Breakpoint Junction.The cosmid DNA library constructed from FLEB14-14, a cellline without the chromosomal translocation, was screenedwith the pKIS probe, and isolated germ-line clones wereanalyzed by restriction mapping and nucleotide sequencing.Comparison of nucleotide sequences surrounding the junc-tion indicated that the breakpoint on chromosome 14 waslocated 265 base pairs downstream from the 3' border of theJH6 segment. The 5' and 3' sides of the breakpoint werehomologous to chromosome 9 and chromosome 14 se-

3. ........*****

q2 2

1 -----

3 234 *

FIG. 4. Distribution of silver grains on chromosome 9. For in situchromosomal hybridization, we used a 3.0-kb Bgl I-Sac I fragment(p8.5-1) and an 8.6-kbBamHI fragment (pBS8.6BB) in addition to thepKIS probe (Fig. 3). Of 100 metaphase cells examined from thehybridization of the pBS8.6BB probe, 27 (27%) were labeled onregion pl or p2 of one or both chromosome 9 homologues. Of 186total labeled sites observed, 48 (26%) were located on chromosome9. These sites were clustered at bands p12-21, and this clusterrepresented 21% (39/186) of all labeled sites (cumulative probabilityfor the Poisson distribution is <<0.0005). The largest number ofgrains was observed at 9p13. Similar results were obtained in asecond hybridization experiment using this probe and in experimentsusing the pKIS and p8.5-1 probes.

quences, respectively. No additional nucleotides were in-serted at the junction (Fig. 5).Northern Blot Analysis. The pKIS probe detected -11-kb

transcripts in three other human B-cell lines as well as KIS-1cells. Several smaller transcripts were also observed in theKIS-1 cells (Fig. 6A). The structure of the IGH locus of theKIS-1 cells suggested that transcription might proceed acrossthe translocation junction into the Ca2 coding region. The Ca2locus was actively transcribed in the KIS-1 cell line, and1.3-kb sterile transcripts were the most abundant (Fig. 6C).Two additional, less abundant transcripts ofthe Ca gene werealso labeled with the pKIS probe. These results suggest thathybrid transcripts containing both pKIS and Ca2 sequencesmay be produced, although these transcripts seem to beminor RNA species.

DISCUSSIONThe close association between the translocation ofoncogenesinto the IGH locus and their expression in lymphoma sug-gests that functional genes, most likely involved in transfor-mation, may be located adjacent to the breakpoint junctionsin other translocations involving 14q32 (4-6). Northern blotanalysis of RNA from the KIS-1 cell line demonstrated atranscriptionally active locus at 9p13 flanking the breakpointof the newly identified t(9;14)(pl3;q32); the presence ofchimeric transcripts with the Ca2 gene suggested that analteration of this gene caused by the translocation might playan important role in the pathogenesis of the original lym-phoma. To our knowledge, no protooncogenes or genes forgrowth factors have been mapped to 9pl3, indicating that thesequences we have isolated at this band may represent anovel gene that might be involved in lymphomagenesis.

-40 -20

CHR. 9 G~GfAA****************** ****************************

T (9; 14) GGCTATIG'..A.A'I* * * * * ** * * ** * **

CHR. 14 _±

AG876+ KIS-1+1 +20 +40

TrFITTTGAGCIGT GGCAZIJ t.L mGGG ATCG AVTATTCAT1G********* * * * ** ** **I* * *

**************** ************************

FIG. 5. Nucleotide sequence analysis of the breakpoint junctionof the t(9;14)(pl3;q32). The breakpoint on chromosome 14 of thet(8;14)(q24;q32) in the Ag876 cell line (27) is also indicated. Se-quences are oriented 5' to 3' from left to right. Asterisks representnucleotide homology.

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Illinois Division (to T.W.M.). M.M.L. is a Scholar of the LeukemiaSociety of America.

28S-

18S-

B 'T28S- " ma

18S-

28S-

18S-

pKIS Cac

FIG. 6. Northern blot analysis. Expression of the pKIS se-quences was studied in the KIS-1 cell line and in three additional celllines of B-cell lineage: LCL, an Epstein-Barr virus-transformedlymphoblastoid cell line having a normal karyotype (LCL-AMKline); K, a Burkitt lymphoma cell line containing a t(8;14)(q24;32)(Kobayashi line); and FL-18, a follicular lymphoma cell line char-acterized by a t(14;18)(q32;q21) (FL-18 line). (A) Poly(A)+ RNA-(5,.g per lane) from cells indicated was hybridized with the pKIS probe.(B) The same filter was rehybridized with the MYC first-exon probe.(C) Poly(A)+ RNA of KIS-1 cells was hybridized with pKIS or C"2probe. Size markers are 28S and 18S ribosomal RNAs.

It has been suggested that the high frequency of chromo-some translocations involving the IGH locus is due to errorsin the V-D-J recombinase-mediated process (28). However,our results do not support this hypothesis, as heptamer-nonamer recombination signals were not found in proximityto the junction on either chromosome 14 or 9. The junctionof a t(8;14)(q24;q32) of an endemic Burkitt tumor cell line,Ag876, was located only 25 base pairs upstream from thebreakpoint of the KIS-1 cell line (27).

It is generally accepted that diffuse large-cell lymphoma isa heterogeneous group of non-Hodgkin lymphoma (29). Anewly described subgroup of large-cell lymphoma, Ki-1 lym-phoma, is characterized by large anaplastic cells that expressthe Ki-1 antigen (CD30) (11, 30). The majority of casesexpress T-cell-related antigens; however, in some cases, theneoplastic cells are of B-cell origin, although antigen expres-sion is often incomplete. There is evidence that the t(2;5)(p23;q35) is a characteristic chromosome abnormality in thissubtype of non-Hodgkin lymphoma (31-34). The malignantcells in our patient had the characteristic features of a Ki-1lymphoma, including anaplastic Ki-1-positive neoplasticcells and incomplete antigen expression on their cell surface,although they lacked the t(2;5)(p23;q35). At present, it is notclear whether the t(9;14)(p13;q32) is associated with a mi-nority of Ki-1 lymphoma cases of B-cell origin.

Cytogenetic analysis of lymphoma cells is often difficultbecause of inadequate tissue, low mitotic index, or poorchromosome morphology; thus, the presence of the t(9;14)(pl3;q32) may have been missed in previously examinedcases of lymphoma. Therefore, DNA-rearrangement studiesusing our probe, in combination with histologic and immu-nologic analyses, would be useful for the subcategorization ofdiffuse large-cell lymphoma as well as the characterization ofKi-1 lymphomas.

After this work was completed, Pellet et al. (35) reported themolecular cloning of a t(9;14)(pll;q32) observed in a heavy-chain disease; however, the reported breakpoint in the shortarm of chromosome 9 is different from that found in KIS-1.

We thank Dr. Janet D. Rowley for helpful discussions and RafaelEspinosa III and Roger L. Eddy, Jr., for technical assistance. Thiswork was supported by grants from the Ministry of Education,Science, and Culture and the Ministry of Health and Welfare ofJapan, by Grant CA42557 from the National Cancer Institute (to Dr.Rowley) and by Grant 88-23 from the American Cancer Society,

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