Molecular Analysis of a Chromosomal Translocation, T(9;14)

Home , Chromosome 14

Proc. Natl. Acad. Sci. USA Vol. 87, pp. 628-632, January 1990 Medical Sciences Molecular analysis of a chromosomal translocation, t(9;14)(p13;q32), in a diffuse large-cell lymphoma cell line expressing the Ki-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) KIS-1 cells. A transcriptionally active locus has been iden- (pl3;q32), in a diffuse large-cell lymphoma cell line, KIS-1, tified at 9p13, flanking the breakpoint of the translocation. that expresses the Ki-1 (CD30) antigen. Molecular cloning of the immunoglobulin heavy-chain locus (IGH) of this cell line revealed an unknown segment linked 5' to IGH. The break- MATERIALS AND METHODS point on chromosome 14 was 265 base pairs downstream from KIS-1 Cell Line. The KIS-1 cell line was established from the 3' border of the JH6 joining gene segment. Class switch the malignant cells of a patient with non-Hodgkin diffuse recombination deleted most of the constant genes of IGH (CH) large-cell lymphoma (9), which expressed the Ki-1 (CD30) and juxtaposed the Ca2 gene downstream of the translocation antigen (10, 11). The karyotype of the KIS-1 cell line was junction. Analysis of somatic cell hybrids and in situ chromo- complex. Each of 20 cells analyzed had a characteristic somal hybridization demonstrated that the translocated seg- translocation, t(9;14)(p13;q32); a translocation with these ment was normally located at band p13 of chromosome 9. The breakpoints has not been reported in other lymphoid neo- chromosome 9 sequences were transcriptionally active, giving plasms. The rearranged chromosome 14 (14q+) that resulted rise to transcripts of =11 kilobases. The KIS-1 cells seemed to from the reciprocal translocation was present in duplicate or have a small quantity of chimeric transcripts containing both triplicate; the additional 14q+ homologues had chromosomal chromosome 9 and Ca2 sequences. material of unknown origin translocated to the short arm, resulting in a 14p+ and q+ chromosome. The normal homo- A significant correlation has been observed between recur- logue was absent (Fig. 1). According to the ISCN nomen- rent chromosomal translocations involving band 14q32, and clature (12), a representative karyotype was as follows: 50,X, the histologic and immunologic subtypes of human lymphoid + der(X)t(X;?)(p11.4;?),Y, - 1, - 14,- 17, - 22, + lp + (HSR), malignancies (1-3). For example, the t(8;14)(q24;q32) is t(9;14)(p13;q32.3),t(12; 13)(q 13.1;pl 2), + der( 4)t found in most cases of small-noncleaved-cell lymphoma, and (9;14;')(?: :14p12-*14q32.3: :9p13--9pter), + der(l4)t(9;14;?) the t(14;18)(q32;q21) is primarily associated with follicular (?::14p12-* 14q32.3::9p13--9pter),del(16)(q22), + der small-cleaved-cell lymphoma. The lymphoma cells charac- (17)t(17;?)(pll .2;?), + der(22)t(1 ;22)(ql1;q13), + mar, + min. terized by each of these translocations show B-cell immu- The primary lymphoma cells had a similar modal karyotype nophenotypes. The biological significance of translocations including the t(9;14)(p13;q32). involving 14q32 has become apparent through the identifica- Southern and Northern Blot Analyses. DNA filter hybridization of an established protooncogene (4, 5) or a putative tion was carried out according to Southern (13). RNA was transforming gene (6), adjacent to the site of chromosome prepared from cultured cell lines (14) and hybridization was breaks on the reciprocal partner chromosomes. performed as described (14, 15). Probes used for the IGHlocus Although some subtypes of non-Hodgkin lymphomas have were as follows: joining region (JH), an EcoRI-HindIII frag- well-defined clinicopathologic features, the classification of ment of H24 (16) (see Fig. 3); ,u-chain constant region (C,), an lymphomas has remained controversial. The Southern blot EcoRI fragment of H24 (16); C,,. a Sac II fragment of Igyl-10 technique, using DNA probes adjacent to chromosome trans- (17); Ca2, an Xho 1-HindIII fragment of CH4A H Iga25 (18). location breakpoints, can be an alternative to karyotype The MYC probe was a 0.6-kilobase (kb) Kpn I-BamHI frag- analysis in some cases (7) and can be used for the diagnosis ment containing the first exon of the gene (19). of lymphoma (8). Cloning of breakpoint junctions of various Molecular Cloning and Nucleotide Sequencing. Genomic translocations in lymphomas will provide DNA probes for the DNA of the KIS-1 cells was digested with Bgl II. The DNA precise classification of lymphomas. fractions ofinterest were purified and ligated to the BamHI site We report the isolation and characterization of a DNA of Charon 28 phage arms. A cosmid library was constructed fragment surrounding the translocation junction of a diffuse from the FLEB14-14 cell line (20). Libraries were screened large-cell lymphoma cell line, KIS-1, which has a newly with probes indicated in the text. DNA segments of the identified t(9;14)(pl3;q32) (9). The short arm of chromosome isolated bacteriophage and cosmid clones were subcloned into 9 has been translocated to a position 5' ofthe immunoglobulin plasmid vectors (pUC18, pUC19, and Bluescribe). The nucle- heavy-chain locus (IGH) on chromosome 14, band q32, in the Abbreviations: J, joining region; C, constant region; V, variable The publication costs of this article were defrayed in part by page charge region; D, diversity region. payment. This article must therefore be hereby marked "advertisement" IThe sequences reported in this paper have been deposited in the in accordance with 18 U.S.C. §1734 solely to indicate this fact. GenBank data base (accession nos. M30453, M30454, M30455). 628 Downloaded by guest on September 30, 2021 Medical Sciences: Ohno et al. Proc. Natl. Acad. Sci. USA 87 (1990) 629

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C 9 1 4 23.1- 9.4-- _- w 6.7 FIG. 1. Partial karyotype of chromosomes 9 and 14 from a 4.4- G-banded metaphase cell from the KIS-1 cell line, illustrating the translocation between the short arm (p) of chromosome 9 and the long arm (q) of chromosome 14 [t(9;14)(p13;q32.3)]. One normal pKIS JH CO chromosome 9 is on the left and one 9p- chromosome is on the right. Two chromosomes 14 are abnormal: one 14q+ chromosome on the FIG. 2. Genomic Southern analysis of the KIS-1 cell line. Pla- left and one 14p+ and q+ chromosome on the right. The 9p- and cental (P) and KIS-1 DNAs (2 ,ug each) were digested with EcoRI (A), 14q+ chromosomes are the result of a reciprocal translocation BamHI (B), or HindIl1 (C). The same filter was hybridized sequen- between chromosomes 9 and 14. The arrowheads indicate break- tially with chromosome 9-specific pKIS, JH, and C,2 probes. Arrows points involved in the 9;14 translocation: bands 9p13 and 14q32.3, indicate rearranged band of identical size. HindlIl-digested A phage respectively. The 14q+ chromosome is present in duplicate; one of DNA provided molecular size markers. these is involved in a second rearrangement and has additional chromosomal material ofunknown origin on the short arm (14p+ and Comparison of the restriction map of the rearranged 14q+ q+)[der(14)t(9;14;?)(?: :14pl2 >14q32.3: :9pl3}>9pter)] . chromosome with those of the corresponding germ-line regions of chromosome 14 indicated that the breakpoint on otide sequences were determined by the dideoxy DNA se- chromosome 14 had occurred at a site 3' of the JH6 segment. quencing method using double-stranded plasmid vectors (21). The 5' 3.8-kb segment of the 9.9-kb Bgl II fragment did not Somatic Cell Hybrid Analysis. Thirty-five hybrids were hybridize with any VH (variable region gene ofIGH) probes. analyzed (22-24). The hybrids were characterized by kary- Genomic Southern analysis using the pKIS probe (Fig. 3) otype analysis, by mapped enzyme markers, and partly by showed that KIS-1 DNA contained one rearranged band in mapped DNA probes (22, 24, 25). DNA from the hybrid cell addition to the germ-line band. The rearranged band of KIS-1 panel was digested with HindIII and analyzed by Southern DNA was identical in size with the rearranged JH bands blot hybridization using the isolated DNA probe. (Fig. 2). In Situ Chromosomal Hybridization. Human metaphase Chromosomal Mapping of the pKIS DNA. The chromo- cells were prepared from phytohemagglutinin-stimulated pe- somal localization of pKIS in the normal cell was determined ripheral blood lymphocytes. In situ hybridization was per- by Southern analysis of a panel ofhuman-mouse somatic cell formed using 3H-labeled probes with a specific activity of 108 dpm/,ug, as described (26). Autoradiographs were exposed A JH HCHC for 11 days. B G12345 6/ G H E 1 111.111 If I RESULTS J :BS Genomic Southern Blot Hybridization. Southern blot hy- JHi u bridization showed that the KIS-1 DNA had one rearranged HC Sg/°t band that hybridized with the JH probe, and no germ-line JH B G SHCG IH S G band was observed (Fig. 2). Hybridization with the C.1 and C,,1 probes showed complete deletion ofboth copies ofthe C. RS IBS BS or2 gene and all the C. subclass genes (data not shown). The rearranged band that hybridized with the JH probe after C pKIS e H H EcoRI or BamHI digestion was also labeled with the Ca2 *ESH GE*' S B S GG4SHE probe. I l EGGIB

Cloning of the IGH Locus of the KIS-1 Cell Line. Southern I analysis of the KIS-1 DNA after Bgl II digestion showed that BS BS: BS p BS BS BS 9.9- and 4.9-kb bands hybridized to the JH probe. Phage 6d -- p8.5-1 1 kb clones containing the 9.9- and 4.9-kb Bgl II fragments were S pBS8.6BB L4-- isolated by screening with the JH probe. The 4.9-kb Bgl II fragment was identical with the 3' halfofthe 9.9-kb fragment, FIG. 3. Restriction maps of the IGH locus of the KIS-1 cell line indicating that the 9.9-kb fragment resulted from partial Bgl and its germ-line counterparts. Open rectangles represent chromo- II digestion. Restriction mapping and Southern hybridization some 9 sequence (C) and solid lines represent chromosome 14 demonstrated that the 9.9-kb Bgl II fragment contained the sequence (A). Coding (JH, Ca,2) and switch (SE,, Sl/a2) regions are indicated by black and gray boxes, respectively. Restriction sites: E, rearranged gene (Fig. 3). These results indicate that JH-C,2 EcoRI; B, BamHI; H, HindIII; G, Bgl II; S, Sac I; BS, BstEII; HC, the KIS-1 cells have a single rearranged IGH locus that has HincII. EcoRI site marked by asterisk was not identified on rear- undergone class-switch recombination to the Ca2 gene, re- ranged chromosome 14q+ (B). Probes used for hybridization studies sulting in deletion of all of the other CH genes. are indicated (JH, pKIS, p8.5-1, and pBS8.6BB). Downloaded by guest on September 30, 2021 630 Medical Sciences: Ohno et al. Proc. Natl. Acad. Sci. USA 87 (1990) 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 2 hybrids (-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 1 hybrids (-/+) 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 hybridized to Southern blots containing HindIII-digested DNA from the human-mouse cell hybrids. The chromosome assignment ofpKIS was determined by scoring the presence or absence of human bands on the blots. Concordant hybrids have either retained or lost pKIS together with a specific chromosome. 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 symbol denotes the presence or absence of the specific human chromosome. Percent discordancy indicates the degree of discordant segregation for a marker and a chromosome. A 0%o discordancy is the basis for chromosome assignment. hybrids. The pKIS probe hybridized to a 10.4-kb HindIll quences, respectively. No additional nucleotides were in- fragment in human DNA, and to an 8.0-kb HindIII fragment serted at the junction (Fig. 5). in mouse DNA under stringent washing conditions. Hybrid- Northern Blot Analysis. The pKIS probe detected -11-kb ization to 35 cell hybrids showed that the pKIS sequence transcripts in three other human B-cell lines as well as KIS-1 mapped to chromosome 9. All other chromosomes segre- cells. Several smaller transcripts were also observed in the gated discordantly (Table 1). KIS-1 cells (Fig. 6A). The structure of the IGH locus of the To localize the pKIS DNA by an independent method, KIS-1 cells suggested that transcription might proceed across three pKIS probes (Fig. 3) were also hybridized to normal the translocation junction into the Ca2 coding region. The Ca2 metaphase chromosomes. In each case, we observed specific locus was actively transcribed in the KIS-1 cell line, and labeling of chromosome 9 only; the largest number of grains 1.3-kb sterile transcripts were the most abundant (Fig. 6C). was at 9p13 (Fig. 4). Two additional, less abundant transcripts ofthe Ca gene were These data indicate that the phage clones we have isolated also labeled with the pKIS probe. These results suggest that contain the breakpoint junction of the t(9;14)(p13;q32). The hybrid transcripts containing both pKIS and Ca2 sequences short arm of chromosome 9 distal to 9p13 was translocated to may be produced, although these transcripts seem to be the 5' side of IGH, deleting all the VH, D (diversity), and JH minor RNA species. segments on the 14q+ chromosome. This nonproductive rearrangement ofIGH and loss ofthe normal chromosome 14 are presumably responsible for the absence of immunoglob- DISCUSSION ulin expression on the KIS-1 cell surface (9). The close association between the translocation ofoncogenes Nucleotide Sequence Analysis of the Breakpoint Junction. into the IGH locus and their expression in lymphoma sug- The cosmid DNA library constructed from FLEB14-14, a cell gests that functional genes, most likely involved in transfor- line without the chromosomal translocation, was screened mation, may be located adjacent to the breakpoint junctions with the pKIS probe, and isolated germ-line clones were in other translocations involving 14q32 (4-6). Northern blot analyzed by restriction mapping and nucleotide sequencing. analysis of RNA from the KIS-1 cell line demonstrated a Comparison of nucleotide sequences surrounding the junc- at flanking the breakpoint tion indicated that the breakpoint on chromosome 14 was transcriptionally active locus 9p13 of the of the newly identified t(9;14)(pl3;q32); the presence of located 265 base pairs downstream from the 3' border chimeric transcripts with the Ca2 gene suggested that an JH6 segment. The 5' and 3' sides of the breakpoint were the translocation might play 9 and chromosome 14 se- alteration of this gene caused by homologous to chromosome an important role in the pathogenesis of the original lymphoma. To our knowledge, no protooncogenes or genes for 3...... ***** growth factors have been mapped to 9pl3, indicating that the sequences we have isolated at this band may represent a novel gene that might be involved in lymphomagenesis. q2 2 -40 -20 1 ----- CHR. 9 G~GfAA ****************** **************************** T(9; 14) GGCTATIG'..A.A'I 3 23 * ** * * ** * * ** * ** _ 4 * CHR. 14 ± AG876 + KIS-1 FIG. 4. Distribution of silver grains on chromosome 9. For in situ +40 chromosomal hybridization, we used a 3.0-kb Bgl I-Sac I fragment +1 +20 (p8.5-1) and an 8.6-kb BamHI fragment (pBS8.6BB) in addition to the TrFITTTGAGCIGT GGCAZIJ.L tmGGG ATCG AVTATTCAT1G pKIS probe (Fig. 3). Of 100 metaphase cells examined from the ********* * * * ** ***I** * * hybridization of the pBS8.6BB probe, 27 (27%) were labeled on region pl or p2 of one or both chromosome 9 homologues. Of 186 **************** ************************ total labeled sites observed, 48 (26%) were located on chromosome 9. These sites were clustered at bands p12-21, and this cluster represented 21% (39/186) of all labeled sites (cumulative probability FIG. 5. Nucleotide sequence analysis of the breakpoint junction for the Poisson distribution is <<0.0005). The largest number of of the t(9;14)(pl3;q32). The breakpoint on chromosome 14 of the grains was observed at 9p13. Similar results were obtained in a t(8;14)(q24;q32) in the Ag876 cell line (27) is also indicated. Se- second hybridization experiment using this probe and in experiments quences are oriented 5' to 3' from left to right. Asterisks represent using the pKIS and p8.5-1 probes. nucleotide homology. Downloaded by guest on September 30, 2021 Medical Sciences: Ohno et al. Proc. Natl. Acad. Sci. USA 87 (1990) 631

Ao - Illinois Division (to T.W.M.). M.M.L. is a Scholar of the Leukemia & C) Society of America. A -j yU- \ y) C 2 11 1. Fukuhara, S. & Rowley, J. D. (1978) Int. J. Cancer 22, 14-21. 2. Fukuhara, S., Ueshima, Y., Kita, K. & Uchino, H. (1984) Acta Haemat. Jpn. 47, 1579-1590. 28S- 28S - 3. Fifth International Workshop on Chromosomes in Leukemia- Lymphoma (1987) Blood 70, 1554-1564. 18S- 18S- 4. Dalla-Favera, R., Bregni, M., Erikson, J., Patterson, D., Gallo, B R. C. & Croce, C. M. (1982) Proc. Nat!. Acad. Sci. USA 79, 'T 7824-7827. 28S- " ma 5. Taub, R., Kirsch, I., Morton, C., Lenoir, G., Swan, D., pKIS Cac Tronick, S., Aaronson, S. & Leder, P. (1982) Proc. Natl. Acad. 18S- Sci. USA 79, 7837-7841. 6. Tsujimoto, Y., Finger, L. R., Yunis, J., Nowell, P. C. & Croce, FIG. 6. Northern blot analysis. Expression of the pKIS se- C. M. (1984) Science 226, 1097-1099. quences was studied in the KIS-1 cell line and in three additional cell 7. Rowley, J. D. (1988) J. Clin. Oncol. 6, 919-925. lines of B-cell lineage: LCL, an Epstein-Barr virus-transformed 8. Cossman, J., Uppenkamp, M., Sundeen, J., Coupland, R. & lymphoblastoid cell line having a normal karyotype (LCL-AMK Raffeld, M. (1988) Arch. Pathol. Lab. Med. 112, 117-127. line); K, a Burkitt lymphoma cell line containing a t(8;14)(q24;32) 9. Kamesaki, H., Miwa, H., Ohno, Y., Miyanishi, S., Yamabe, (Kobayashi line); and FL-18, a follicular lymphoma cell line char- H., Doi, S., Arita, Y., Ohno, H., Tatsumi, E., Nishikori, M., acterized by a t(14;18)(q32;q21) (FL-18 line). (A) Poly(A)+ RNA-(5 Fukuhara, S., Hatanaka, M. & Uchino, H. (1988) Jpn. J. ,.g per lane) from cells indicated was hybridized with the pKIS probe. Cancer Res. (GANN) 79, 1193-1200. (B) The same filter was rehybridized with the MYC first-exon probe. 10. Schwab, U., Stein, H., Gerdes, J., Lemke, H., Kirchner, H., (C) Poly(A)+ RNA of KIS-1 cells was hybridized with pKIS or C"2 Schaadt, M. & Diehl, V. (1982) Nature (London) 299, 65-67. probe. Size markers are 28S and 18S ribosomal RNAs. 11. Stein, H., Mason, D. Y., Gerdes, J., O'Connor, N., Wainscoat, J., Pallesen, G., Gatter, K., Falini, B., Delsol, G., Lemke, H., It has been suggested that the high frequency of chromo- Schwarting, R. & Lennert, K. (1985) Blood 66, 848-858. some translocations involving the IGH locus is due to errors 12. Harnden, D. G. & Klinger, H. P., eds. (1985) ISCN 1985: An in the V-D-J recombinase-mediated process (28). However, International System for Human Cytogenetic Nomenclature our results do not support this hypothesis, as heptamer- (Karger, Basel). 13. Southern, E. M. (1975) J. Mol. Biol. 98, 503-517. nonamer recombination signals were not found in proximity 14. Ullrich, A., Shine, J., Chirgwin, J., Pictet, R., Tischer, E., Rutter, to the junction on either chromosome 14 or 9. The junction W. J. & Goodman, H. M. (1977) Science 196, 1313-1319. of a t(8;14)(q24;q32) of an endemic Burkitt tumor cell line, 15. Ohno, H., Fukuhara, S., Arita, Y., Doi, S., Takahashi, R., Ag876, was located only 25 base pairs upstream from the Fujii, H., Honjo, T., Sugiyama, T. & Uchino, H. (1988) Cancer breakpoint of the KIS-1 cell line (27). Res. 48, 4959-4963. It is generally accepted that diffuse large-cell lymphoma is 16. Takahashi, N., Nakai, S. & Honjo, T. (1980) Nucleic Acids a heterogeneous group of non-Hodgkin A Res. 8, 5983-5991. lymphoma (29). 17. Takahashi, N., Ueda, S., Obata, M., Nikaido, S. & Honjo, T. newly described subgroup of large-cell lymphoma, Ki-1 lym- (1982) Cell 29, 671-679. phoma, is characterized by large anaplastic cells that express 18. Hisajima, H., Nishida, Y., Nakai, S., Takahashi, N., Ueda, S. the Ki-1 antigen (CD30) (11, 30). The majority of cases & Honjo, T. (1983) Proc. Natl. Acad. Sci. USA 80, 2995-2999. express T-cell-related antigens; however, in some cases, the 19. Taya, Y., Hosogai, K., Hirohashi, S., Shimosato, Y., Tsuch- neoplastic cells are of B-cell origin, although antigen expres- iya, R., Tsuchida, N., Fushimi, M., Sekiya, T. & Nishimura, S. sion is often incomplete. There is evidence that the t(2;5) (1984) EMBO J. 3, 2943-2946. (p23;q35) is a characteristic chromosome abnormality in this 20. Katamine, S., Otsu, M., Tada, K., Tsuchiya, S., Sato, T., Ishida, N., Honjo, T. & Ono, Y. (1984) Nature (London) 309, 369-371. subtype of non-Hodgkin lymphoma (31-34). The malignant 21. Hattori, M. & Sakaki, Y. (1986) Anal. Biochem. 152, 232-239. cells in our patient had the characteristic features of a Ki-1 22. Shows, T. B., Sakaguchi, A. Y. & Naylor, S. L. (1982) in lymphoma, including anaplastic Ki-1-positive neoplastic Advances in Human Genetics, eds. Harris, H. & Hirschhorn, cells and incomplete antigen expression on their cell surface, K. (Plenum, New York), Vol. 12, pp. 341-452. although they lacked the t(2;5)(p23;q35). At present, it is not 23. Shows, T., Eddy, R., Haley, L., Byers, M., Henry, M., Fujita, clear whether the t(9;14)(p13;q32) is associated with a mi- T., Matsui, H. & Taniguchi, T. (1984) Somat. Cell Mol. Genet. nority of Ki-1 lymphoma cases of B-cell origin. 10, 315-318. 24. Shows, T. B., Brown, J. A., Haley, L. L., Byers, M. G., Cytogenetic analysis of lymphoma cells is often difficult Eddy, R. L., Cooper, E. S. & Goggin, A. P. (1978) Cytogenet. because of inadequate tissue, low mitotic index, or poor Cell Genet. 21, 99-104. chromosome morphology; thus, the presence of the t(9;14) 25. Shows, T. B. (1983) in Isozymes: Current Topics in Biological (pl3;q32) may have been missed in previously examined and Medical Research, eds. Rattazzi, M. C., Scandalios, J. G. cases of lymphoma. Therefore, DNA-rearrangement studies & Whitt, G. S. (Liss, New York), Vol. 10, pp. 323-339. using our probe, in combination with histologic and immu- 26. Le Beau, M. M., Westbrook, C. A., Diaz, M. 0. & Rowley, nologic analyses, would be useful for the subcategorization of J. D. (1984) Nature (London) 312, 70-71. 27. Neri, A., Barriga, F., Knowles, D. M., Magrath, I. T. & Dalla- diffuse large-cell lymphoma as well as the characterization of Favera, R. (1988) Proc. Natl. Acad. Sci. USA 85, 2748-2752. Ki-1 lymphomas. 28. Haluska, F. G., Finver, S., Tsujimoto, Y. & Croce, C. M. After this work was completed, Pellet et al. (35) reported the (1986) Nature (London) 324, 158-161. molecular cloning of a t(9;14)(pll;q32) observed in a heavy- 29. Nathwani, B. N., Dixon, D. O., Jones, S. E., Hartsock, R. J., chain disease; however, the reported breakpoint in the short Rebuck, J. W., Byrne, G. E., Jr., Sheehan, W. E., Kim, H., arm of chromosome 9 is different from that found in KIS-1. Coltman, C. A., Jr., & Rappaport, H. (1982) Blood 60, 1068- 1074. We thank Dr. Janet D. Rowley for helpful discussions and Rafael 30. Suchi, T., Lennert, K., Tu, L.-Y., Kikuchi, M., Sato, E., Espinosa III and Roger L. Eddy, Jr., for technical assistance. This Stanfeld, A. G. & Feller, A. C. (1987) J. Clin. Pathol. 40, work was supported by grants from the Ministry of Education, 995-1015. Science, and Culture and the Ministry of Health and Welfare of 31. Fischer, P., Nacheva, E., Mason, D. Y., Sherrington, P. D., Japan, by Grant CA42557 from the National Cancer Institute (to Dr. Hoyle, C., Hayhoe, F. G. & Karpas, A. (1988) Blood 72, Rowley) and by Grant 88-23 from the American Cancer Society, 234-240. Downloaded by guest on September 30, 2021 632 Medical Sciences: Ohno et al. Proc. Nadl. Acad. Sci. USA 87 (1990)

32. Le Beau, M. M., Bitter, M. A., Larson, R. A., Doane, L. A., 34. Kaneko, Y., Frizzera, G., Edamura, S., Maseki, N., Sakurai, Ellis, E. D., Franklin, W. A., Rubin, C. M., Kadin, M. D. & M., Komada, Y., Sakurai, M., Tanaka, H., Sasaki, M., Suchi, Vardiman, J. W. (1989) Leukemia 3, 866-870. T., Kikuta, A., Wakasa, H., Hojo, H. & Mizutani, S. (1989) 33. Rimokh, R., Magaud, J.-P., Berger, F., Samarut, J., Coiffier, Blood 73, 806-813. B., Germain, D. & Mason, D. Y. (1989) Br. J. Haematol. 71, 35. Pellet, P., Berger, R., Bernheim, A., Brouet, J. C. & Tsapis, A. 31-36. (1989) Oncogene 4, 653-657. Downloaded by guest on September 30, 2021