Proc. Natl. Acad. Sci. USAVol. 91, pp. 10962-10966, November 1994Medical Sciences

Hodgkin disease: Hodgkin and Reed-Sternberg cells picked fromhistological sections show clonal immunoglobulin generearrangements and appear to be derived from B cellsat various stages of developmentRALF KOPPERS*t, KLAUS RAJEWSKY*, MIN ZHAOt, GUNTHER SIMONSt, RALF LAUMANN*, ROBERT FISCHER,AND MARTIN-LEO HANSMANN**Institute for Genetics, and tDepartment of Pathology, University of Cologne, 50931 Cologne, Germany

Contributed by Klaus Rajewsky, July 20, 1994

ABSTRACT Hodgkin disease (HD) is characterized by asmall number of putative malignant cells [Hodgkin and Reed-Sternberg (HRS) cells] among a background of lymphocytesand histiocytes. The lineage of HRS cells is still elusive and aclonal origin of these rare cells has not formally been demon-strated. We isolated HRS cells by micromanipulation fromhistological sections of three cases of Hodgkin lymphoma (eachrepresenting a distinct subtype of the disease) and analyzedindividual cells for immunoglobulin variable (V) gene rear-rangements by PCR. In each of the three cases a singieheavy-chain V (VH) (and in one case, in addition, a K light-chain) gene rearrangement was amplified from the HRS cells,identifying these cells as members of a single done. A poten-tially functional VH rearrangement was obtained from a case ofnodular sclerosis HD. Somatic mutations and intraclonal di-versity in the VH genes indicate a germinal center B-cell originof the HRS cells in a case of lymphocyte-predominant HD,whereas in a case of mixed-cellularity HD the sequence analysisrevealed only nonfunctional V gene rearrangements, suggest-ing a pre-B-cell origin. This indicates that HRS cells canoriginate from B-lineage cells at various stages of development.

Hodgkin disease (HD), one of the most common malignantlymphomas, is characterized by a small number of putativemalignant mononuclear Hodgkin and multinucleated Reed-Sternberg cells (HRS cells; usually <1% ofthe tumor) amidsta major population oflymphocytes, plasma cells, histiocytes,neutrophils, eosinophils, and stromal cells. Four subtypes ofHD have been described: lymphocyte predominant (LP),nodular sclerosis (NS), mixed cellularity (MC), and lympho-cyte depletion (LD) (1). This subclassification is based on themorphology of the HRS cells as well as on differences of thecellular background. HRS cells in the NS, LD, and MCsubtypes ofHD are characterized by expression of the CD30and CD15 antigens, whereas HRS cells in the LP subtypeoften lack these antigens but express B-cell antigens such asCD20 (2).Although HD has been known for a long time, the lineage

derivation ofthe putative malignant HRS cells is still a matterof controversy. Immunohistochemical studies detected Band/or T-cell or monocyte markers on HRS cells (2), and arecent analysis of gene expression in HRS cells at thesingle-cell level demonstrated the coexpression of genescharacteristic of several hematopoietic lineages (3). The lowfrequency of HRS cells in the tumor and the difficulty ofisolating these cells make molecular biological studies diffi-cult to interpret. Southern blot analysis for immunoglobulin(Ig) or T-cell antigen receptor (TCR) gene rearrangements in

HD tumor tissue (4) does not allow one to determine whetherclonal gene rearrangements detected in HD biopsies arederived from the HRS or from other cells.The detection of Ig and/or TCR gene rearrangements in 11

of 12 HD-derived cell lines thought to originate from HRScells (5-7) suggests that HRS cells may be of B- and/orT-lineage origin, but the limited success in establishing celllines from HD precludes generalization of those observa-tions.A monoclonal origin ofHRS cells in at least some instances

is suggested by karyotype analysis (8, 9) and by the findingof monoclonal Epstein-Barr virus DNA in some HD tissuesin which HRS cells harbor the virus (10, 11). To confirm themonoclonality of HRS cells and to analyze these cells for apossible B-lineage origin we isolated single HRS cells fromhistological sections by micromanipulation and analyzedthem for immunoglobulin heavy-chain and K light-chain vari-able (VH and VJ gene rearrangements by polymerase chainreaction (PCR) (12). The amplification ofrearranged Ig genesallows one to unambigiously determine the clonality of thecells (13, 14). In addition, the sequence determination ofVHDHJH and VKJK gene rearrangements obtained by PCRgives a detailed picture of Ig gene rearrangements in HRScells and discriminates VHDHJH from DHJH rearrangements.§Such a discrimination is important, since cross-lineage rear-rangements of Ig heavy-chain genes have been described innon-B-cell leukemias (15) but appear to be restricted to DHJHjoints (16-20). Therefore, the present approach of amplifyingVHDHJH rearrangements tests HRS cells for a possible B-lin-eage origin.

MATERIALS AND METHODSTissues and Clinical Data. The patients were a 39-year-old

man with LP HD at relapse, a 73-year-old man with NS HD,and a 26-year-old man with MC HD. Inguinal lymph nodeswere available from the former two patients and a cervicallymph node from the latter.Immunostaining and Micromanipulation. Eight- to 10-,um-

thick frozen sections were stained with either anti-CD20(L26), anti-CD3 (OKT3), or anti-CD30 (BerH2) monoclonalantibodies (21). Stained cells were mobilized and aspiratedwith the help of a micropipette fixed to a hydraulic micro-manipulator (12). Multiple cells were picked from each sec-tion.

Abbreviations: HD, Hodgkin disease; HRS, Hodgkin and Reed-Sternberg; MC, mixed cellularity; LP, lymphocyte predominant;NS, nodular sclerosis; LD, lymphocyte depletion; CDR, comple-mentarity-determining region; TCR, T-cell antigen receptor.tTo whom reprint requests should be addressed at: Institute forGenetics, Weyertal 121, 50931 Cologne, Germany.§Sequences repeatedly obtained from HRS cells have been depositedin the GenBank data base (accession nos. X77415-X77421).

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Single-Cell PCR. Rearranged VH- and VK-region geneswere amplified from single cells by using V-gene family-specific primers and JH and JK primers as previously de-scribed (12). Both strands of the PCR products were directlysequenced.

RESULTS

Immunohistochemical Analysis. Single HRS cells were mi-cromanipulated from frozen sections stained with anti-CD30antibodies for HRS cells in a case of NS and a case of MCtype (Fig. 1) and anti-CD20 antibodies for HRS cells in a caseof LP HD. HRS cells of the cases of NS and MC type werepositive for CD30 and CD15 and negative for CD3 and CD20,whereas in the case of LP HD the HRS cells were CD3,CD15, and CD30 negative and CD20 positive (data notshown).

Validity of the Experimental Approach. In all three cases arepeat experiment was carried out, and in four of the sixexperiments T and B cells were picked on the same day asHRS cells and analyzed together with the HRS cells in a blindfashion. For a total of 50 T cells only one rearranged V genewas obtained, whereas 50 B cells gave rise to 17 PCRproducts, 13 of which were sequenced (Table 1). These 13V-gene sequences were all unique and clonally unrelated torearrangements amplified from HRS cells.

Clonal Heavy-Chain Gene Rearrangements in a Case of NSHD. From 30 single HRS cells of the NS case 12 PCRproducts indicating a heavy-chain rearrangement were ob-tained, 11 with a VH3 family-specific primer and one with aVH4 primer (Table 1). Sequencing of 8 of these productsrevealed an identical VHDHJH sequence for 7 cells. (In this

FIG. 1. (Upper) Frozen section of a lymph node with a binucle-ated CD30-positive Reed-Steinberg cell in the middle of the picture.(x240.) (Lower) Frozen section after picking, showing a hole wherethe Reed-Sternberg cell was located, with a small rim of immuno-stained cell membrane and cytoplasm.

Table 1. Summary of single-cell analyses of three cases ofHodgkin lymphoma for Ig V gene rearrangements

Cells PCR products RearrangementsCase Exp. positive Total Sequenced Repeated Unique

HRS cellsNS 1 7/20 9 5t 4 VH3+ 1 VH3-

2* 3/10 3 3 3 VH3+MC 1 12/20 30 18* 4 VH1- 1 VH1+

6 VH3- 1 VK4-3 VK1- 1 VH3+2 VA-

2* 3/5 7 4* 1 VH3- 1 VK4+2 Vj-

LP 1* 3/11 3 3 3 VHS-2* 4/17 6 6 3 VHS-

3 VH4+ControlsNS 2 2/10 B 2 2 1 VH1?

0/10 T 1 VH4+MC 2 1/10 B 1 1 1 VH1+

0/10 TLP 1 2/10 B 6 3 1 VK2+

0/10 T 2 VH3+2 6/20 B 8 7 Multiple§

1/20 T 1 1 1 VH4+For analysis of HRS cells, two independent experiments were

carried out for each case. Some experiments (*) were performed asdouble-blind experiments meaning that HRS cells as well as control(B and T) cells isolated on the same day were analyzed coded in ablinded fashion. The numbers of PCR products obtained and se-quenced are indicated. The sequences repeatedly obtained from HRScells were deposited in the EMBL/GenBank data library underaccession nos. X77415-X77421. In each of the three cases the samerepeated rearrangements were obtained in the first and secondexperiments. It is indicated whether the rearrangements are non-functional (-) or potentially functional (+). The functionality of therearrangement marked by the question mark is unknown due to asequence ambiguity in complementarity-determining region (CDR)III. For several PCR products no sequences were obtained becausePCR products were lost upon purification.tThree of the four PCR products that were not sequenced representVH3 rearrangements.tAmong 15 PCR products that were not sequenced, 8 VH1 familyrearrangements with a deletion of about 50 bp and 5 V generearrangements belonging to the VH3, VJ, and VA families wereobtained.§Clonally unrelated VH and VK gene rearrangements of various Vgene families (2 VH3, 1 VH4, 1 Vj1, 2 VK2, and 1 VA) were obtained.

and the other two cases studied, sequences were not obtainedfor some PCR products, because DNA was lost upon puri-fication; see Table 1.) This rearrangement is potentiallyfunctional, because the correct reading frame for translationof the Ig gene was preserved in the VHDHJH rearrangementprocess. A nonfunctional (out-of-frame) VH3 rearrangementand a VH4 PCR product were each obtained only once andcould potentially represent cellular or other contamination.

Clonal Nonfunctional Heavy- and Light-Chain Gene Rear-rangements in a Case ofMC HI). Twenty-five HRS cells wereanalyzed for the case ofMC HD, with 15 of these cells givingrise to a total of 37 PCR products, 22 of which weresequenced (Table 1). Two nonfunctional VH and two non-functional VK rearrangements were obtained repeatedly, withfive cells harboring two to four of these rearrangements. TheVH3 rearrangement, amplified from seven cells, uses a VH3gene with 98% homology to the VH3 germline gene DP42 andis nonfunctional because ofa stop codon in CDR III. The VH1rearrangement, obtained from four cells, uses a VH1 genewith 97% homology to the VH1 germline gene DP15 and hasa 47-bp deletion of codons 56-71 which leads to loss of the

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reading frame for translation (Fig. 2). Interestingly, close tothe 5' end of the deletion within the VH gene, a sequencestretch of six nucleotides homologous to the conservedheptamer recombination signal sequence (13) was found (Fig.2). The deletion might thus be explained by an "open-and-shut" rearrangement near that heptamer, a process whichcan lead to the addition or deletion of nucleotides at aheptamer signal sequence (27, 28). In addition, at the 3' endof the deletion a tetranucleotide motif (CCAG) is present asis often seen near nonhomologous recombination break-points in Ig genes (26). Four additional V-gene sequenceswere each obtained only once (two of them from the samecell) and might represent cellular or other contamination.

Clonal Heavy-Chain Gene Rearrangements and SomaticMutation in a Case of LP HD. For the case of LP HD, 28 HRScells were analyzed. From 6 cells a nonfunctional VHS generearrangement was obtained, harboring two stop codonswithin the VH gene. One of the six sequences (LPVH5.6; Fig.3a) differs by three nucleotides from the others. The VHsequence has 85% sequence homology to the VH5-251 germ-line gene and has a deletion of six nucleotides (codons 28 and29, see Fig. 3a). Since the VH5 family is composed of onlythree members, which appear to be nonpolymorphic withinthe human population (29), it is likely that the sequencedifferences to the corresponding germline gene are the resultof somatic mutation. Interestingly, a tetranucleotide motifcommonly found near nonhomologous recombination break-points in Ig genes (26) is seen at three positions in theVH5-251 germline gene near the deletion (Fig. 3a).For three cells (two of which had also given rise to a VHS

PCR product) a potentially functional VH4 rearrangementwas obtained. The VH4 gene in this structure has 88%sequence homology to the DP64 germline gene (Fig. 3b) andcontains a 6-bp insertion between codons 49 and 50. One ofthe three sequences (LPVH4.3; Fig. 3b) differs in two posi-tions from the other two sequences. To test whether the

25 30 CDR ISer Gly Tyr Thr Phe Thr Ser Tyr Asp Val Asn Trp Val Arg Gin

MCVH1 T TCT GGA TAC ACC TTC ACC AGT TAT GAT GTC AAC TGG GTG CGA CAGDP15 - --- --- --- --- --- --- --- --- --- A-- --- --- --- --- ---

40 50 CDR IIAla Thr Gly Gln Gly Leu Glu Trp Met Gly Trp Met Asn Pro Asn Ser

MCVH1 GCC ACT GGA CAA GGG CTI GAG TGG ATG GGA TGG ATG AAC CCT AAC AGTDP15 --- --- --- __

55Gly

MCVH1 GGTDP15 --- AAC ACA GGC TAT GCA CAG AAG TTC CAG GGC AGA GTC ACC ATG ACC

72 80Glu His Leu His Lys Ser Ser Leu His Gly Ala Gly Gin Pro Glu

MCVH1 GAA CAC CTC CAT AAG TCC AGC CTA CAT GGC GCT GGG CAG CCT GAGDP15 AG --- --- - --- --- CA- --- --- --- --A--- -A- --- --- ---

90 CDR IIIIle *** Gly His Gly Arg Val Lou Leu Ser Glu Ala Ser Leu Glu *

MCVH1 ATC TGA GGA CAC GGC CGT GTA TTA CTG TCC GAG GCA TCT CTG GAG TGAW1.5-- --- --- --- --- --- --- --- --- -G- ---

DFS-1100 a h 102Leu Ser Tyr ** Gly Lou Leu Arg Tyr Gly Arg

MCVH1 TTA TCA TAT TAG 6GA CTA CTA CGA TAT GGA CGTDFS-l ---- - --- --- --G--- --- ---

JH6

FIG. 2. V gene segments derived from single HRS cells of a caseof MC HD, compared with the most homologous germline genes.Codons are numbered according to Kabat et al. (22). Correspondingamino acids are shown above each codon; asterisks indicate stopcodons. CDRs I-III ofthe V-region gene are indicated. PCR productsof single HRS cells with a VH1 rearrangement (MCVH1) are com-pared with germline VH1 gene DP15 (23), rearranged DH gene DFS-1(24), and the JH6 gene (25). The VH1 rearrangement harbors adeletion of 47 bp from codons 56-71. PCR products of four cellsshowed an identical sequence. A sequence stretch of six nucleotideshomologous to the conserved heptamer recombination signal se-quence for Ig genes close to the 5' end of the deletion is underlined.At the 3' end ofthe deletion a tetranucleotide motif(CCAG; see text)(26) is underlined.

nucleotide differences of the VH rearrangements amplifiedfrom this case of LP HD compared with the putative germlinegenes were due to somatic point mutations, a PCR analysiswas performed using primers specific for the VHS and VH4sequences rearranged in the HRS cells (see Fig. 3). A PCRproduct was obtained from DNA of the HD lymph node butnot from peripheral blood DNA of the patient, stronglyindicating that the VH genes in these rearrangements harborsomatic mutations. This was confirmed for the VHS rear-rangement by sequencing the VH5-251 germline gene fromDNA isolated from peripheral blood of the patient, whichturned out to be identical to the published VH5-251 sequence(data not shown). In addition, the JH genes in both rearrange-ments carry a single point mutation each (Fig. 3). Nucleotidedifferences seen between the sequences obtained from singlecells and from DNA of another section of the same lymphnode (amplified with the sequence-specific primers) indicateintraclonal diversity due to a low level of ongoing somatichypermutation (Fig. 3). This is further supported by threepositions within the sequences obtained from lymph nodeDNA at which two different nucleotides were seen in thesequence analysis (Fig. 3).

DISCUSSIONReliability and Efficiency of Molecular Single-Cell Analysis.

Due to the high sensitivity of the technique-allowing am-plification of single target molecules-contamination is amajor technical risk in the single-cell analysis. Nevertheless,as we had already shown in a recent analysis of B-celldevelopment in human germinal centers (12), the methodyields reliable results if appropriate precautions are taken.Several lines of evidence indicate that in the present analysisa reliable picture of V gene rearrangements in the microma-nipulated cells was obtained. (i) In all three cases a repeatexperiment confirmed the results of the first experiment, andwe never obtained the same sequence from different patients.(ii) In four of the six experiments (Table 1) B, T, and HRScells were micromanipulated on the same day and analyzedin a double-blind fashion. We never obtained a HRS cellsequence for B or T cells. Only one VH gene rearrangementwas obtained from 50 T cells, and 13 PCR products se-quenced from 50 B cells were all unique and clonally unre-lated. (iii) Intraclonal diversity seen for both VH rearrange-ments of the case of LP HD cannot be explained by contam-ination.That PCR products were not amplified from all of the HRS

and B cells represents a methodological limitation that canhave several reasons (see also ref. 12): (a) Since cells weremicromanipulated from frozen sections with a thickness ofabout 10 pim, in most instances part of the cell nucleus wasmissing, which is especially likely for the large HRS cells. (b)The primers used may not amplify all VH and VK rearrange-ments, due to either somatic mutations or usage ofpreviouslyunknown V genes. (c) The lack of detectable K light-chainrearrangements in two of the three cases of HD could beexplained if the HRS cells in these cases harbor a VA generearrangement (which was not analyzed).

HiD Can Result from Manant Transformation of B-Lin-eage Cells at Various Stages of Development. For the case ofMC HD two nonfunctional VH and VK gene rearrangementswere obtained from the HRS cells (Table 1; Fig. 2). Whereastwo nonfunctional VK rearrangements might occur in A light-chain-expressing B cells (30), the identification of a humanB-cell clone with two nonfunctional VH rearrangements hasto our knowledge not been described before. Gene targetingexperiments in mice have shown that B-cell progenitorsunable to express membrane-bound 1L heavy chains arearrested in development and presumably die still within thebone marrow (31, 32). In recent work a cellular compartment

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a21 27 30 CDR I

Ser Cys Lys Ala Tyr Gly Tyr Asn Asn Tyr Trp Ile Ala TrpLPVH5 OG TGT AAG GCT TAT OGA TAC AAC AAC TAC TGO ATC GCC TGOLPVH5.6 . -.------ .LPVH5S - N-- - --- --- --- --- --- ---

5-251 T- -- G----------

40 50Lou Arg Gln Met Pro Oly Lys G1y Leu Glu Trp Met Oly Ile Ph. Ph.

LPVHS CTG 00G CAG ATO 00C 00 AAA 00C CTG GAG TGO ATG GGG ATC Tm,,LPVH5.6 --- --- - --------- - -.CLPVH5S --- --- --- --- --- --- --- P-_ --- ___5-251 G-- --C --- --- --- --- --- --- --- --- --- -- --- --- A-C -A-

CDR II 60Pro Gly Asp Ser Olu Thr Ilei * Ser Pro Ser Phe ** Gly Gln Val

LPVH5 CCT GOT GAC TCT GAA ACC ATT TAG AGC COG TCC TTC TA0G0C CAG GTCLPVH5.6 --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --

LPVH5S - .-.5-251 --- --- --- --- T-- -GA --C-C-- -

70 80Ser Ile Ser Pro Asp Lys Ser Ile Asn Thr Ala Tyr Lou Oln Trp Ser

LPVH5 AGC ATT TCA CCC GAC AAG TCC ATC AAC ACC 0CC TAC CTG CAG TGO AGTLPVH5.6 - - - --- --- --- -.- --- ---

LPVH5S - --- --- --- --- --- --- --- -- ---

5-251 - C ---C -- --- --- --- --- .----- --- --C

90Ala Lou Lys Ala Ser Asp Thr Ala Met Tyr Cys Cys Ala Arg d1-nWr

LPVH5 G0C CTG AAG 00C TOG GAC ACC 0CC ATG TAT TGC TOT 0G0 AGA CAG TCTLPVH5.6 ---5-251 AG---- ------------ ------ -------A---

100 a CDR III i 102Arg Ala Ala Tyr Arg Val Gly Pro Pro Oly Gly Ser Thr Phe Thr Thr

LPVH5 C0C 0CCGCC TAT CGC OTT 00G CCA 0CC 00C G6A AGT ACT TTC ACT ACTLPVH5.6 --- -------- --- --- ---

JH4 .___ -+ ___

b

LPVH4LPVH4.3LPVH4SDP64

LPVH4LPVH4.3LPVH4SDP64

LPVH4LPVH4.3LPVH4SDP64

LPVH4LPVH4.3LPVH4SDP64

LPVH4LPVH4.3DP64

LPVH4LPVH4.3JH3b

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30 CDR I 40Ala Ser Ile Ser Ser Gly Gly Asp Ser Trp Ala Trp Ile Arg Gin Pro0CC TOC ATC AGC AGT GOT GOT GAC TOG TOG GCC TOG ATC C00 CAG OCA

-6- --- - T-- - --- A- -- A

49 50Pro Glu Lys Gly Lou Glu Trp Ile Oly Val Lou Lys Ile Tyr Arg SerCCA GAGAAGG GC CTG GAG TGO ATT G6G GTT CT MG ATC TAT COO AGT

--- 0 -- --- --- --- --- --- --- T-C- -AT ---

CDR II 60 70Pro Arg Thr Val Tyr Asn Pro Ser Leu Lys Ser Arg Val Thr Ile SerOCA AGO ACA GTC TAC AAC CCG TCT CTC AAG AGT COA GTC ACC ATA TCA---T-- ----------GI;G -< ---

GGGO--C --C TA---------C--

80Val Glu Thr Ala Lys Asn Gln Phe Ser Lou Ile Lou Asn Ser Val SerOTT GAG ACT GCC AAG AAC CAG TTC TCC CTG ATT lTG AAC TCT GTG AGC

--A --C -G T-- -AG C----- -C-

90 CDR IIIAla Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg Gly His Val Gly GluGCC 000 GAC ACG 00C GTG TAT TAC TGT 0C0 AGA 000 CAC GOT GOT GAA

DIR11

100 a b 103Asp Ala Phe Asp Met TrpGAT GCT TmT GAT ATG TGO

--- -- C---

FIG. 3. VH rearrangements derived from a case of LP HD, compared with the most homologous germline genes. The sequence format isthe same as in Fig. 2. Positions where two nucleotides were found in the sequence are indicated as 0, for A and C, and P, for G and A. Thesequences of the primers used for amplification of the mutated VH genes (see Results) are underlined in a and b (for the forward primer of theVHS rearrangement, 11 additional nucleotides at the 5' end were chosen from the VH5-251 germline sequence). (a) Nonfunctional VHSrearrangement. The sequence amplified from five single cells (LPVH5) is compared with the sequence of a sixth cell (LPVH5.6) and with thesequence obtained from purified DNA of a whole section (LPVH5S). The HRS genes are compared with a VHS germline gene (VH5-251) (29)and the JH4 gene (25). No DH gene could be identified in CDR III. Within the VH gene there is a deletion of codons 28 and 29. Close to thedeletion a tetranucleotide motif (5'-CCAG-3'/5'-CTGG-3') is present in the VH5-251 gene (see text). (b) In-frame VH4 rearrangement. Thesequence amplified from two single cells (LPVH4) is compared with the sequence ofa third single cell (LPVH4.3) and with the sequence obtainedfrom purified DNA of a whole section (LPVH4S). Between codons 49 and 50 there is an insertion of six nucleotides that is not present in anyof the published VH4 germline genes. The HRS genes are compared with the VH4 germline gene DP64 (23), the DH-like gene DIRlinvers (14),and the JH3b gene (25).

in the bone marrow has been identified in which such cells(i.e., cells carrying nonproductive VHDHJH joints on both Igheavy-chain loci) accumulate (33). The disease in the MC HDpatient may well have resulted from the malignant transfor-mation of a cell in this cellular compartment which is distinctfrom that of the classical, cytoplasmic-,u-chain-expressingpre-B cells.The target of malignant transformation was clearly differ-

ent in the patient with LP HD. Although no K light-chainrearrangements were detected, the HRS cells resemble ma-ture B cells in that they carry a productive heavy-chainrearrangement, and there is evidence for ongoing somaticmutation of the rearranged V-region genes within the tumor.The detection of somatic mutations and intraclonal diversityidentifies a B cell as the progenitor of the tumor in thispatient, and more specifically a germinal-center B cell, sincesomatic hypermutation is largely restricted to B cells prolif-erating in the germinal-center microenvironment (12, 34, 35).For LP HD a germinal-center B-cell origin of the HRS cellshas previously and independently been suggested on the basisof surface antigen expression and association with folliculardendritic and germinal-center-specific T helper cells (36, 37).Besides expression of B-cell antigens like CD19 and CD20(2), the detection of the B-cell-specific J chain (38), of theIg-associated mb-i protein (39), and of Ig light-chain mRNA(40) support a B-cell origin of the HRS cells in LP HD.LP HD thus appears related to follicular lymphoma, for

which a similar cellular composition and ongoing somaticmutation of V-region genes are main characteristics (41).

The HRS cell origin is less clear in the case of NS HD inwhich only a potentially functional heavy-chain joint wasidentified, in the absence of evidence for somatic mutation.Since we may have missed a corresponding light-chain generearrangement, it cannot be decided whether the transformedprogenitor cell was a pre-B or a B cell in this patient.Taken together, the gene rearrangement data indicate that

HD can result from malignant transformation of B-lineagecells at various stages ofdevelopment. More cases have to beanalyzed before it can be decided whether the developmentalstage of the transformed progenitor cell determines whichsubtype of HD arises in the patient and whether V generearrangements with unusual deletions or insertions-seen inthree ofthe five VH gene rearrangements described here-arecharacteristic for HRS cells.Concluding Remarks. The present results demonstrate the

clonal expansion of HRS cells in each of the three patientsanalyzed. The most straightforward interpretation of thisresult is that in each case the disease resulted from thetransformation of a single HRS cell. On the other hand,heterogeneity of HRS cells within a patient has been ob-served in the past at the level ofimmunophenotype or patternof gene expression (3, 42). By using specific Ig gene rear-rangements as markers, it should now be possible to decidewhether such heterogeneity reflects intra- or interclonalcellular diversity. Indeed, the main strength of the presentapproach for future work lies in the possibility to use HRScell-specific V-region gene rearrangements as markers in thesearch for members of the same clone in the various lym-phocyte compartments in the body. Perhaps this approach

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will allow us to trace HRS cells back to their progenitors andalso to follow disease progression in the patient.While the present results demonstrate that HRS cells can

be derived from the B-cell lineage, they do not exclude thepossibility of other lineage origins in other cases of HD. Thisis also indicated by a recent study of single HRS cells from13 patients, isolated from cell suspensions (43). However, thefailure to obtain VHDHJH PCR products from the HRS cellsin the latter analysis could at least in part be due to technicalmatters. A primer mix hybridizing near to the 5' end of the JHsegment was used which may fail to amplify VH rearrange-ments using short JH segments, and a consensus oligonucle-otide used as hybridization probe to detect VHDHJH PCRproducts harbors two or more mismatches to several VHgermline genes. In fact, two of the five VH rearrangementsdetected in our analysis may have been undetectable with theprobe used by Roth et al. (43). In addition, unequivocalidentification ofHRS cells is more difficult in suspension thanon tissue sections.

T-cell-specific surface antigens have been demonstrated onHRS cells in cases of NS, MC, and LD HD, in the absenceof B-cell markers (2). In addition, TCR gene rearrangementsare present in some HD-derived cell lines (5). These dataindicate that HRS cells may also originate from the T-celllineage. An analysis ofTCR gene rearrangements at the levelof single HRS cells is needed to further investigate thisproblem.

We thank Andrea Klockner for technical help and Ulf Klein andDiane Mathis for helpful discussions. This work was supported bythe Deutsche Forschungsgemeinschaft (Di184, Di1284/1-5, and SFB243), the Dr. Mildred Scheel Stiftung, the Deutsche Krebshilfe, theFazit Foundation, and the Land Nordrhein Westfalen.

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Griesser, H., Wagman, R., Braziel, R., Gascoyne, R. D.,Vicini, S., Cossman, J. & Mak, T. (1993) Blood 81, 3097-3115.

4. Weiss, L. M. & Chang, K. L. (1992) Semin. Diagn. Pathol. 9,272-278.

5. Diehl, V., von Kalle, C., Fonatsch, C., Tesch, H., Jucker, M.& Schaadt, M. (1990) Semin. Oncol. 17, 660-672.

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