, . 181: 387–393 (1997)

IgH AND TcR-ã GENE REARRANGEMENTSIDENTIFIED IN HODGKIN’S DISEASE BY PCR

DEMONSTRATE LACK OF CORRELATION BETWEENGENOTYPE, PHENOTYPE, AND EPSTEIN–BARR

VIRUS STATUS

*, , , , , .

Department of Pathology and CIGH/CNRS, CHU Purpan, Toulouse, France

SUMMARY

Analysis of IgH and TcR-ã genes using consensus primers identifying junctional regions of rearranged genes by polymerase chainreaction (PCR) was performed on tissues involved by Hodgkin’s disease (HD) in 90 cases and was correlated with the immunophenotypeof Hodgkin and Reed–Sternberg (HRS) cells and the presence of Epstein–Barr virus (EBV) within these cells. Clonal IgH generearrangements were found in 1/5 cases of lymphocyte predominance (LP) subtype and none was positive for EBV. In 85 cases of classicHD, no IgH or TcR-ã gene rearrangements were found in 51 (60 per cent) cases. A similar percentage, but not the same cases, were ofnull (non-B, non-T) phenotype. Of 30 cases where a B phenotype was assigned to HRS cells, nine had IgH gene rearrangements, threehad TcR-ã gene rearrangements, and two had both genes rearranged. None of the five cases assigned to T phenotype of HRS cellsshowed rearrangement of TcR-ã genes, but two cases showed rearranged IgH genes. Among 41 cases of null phenotype, ten had IgH generearrangements, five had TcR-ã gene rearrangements, and three cases had both genes rearranged. Whereas EBV was detectable in HRScells in 17/43 classic HD cases of assigned B phenotype, EBV was also detectable in 2/5 cases of assigned T phenotype and in 21 caseswith the null phenotype. Furthermore, there was no correlation of EBV with the presence or lack of IgH or TCR-ã gene rearrangements.Of the remainder, half (30 per cent) expressed antigens associated with lymphocytes without an appropriate genotype. The resultsconfirm lymphocyte-lineage committed cells at the origin of HRS cells in 40 per cent of cases. Any hypothesis of a non-lymphocyticorigin of HRS cells will require the inducibility of CD30 on candidate precursors and the methodology for probing genetic events in suchcells to be addressed. ? 1997 by John Wiley & Sons, Ltd

J. Pathol. 181: 387–393, 1997.No. of Figures: 6. No. of Tables: 0. No. of References: 46.

KEY WORDS—Hodgkin’s disease; polymerase chain reaction; IgH gene rearrangement; TcR-ã gene rearrangement; immunophenotype;Epstein–Barr virus (EBV)

INTRODUCTION

Hodgkin’s disease (HD) is a malignancy occurringprimarily in lymph nodes, characterized by the presenceof a few (<5 per cent) Hodgkin and Reed–Sternberg(HRS) cells. The non-neoplastic background consists oflymphocytes and other cells reactive to the cytokinesecretory properties of the neoplastic cells.1 With theexception of lymphocyte predominance Hodgkin’s dis-ease, recognizable as a distinct B-cell neoplasm,2 theorigin of HRS cells remains controversial despite exten-sive molecular biological studies using Southern blottingand detailed evaluation of phenotype.3,4 The lowdetection rate of Ig and TcR gene rearrangements bySouthern blotting has been attributed to either the

paucity of tumour cells below the detection threshold orthe actual absence of such gene rearrangements, leavingwidely divergent possibilities for the derivation ofHRS cells. With the use of the more sensitive andspecific polymerase chain reaction (PCR), rearranged Igheavy chain (IgH) genes have been detected recently in25–50 per cent of classic HD (non-lymphocyte pre-dominant subtypes),5–8 supporting a clonal lymphocyticorigin in a proportion of HD cases. While the currentinvestigation on the study of HD by PCR using consen-sus primers was in progress, two groups confirmed thepresence of Ig gene rearrangements in selected cases ofHD in single HRS cells isolated by micromanipulationfrom frozen sections.9,10 We have explored the relation-ship between rearrangements of IgH and TcR-ã chaingenes, the phenotype of diagnostic cells, and the EBVstatus in 90 cases of HD.

MATERIALS AND METHODS

Tissue sourcesNinety cases of Hodgkin’s disease (HD) of various

subtypes involving lymph nodes for which frozen tissue

*Correspondence to: Dr Talal Al Saati, MD, PhD, Laboratoired’Anatomie Pathologique, CHU Purpan, Place du Dr Baylac, 31059,Toulouse Cedex, France.

Contract grant sponsor: Association pour la Recherche Contre leCancer (ARC).

Contract grant sponsor: Délégation à la Recherche Clinique (PHRC).

Contract grant sponsor: French Ministry of Education.

CCC 0022–3417/97/040387–07 $17.50 Received 31 May 1996? 1997 by John Wiley & Sons, Ltd. Accepted 11 September 1996

was available in our tissue bank were studied by PCRmethods (described below). The phenotype and EBVstatus of many of these cases were included in ourprevious reports,11–14 but additional immunostainingwas performed with newer generation antibodies toascertain the phenotype. All cases were negative for HIVserology. Control specimens consisted of 30 reactivelymph nodes and tonsils in our tissue bank from caseswith negative HIV serology, and negative for auto-immune and neoplastic disease by clinical history. Thediagnosis of HD subtypes was based on previous andrenewed immunomorphological criteria.11,15 The distri-bution of cases was as follows: five cases of lymphocytepredominance (LP), 35 cases of nodular sclerosis (NS),and 50 cases of mixed cellularity (MC). Examples ofknown clonal B-cell line (LIB) (established in ourlaboratory) or T-cell lines (CEM and HSB-2) (ATCCCCL 119 and ATCC CCL 120) were used as positivecontrols in gene rearrangement studies. In addition,standard precautions were taken to guard against cross-contamination of the amplified DNA.16 In each exper-iment, polyclonal (reactive lymph node) and negative[sterile water (blank)] controls were systematicallyincluded.

DNA isolation and PCR analysis of HD samples

DNA extraction —Total cellular DNA was extractedfrom 10 frozen sections (10 µm thick) by proteinase Kdigestion, phenol–chloroform extraction, and ethanolprecipitation.17 An amplification of a fragment of thehousekeeping gene c-raf-1 (258 bp fragment)18,19 wasused as a positive control for successful amplification ofthe extracted DNA.

Primer design—Analysis of Ig heavy chain (IgH) generearrangements was performed by semi-nested PCRaccording to Trainor et al.20 and Ramasamy et al.21using FRIII/VH or FRII/VH specific primer amplifi-cations. The demonstration of TCR-ã gene rearrange-ments was performed by using two rounds of PCR witha set of seven primers,22 four of which were complemen-tary to sequences common to Vã-I (VãIcons primer:5*-CTG GTA CCT ACA CCA GGA GGG GAA-3*),Vã-II (Vã9.2 primer: 5*-GAA AGG AAT CTG GCATTC CG-3*), Vã-III (Vã10 primer: 5*-GCA GCA TGGGTA AGA CAA GC-3*), and Vã-IV (Vã11 primer:5*-GAT TGC TCA GGT GGG AAG AC-3*) genefamilies. The remaining three primers were complemen-tary to a common sequence in the Jã1 and Jã2 (Jã2S2primer: 5*-CCT GTG ACA ACA AGT GTT GT-3*), acommon sequence in the JP1 and JP2 (JP1/2 primer:5*-CCA GGT GAA GTT ACT ATG AG-3*), and onefor JP (JP primer: 5*-TTG TTC CGG GAC CAA ATACC-3*) gene segments, respectively. These primers wereused as two mixes: mix I containing VãI cons in con-junction with Jã2S2, JP, and JP1/2, and mix II containedVã9.2, Vã10, Vã11 in association with Jã2S2, JP, andJP1/2. For each sample, two PCR amplifications wereperformed using mix I and mix II, respectively. Thereamplification was done with the same mix as the firstcycle.

PCR conditions—For each initial amplification, 0·5 ìgof DNA template was subjected to 35 cycles of PCRamplification as described previously,23 using conditionsadapted for each set of primers. The cycles consistedof denaturation at 95)C and annealing at 55)C forc-raf-1 and TcR-ã, at 54)C for Fr3a/JH, and at 50)Cfor Fr2a/JH primers, followed by extension at 72)C. Thelength of each step of the cycle was 1 min, exceptfor Fr2a/JH primers for which the cycle was of 2 min ateach step. For reamplification of the PCR products,1 per cent of the first round was transferred as atemplate and subjected to 25–30 cycles of amplification.Twenty microlitres of each PCR product was appliedto a polyacrylamide (8 per cent for Fr3a/JH, 6 per centfor Fr2a/JH and TcR-ã PCR amplifications) gel electro-phoresis and visualized by staining with ethidiumbromide.

Sensitivity of PCRTo evaluate the sensitivity of our PCR, DNA from the

LIB cell line (positive for FR3a/JH and Fr2a/JH ampli-fications), CEM cell line (positive for Vã/Jã-mix I ampli-fication) or HSB-2 cell line (positive for Vã/Jã-mix IIamplification) was serially diluted in genomic humanDNA from normal lymphoid tissue (dilution from 100to 0·1 per cent). A clonally rearranged B-cell populationof §0·2 per cent of total cells was detected usingFr2a/JH (Fig. 1A) and Fr3a/JH primers. Beyond thisvalue, the smear from the polyclonal lymphoid cellDNA obscured the clonal band. Clonally rearrangedTcR-ã genes representing §1 per cent and §5 per centof the total cell population were detectable using mix II(Fig. 1B) and mix I, respectively.

ImmunohistochemistryThe diagnosis of the subtypes of HD was based on

immunomorphological criteria on paraffin sectionsusing CD15/ION1 (Immunotech, Marseilles, France),CD30/HSR4 (Immunotech), and Dako-EMA/E29(Dako, Copenhagen, Denmark) antibodies.11 Immuno-staining was performed by the streptavidin–biotin–peroxidase complex (ABC) method using the DakoStreptABComplex/HRP Duet (mouse/rabbit) kit(Dakopatts, code No. K492)24 or the APAAP method.25The B- or T-cell phenotype of HRS cells was analysed ineither cryostat, ModAMeX (cryostat equivalent),26 orroutine paraffin sections, as warranted by the reactivityof the antibodies directed against B-cell and T-cell-associated antigens. CD19/HD37, CD20/L26, andCD22/KB128 were purchased from Dako. CD79a/JCB117 was a kind gift of Dr D. Y. Mason, Oxford,U.K.27 CD3/Dako-CD3 was purchased from Dako;CD4/anti-Leu3a from Becton Dickinson, Mountain-view, CA, U.S.A.; and CD8/IOT8 from Immunotech.The number of sections was equal to the number ofantibodies. Immunohistochemistry on paraffin sectionswas often performed by microwave oven heating oftissue sections.28

Detection of Epstein–Barr virusThe methods for detecting EBV nucleic acids in

involved tissues using fluorescein isothiocyanate

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(FITC)-labelled EBER oligonucleotides and immuno-histochemistry with anti-LMP1 antibody (CS1–4)(Dako) on routine paraffin sections was performedaccording to our previous reports.12,14

RESULTSReactivity of HRS cells with anti-B and anti-TantibodiesImmunophenotyping was performed by the same

pathologist (GD) for all cases. In LPHD, the lympho-cytic and histiocytic (L&H) cells uniformly expressed allB-cell markers. On routine paraffin sections, consistentand uniform reactivity of HRS cells was observed forCD30 in all cases of classic HD. Rare cases werenegative for the CD15 antigen . Cases were assigned asB-cell or T-cell phenotype when clearly identifiablestaining of one or more atypical cells was obtainedby respective specific antibodies. Variable reaction withone or more anti-B antibodies (CD19, CD20, CD22,

and CD79a) was seen in 30/85 (35 per cent) cases.Heterogeneous expression of CD20 or CD79a on HRScells (greatly varying from cell to cell in a given case) wasobserved in 17/51 (33 per cent) and 13/73 (18 per cent),respectively. Of these cases, combined reactivity forCD20 and CD79a was found in 4/47 (9 per cent) cases,which was thought to be stronger evidence for possibleB-lineage. Immunostaining of HRS with the CD20/L26antibody was mainly membrane-associated (Fig. 2),whereas CD79a staining was mainly cytoplasmic, andusually weaker than that of small surroundingB-lymphocytes (Fig. 3). Cases were assigned asT-phenotype when CD3 was expressed by a few cellswith or without CD4 or CD8. Only 5/85 (6 per cent)could be assigned a T-phenotype. Focal but clearlyidentifiable expression of both B- and T-cell antigenswas found on a few cells in nine cases (11 per cent).In approximately 50 per cent (41/85 cases), the HRScells were clearly negative for all anti-B and anti-Tantibodies, irrespective of the fixation employed.

Fig. 1—Sensitivity of PCR analysis of IgH and TcR-ã gene rearrangements. A showsDNA from a B-lymphoma cell line (LIB) mixed in varying proportions (0·1–100 per cent)with DNA obtained from reactive lymph node tissue and subjected to FRII/JH amplifi-cation. B shows DNA from a T-cell line (HSB2) mixed in varying porportions (0·1–100per cent) with DNA obtained from reactive lymph node tissue and subjected to TcR-ã/mixII PCR amplification. The amplified DNA was separated by 6 per cent polyacrylamide gelelectrophoresis and visualized by staining with ethidium bromide. Ö’s are size markers;Bl (blank) is PCR amplification without DNA template to rule out contamination; and Pis the polyclonal control (reactive lymph node). Numbers at the top refer to thepercentages of clonal cell line DNA in the sample. A clearly visible rearranged band isseen for §0·2 per cent of the total clonal B-cell population in A. A TcR-ã clonallyrearranged cell population representing §1 per cent of the total DNA was detectable asshown in B

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Detection of rearranged IgH chain and TcR-ã genesin HD

The control cases of reactive lymphoid tissues showeda polyclonal pattern. In HD, rearranged IgH genes werefound in 1/5 cases of LPHD and all five were negativefor TcR-ã chain gene rearrangements. IgH genes wererearranged in 26/85 cases (31 per cent) of classic HD[7/35 (20 per cent) NSHD and 19/50 (38 per cent)MCHD]. Clonal rearrangements of IgH chain genesresulted in one or two predominant amplification prod-ucts within the expected range of size, i.e., 230–270 bpand 70–110 bp for FRII/JH (Fig. 4) and FRIII/JHprimers, respectively. TcR-ã chain gene rearrangementswere found in 13/85 (15 per cent) cases of classic HD[4/35 (11 per cent) NSHD and 9/50 (18 per cent)MCHD]. As expected, PCR products for TcR-ã chaingene rearrangements were approximately of 125 and230 bp for the mix II (Fig. 5) and mix I primer pairs,respectively. IgH genes were also rearranged in 5 of the13 cases displaying the TcR-ã rearranged genes. NoIgH or TcR-ã gene rearrangements were found in theremaining 51 (60 per cent) of the 85 cases of classic HD.

Correlation between phenotype and genotype

No clear correlation was found between the presenceof IgH or TcR-ã gene rearrangements and the immu-nophenotype of HRS cells. The lack of correlation isshown in Fig. 6, which also shows the EBV statussuperimposed on genotype and phenotype. The 21 caseswith only IgH gene rearrangement showed divergent

phenotypes (B-cell phenotype in nine cases; T-cellphenotype in two cases; null-cell phenotype in ten cases).The situation was similar in the eight cases with TcR-ãgene rearrangement only (B-cell phenotype in threecases; null-cell phenotype in five cases). The five caseswith combined IgH and TcR-ã gene rearrangementswere of B-cell phenotype in two cases and null-cellphenotype in three cases.

Correlation between the detection of EBV and thephenotype and genotype of HRS cells

EBV was not detected in any case of LPHD. EBVwas detected in HRS cells in 43/85 (51 per cent) casesof classic HD [6/35 (17 per cent) NSHD and 37/50(74 per cent) MCHD]. The 43 EBV-positive casesshowed widely divergent phenotypes and genotypes(Fig. 6). Thus, EBV was detected in 17 cases with B-cellphenotype, of which six had IgH genes rearranged, threehad TcR-ã genes rearranged, and one had both genesrearranged. EBV was present in seven cases withoutrearrangement of either IgH or TcR-ã genes. EBV wasdetectable in two cases of T-cell phenotype with nullgenotype. EBV was detectable in three cases of dualT/B phenotype with null genotype. EBV was alsodetected in 21 cases with null phenotype (IgH genesrearranged in seven; TcR-ã rearranged in four; IgH andTcR-ã rearranged in one). The remaining nine EBV-positive cases with a null phenotype had a null genotypeas well. There was no correlation of the subtype ofclassic HD with genotype, phenotype, or EBV status(not shown).

Fig. 2—Reactivity of the CD20/L26 antibody with Hodgkin andReed–Sternberg (HRS) cells in a case of mixed cellularity Hodgkin’sdisease on a paraffin section with heterogeneous staining of largeHRS cells. Positive small lymphocytes serve as an internal control.(Immunoperoxidase technique with nuclear counterstaining)

Fig. 3—Immunostaining of some HRS cells in a case of mixedcellularity Hodgkin’s disease with the CD79a/JCB117 monoclonalantibody on a paraffin section. Small B-cells are an internal control.(Immunoperoxidase technique with nuclear counterstaining)

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DISCUSSION

Genotypic analysis was performed on whole tissuesinvolved by HD using PCR with consensus primersidentifying junctional regions of rearranged genes of theIgH chain and TcR-ã receptor. The technique offersgreater sensitivity and specificity than previous methodsof standard Southern blotting.5,9 The specificity andsensitivity of nested or semi-nested PCR using aninternal oligonucleotide probe(s) are now established asequal to radiolabelled internal oligonucleotide probehybridization.29,30 PCR has widely and effectivelyreplaced Southern blot hybridization because of its

obvious advantages.29 Using this technique, we andothers have been capable of amplifying a single copy ofa DNA sequence, as is the case in single cell PCRmethodology.31 Both sensitivity and specificity wereconsolidated by using reative lymph nodes and B- andT-cell lines as controls. Approximately 30 per cent ofcases of classic HD showed IgH chain gene rearrange-ment, in overall agreement with reports using similartechniques but with a smaller number of cases.5–8 Com-plete rearrangement of the IgH gene signifies commit-ment to B-lymphocyte lineage.9 The study of TcR-ã generearrangements has practical implications, since TcR-ãgenes have a limited V gene repertoire, despite extensive

Fig. 4—Example of PCR analysis of IgH chain gene rearrangement using Fr2a and JH primers in 12 casesof HD (1–12). Ö’s are size markers; Bl (blank) is PCR amplification without a DNA template. C is a positivecontrol (B-cell lymphoma) and P is a polyclonal control (reactive lymph node). The HD samples 1, 2, 3, 6,and 7 show rearranged bands between 230 and 270 bp, indicating a clonal IgH-rearranged cell population.The other HD samples show polyclonal smears in the same zone

Fig. 5—Example of PCR analysis of TcR-ã gene rearrangement (using TcR-ã/mix IIPCR amplification) in nine cases of HD (1–9). C is a positive control (T-celllymphoma); Ö’s are size markers; Bl (blank) is PCR amplification without a DNAtemplate; P is a polyclonal control (reactive lymph node). The HD samples 1, 2, 6,7, 8, and 9 show the presence of a clonal TcR-ã-positive cell population, indicatedby the presence of one or two bands of approximately 125 bp. Other HD samplesshow polyclonal smears in the same zone

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junctional diversity, requiring the least number ofconsensus primers.23,32–34 In addition, TcR-ã generearrangement occurs at an early stage of T-lymphocytedevelopment and can be exploited as a marker ofclonality in almost all T-lymphoid neoplasms.35,36Rearranged TcR-ã genes were found in approximately15 per cent of cases of classic HD, which is much lowerthan by Southern blotting by Greisser et al.,37,38 butsimilar to results obtained by Herbst et al.39 Dual IgHand TcR-ã chain gene rearrangements were found in6 per cent of classic HD cases. The presence of IgHand/or TcR-ã gene rearrangements supports derivationfrom, and clonal presence of, cells committed to lym-phocyte lineage. The dual rearrangements in a fractionof these cases could suggest progression of a pathologi-cal state in circumstances analogous to some cases ofhigh-grade B-cell lymphomas.37,40Based on the assigned phenotype, a possible B-cell

origin of HRS cells can be deduced from the expressionof B-cell antigens. The present study corroborates CD20expression by HRS cells in 15–30 per cent of cases ofHD,41–43 as well as the 20 per cent range of the expres-sion of CD79a.8,43 Earlier studies have suggested thatclonal IgH gene rearrangements were more frequentlyfound in HD with HRS of B-cell phenotype5,7,8 but wefound no clear correlation between IgH gene rearrange-ment and the expression of B-cell-associated antigensand vice versa, a finding also reported by Manzanalet al.6 The discordance of genotype and phenotype (i.e.,no clonal gene rearrangement despite the expression ofB- and T-cell markers in 30 per cent of our classic HDcases) has led to proposals of activation events in an

immature lymphoid cell.39,44 The situation is also anal-ogous to the experiments of Gregory et al.45 on fetalbone marrow and liver cells, where a matureB-phenotype was induced on cells in germ-line config-uration by EBV. EBV is a potential candidate forinducing activation in precursor HRS cells as well asprogression of the disease.39,44,45 EBV was associatedwith half of our cases of classic HD without correlationwith genotype or phenotype. However, the absence ofEBV in approximately half the cases cannot support asingular role in either the aetiology or the pathogenesisof HD.3There are no apparent clues to the identity of HRS

cells in approximately 30 per cent of cases of HD, eitherby genotype or by phenotype (no clonal gene rearrange-ment and absence of B- and T-cell markers). It is in thisgroup of cases that other techniques, such as single cellPCR, will be required to resolve the issue of clonalityversus polyclonality and the question of involvement ofnon-lymphocytic cells.13,46 However, any hypothesis ofthe origin of HRS cells from cells other than thosecommitted to lymphocyte lineage will have to confronttwo main issues: the inducibility of CD30 expression incandidate non-lymphocytic cells and the methodologyfor the detection of genetic events in such cells.

ACKNOWLEDGEMENTS

We thank the staff of the Pathological AnatomyLaboratory and CIGH/CNRS, in particular Ms JeanineBoyes and Mr Michel March, for excellent technicalassistance. Dr S. M. Chittal is grateful for partialsupport from the French Ministry of Education. Thiswork was supported by grants from Association pour laRecherche Contre le Cancer ‘ARC’ and Délégation à laRecherche Clinique (PHRC).

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Fig. 6—A cytodiagram of findings in 85 cases of classic Hodgkin’sdisease showing lack of correlation between genotype, phenotype, andEpstein–Barr (EBV) status. The inner circle shows the genotype withpresence or absence of IgH and/or TcR-ã gene rearrangements. Thephenotype is in the middle circle reflecting percentages of the genotype.EBV status (51 per cent of total) is superimposed on the genotype andphenotype

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393GENOTYPE, PHENOTYPE, AND EBV IN HODGKIN’S DISEASE

? 1997 by John Wiley & Sons, Ltd. J. Pathol. 181: 387–393 (1997)