B-Cell Clonality Determination Using anImmunoglobulin � Light Chain Polymerase ChainReaction Method

Reetesh K. Pai,* Artemis E. Chakerian,*John M. Binder,† Mitual Amin,† andDavid S. Viswanatha*‡

From the Department of Pathology,* Experimental Pathology

Laboratory, University of New Mexico; Tricore Reference

Laboratories,‡ Albuquerque, New Mexico; and the Department of

Pathology,† William Beaumont Hospital, Troy, Michigan

To augment the detection of clonality in B-cell malig-nancies, we designed a consensus primer � lightchain gene (Ig�) polymerase chain reaction (PCR)assay in combination with a consensus primer immu-noglobulin heavy chain gene (IgH) PCR assay. Its ef-ficacy was then evaluated in a series of 86 paraffintissue samples comprising neoplastic and reactivelymphoproliferations. Analysis after PCR was accom-plished by 10% native polyacrylamide gel electro-phoresis after heteroduplex pretreatment of PCRproducts and by a post-PCR chip-based capillary elec-trophoresis analytic method. Overall , 49 of 68 (72%)of mature B-cell neoplasms yielded discrete Ig� gelbands within the predicted size range with no clono-typic Ig� products observed among reactive lymphoidor T-cell proliferations. The application of Ig� PCRimproved overall sensitivity from 81% with IgH PCRalone to 90% with combined Ig�/IgH PCR, with thiseffect being most notable in germinal center-relatedlymphomas. Sequencing of positive Ig� rearrange-ments revealed that most rearrangements involvedmembers of the V�1 (40%) and V�2 (34%) gene fam-ilies along with J�1 (26%), J�2 (23%), and J�4 (51%)gene segments. Involvement of V� pseudogenes wasidentified in 24% of cases with V�-KDE rearrange-ments. Our results demonstrate the efficacy of Ig�PCR in improving the detection rate of clonality inB-cell neoplasms and further introduce a novel post-PCR chip-based capillary electrophoresis analyticmethod for rapid PCR fragment size evaluation. (JMol Diagn 2005, 7:300–307)

Consensus primer polymerase chain reaction (PCR) am-plification of the immunoglobulin heavy chain (IgH) genecomplementary-determining region 3 (CDR3) is a routineancillary diagnostic technique for evaluating clonality inB-cell lymphoproliferative disorders.1–4 Although PCR

methods have the advantage of rapidity and the require-ment for minimal sample material (compared to genomicSouthern blot analysis), the potential false-negative rateis a considerable shortcoming. A significant proportion ofB-cell lymphomas do not demonstrate clonotypic IgHamplification mainly due to suboptimal primer binding,either from a lack of consensus target sequences ortarget site alteration as a result of somatic hypermutation.Alternative strategies for detecting IgH clonality usingconsensus primers directed against IgH framework re-gion 2 (FR2) or framework region 1 (FR1) have beendescribed,5–7 but these approaches are also subject topitfalls. For example, IgH PCR using FR2 consensusprimers may be subject to false-negative amplificationsdue to the lack of highly conserved sequences amongmany VH-FR2 regions, necessitating the use of highlydegenerate consensus primers. The use of multiple IgHFR1 family-specific consensus primers can achieve ahigh clonal detection rate, but results in larger sized PCRproducts that are often not well amplified from paraffin-embedded tissue samples.8,9 In light of these issues, theimmunoglobulin light chain genes can present attractivealternative targets for B-cell clonality determination.

During normal B-cell differentiation, IgH gene rear-rangements precede immunoglobulin � light chain (Ig�)gene rearrangements, which in turn occur before immu-noglobulin � light chain (Ig�) gene rearrangements.10 Fora particular allele, if the Ig� gene rearrangement pro-duces a nonfunctional V�-J� product, the locus may un-dergo segmental deletion via rearrangement with thedownstream �-deleting element (KDE).10–12 In fact, thevast majority of phenotypic �-expressing B cells and asubset of �-expressing B cells have rearrangements in-volving the KDE. Similar to V�-J� joinings, KDE-mediatedIg� gene rearrangements occur via recombination signalsequences located in either the J�-C� intron (ie, intronrecombination signal sequences) or immediately 3� to theV� gene segments, and these often exhibit junctionaldiversity with the addition of nontemplated (N) nucleo-tides. Both V�-J� and KDE rearrangements thus offeradditional targets for detection of B-cell clonality by PCR.

Accepted for publication November 30, 2004.

Address reprint requests to David S. Viswanatha, M.D., University ofNew Mexico Health Sciences Center, Department of Pathology, BiomedRes Facility 337C, 915 Camino de Salud, NE, Albuquerque, NM 87131.E-mail: dviswanatha@salud.unm.edu.

Journal of Molecular Diagnostics, Vol. 7, No. 2, May 2005

Copyright © American Society for Investigative Pathology

and the Association for Molecular Pathology

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To further augment the ability to detect clonal B-cellpopulations in formalin-fixed, paraffin-embedded diag-nostic tissue biopsies, we developed and applied a mul-tiplex PCR approach to detect V�-J� and V�-KDE rear-rangements in 68 cases of diverse types of mature B-cellneoplasms, as well as 18 other lymphoid proliferations.We demonstrate that a comprehensive Ig� PCR ap-proach to identify both V�-J� and V�-KDE gene rear-rangements significantly augments consensus primerIgH PCR for the detection of B-cell clonality and is suit-able for paraffin-embedded tissue sources. This studyfurther provides preliminary data regarding the utility of apost-PCR chip-based capillary electrophoresis (CBCE)analytic method that is capable of superior resolution foramplicon detection and sizing compared to standard gelanalysis.

Materials and Methods

Patients and Samples

Paraffin blocks from 68 cases of B-cell neoplasms (25diffuse large B-cell lymphomas, 13 follicular lymphomas,10 small lymphocytic lymphomas, 9 mantle cell lympho-mas, 8 marginal zone lymphomas, and 3 multiple myelo-mas), along with 7 Hodgkin lymphomas, 2 peripheralT-cell lymphomas, and 9 reactive lymphoid proliferationswere retrieved from the Pathology Departments of theUniversity of New Mexico and William Beaumont Hospi-tals. All of the samples were clinical diagnostic casesobtained between 2001 to 2003 and were classified us-ing conventional histopathological and clinical criteria inaccordance with the World Health Organization classifi-cation of hematopoietic neoplasms.13 The histologicaldiagnosis was reviewed in each case by two of the in-vestigators (R.P., D.V.), along with details of availableflow cytometry data. This study was approved by theUniversity of New Mexico Human Research ReviewCommittee.

DNA Preparation and Polymerase ChainReaction Analysis

Genomic DNA from all cases was extracted from forma-lin-fixed, paraffin-embedded tissue sections, accordingto the manufacturer’s directions (DNEasy kit; Qiagen,Santa Clarita, CA) and quantitated by UV absorbancespectrophotometry. Three consensus family-specificV�FR3 region primers (designed to target the V�1through V�6 gene families of Ig�), one consensus J�primer, and one KDE primer were used based on slightmodifications of reported V�-FR3, J�, and KDE sequenc-es.14–16 Less degeneracy was used in the family-specificV�-FR3 and J� primers at the 3� end compared to previ-ous studies.14–16 The three forward V� primers were des-ignated as follows: V�I/VI targeting the V�I and V�VI genefamilies; V�III/IV targeting the V�III and V�IV gene fami-lies; and V�II targeting the V�II gene family. One consen-sus J� reverse primer was designed to target J�I, J�II,J�III, and J�IV genes, and one reverse primer was de-

signed to target the KDE locus. Nucleotide sequences forV�, J�, and KDE primers, as well as the schematics of theIg� PCR strategy, are depicted in Figure 1.

The Ig� PCR approach consisted of three different reac-tion tubes per sample (Figure 1). In tube 1, two V� primers(V�I/VI at 10 pmol/reaction and V�II at 20 pmol/reaction)were combined with the J� primer (at 10 pmol/reaction). Intube 2, one V� primer (V�III/IV at 30 pmol/reaction) wascombined with the J� primer (at 20 pmol/reaction). In tube3, two V� primers (V�I/VI at 10 pmol/reaction and V�III/IV at30 pmol/reaction) were combined with the KDE primer (at10 pmol/reaction). The primer combinations were arrivedat based on preliminary experiments to optimize amplifi-cation efficiency and minimize primer interference andnonspecific product generation.

For the Ig� PCR tubes, 200 ng of genomic DNA weresubjected to amplification in a reaction mixture contain-ing 1� GeneAmp PCR buffer (Applied Biosystems, Fos-ter City, CA), 1.5 mmol/L MgCl2, 200 �mol/L of eachdNTP, 2.5 U of Taq polymerase (Applied Biosystems),and the V�, J�, and KDE primer combinations as desig-nated above, and shown in Figure 1. A touchdown PCRwas used involving a denaturing period of 95°C for 5minutes followed by eight cycles of 95°C for 30 seconds,1 minute at 64°C, then decreasing the annealing temper-ature by 0.5°C each subsequent cycle, and 1 minuteextension at 72°C. This was followed by 32 cycles of 95°Cfor 30 seconds, 60°C for 1 minute, and 72°C for 1 minute,with a final primer extension at 72°C for 5 minutes.

Immunoglobulin heavy chain (IgH) gene rearrange-ments were amplified by PCR, using consensus oligonu-cleotide primers for VH-FR3 and JH regions, as previ-ously described.17 For IgH PCR, 400 ng of genomic DNAwere subjected to amplification in reaction mixtures con-

Figure 1. Schematic diagram of PCR strategy to detect V�-J� and V�-KDErearrangements of the Ig� gene. Primer set combinations for each of the threePCR tubes are as indicated: tubes 1 and 2 identify V�-J� gene rearrange-ments and tube 3 amplifies V�-KDE gene rearrangements. Nucleotide basesin parentheses indicate degenerate sites in the primers. Use of a touchdownPCR method promotes amplification of dominant rearrangement(s) despitesome consensus primer degeneracy or minor mismatching.

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taining 1� GeneAmp PCR buffer (Applied Biosystems),1.5 mmol/L MgCl2, 200 �mol/L of each dNTP, 10 pmol ofeach primer, and 2.5 U of Taq polymerase (Applied Bio-systems). PCR amplification was performed by denatur-ation at 95°C for 5 minutes followed by 40 cycles of 95°Cfor 30 seconds, 1 minute at 58°C, and 1 minute at 72°C.This was followed by a final primer extension at 72°C for5 minutes. In all PCR experiments, genomic DNA fromknown monoclonal neoplastic B cells and reactive tonsiltissue were included as positive and negative controls,respectively. In addition, no DNA template (blank) con-trols were included with each sample run to excludepotential contamination. Amplification of a 165-bp seg-ment of the �-globin gene (forward primer, 5�ACA-CAACTGTGTTCACTAGC3�; and reverse primer, 5�TG-GTCTCCTTAAACCTGTCTTG3�) was performed in everysample as an internal control for DNA integrity. An addi-tional larger fragment size �-globin gene amplification(325 bp) was subsequently performed (same forwardprimer with reverse primer 5�ATCAGGAGT GGACA-GATCC3�) in a subset of cases found to be negative forV�-KDE amplification, to verify DNA integrity in this largeramplicon range. For Ig� PCR assay sensitivity determi-nation, serial 10-fold dilutions of DNA from a patient sam-ple positive for an Ig� V�-J� clonal rearrangement weremade into tonsil DNA (polyclonal B-cell background).Each dilution was kept at 200 ng of total DNA. PCRamplification was performed as described for standardIg� PCR.

Gel Electrophoresis of PCR Products

All Ig� PCR products were subjected to a heteroduplex(HDX) procedure, which involved heating the reaction for5 minutes at 94°C followed by immediate immersion onice for 60 minutes.18 Ten �l of the Ig� PCR products werethen analyzed by 10% nondenaturing polyacrylamide gelelectrophoresis (PAGE) in 1� TBE buffer for 40 minutes(Bio-Rad Mini-Protean II system; Bio-Rad, Hercules CA)at room temperature, stained with ethidium bromide, andvisualized under UV light. The V�-J� PCR is predicted toamplify rearranged products of �130 to 150 bp, whereasthe V�-KDE PCR was expected to amplify products of�250 to 300 bp. IgH PCR products (predicted size range�70 to 140 bp) were analyzed by 10% PAGE, but withoutHDX pretreatment. Samples with an unequivocal domi-nant band in the expected size ranges were interpretedas monoclonal, and those with a minimal smear pattern orabsence of products were interpreted as polyclonal or neg-ative, respectively. �-Globin controls were assessed con-currently with Ig� and IgH PCR product gel electrophoresis.

Sequencing Analysis of Ig� PCR Products

To confirm the specificity of amplified Ig� gene rear-rangements and determine patterns of gene segmentusage, the clonal bands from all positive Ig� PCR caseswere sequenced. PCR products were gel purified from4% agarose gels using the Qiagen gel extraction kit,(Qiagen). The extracted PCR products were directly se-

quenced in one or both directions with V�, J�, or KDEprimers, using the dye terminator method on an ABI 3100capillary sequencer (Applied Biosystems). The derivedsequences were compared to known germline DNA se-quences of the Ig�V- and J-regions using the V-BASE(CPE Cambridge, UK; http://www.mrc-cpe.cam.ac.uk/vbase-ok) and BLAST (NCBI/GenBank; http://www.ncbi.nlm.nih.gov/blast) computer programs. Individual positivecases, particularly with similar V� region use, were furthercompared to each other by pair-wise BLAST nucleotidealignment, to ensure unique sequence identity. From thenucleotide sequence information, data regarding V� andJ� gene segment usage in each Ig�-positive case wereobtained. These results were compared to establishedgermline frequencies for V� and J� gene segments using�2 statistical analysis.

Product Size Determination by CBCE

Analysis after PCR was also performed by a CBCE ana-lytic method in 29 cases comprising a subset of V�-J� (21cases) and V�-KDE (8 cases) clonal-positive cases, aswell as several PCR-negative cases. For CBCE, the pre-viously heteroduplexed PCR products were analyzed ona model 2100 bioanalyzer using DNA 500 chips (AgilentTechnologies, Palo Alto, CA). The 16-well chips wereprepared for analysis according to the manufacturer’sdirections and using 1 �l of PCR product per sample. Arun time of �40 minutes was required for one completechip (12 samples total), with real-time software renderingand display of the data. A clonal population was definedin this study as a single peak of greater than 15 relativefluorescence units on the y axis (for a 1-�l PCR productamount), falling in the expected product size range forIg� amplicons. A low-intensity bell-shaped pattern or lackof products indicated polyclonal or negative results, re-spectively. The CBCE criteria were derived from our pre-liminary experience with this platform.19 The fragmentsize and relative intensity of each Ig� amplicon was com-pared with HDX-PAGE results.

Results

Evaluation of Patient Samples with Ig� and IgHPCR

The Ig� PCR approach consisted of three different reac-tion tubes (Figure 1) to detect both V�-J� and V�-KDErearrangements. Using this PCR strategy and post-PCRHDX-PAGE analysis, Ig� clonal products could be de-tected in 72% of mature B-cell non-Hodgkin lymphomas(Table 1). In each positive (clonal) case, a discrete bandwas identified by HDX-PAGE within the expected sizerange of 130 to 150 bp for V�-J� rearrangements and 250to 300 bp for V�-KDE rearrangements (Figure 2). Largersized, nonspecific HDX bands were also noted in theV�-J� PCR.

For Ig� PCR, the highest detection rates were found indiffuse large B-cell lymphomas (76%), small lymphocyticlymphomas (80%), and marginal zone lymphomas (88%).

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The lowest detection rates were obtained in follicularlymphomas (62%), mantle cell lymphomas (67%), andmyelomas (33%) (Table 1). The addition of Ig� PCR sig-nificantly increased the overall detection of clonality inmature B-cell tumors from 81% (IgH PCR alone) to 90%(combined Ig�/IgH PCR). This effect was most notable inthe germinal center-associated lymphomas. For diffuselarge B-cell lymphomas, the additional analysis of Ig�rearrangements improved the overall detection rate from84% with IgH PCR alone to 100% with combined IgH/Ig�PCR (Tables 1 and 2). For follicular lymphomas, the ad-ditional analysis of Ig� rearrangements improved theoverall detection of clonality from 46% with IgH PCRalone to 62% with combined IgH/Ig� PCR (Tables 1 and2). For small lymphocytic lymphoma/chronic lymphocyticleukemia, marginal zone lymphoma, and mantle cell lym-phoma, additional analysis by Ig� PCR did not signifi-cantly impact the overall detection rate of clonality. Ig�PCR also detected clonal rearrangements in two of sevencases of classical Hodgkin lymphoma, with one of thesecases also demonstrating monoclonality by IgH PCR (Ta-ble 1); these cases were notable for syncytial histologicalfeatures. No clonal Ig� products were observed in any ofthe 11 reactive lymphoid proliferations or T-cell lympho-mas. The dilutional sensitivity of Ig� PCR was found to be10%, using DNA from a V�-J� clonal lymphoma casediluted into a polyclonal B-cell background (Figure 3).

Examination of the positive Ig� cases revealed thatV�-J� rearrangements generally predominated over V�-KDE joinings, with the exception of an equal V�-KDEfrequency in follicular lymphomas, and only V�-KDE usein the positive Hodgkin lymphoma and myeloma samples(Table 1). The surface light chain expression of 36 matureB-cell lymphomas was known by prior flow cytometricanalysis. Of the 23 �-expressing B-cell lymphomas, 10cases (44%) had only a V�-J� rearrangement, 4 cases

Figure 2. HDX-PAGE detection of Ig� gene rearrangement with V�-J� andV�-KDE PCR. A: Representative set of various B-cell lymphoma cases withpositive (monoclonal) V�-J� Ig� gene rearrangements (lanes 1 to 7). Whitearrow indicates expected size region for positive bands (�130 to 150 bp).Note slight variation in PCR product sizes for each sample. Some highermolecular size, nonspecific amplicons are present in most cases. Lanes 8 and9 represent reactive (polyclonal) B-cell and water-only (no template) con-trols, respectively. L indicates the 100-bp size ladder. B: Illustration of V�-J�and V�-KDE PCR in combination with IgH PCR for a representative set ofcases as follows: diffuse large B-cell lymphoma (lane 1, IgH; lane 2, V�-J�;lane 3, V�-KDE), follicular lymphoma (lane 4, IgH; lane 5, V�-J�; lane 6,V�-KDE), extranodal marginal zone lymphoma (lane 7, IgH; lane 8, V�-J�;lane 9, V�-KDE), and Hodgkin lymphoma, syncytial nodular sclerosis type(lane 10, IgH; lane 11, V�-J�; lane 12, V�-KDE). Expected size ranges forPCR products: IgH, �70 to 140 bp; V�-J� Ig�, �130 to 150 bp; V�-KDE Ig�,�250 to 300 bp. L indicates the 100-bp size ladder. For all samples, DNAintegrity was confirmed by amplification of a segment of the �-globin gene(not shown).

Table 1. Frequencies of IgH and Ig� Rearrangements in B-Cell Neoplasms

Lymphoma type IgH PCR V�-J� PCR V�-KDE PCR Ig� PCR* overall IgH/Ig� overall

Diffuse large B-cell lymphoma 21/25 (84%) 15/25 (60%) 6/25 (24%) 19/25 (76%) 25/25 (100%)Follicular lymphoma 6/13 (46%) 4/13 (31%) 4/13 (31%) 8/13 (62%) 8/13 (62%)Small lymphocytic lymphoma 10/10 (100%) 7/10 (70%) 1/10 (10%) 8/10 (80%) 10/10 (100%)Mantle cell lymphoma 9/9 (100%) 6/9 (67%) 2/9 (22%) 6/9 (67%) 9/9 (100%)Marginal zone lymphoma 7/8 (88%) 6/8 (75%) 3/8 (38%) 7/8 (88%) 7/8 (88%)Multiple myeloma 2/3 (67%) 0/3 (0%) 1/3 (33%) 1/3 (33%) 2/3 (67%)Hodgkin lymphoma 1/7 (14%) 0/7 (0%) 2/7 (29%) 2/7 (29%) 2/7 (29%)†

Reactive hyperplasias and T-cellneoplasms

0/11 (0%) 0/11 (0%) 0/11 (0%) 0/11 (0%) 0/11 (0%)

Overall B-cell non-Hodgkinlymphoma cases

55/68 (81%) 38/68 (56%) 17/68 (25%) 49/68 (72%) 61/68 (90%)

*A case is defined as being positive for Ig� PCR overall if either a V�-J� or V�-KDE rearrangement was identified.†Positive Hodgkin lymphoma cases with syncytial histology.

Table 2. Comparison of IgH and Ig� PCR for Diffuse LargeB-Cell Lymphoma and Follicular Lymphoma

IgH/Ig� DLBCL (n � 25) FL (n � 13)

IgH�/Ig�� 15 6IgH�/Ig�� 4 2IgH�/Ig�� 6 0IgH�/Ig�� 0 5

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(17%) had a V�-KDE rearrangement only, 1 case (4%)had both V�-J� and V�-KDE rearrangements, and 8cases (35%) had no rearrangements detected by Ig�PCR. Of the 13 �-expressing B-cell lymphomas, 5 cases(39%) had only a V�-J� rearrangement, 3 cases (23%)had a V�-KDE rearrangement only, 3 cases (23%) hadboth V�-J� and V�-KDE rearrangements, and 2 cases(15%) had no rearrangements detected by Ig� PCR.These findings are summarized in Table 3. Of note, DNAsamples from Vk-KDE-negative cases were successfullyreamplified with primers encompassing a larger segmentof the �-globin gene (325 bp), to ensure that insufficientDNA integrity was not the cause for the negative PCRresults (data not shown).

Sequence Data of Ig� PCR for Determination ofVk and Jk Gene Usage

A total of 56 Ig� PCR clonotypic alleles were sequenced.V� and J� gene use was assigned by comparison withknown germline sequences using the V-BASE andBLAST computer programs. The prevalence of V� genefamilies in V�-J� gene rearrangements generally re-flected the number of available germline V� gene seg-ments within each family (Figure 4). Most V�-J� rear-

rangements involved members of the V�1 (40%) and V�2(34%) gene families. There was a significant relative over-representation of V�4 gene family usage (14%) whencompared to the number of available germline V�4 genesusing �2 statistical analysis (P � 0.015, �2 � 5.92). Re-arrangements involving V� pseudogenes were not iden-tified in any of the V�-J� rearrangements. For V�-KDErearrangements, members of the V�1 (35%) and V�2(29%) gene families were most often used (Figure 4).Rearrangements involving V� pseudogenes were identi-fied in 4 of 17 (24%) of the V�-KDE-positive cases andprimarily involved V�2 and V�7 pseudogenes. Concern-ing the J� region, J�1 (26%), J�2 (23%), and J�4 (51%)gene segments were used most frequently (Figure 5).However, no involvement of the J�3 segment wasobserved.

CBCE Evaluation of Ig� PCR Products

CBCE provided highly accurate PCR amplicon sizing in29 clonal Ig�-positive samples tested, with representativeresults shown in Figure 6, A to C. By CBCE, the sizerange for V�-J� rearrangements was between 120 to 145bp. An additional nonspecific peak was sometimes iden-

Figure 3. Dilutional sensitivity of the Ig� PCR assay. Genomic DNA from aB-cell lymphoma with a known monoclonal Ig� rearrangement was seriallydiluted into DNA from reactive tonsil tissue and analyzed by multiplex V�-J�PCR and HDX-PAGE. Dilutions are as indicated: lanes 1 to 5: 200 ng, 20 ng,2 ng, 0.2 ng, and 0.02 ng of target DNA, respectively; lane 6: tonsil DNAalone; lane 7: water-only (no template) control. L denotes a 100-bp ladder.A clonal PCR amplicon could be detected in the dilution containing 20 ng ofsample DNA, representing detection of a 10% dilution (indicated by whitearrow).

Table 3. Ig� Rearrangement Frequencies by Light-ChainExpression

Ig� rearrangement type � (n � 23) � (n � 13)

V�-J� only 10 5V�-KDE only 4 3Both V�-J� and V�-KDE 1 3None 8 2

Figure 4. Observed relative frequencies of V� gene segments in clonal V�-J�and V�-KDE rearrangements. The relative frequencies of V�-J� rearrange-ments (black bars) and V�-KDE rearrangements (gray bars) were derivedfrom sequence analysis of V�-J� and Vk-KDE PCR amplicons. Note that V�pseudogene use was not encountered with V�-J� rearrangements, but wasobserved in occasional V�-KDE rearrangements.

Figure 5. Observed relative frequencies of J� gene segments in clonal V�-J�rearrangements. The relative frequencies were derived from sequence anal-ysis of V�-J� PCR amplicons. J�3 segments were not detected among thesecases.

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tified with V�-J� PCR at �210 bp. By CBCE, the sizerange for V�-KDE rearrangements was between 260 to290 bp. PCR-negative samples showed either a minimalpolyclonal pattern in the expected size ranges, or nodeviation from baseline. The data from CBCE agreed wellwith HDX-PAGE results. CBCE was also more rapid andprovided highly resolved histogram data for clonal re-sults. Dilutional sensitivity of CBCE using the same clonalV�-J� lymphoma sample as in Figure 3 was comparableto HDX-PAGE at 10% detection in a polyclonal B-cellbackground (Figure 6; D to F).

Discussion

The recent publication of the European BIOMED-2 con-sortium underscores the value of using comprehensivePCR primers and multiple antigen receptor gene targetsto establish the presence of a clonal lymphoid prolifera-tion.20 Ig� gene rearrangements have been previouslystudied as additional markers of B-cell clonality.14–16,21

Although some investigators have thoroughly character-ized the specific nature of V�-J� and V�-KDE rearrange-

ments for use in minimal residual disease detection inB-lineage acute leukemias,14,21 relatively few efforts haveevaluated the potential of using Ig� PCR to augment IgHPCR for B-cell clonality determination in the routine mo-lecular hematopathology laboratory setting.15,16 Ourstudy is complementary to work from other groups andenhances previous data by investigating a broad rangeof mature B-cell neoplasms, using a simplified Ig� PCRstrategy.

This study is unique in also evaluating the frequencyand nature of the KDE (specifically V�-KDE) locus generearrangements in a series of commonly encounteredmature B-cell malignancies. The addition of V�-KDE anal-ysis is particularly helpful in PCR clonality detection ofboth �- and �-expressing B-cell lymphomas. In our groupof 68 well-characterized mature B-cell neoplasms, appli-cation of Ig� PCR alone resulted in the demonstration ofclonality in 72% of cases. Significantly, by combining theIg� and IgH PCR methods, the detection rate for clonalityin B-cell lymphomas increased from 81 to 90%, with thiseffect being most evident among follicular lymphomasand diffuse large B-cell lymphomas. We attribute therelatively high overall rate of IgH clonality observed in thisseries to selection bias, as well as the inclusion of arelatively pronounced number of small B-lymphocyteneoplasms, which are typically positive in IgH PCR anal-ysis using FR3-JH consensus primers. Both V�-J� andV�-KDE rearrangements were observed in Ig� PCR-pos-itive cases, indicating the utility of the latter locus forclonality determination. Furthermore, comparison ofV�-J� and V�-KDE by surface light chain immunoglobulintype demonstrated � locus clonal rearrangements in 85%of �-expressing B-cell lymphomas, emphasizing thebroad applicability of this approach. Of note, V�-KDEclonal bands were also detected in two of seven cases ofHodgkin lymphoma, one of which also harbored a clonalIgH rearrangement. Pathological review of these twocases revealed syncytial histology, suggesting that sam-pling of these tissue areas may have enriched for detec-tion of neoplastic B-lineage Reed-Sternberg cells.Among 18 cases of reactive B-cell proliferations andT-cell lymphomas, none yielded discrete bands with theIg� PCR. The dilutional sensitivity for this Ig� PCR assaywas found to be 10%; although better sensitivity is gen-erally expected with PCR-based methods, this finding isin keeping with the experience of other investigators,20

particularly when using a polyclonal B-cell backgrounddiluent. Nonetheless, this level of sensitivity should beconsidered when analyzing tissues only partially involvedby a B-cell lymphoma using Ig� PCR.

Despite substantially improving the overall clonal de-tection in B-cell neoplasms (when combined with IgHPCR), our Ig� PCR assay is nevertheless associated witha false-negative rate. A number of factors account for thisissue and the extensive work of the BIOMED-2 ConcertedAction group is also instructive in this regard.20 Firstly,the primer strategy used in our approach was relativelylimited compared to the BIOMED-2 protocol for Ig�PCR.20 However, we opted to use a simplified set ofprimers targeting only V� FR3 regions and fewer totalprimers, to reduce the complexity inherent in multiprimer

Figure 6. Analysis after PCR by CBCE. One �l of heteroduplexed PCRproducts was analyzed on a 2100 Bioanalyzer (Agilent Technologies) withreal-time generation of electropherogram data. Peaks labeled with an aster-isk at far left and right ends of the electropherogram images represent15-bp and 600-bp size standards, respectively. The x axis parameter is samplerun time in seconds, however the software detects and calculates samplepeaks in bp size relative to the standards. The y axis represents relativefluorescence units (RFU). A clonal population was defined as a single peakof greater than 15 RFU. The electropherograms are as shown: A: diffuse largeB-cell lymphoma positive for a V�-J� clonal rearrangement with a PCRamplicon size of 139 bp; B: follicular lymphoma positive for a V�-KDE clonalrearrangement with a PCR amplicon size of 267 bp; C: polyclonal (negative)PCR result; D through F: dilution sensitivity assay with PCR products fromsame Ig�-positive lymphoma sample as in Figure 3 (D: 200 ng tumor DNA;E: 20 ng tumor DNA; F: 2 ng of tumor DNA). Note that CBCE produces highlyaccurate fragment sizing for the positive cases, however, the dilutionalsensitivity of this platform with a consensus Ig� PCR primer method isessentially similar to PAGE results (ie, 20 ng of target template or 10%; Figure3). Peaks immediately to the right of the 15-bp size standard in electro-pherograms represent primer dimers.

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PCR techniques. Second, the single consensus J� primersequence used in our assays was slightly more degen-erate, or mismatched against the J�3 gene segment,which may account for the absence of J�3 among theV�-J� gene rearrangements in Ig�-positive cases. Thiseffect, however, should have been mitigated in part bythe use of the touch down PCR method as described.Similarly, the consensus J� primer was not homologouswith the J�5 gene segment, although this segment isinfrequently used overall in V�-J� joinings. Third, we didnot use an intron recombination signal sequence regionprimer to detect a subset of intron recombination signalsequence-KDE � rearrangements known to occur at leastin chronic B-cell leukemias.21 Lastly, the Ig� loci may begermline in some �-expressing mature B cells due to rarereversals of the usual, orderly gene rearrangement hier-archy, such that successful � light chain gene rearrange-ments precede � light chain rearrangements.10,22 As atechnical footnote, the upper limit of the PCR product sizerange for V�-KDE amplicons approaches 300 bp, whichmay prove problematic when amplifying this target insamples with excessive DNA degradation, although thissituation was not encountered in our study. In short, ourIg� PCR primer strategy sought to minimize complexity infavor of robust and unequivocal amplification results,while accepting a modest compromise in overall detec-tion rate. As is evident from the data in this series of B-cellneoplasms, combined Ig� and IgH PCR substantiallyimproves the overall clonal detection rate, particularly forgerminal center-related subcategories of lymphoma. Inthe cases of follicular lymphoma, the combined overallresults, although improved, indicate the need for yet ad-ditional evaluation of BCL2/IgH rearrangements to furtheraugment the clonality detection rate.

We assessed 56 cases by direct sequencing to deter-mine the distribution of V� and J� gene families in V�-J�and V�-KDE rearrangements and found the frequency ofgene segment use generally reflected their representa-tions in the genome. Most Ig� rearrangements involvedmembers of the V�1 and V�2 gene families, which ac-count for 44% and 39% of functional germline V� genes,respectively.23–25 In our analysis, there was a statisticallysignificant relative overrepresentation of V�4 gene familyusage (14%), which only accounts for �2% of germlineV� genes. In keeping with the previous observations ofothers, V� pseudogenes were not involved in any of theV�-J� rearrangements.23 The distribution of V� gene us-age demonstrated in this study is also similar to otherpublished series involving both normal and malignant Bcells, with the exception of a comparatively greater de-gree of V�2 gene prevalence seen in our series.23,26–28

The V�2 gene family has been previously shown to beused more frequently than other V� gene segments innonproductive V�-J� rearrangements.27 The relativeprominence of V�2 gene usage seen in our series maythus in part be the result of nonproductive V�-J� rear-rangements that favored the more frequent use of the V�2region. Among these mature B-cell neoplasms, the dis-tribution of J� genes in V�-J� gene rearrangements wassimilar to other published series involving both normaland malignant B cells,22,23,26,27 with the J�1, J�2, and

J�4 gene segments used most frequently. The J�3 seg-ment was not identified in any of our cases; however, atleast in normal B cells, this J� gene is only present in�5% of V�-J� gene rearrangements.27 Nevertheless, wecannot be completely certain that the inability to detectJ�3 segment rearrangements in our cases represents astatistical artifact of sample size, or inefficient PCR de-tection due to compromised primer-template homology.Notably, no significant association was identified be-tween subtypes of non-Hodgkin B-cell lymphoma andpreferential use of � gene families, although a largerstudy would likely be needed to adequately address thisissue.

The distribution of V� genes in V�-KDE gene rear-rangements in mature B-cell neoplasms was also approx-imately representative of their presence in the genome.Of interest, we documented occasional rearrangementsinvolving V� pseudogene segments (primarily V�2 andV�7 families) in the V�-KDE-positive cases. As noted, theuse of pseudogenes does not appear to occur in V�-J�rearrangements in either �- or �-positive B cells, but mayoccur in V�-KDE joinings, perhaps as a mechanism toexclude unwanted or nonfunctional rearrangements.23,27

Regardless, the presence of such rearrangements re-mains a valuable clonotypic marker that can be detectedwith the PCR primers described in this study.

Finally, although PAGE was used in our cases to eval-uate heteroduplexed PCR products, we compared stan-dard PAGE analysis to a CBCE platform, to determine theutility of the latter method in detecting V�-J� and V�-KDErearrangements. CBCE analysis resulted in superior res-olution for amplicon detection and permitted more accu-rate and rapid molecular fragment sizing than PAGE, withcomparable dilutional sensitivity. In conclusion, this studydemonstrates that the combined approach of IgH andIg� gene rearrangement analysis can significantly in-crease the detection rate of B-cell clonality in matureB-cell lymphomas. Multiplex Ig� PCR followed by HDX-PAGE or CBCE is thus a useful ancillary diagnostic toolfor detection of B-cell clonality in formalin-fixed paraffin-embedded tissues.

Acknowledgments

We thank Terry Mulcahy in the Center for Sequencing atthe University of New Mexico for his assistance in se-quencing of PCR products, Michael Grady for excellenttechnical assistance in preparation of the manuscriptimages, and Dr. Sarah Lathrop for help with statisticalanalysis.

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