Automated High Resolution PCR Fragment Analysis for Identification

Automated high resolution PCR fragment analysis for identiﬁcation of clonally rearranged immunoglobulin heavy chain genes B Linke1, I Bolz1, A Fayyazi2, M von Hofen1, Ch Pott1, J Bertram, W Hiddemann1 and M Kneba1

1Department of Internal Medicine, Division of Hematology/Oncology and 2Institute of Pathology, Georg-August University, Robert-Koch-Str 40, D 37075 Goettingen, Germany

The development of rapid polymerase chain reaction (PCR) region (CDR3).2,6–10 Clonal expansions of B cells carry ident- protocols for amplification of rearranged heavy chain immuno- ical copies of unique V -N-D -N-J junctional region globulin (IgH) gene sequences has facilitated the identification H H H of clonal IgH rearrangements in non-Hodgkin’s lymphomas sequences. Therefore, rearranged IgH genes are used as mark- (NHL) and leukemias of B lineage. In the present report we have ers for lineage and clonality in human lymphoid neoplasms explored the recently described improved strategy for assess- and DNA sequences at the junction of VH,DH and JH segments ment of clonality of rearranged immunoglobulin heavy chain can be used as clone-specific markers in individual (IgH) genes in more detail in a series of 101 B cell malignancies patients.6,9–20 and 50 polyclonal controls. The assay is based on an IgH-PCR In the past Southern blotting techniques with radiolabeled with an automated fluorescence-based strategy for PCR detec- probes for specific immune gene segments have been exten- tion of IgH gene rearrangements. Third complementarity 19,21–23 determining region (IgH-CDR3) sequences were amplified sively applied to the study of ALL and NHL patients. using fluorescent dye labeled consensus primers homologous However, this technique is time-consuming, labour-intensive, to the corresponding variable (VH) and joining (JH) gene seg- relatively insensitive and impractical for clinical studies. The ments in combination with a thermostable proofreading DNA development of PCR has markedly facilitated the characteriz- polymerase. PCR products were size separated on a high resol- ation of IgH junctions by direct DNA-sequencing and ution polyacrylamide gel and analyzed for clonality by exact size determination and fluorescence quantification in an auto- enhanced the sensitivity for the detection of these clonal mar- mated DNA sequencer. PCR findings obtained with the optim- kers. We recently showed that the amplification efficacy of ized IgH-CDR3-PCR assay showed an overall monoclonality IgH–CDR3 junctions with consensus PCR primers and the detection rate of 97% (97 of 101 cases with B cell neoplasms). accuracy of PCR results is significantly improved by appli- The specificity was 100% as determined by analysis of 50 con- cation of the thermostable UITma DNA polymerase with an trols, all of which gave polyclonal PCR results. We found a high inherent 3′ to 5′ exonuclease activity.24 In the present report rate of monoclonal IgH-CDR3-PCR results not only in the leukemias and diffuse lymphoma but also in the group of follicular we have explored this new strategy for detection of clonally lymphoma, where a high rate of false negative results is fre- rearranged IgH-CDR3 junctions in detail in a large series of B quently reported in the literature. In summary, we identified cell malignancies. Clone-specific IgH CDR3 sequences were monoclonal IgH-CDR3 junctions in 55 out of 59 cases (93%) amplified by the polymerase chain reaction (PCR) using flu- with B cell lymphoma and in 42 of 42 (100%) cases with leuke- orescent dye labeled consensus primers. PCR products were mia, immunocytoma and multiple myeloma. The results demon- size separated on a high resolution polyacrylamide gel and strate that automated fluorescence detection of IgH-CDR3-PCR products is an ideal tool for detection of clonal and polyclonal analyzed by fluorescence quantification and size determi- lymphoid B cells. In combination with allele-specific primers nation in an automated DNA sequencer. The gene scanning the procedure may improve current experimental approaches results were verified by sequencing randomly selected PCR to detect occult malginant B cells during initial staging and products. The results demonstrate that automated fluor- follow-up of NHL and ALL patients. escence detection of IgH-CDR3-PCR products is a powerful Keywords: lymphoma; PCR; IgH-CDR3; sequencing; gene tool for the detection of clonal and polyclonal B cells. scanning

Materials and methods Introduction Clinical samples and cell lines During B cell development, IgH genes undergo a complex rearrangement process of their variable (VH), diversity (DH) A total of 101 newly diagnosed patients with B cell non-Hodg- 1–5 and joining (JH) gene segments. The recombination sites of kin’s lymphomas and B lineage acute or chronic leukemias the IgH genes form the structure of the highly diverse VH– were selected for this study (Table 1). All patients were admit- DH–JH junctions known as third complementarity determining ted to the Department of Internal Medicine of the University of Go¨ttingen between October 1985 and June 1996 (19 cases of chronic B cell leukemia (B-CLL), three hairy cell leukemia Correspondence: M Kneba (HCL), 12 acute B cell leukemias (ALL), two cases with mul- Received 8 October 1996; accepted 10 April 1997 tiple myeloma, six stage IV immunocytomas (IC), 19 cases of Technical report B Linke et al 1056 Table 1 IgH-CDR3, t(14;18) mbr and t(11;14) results

Diagnosis n t(14;18) mbr- t(11;14) PCR IgH-CDR3-PCR/Gene-scanning results PCRa positive cases + positive cases n /nt(%) + p 1R 2R Total number of n /nt(%) positive cases

B-NHL CB-CC 19 10/19 (53) 0/19 (0) 1/19 (5) 16/19 (84) 2/19 (11) 18/19 (95) CC 11 0/11 (0) 3/11 (27) 0/11 (0) 9/11 (82) 2/11 (18) 11/11 (100) DLC (IB, CB) 24 2/24 (8) 0/24 (0) 2/24 (8) 15/24 (63) 7/24 (29) 22/24 (92) Burkitt 5 0/5 (0) 0/5 (0) 1/5 (20) 4/5 (80) 0/5 (0) 4/5 (80) Total 59 13/59 (22) 3/59 (5) 4/59 (7) 44/59 (74) 11/59 (19) 55/59 (93)

B cell leukemia and multiple myeloma CLL, ALL, IC, 40 ND ND 0/40 (0) 26/40 (65) 14/40 (35) 40/40 (100) HCL Myeloma 2 ND ND 0/2 (0) 2/2 (100) 0/2 (0) 2/2 (100) Total 101 4/101 (4) 72/101 (72) 25/101 (25) 97/101 (97)

Controls 50 ND ND 50/50 (100) 0/50 (0) 0/50 (0) 0/50 (0)

+ + n, the number of tested cases in this diagnosis group; n 2/nt, denotes the number of cases with a monoclonal gene scanning result (n ) per number of tested cases (nt); DLC, diffuse large cells lymphoma, for the other diagnoses see Materials and methods; p, polyclonal; 1R and 2R, indicate the number of monoclonally rearranged alleles in individual cases. ND, not determined. aOnly the major breakpoint region of the bcl-2 gene was analyzed.

low-grade centroblastic-centrocytic (CB-CC), 11 mantle cell PCR conditions (centrocytic, CC) lymphomas, 24 cases of high-grade centroblastic (CB) and immunoblastic NHL (IB), and five cases with The PCR primers and cycling conditions used in this study Burkitt’s lymphoma). The lymphomas were categorized followed the recently described protocol.24 Briefly, amplifi- 25 according to the updated Kiel classification, using conven- cation reactions with consensus VH and JH primers were per- tional morphological and immunohistological techniques. formed in an automated thermocycler (model 480; Perkin The B cell leukemia cases and the polyclonal blood and bone Elmer) with 0.5–1.0 ␮g of sample DNA in a final volume of marrow samples were classified by flow cytometry. As poly- 30 ␮l reaction buffer containing 40 ␮M of each deoxynucleo- clonal controls DNA samples from normal peripheral blood tide, 5 pmol of each primer and 2 units of U1Tma DNA poly- 24 lymphocytes (18 cases), patients with nonmalignant disorders merase (Perkin Elmer) for 35 cycles with primers VH and JH. (10 bone marrow samples) and diagnostic lymph node biopsy Cycle conditions were: denaturation at 96°C for 1 min. with specimens of patients with unspecific lymphadenitis (seven annealing/extension at 55°C for 45 s). Hot-start-initiated PCR cases), three cases with toxoplasmosis, Hodgkin’s disease was performed by addition of BioWax (Biozym, Hameln,

(HD; five cases) and peripheral T cell NHL (seven cases) were Germany) to the reaction tubes. The VH primer was HPLC- studied. Fifteen of these negative controls were tested by purified and labeled at its 5′ end with 6-FAM (6- Southern analysis and all showed a germline JH-configuration. carboxyfluorescein) (ABI) generating blue fluorescence signals All patients were admitted to the Department of Internal Medi- after excitation with an argon ion laser in the automated cine of the University of Goettingen for diagnosis and treat- sequencer (model 373A; ABI). ment. The B cell line SKW 6.4 (American Type Culture Collec- tion TIB 215) served as monoclonal control. IgH Southern and IgH PCR results of 44 patients with lymphoma or leukemia of Fluorescent fragment analysis (gene scanning) B lineage have been reported recently in a short communication.24 For automated fluorescent fragment analysis, aliquots of the PCR products (1–2 ␮l) were mixed with equal amounts of DNA preparation loading buffer (5 volumes formamide:1 volume EDTA; 10% dextraneblue) and 0.5 ␮l of the internal size standard Genes- High molecular weight DNA was extracted using an auto- can-350-ROX (ABI). After denaturation for 3 min at 95°C pro- mated DNA extractor (model 341; Applied Biosystems (ABI), ducts were analyzed by automatic fluorescence quantification Weiterstadt, Germany) from snap-frozen peripheral blood, and size determination using the computer program GENES- bone marrow, or lymph node biopsy specimens that were col- CAN 672 (ABI). Typical running times were 2–3 h with a 12- lected during routine diagnostic procedures. In 13 cases DNA cm long electrophoresis gel. for PCR was extracted from paraffin-embedded lymph node biopsy specimens as described.17 Cloning and DNA sequencing Southern blot analysis Cloning of PCR products was performed as previously Southern blot analysis of genomic DNA were performed as described.17 Recombinant plasmids were sequenced by the 23 previously described with a radiolabeled JH-probe after Taq cycle sequencing method involving the universal forward EcoRI and BamHI restriction enzyme digestion. sequencing primers for pUC 19 and the Ready Reaction Dye Technical report B Linke et al 1057 Deoxy Terminator Cycle Sequencing kit (ABI). For each DNA size range of 80 and 150 bp which is predicted for IgH–CDR3 sample, three to 10 randomly chosen clones were sequenced. junctions with the primers used. The most abundant FR3A- Sequencing reaction products were analyzed on the DNA CDR3-PCR fragments of the polyclonal samples cluster

sequencer with the SeqEd 675 DNA Sequence Editor (ABI). JH around 105–115 bp. PCR products derived from polyclonal elements were identified by comparison with published germ- samples showed a ‘spectrum’ of fragments with a 3 bp size 4,18 line sequences. Individual DH segments were identified difference between individual bands. This indicates that the by comparison with 32 published germline DH-DNA amplified polyclonal CDR3 junctions are mostly functional sequences.17 Criteria for sequence identity were as reported and conserve an intact open reading frame and therefore differ 2 by Sanz. Since the FR2A VH consensus primer binds too close in size by multiples of one codon as already observed by ′ 29,30 to the 3 VH recombination site, the specific VH gene cannot others. In all polyclonal cases none of the detected frag- be identified. ments indicated the presence of a predominant B cell clone and no peak showed an extraordinary peak height in comparison with the intensity of the normally distributed peaks. A Direct sequencing polyclonal nature as in representative cases 1–3 of Figure 1 was consistently demonstrated in all normal and reactive For direct sequencing, PCR products were diluted 1:100 in lymphoid B cell samples, ie normal bone marrow; normal ton- ␮ autoclaved H2O. 2 l of the dilution mixture were reamplified sils; normal peripheral blood lymphocytes, as well as in Hodg- ␮ with the T7-Vcon primer and RSP-JH primer set in 30 l reac- in’s disease and T-NHL. tion buffer (see IgH-CDR3 PCR) for 30 cycles (cycle con- In contrast to polyclonal cell populations, PCR products of ditions: initial denaturation at 96°C for 7 min followed by 15 the group of leukemias and high-grade lymphomas displayed cycles with denaturation at 96°C for 1 min, annealing at 58°C one or two monoclonal peaks (depending on the number of for 1 min and extension at 73°C for 2 min followed by 15 rearranged IgH alleles) with relatively weak background sig- additional cycles with denaturation at 96°C for 1 min, nals. Representative examples are shown in Figure 1 (cases 4, annealing at 55°C for 1 min and extension at 73°C for 2 min). 5, 10, 11 and 12). The diagnostic samples of patients with The products were purified for sequencing by separation on these B cell malignancies often contain a relatively high per- low-melting point agarose or by differential precipitation of centage of tumor cells. Therefore these PCR products were DNA.17,24 Five to 8.5 ␮l of the purified PCR product were sub- highly homogeneous and contained no significant polyclonal jected to a flourescent dye terminator cycle sequencing reac- background derived from admixed nonmalignant B cells. tion (kit purchased from ABI) with either 10 pmol T7 (ABI) or However, a completely different gene-scanning profile was RSP (reverse sequencing primer; Pharmacia, Freiburg, observed in the group of follicular lymphoma. The admixture Germany). The sequencing reaction product was analyzed on of significant numbers of polyclonal B cells in these cases pro- the automated sequencer. duced a characteristic scanning profile with usually one dominant peak of fluorescence belonging to the lymphoma-related monoclonal CDR-3 PCR product superimposed over a back- t(11;14) and t(14;18) PCR ground of the fluorescence-intensity size-distribution pattern which is characteristic for polyclonal B cells. Typical

The t(14;18) and t(11;14) chromosomal translocations with examples are shown in Figure 1, cases 7–9. Specific VH-N- involvement of the major breakpoint region (MBR) or the DH-N-JH products were considered as monoclonal and to rep- major translocation cluster (MTC) region were analyzed by resent the malignant cell clone if they showed one or two PCR.26,27 These translocations are characteristic for follicular clearly dominant fluorescence peaks. This criterion permitted (CB-CC) and a subgroup of diffuse large cell, t(14;18), and us to distinguish between mono- and polyclonal PCR products mantle cell, t(11;14), lymphomas.26–28 not only in the leukemias and diffuse lymphomas but also in the follicular lymphomas. As demonstrated by sequencing the cloned PCR products (see also Ref. 17) this fluorescence pat- Results tern is caused by coamplification of monoclonal as well as

polyclonal VH-N-DH-N-JH junctions of neoplastic and A total of 101 DNA samples extracted from diagnostic admixed ‘contaminating’ nonmalignant B cells which can material of patients with B cell malignancies and 50 make up a substantial cell fraction in some of the follicular B polyclonal controls were studied. Sixty-four of the cases had cell lymphomas. Because the dominant clonal peaks were been previously analyzed by Southern hybridization with a clearly set off to advantage against the polyclonal fragment 23,24 radiolabeled JH probe for IgH gene rearrangements. spectrum they could easily be discriminated from the polyclonal background. Thus, with the approach described here we were able to identify a monoclonal junction in 18/19 Gene scanning of IgH-CDR3-PCR products (95%) of the cb-cc NHL cases even in the presence of a significant background of polyclonal B cells as in cases 7–9. PCR products were size separated on a high resolution polyac- Case 6 which represents a c-ALL at diagnosis was rylamide gel and analyzed by automatic fluorescence quanti- exceptional as it showed three predominant sharp peaks fication and accurate size determination as described under of fluorescence and in addition six minor bands in the experimental conditions. Representative scanning results are range between 100 and 120 bp in the absence of other back- shown in Figure 1. Because of the hypervariable character of ground bands. As an explanation for this unexpected finding, rearranged IgH–CDR3 junctions, the fluorescence-intensity clonal microheterogeneity due to ongoing rearrangement or size-distribution pattern of a given PCR product characterizes somatic maturation processes at the site of the V–N–D the corresponding B cell population. DNA extracts from poly- junction,3,8–10,14,17,31–34 or true oligoclonality were considered. clonal controls yielded a typical fluorescence peak pattern For clarification, these PCR products were cloned and with a normal Gaussian-like PCR fragment distribution in the sequenced. As shown in Figure 2, seven out of nine randomly Technical report B Linke et al 1058

Figure 1 Electrophoretic (gene scanning) profiles of fluorescent IgH-CDR3-PCR products generated from DNA of three polyclonal controls (cases 1–3) and nine patients with malignant B cell proliferations analyzed with the GENESCAN 672 software on the automated sequencer. Relative fluorescence intensities (y-axis) are plotted as a function of PCR fragment size in basepairs (x-axis) for each PCR product. Case numbers and conditions were: normal bone marrow (1); tonsil (2); normal peripheral blood (3); B-ALL (4); B-CLL (5); c-ALL (6); CB-CC (cases 7–9); CB (10); mantle cell NHL (11) and Burkitt NHL (12). Technical report B Linke et al 1059

Figure 2 IgH-CDR3 junctional sequences deduced from cloned PCR products of case 6 (Figure 1) are shown. Nine randomly chosen clones were sequenced. The PCR-derived sequences are aligned to the known germline DH and JH sequences as shown at the top. DH elements ﬂanked by template independent N-region (N1 and N2; indicated by lower case letters) are written in bold letters. In seven tumor-related IgH–CDR3 ′ ′ junctions a DH-LR2 element was found. The 3 VH and 5 JH primer annealing sites are underlined.

chosen clones used identical DH and JH elements (LR2 Assessment of B cell clonality by IgH-CDR3-gene and JH4B, respectively), had identical breaks on DH and JH scanning and identification of t(14;18)- and t(11;14) and carried identical N-nucleotides at the site of the DH–JH chromosomal translocations by PCR junction. These clones differed only by partial nucleotide changes within the VH–DH junction due to different nucleotide The IgH-CDR3-gene scanning data together with the t(14;18)- ′ deletions at the 3 VH site and diverse N-regions at the VH–DH and t(11;14)-PCR results are summarized in Table 1. In 10 of junction. Therefore in this case these junctions correspond to 19 cases (53%) of CB-CC NHL which were additionally tested malignant B cell subpopulations with a common origin. for the presence of a mbr bcl-2/JH rearrangement, a t(14;18) could be demonstrated by PCR. Of these 19 cases the IgH- PCR assay was positive in 18 (95%). The positive cases showed in 16 of 19 CB-CC cases (84%) a monoallelic PCR Sequence analysis of IgH-CDR3-PCR products product and in two of the nine t(14;18)-negative cases a biallelic PCR product. In three of 11 (27%) cases with mantle cell To confirm the specificity of the PCR/gene scanning results, lymphoma PCR identified a t(11;14) translocation, whereas a we sequenced 17 PCR products derived from high- and low- monoclonal IgH-CDR3-PCR product pattern was observed in grade B-NHL and B cell leukemias. The sequencing results of 11/11 (100%) of the cases. Of these 82% including the three 17 directly sequenced alleles are shown in Figure 3. The cor- t(11;14)-PCR-positive cases showed a monoallelic rearrange- rect interpretation of the PCR/gene scanning results was dem- ment. In Burkitt’s lymphoma, a monoallelic IgH-CDR3-PCR product was detected in all four cases that were PCR-positive. onstrated by the identification of clone-specific VH-N-DH-N- In summary we identified monoclonal IgH-CDR3 junctions in JH junctions in each case. The sizes of the PCR fragments cal- culated from the sequence data were identical to the size 55 out of 59 cases (93%) with B cell lymphoma and in 42 of determined by the DNA fragment analysis within ±1 bp. We 42 (100%) cases with leukemia, immunocytoma and multiple 24 obtained only directly readable sequences in those cases myeloma (Table 1). As we have shown previously, complete where the automated fluorescent DNA profiling showed less agreement with respect to clonality detected by Southern blot- than approximately 15% of polyclonal background signal in ting and fragment analysis was achieved in all of the 44 B relation to the specific monoclonal product. Remarkably, a cell malignancies and 15 germline controls which had been ′ studied by both methods. mismatch at the 3 end of the VH PCR primer site was seen in seven of the 17 cases sequenced (G to T alteration in six cases and a G to C alteration in one case). These single nucleotide ′ mismatches at the 3 VH primer site provide a further expla- Allele-specific IgH-CDR3 PCR nation for the improvement of the monoclonality detection rate with UITma DNA polymerase24 as the amplification of To assess the sensitivity of the technique, mixing experiments these junctions could be assigned to the proofreading activity with SKW 6.4 cells diluted in peripheral blood mononuclear of this enzyme. cells were performed. The detection limit for SKW 6.4 cells A total of 33 PCR products which were considered unsuit- was 1:102 in a single 35 cycle PCR. In a semi-nested allele- able for direct sequencing due to admixed polyclonal side specific PCR with a specific N-JH primer and the VH consensus bands on gene scanning were sequenced after cloning. The primer the sensitivity was 1:105 (not shown). sequencing results of 17 samples have been reported else- where.17 In each case a good correlation with respect to clonality and size of the dominant monoclonal product was Discussion observed when gene scanning and sequencing data were 21 compared. A DH and JH element was found in all except one Since the original reports by Arnold et al and Clearly and case and the junctional regions showed a remarkable diversity colleagues22 Southern analysis for analysis of IgH gene and represent clone-specific identification sequences. rearrangements was instituted in many laboratories.18 In the Technical report B Linke et al 1060

Figure 3 IgH CDR3 junctional sequences are shown. The PCR products were generated from genomic DNA under described conditions (see Materials and methods), reamplified with modified PCR primers including artificial sequencing primer sites (T7 and RSP), and sequenced

by the Taq cycle sequencing method with sequencing primers T7 and RSP. The PCR-derived sequences are aligned to the known germline DH and JH sequences as shown at the top. DH elements ﬂanked by template independent N region (N) sequences are written in bold letters. Indistinct ′ ′ ′ nucleotides are written as N. The 3 VH and the 5 JH PCR primer annealing sites are underlined. The asterisks indicate a mismatch at the 3 end of the VH PCR primer site (G to T alteration in six cases and a G to C alteration in one case).

last years this technique for detection of clonality was increas- characteristic fluorescence-intensity size-distribution pattern. ingly replaced by PCR.6–12,15–17,24,29–31,35–40 In order to over- In polyclonal B cell populations the size and frequency of the come limitations of conventional detection systems with vis- IgH-CDR3 PCR products follows a normal distribution due to ual interpretation of the size-separated PCR products for imprecise V-D-J joining (recombinatorial diversity) and to assessment of clonality, we recently developed an improved insertion of nontemplated N nucleotides and variations in FR3A-IgH-CDR3-PCR assay which employs a proofreading deletions of the IgH elements at the recombination site DNA polymerase in combination with a new automated flu- (junctional diversity). Previous reports described the pro- orescence-based strategy for detection of PCR products of duction of a smear or ‘ladder’ of fragments with DNA from immunoglobulin gene rearrangements.24 This detection strat- normal peripheral blood upon analysis of PCR products by egy relies on PCR with fluorescent primers and high resolution high resolution electrophoretic separation with visual scanning of size-separated products in an automatic interpretation of stained bands.12,15,16,30,39 With the improved sequencer and was first described in a case report by Shiach analysis method we also observed a size-difference of 3 bp et al29 and in the brief report by our laboratory.24 Recently between single peaks of polyclonal IgH-CDR3-PCR products we applied the gene scanning technique for the analysis of in agreement with these reports. rearranged TCR␤ genes.41 The evaluation of this new We found a high rate of monoclonal IgH-CDR3-PCR results approach for detection of monoclonal and poyclonal B cell not only in the leukemias and diffuse lymphomas but also in populations in our series of patients with B cell malignancies the group of follicular CB-CC lymphomas. In contrast to the and polyclonal controls further demonstrates the usefulness of leukemias and to the diffuse high-grade and low-grade (ie this technique. mantle cell and immunocytoma) lymphomas, biopsy materials Each IgH-CDR3 PCR product produces an individual and of most of the follicular CB-CC lymphomas contained signifi- Technical report B Linke et al 1061 cant amounts of admixed non-neoplastic B cells in addition absent in a significant proportion of low-grade and in the to the malignant B cell clone. Consequently a mixture of unre- majority of high-grade lymphomas.26–28 On the other hand, lated IgH–CDR3 junctions is usually amplified by PCR clonal rearrangements of the immunoglobulin heavy chain together with the clonal junction. In these cases the analysis (IgH) gene can be detected in the majority of B cell lym- with conventional agarose and polyacrylamide gel electro- phomas and leukemias.19,21,22 The simple method described phoresis and visual interpretation of the band pattern with of detecting monoclonal rearranged IgH genes and clonotypic ethidium bromide staining gives unreliable results.17,35,37,40 CDR3-IgH sequences will significantly simplify current experi- Thus a high rate of false negative IgH CDR3 PCR results is mental approaches to detect and to quantitate malignant B frequently reported for these lymphomas in the literature. In cells during initial staging and follow-up of NHL and ALL these cases gene scanning clearly detected the monoclonal patients and might therefore become useful as a broadly appli- IgH product which had previously been demonstrated only by cable diagnositic tool in the study of NHL and ALL patients. PCR cloning.17 However it has to be noted, that in other cases with lower numbers of tumor cells or in cases with very small numbers of samples the interpretation of the data may be Acknowledgements much less clear cut. We noticed a characteristic scanning pattern of IgH PCR products in follicular lymphoma. As could This work was supported by Deutsche Krebshilfe grant 70523. be demonstrated by sequencing of cloned PCR products (see Ref. 17) this fluorescence pattern is caused by coamplification of monoclonal as well as polyclonal V -N-D -N-J junctions H H H References of neoplastic and admixed ‘contaminating’ nonmalignant B cells which can make up a substantial cell fraction in some 1 Tonegawa S. Somatic generation of antibody diversity. Nature of the follicular B cell lymphomas. These admixed polyclonal 1983; 302: 575. products can readily mask a monoclonal band when the 2 Sanz I. Multiple mechanisms participate in the generation of diver- results are obtained only by visual inspection of stained aga- sity of human H chain CDR3 regions. J Immunol 1991; 147: 1720. rose and polyacrylamide gels and the PCR is interpreted as 3 Stewart AK, Schwartz RS. Immunoglobulin V regions and the B negative. In the target groups (B-NHL and B lineage ALL) we cell. Blood 1994; 83: 1717. 4 Ravetch JV, Siebenlist U, Korsmeyer S, Waldmann T, Leder P. detected monoclonal IgH-CDR3 rearrangements in 93 and Structure of the human immunoglobulin locus: characterization of 100% of the cases. This overall monoclonality detection rate embryonic and rearranged J and D genes. Cell 1981; 27: 583. with one consensus FR3-VH and one consensus JH primer is 5 Ichihara Y, Matsuoka H, Kurosawa Y. Organization of human much higher than the published frequency of 60– immunoglobulin heavy chain diversity gene loci. EMBO J 1988; 85%17,35,37,40 and confirms our previous observation that the 7: 4141. problem of the high false negativity rate which hampered most 6 Yamada M, Hudson S, Tournay O, Bittenbender S, Shane SS, Lange B, Tsujimoto Y, Caton AJ, Rovera G. Detection of minimal published IgH-CDR3 strategies is considerably reduced by the disease in hematopoietic malignances of the B cell lineage by combined use of U1Tma polymerase and high resolution gene using third-complementarity-determining region (CDR-III)-specific scanning of PCR products.24 probes. Proc Natl Acad Sci USA 1989; 86: 5123. As expected, we found no biallelic CDR3 IgH rearrange- 7 Kiyoi H, Naoe T, Horibe K, Ohno R. Characterization of the ment in cases in which a t(14;18) and t(11;14) with fusion of immunoglobulin heavy chain complementarity determining the bcl-2 or bcl-1 genes to one of the J elements could be region (CDR)-III sequences from human B cell precursor acute H lymphoblastic leukemia cells. J Clin Invest 1992; 89: 739. identified by PCR. A biallelic IgH-CDR3 junction was signifi- 8 Steward CG, Potter MN, Oakhill A. Third complementarity cantly less frequently observed in CB-CC, mantle cell and Bur- determining region (CDR III) sequence analysis in childhood B- kitt lymphoma in which one JH is commonly fused to bcl-1, lineage acute lymphoblastic leukaemia: implications for the design bcl-2 or c-myc.28 Thus the IgH-PCR analysis described here of oligonucleotide probes for use in monitoring minimal residual could be useful as a first screening test for lymphomas in disease. Leukemia 1992; 6: 1213. which a chromosomal translocation with fusion of an IgH 9 Steenbergen EJ, Verhagen OJ, van Leeuwen EF, von dem Borne AE, van der Schoot CE. Distinct ongoing Ig heavy chain rearrange- gene on one allele to an unidentified fusion partner is ment processes in childhood B-precursor acute lymphoblastic leu- expected. The procedure could identify those monoallelic kemia. Blood 1993; 82: 581. cases suitable for the JH-directed vectorette PCR approach for 10 Kitchingman GR. Immunoglobulin heavy chain gene VHD junc- molecular exploration of 14q+ chromosomes with unknown tional diversity at diagnosis in patients with acute lymphoblastic fusion partners that are not amplified by standard PCR proto- leukemia. Blood 1993; 81: 775. cols as described by Lee et al.42 11 Trainor KJ, Brisco MJ, Story CJ, Morley AA. Monoclonality in B- lymphoproliferative disorders detected at the DNA level. Blood Studies in ALL demonstrated a marked heterogeneity in the 1990; 75: 2220. leukemia-specific VH–DH junction in up to 40% of the 12 McCarthy KP, Sloan JP, Wiedemann LM. Rapid method for dis- samples.8–10,31 As shown here in one case with cALL (case 6, tinguishing clonal from polyclonal B cell populations in surgical Figures 1 and 2), PCR-gene-scanning can identify such cases biopsy specimens. J Clin Pathol 1990; 43: 429. with remarkable simplicity. 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