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Blood, 15 May 2004, Vol. 103, No. 10, pp. 3798-3804. Prepublished online as a Blood First Edition Paper on December 4, 2003; DOI 10.1182/blood-2003-08-2952. This Article Abstract Full Text (PDF) All Versions of this Article: 2003-08-2952v1 103/10/3798    most recent Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Rights and Permissions Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Szczepanski, T. Articles by van Dongen, J. J. M. Search for Related Content PubMed PubMed Citation Articles by Szczepanski, T. Articles by van Dongen, J. J. M. Related Collections Immunobiology Neoplasia Social Bookmarking                   What's this?  Previous Article  |  Table of Contents  |  Next Article  IMMUNOBIOLOGY V2-J rearrangements are frequent in precursor-B–acute lymphoblastic leukemia but rare in normal lymphoid cells Tomasz Szczepaski, Vincent H. J. van der Velden, Patricia G. Hoogeveen, Maaike de Bie, Daniëlle C. H. Jacobs, Elisabeth R. van Wering, and Jacques J. M. van Dongen From the Department of Immunology, Erasmus MC, University Medical Center Rotterdam, the Netherlands; Department of Pediatric Hematology and Oncology, Silesian Medical Academy, Zabrze, Poland; and Dutch Childhood Oncology Group, The Hague, the Netherlands.    Abstract Top Abstract Introduction Patients, materials, and methods Results Discussion References   The frequently occurring T-cell receptor delta (TCRD) deletions in precursor-B–acute lymphoblastic leukemia (precursor-B–ALL) are assumed to be mainly caused by V2-J rearrangements. We designed a multiplex polymerase chain reaction tified clonal V2-J rearrangements in 141 of 339 (41%) childhood and 8 of 22 (36%) adult precursor-B–ALL. A significant proportion (44%) of V2-J rearrangements in childhood precursor-B–ALL were oligoclonal. Sequence analysis showed preferential usage of the J29 gene segment in 54% of rearrangements. The remaining V2-J rearrangements used 26 other J segments, which included 2 additional clusters, one involv ing the most upstream J segments (ie, J48 to J61; 23%) and the second cluster located around the J9 gene segment (7%). Real-time quantitative PCR studies of normal lymphoid cells showed that V2 rearrangements to upstream J segments occurred at low levels in the thymus (10–2 to 10–3) and were rare (generally below 10–3) in B-cell precursors and mature T cells. V2-J29 rearrangements were virtually absent in normal lymphoid cells. The monoclonal V2-J rearrangements in precursor-B–ALL may serve as patient-specific targets for detection of minimal residual disease, because they show high sensitivity (10–4 or less in most cases) and good stability (88% of rearrangements preserved at relapse).    Introduction Top Abstract Introduction Patients, materials, and methods Results Discussion References   Rearrangements of T-cell receptor (TCR) delta (TCRD) genes represent one of the earliest events in normal T-cell development.1-3 However, recombinations in TCRD genes are not fully restricted to the T-cell lineage. The presence of cross-lineage TCRD gene rearrangements is a frequent phenomenon both in childhood and adult precursor-B–acute lymphoblastic leukemia (precursor-B–ALL).4-6 Nevertheless, the spectrum of TCRD gene rearrangements in precursor-B–ALL is very limited, with 80% of detected rearrangements representing incomplete V2-D3 or D2-D3 joinings.5,7,8 Similarly, only D2-D3 and V2-D3 joinings can be found in normal B-cell precursors or even in mature B cells.9,10 Moreover, exactly the same types of incomplete TCRD gene rearrangements can be induced in nonlymphoid tissues transfected in vitro with basic helix-loop-helix transcription factors.11 Interestingly, V2-D3 rearrangements in precursor-B–ALL are prone to continuing rearrangements, particularly to J gene segments with concomitant deletion of the C exons and subsequent V-J recombination (Figure 1A).4,5,9,12-14 Our detailed Southern blot study indicated that at least 40% of TCRD alleles in precursor-B–ALL are deleted, which might be largely due to V2-J rearrangements.5 Limited, mainly qualitative data indicate that V2-J rearrangements are infrequent in normal lymphoid tissues.15,16 Other immunobiologic characteristics of V2-J rearrangements in normal and malignant lymphoid cells are largely unknown. View larger version (48K): [in this window] [in a new window]   Figure 1.. TCRD/A gene rearrangements in precursor-B–ALL. (A) Consecutive rearrangements in the TCRD/A locus involving the V2 gene segment that are characteristic for precursor-B–ALL. The main pathway concerns consecutive V2-D3 V2-D3-J recombinations. D2-D3 and D2-J gene rearrangements can also occur, albeit at much lower frequencies. Solid boxes below the gene segments represent the probes used for Southern blot hybridization. (B) Southern blot analysis with TCRDV2 probe in 10 precursor-B–ALL samples. V2-D3 and/or V2-J29 gene rearrangements in cases 5602, 5675, 5683, and 5696 are monoclonal. The presence of several rearranged bands of different densities in cases 5515, 5647, 5662, and 5698 is consistent with oligoclonality. Both V2 alleles in case 5670 are deleted, while case 5565 has both V2 alleles in germline (G) configuration.   We developed a multiplex polymerase chain reaction (PCR) strategy for easy identification and characterization of clonal V2-J gene rearrangements in a large series (n = 361) of precursor-B–ALL. Subsequently, we investigated the presence of the most frequent V2-J rearrangements in various types of normal lymphoid tissues. Finally, we evaluated the sensitivity and stability of V2-J rearrangements as real-time quantitative (RQ)–PCR targets for detection of minimal residual disease (MRD).17    Patients, materials, and methods Top Abstract Introduction Patients, materials, and methods Results Discussion References   Patients Bone marrow (BM) or peripheral blood (PB) samples from 339 children with precursor-B–ALL were obtained at initial diagnosis (age range, 1.5 months to 15.9 years). Immunologic marker analysis revealed 12 pro-B-ALL, 226 common ALL, and 101 pre-B–ALL.18 In addition, diagnosis samples from 22 adult precursor-B–ALL were analyzed. The clinical, immunophenotypic, and immunogenotypic characteristics of these adult patients were reported previously.6 Patient samples were obtained after informed consent according to the guidelines of the Medical Ethics Committee of the Erasmus MC, Rotterdam. Southern blot analysis Mononuclear cells (MNCs) were isolated from BM or PB samples by Ficoll-Paque centrifugation (density, 1.077 g/cm3; Pharmacia, Uppsala, Sweden). DNA was isolated from fresh or frozen MNC fractions as described previously.19,20 Fifteen micrograms of DNA were digested with the appropriate restriction enzymes (Pharmacia), size-separated in 0.7% agarose gels, and transferred to Nytran-13N nylon membranes (Schleicher & Schuell, Dassel, Germany) as described.19 The configuration of the TCRD genes was analyzed with the TCRDJ1 and TCRDV2 probes (DAKO, Carpinteria, CA) in BglII, EcoRI, or HindIII digests.8 Southern blot analysis was successfully performed in 208 precursor-B–ALL. Primer design and heteroduplex PCR analysis V2 and D2 primers have been developed by the BIOMED-2 Concerted Action BMH4-CT98-3936 "PCR-based clonality studies for early diagnosis of lymphoproliferative disorders."21 Based on the available nucleotide sequence of the human 3' terminal end of the TCRA/D locus (European Molecular Biology Laboratory [EMBL] accession no. M94081 [GenBank] ),22 61 J primers compatible with the V2 primer were designed using OLIGO 6.0 software (developed by Dr W. Rychlik; Molecular Biology Insights, Cascade, CO) and applying previously described guidelines.23 The sequences of the primers and the composition of 7 V2-J multiplex PCR tubes are available upon request. The multiplex V2-J PCR analyses were performed in all 339 patients, essentially as described previously.6,23 In each 50 µL PCR reaction, 100 ng DNA sample, 10 pmol of the 5' and 3' oligonucleotide primers, and 1 unit AmpliTaq Gold polymerase (Applied Biosystems, Foster City, CA) were used. PCR conditions were initial denaturation for 10 minutes at 94° C, followed by 35 cycles of 45 seconds at 92° C, 90 seconds at 60° C, and 2 minutes at 72° C using a Perkin-Elmer 480 thermal cycler (Applied Biosystems). After the last cycle an additional extension step of 10 minutes at 72° C was performed. Appropriate positive and negative controls were included in all experiments.23 Heteroduplex analysis of PCR products was performed as described previously.24 The presence of clonal V2-D3 and D2-D3 gene rearrangements was tested using our classical monoplex approach.23 Multiplex D2-J PCR was performed in 11 patients, preselected based on Southern blot and PCR information (ie, germline V2 allele with deleted D3/J1 area and absence of clonal V2-J rearrangements). Comparative heteroduplex analysis of PCR products Comparative heteroduplex analysis of V2-J PCR products at diagnosis and relapse concerned 43 of 91 relapsed precursor-B–ALL patients,25 selected for the presence of V2-J rearrangements at diagnosis. The relapse samples were first analyzed in a monoplex PCR with those primer combinations that showed clonal PCR products at diagnosis. When the clonal PCR product was also found at relapse, its identity was subsequently compared with the PCR product found at diagnosis by mixed heteroduplex analysis—that is, mixing of the diagnosis and relapse PCR products followed by heteroduplex analysis.25,26 When clonal PCR products found at diagnosis were undetectable at relapse, the relapse sample was analyzed with all 7 V2-J multiplex tubes. Sequence analysis of V2-J rearrangements Direct sequencing of V2-J rearrangements was performed with the V2 primer using the dye-terminator cycle sequencing kit with AmpliTaq DNA polymerase FS on an ABI 377 sequencer (Applied Biosystems) as previously described.27 When heteroduplex PCR analysis revealed more than 2 clonal bands (ie, 2 homoduplexes or an additional upper band resulting from extension to a downstream J segment), the bands were excised from the polyacrylamide gel, eluted, and directly sequenced as described before.28 Recognition of D2 and D3 segments in V2-J junctional regions required at least 4 and 5 consecutive matching nucleotides, respectively.29 RQ-PCR detection of V2-J rearrangements in normal tissue samples Normal tissue samples tested for the presence of V2-J rearrangements included normal PB, E-rosette–positive PB cells (T cells), E-rosette–negative PB cells (B cells, natural killer [NK] cells, and monocytes), normal BM, sorted BM B cells and B-cell precursors, tonsils, lymph nodes, thymuses, and postchemotherapy regenerating BM samples, which are known to contain high frequencies of normal precursor-B-cells.30,31 Whenever possible, at least 2 different samples were tested per category, each sample in triplicate. To analyze the presence of V2-J gene rearrangements in normal tissue samples, the germline V2 TaqMan probe (5'-AGACCCTTCATCTCTCTCTGATGGTGCAAGTA-3') and the germline V2 forward primer (5'-TGCAAAGAACCTGGCTGTACTTAA-3') were used together with a germline reverse J primer. Based on the frequencies of particular V2-J gene rearrangements in precursor-B–ALL ("Results"), J9, J29, J58, and J61 primers were selected for analysis in normal lymphoid cells. To determine the efficiency of amplification and sensitivity of the RQ-PCR, diagnostic DNA from precursor-B–ALL containing the same V2-J gene rearrangements was 10-fold serially diluted (10–1 down to 10–6) into DNA from the cell line CEM, known to have 2 deleted TCRD alleles. To correct for the quantity and quality (amplifiability) of DNA, RQ-PCR analysis of the albumin gene was used.32 Bovine serum albumin was added to each RQ-PCR reaction to prevent inhibition.33 RQ-PCR detection of patient-specificV2-J rearrangements RQ-PCR–based detection of clonal V2-J rearrangements relied on the allele-specific oligonucleotide (ASO) primer approach as described previously.34-36 The above-described germline V2 TaqMan probe and V2 forward primer were combined with the ASO primers positioned at the junctional regions, preferably covering the D3-J, and sometimes also the V2-D3 junction. A standard annealing temperature of 60° C was used. The serial dilutions (10–1 down to 10–6) of diagnostic DNA into control MNC DNA were subjected to RQ-PCR analysis together with negative controls (H2O and control MNC DNA). Each dilution step was analyzed in triplicate. RQ-PCR data were analyzed as described previously.37    Results Top Abstract Introduction Patients, materials, and methods Results Discussion References   Clonal TCRD gene rearrangements in childhood precursor-B–ALL Based on the combined Southern blot and PCR heteroduplex results, clonal V2 rearrangements were found in 69% (144 of 208) of childhood precursor-B–ALL cases (Figure 1B). Clonal V2-D3 rearrangements were detected in 40% (83 of 208) of leukemias. In an additional 7% (14 of 208) of cases, Southern blot indicated the presence of a clonal V2-D3 recombination that turned out to be oligoclonal/polyclonal by PCR analysis.6,38 V2-J rearrangements were found by PCR in 46% (95 of 208) of cases. Consequently, 27% (57 of 208) of precursor-B–ALL had both V2-D3 and V2-J rearrangements (Figure 1). In 56% (53 of 95) of V2-J-positive precursor-B–ALL, V2-J joinings were monoclonal (in 83% monoallelic), but in a significant proportion (42 of 95; 44%) the V2-J rearrangements were oligoclonal. Oligoclonality was assumed either when the Southern blot revealed presence of rearranged bands of different densities (22 cases) or when the number of PCR-detected clonal V2-J and V2-D3 recombinations exceeded the number of V2 rearrangements detected by Southern blot analysis (20 cases). Clonal D2-D3 rearrangements were detected in 10% (20 of 208) of precursor-B–ALL cases. Monoclonal D2-J rearrangements were found in only 3 of the 11 leukemias with a germline V2 allele but a deleted D3/J1 region. Spectrum of V2-J rearrangements in childhood precursor-B–ALL In the group of 339 childhood precursor-B–ALL cases studied with our multiplex PCR strategy, a total of 172 clonal V2-J rearrangements were detected in 141 cases (42%). The frequency of V2-J joinings was slightly lower in pro-B-ALL (3 of 12; 25%) as compared with common ALL (95 of 226; 42%) and pre-B–ALL (43 of 101; 43%), but this was not statistically significant. Sequence analysis of the 172 clonal V2-J PCR products revealed that 27 different J segments were used (Figure 2A). Surprisingly, the J29 gene segment was present in 54% (93 of 172) of all clonal V2-J joinings (Figure 2). Together with J30 and J31 segments, the J29 segment formed a first cluster comprising 59% of V2-J gene rearrangements. A second cluster frequently involved in V2-J recombination concerned the J segments most proximal to the TCRD locus. Altogether, 10 of the most upstream J segments were found in 23% (40 of 172) of V2-J joinings, with J48, J54, J58, and J61 segments used most frequently (Figure 2). The third and most downstream cluster was located around the J9 segment and comprised 7% (12 of 172) of V2-J rearrangements. In line with these results, the 3 identified D2-J rearrangements contained the J9, J29, and J58 gene segments, respectively. View larger version (23K): [in this window] [in a new window]   Figure 2.. Spectrum of V2-J rearrangements in childhood precursor-B–ALL. (A) Bar diagram summarizing the usage of J segments in V2-J rearrangements in precursor-B–ALL. (B) Schematic diagram of the V2 gene segment joined to the J29 gene segment via a junctional region. The presented V2-J29 junctional region sequences are derived from precursor-B–ALL and illustrate the deletion of nucleotides from the germline sequences as well as the size and composition of the junctional regions. D gene segments and inserted nucleotides are indicated by uppercase and small uppercase letters, respectively. (C) Multiplex heteroduplex PCR analysis with the V2 primer in combination with 8 J primers (mix 3) showed clonal V2-J homoduplexes (ho) in all samples tested. Sequence analysis (B) showed that all these rearrangements involved the J29 gene segment. The presence of heteroduplexes (he) in cases 5199, 5504, and 5609 indicated the presence of double V2-J29 rearrangements.   Most of the V2-J rearrangements (79%; 132 of 167 fully sequenced clonal PCR products) contained a part of the D3 gene segment. In striking contrast, remnants of the D2 gene segment were found in only 8% (13 of 167) of the V2-J sequences. Overall sizes of the V2-J junctional regions were extensive, with 18.6 nucleotides on average. V2-J rearrangements in adult precursor-B–ALL Heteroduplex PCR analysis showed a total of 9 clonal V2-J rearrangements in 8 of 22 (36%) adult precursor-B–ALL cases. Interestingly, 7 of 9 V2-J junctions (78%) contained the J29 gene segment. The remaining 2 V2-J rearrangements contained J48 and J54, respectively. A D3 gene segment was identified in 6 V2-J junctions. Based on combined Southern blot and PCR assessment, monoclonality in the TCRD/A locus was assumed in all except 1 precursor-B–ALL case with a subclonal V2-J rearrangement. V2-J rearrangements in normal lymphoid tissues Using RQ-PCR assays with the germline V2 forward primer, the germline V2 TaqMan probe, and 1 of 4 reverse germline J primers (J61, J58, J29, and J9), according to the most frequent V2-J rearrangements in precursor-B–ALL, we demonstrated that such preferential J usage is not characteristic for normal lymphoid tissues. Relatively high levels of V2-J58 and V2-J61 rearrangements (10–3 to 10–2) were only found in thymus samples (Table 1 and Figure 3). Ten-fold lower levels (10–4 to 10–3) were repeatedly detected in PB, particularly in a fraction of E-rosette–selected T cells. Lower frequencies (generally 10–4 or less) of V2-J58 and V2-J61 rearrangements were detected in normal BM, lymph nodes, and tonsils. V2-J29 rearrangements were found in thymus, lymph node, and tonsil samples at very low levels (10–5 to 10–4) but were virtually absent in BM and PB. V2-J9 rearrangements were virtually undetectable in all tested normal lymphoid samples, including the thymus (Table 1). View this table: [in this window] [in a new window]   Table 1.. V2-J rearrangements in normal lymphoid tissues as compared with precursor-B—ALL   View larger version (32K): [in this window] [in a new window]   Figure 3.. RQ-PCR analysis of V2-J rearrangements. (A) Schematic representation of RQ-PCR analysis of V2-(D3)-J rearrangements. The positions and sequences of the germline V2 TaqMan probe, germline V2 forward primer, and 4 germline J primers are indicated. (B) Real-time amplification plots of the serial dilutions of a precursor-B–ALL DNA containing clonal V2-J61 gene rearrangement into DNA of the cell line CEM, known to have 2 deleted TCRD alleles. RQ-PCR analysis was performed using the germline V2 TaqMan probe, the V2 forward primer, and the J61 primer. Relatively high levels of V2-J61 rearrangements were found in thymus (6 x 10–3). Such rearrangements were also detectable in normal BM, albeit at low levels (less than 10–4). (C) Real-time amplification plots of the serial diagnosis DNA dilutions into MNC DNA in precursor-B–ALL. RQ-PCR analysis by use of the TaqMan technique was performed using a V2-J56 rearrangement with the junctional region-specific primer approach. The reproducible sensitivity in this case reached 10–5.   Stability of V2-J rearrangements in 43 precursor-B–ALL at relapse Forty-three relapsed precursor-B–ALL cases with a total of 56 clonal V2-J rearrangements at diagnosis were also evaluated at relapse. Altogether, 34 of the 56 (61%) clonal V2-J rearrangements found at diagnosis were stable at relapse. In 28 precursor-B–ALL (65%), at least 1 V2-J rearrangement was preserved at relapse. The stability was markedly different between monoclonal and oligoclonal V2-J gene rearrangements, with 21 of 24 monoclonal V2-J joinings being stable (88%) as compared with only 13 of 32 oligoclonal rearrangements (41%). Owing to clonal evolution phenomena, 22 V2-J rearrangements were lost in 18 precursor-B–ALL. In 13 cases, this concerned either "regression" of (sub)clonal rearrangements to germline configuration or disappearance (deletion) of the V2-J joinings, probably owing to secondary V-J recombinations. In 5 precursor-B–ALL, new V2-J rearrangements were detected at relapse. In 1 of these 5 cases, the V2-J23 sequence at diagnosis and the V2-J29 sequence at relapse shared a common V2-D3 stem, confirming their origin from a common (pre)leukemic progenitor cell with a V2-D3 rearrangement. In the remaining 4 cases, the V2-D3 junctional regions and the J segments of the V2-J rearrangements at diagnosis and at relapse were completely different, suggesting that the common leukemic progenitor probably had germline TCRD genes. It is tempting to speculate that some of the new V2-J rearrangements at relapse might have been already present at diagnosis at low frequency as has been described in literature for other immunoglobulin (Ig)/TCR gene rearrangements.39-41 V2-J rearrangements as MRD-PCR targets in precursor-B–ALL V2-J rearrangements were tested as MRD-PCR targets in TaqMan-based RQ-PCR assays employing the germline V2 forward primer and the germline V2 TaqMan probe together with ASO reverse primers. In 21 of 32 cases (66%), a quantitative range of 10–4 was achieved at the routine annealing temperature of 60° C (ie, requiring no optimization). In 27 of 32 cases (84%) the sensitivity was at least 10–4. Very low levels of background amplification in normal MNCs was found in only 7 cases (22%). If observed, limited sensitivity of the MRD-PCR assay was mainly due to the presence of the V2-J rearrangement in a subclone only, as indicated by the relatively high threshold cycle (CT) value of the 10–1 dilution.    Discussion Top Abstract Introduction Patients, materials, and methods Results Discussion References   Our study indicates that the V2 gene segment is a "hot spot" for V(D)J recombination in precursor-B–ALL. This single gene segment is involved in various gene rearrangements in approximately 70% of precursor-B–ALL. By combined Southern blot and PCR analyses, V2-D3 joinings were found in 40%, whereas V2-J rearrangements were found in 42% of precursor-B–ALL. In 27% of cases, V2-D3 and V2-J joinings occurred simultaneously. The junctional regions of most (79%) V2-J rearrangements contained the D3 segment, which indicates that recombination to J was preceded by a V2-D3 rearrangement (Figure 1). D2-D3 rearrangements were found in only 10% of precursor-B–ALL, and the D2 segment was found in only 8% of V2-J junctional regions. Clonal D2-J rearrangements occur even more seldom, because we were able to detect clonal D2-J PCR products in only 3 precursor-B–ALL (less than 2%). Thus, V2, D3, and several J genes are preferentially involved in recombinations in the TCRD/A locus in precursor-B–ALL, with the main pathway being V2-D3 V2-D3-J (Figure 1). The next step might concern secondary V-J rearrangements, deleting the whole TCRD locus as well as preexisting V2-J joinings.5,14 The limited number of TCRD/A gene segments involved in these rearrangements may be explained by differential accessibility of gene segments within the TCRD locus in precursor-B–ALL. Some TCRD regions, particularly V1 and all J gene segments, seem to be fully closed for the persistent activity of the V(D)J recombinase in precursor-B–ALL, because rearrangements involving these gene segments were reported only anecdotally.42-44 The spectrum of J segment usage in V2-J rearrangements in precursor-B–ALL was not random. The single J29 segment was found in 54% of all V2-J joinings. Such nonrandom usage of J segments was previously suggested by Southern blot data but was never confirmed at the PCR and sequence level.4,13 The remaining V2-J sequences contained 26 different J segments, most of them belonging to 2 additional clusters. The preferential usage of J29 might be related to the fact that the recombination signal sequence (RSS) of J29 is fully identical to the consensus RSS. However, no preferential usage was found for the other 2 J gene segments with a full consensus RSS (ie, J15 and J34). Apparently, a combination of several factors determines the preferential usage of several J gene segments, such as (1) proximity to the TCRD locus (eg, for J61, J58, J54, and J48); (2) leukemia-associated differential accessibility, potentially related to specific (yet unknown) transcription factors; and (3) presence of a consensus RSS. V2-J rearrangements can occur at low levels in normal lymphoid tissues (Table 1). They are relatively frequent in the thymus, where they might represent an infrequent TCRD deletion pathway for commitment to the TCR lineage.15,16,45 Most of the V2-J gene rearrangements in the thymus involved the most proximal J genes (in our study represented by J58 and J61),15,16 and the frequency of such recombinations ranged from 10–3 to 10–2. The same spectrum of rearrangements was detectable at more than 10-fold lower levels (ie, less than 10–3) in other lymphoid tissues, including PB MNCs, BM, lymph nodes, and tonsils. In striking contrast, V2-J29 and V2-J9 joinings were virtually undetectable in normal lymphoid cells (Table 1). This suggests that the preferential usage of the J9 and J29 clusters is a leukemia-specific characteristic in precursor-B–ALL. Our multiplex PCR strategy can easily identify clonal V2-J rearrangements that can be applied as PCR targets for MRD monitoring.46 In fact, based on the limited spectrum of V2-J rearrangements in precursor-B–ALL, the assay for V2-J detection can be further simplified. Using 2 tubes, one with V2 and J29 primers and the second with V2 and 12 J primers (J9, J30, J48, J49, J52, J54, J55, J56, J57, J58, J59, J61), we could reliably detect 87% of V2-J rearrangements (data not shown). Because the junctional regions of V2-J joinings are extensive, it is relatively easy to design optimal patient-specific oligonucleotides reaching sensitivities of at least 10–4, which is required for reliable recognition of the MRD-based risk groups.47,48 Another advantage of V2-J rearrangements as MRD-PCR targets is the extremely low background of polyclonal V2-J joinings in normal BM and PB, irrespective of the treatment phase. Many V2-J joinings (about 45%) were oligoclonal, comparably to Ig heavy chain gene rearrangements (30% to 40%) and TCRD gene rearrangements (about 25%) in precursor-B– ALL.49,50 Monoclonal V2-J gene rearrangements are excellent MRD-PCR targets with good stability (88% of monoclonal rearrangements preserved at relapse) and high sensitivity of at least 10–4 in virtually all cases. The usage of oligoclonal V2-J rearrangements as MRD-PCR targets is not recommended owing to their low stability at relapse (41%). When the applied MRD-PCR strategy does not include Southern blotting for detection of oligoclonality, one might decide to use our germline V2-J RQ-PCR as used for characterization of polyclonal V2-J gene rearrangements in normal tissues (Figure 3). Based on the obtained high CT values (compared with monoclonal V2-J controls), it is possible to identify subclonal V2-J joinings when they contribute to less than 10% of the tumor load (data not shown). In conclusion, V2-J rearrangements are frequent cross-lineage recombinations in precursor-B–ALL, which is in striking contrast to their infrequent occurrence in normal B cells and B-cell precursors. The spectrum of V2-J rearrangements in precursor-B–ALL is not random with preferential usage of the J29 gene segment. The extensive junctional regions, the low background in normal BM and PB, and the high stability (88%) of monoclonal rearrangements are the features that favor the usage of monoclonal V2-J rearrangements as principal MRD-PCR targets in approximately 25% of precursor-B–ALL.    Acknowledgements   We are grateful to Prof dr R. Benner and Prof dr D. Sota-Jakimczyk for their continuous support; Dr A.W. Langerak for critical reading of the manuscript; Mrs J. M. Wijkhuijs, Mrs P. Hart, and Mrs I. L. M. Wolvers-Tettero for excellent technical assistance; and Mrs W. M. Comans-Bitter for preparation of the figures. We thank the Dutch Childhood Oncology Group for kindly providing childhood precursor-B–ALL cell samples. The clinicians of HOVON-17 study are acknowledged for providing adult precursor-B–ALL samples.    Footnotes   Submitted August 29, 2003; accepted November 30, 2003. Prepublished online as Blood First Edition Paper, December 4, 2003; DOI 10.1182/blood-2003-08-2952. Supported by the Dutch Cancer Foundation/Koningin Wilhelmina Fonds (grants SNWLK 97-1567 and SNWLK 2000-2268). The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734. Reprints: J. J. M. van Dongen, Dept of Immunology, Erasmus MC, University Medical Center Rotterdam, Dr Molewaterplein 50, 3015 GE Rotterdam, the Netherlands; e-mail: j.j.m.vandongen{at}erasmusmc.nl.    References Top Abstract Introduction Patients, materials, and methods Results Discussion References   Davis MM, Björkman PJ. T-cell antigen receptor genes and T-cell recognition. Nature. 1988;334: 395-402.[CrossRef][Medline] [Order article via Infotrieve] Van Dongen JJM, Comans-Bitter WM, Wolvers-Tettero ILM, Borst J. Development of human T lymphocytes and their thymus-dependency. 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