Comparative analysis of T-cell receptor gene rearrangements at diagnosis and relapse of T-cell acute lymphoblastic leukemia (T-ALL) shows high stability of clonal markers for monitoring of minimal residual disease and reveals the occurrence of second T-ALL

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Abstract

A total of 28 children and nine adults with relapsed T-ALL were analyzed for the configuration of their T-cell receptor (TCR) and TAL1 genes at diagnosis and relapse to evaluate their stability throughout the disease course. A total of 150 clonal TCR and TAL1 gene rearrangements were identified in the 37 patients at diagnosis. In 65% of cases all rearrangements and in 27% of cases most rearrangements found at diagnosis were preserved at relapse. Two children with unusually late T-ALL recurrences displayed completely different TCR gene rearrangement sequences between diagnosis and relapse. This indicates that a proportion of very late T-ALL recurrences might represent second T-ALL. Specifically, 88% of clonal rearrangements identified at diagnosis in truly relapsed T-ALL were preserved at relapse. This is significantly higher as compared to previously studied precursor-B-ALL (70%). Thus, from biological point of view, immunogenotype of T-ALL is more stable as compared with precursor-B-ALL. The overall stability of TCR gene rearrangements was higher in adult T-ALL (97%) than in childhood T-ALL (86%). Based on the stability of TCR gene rearrangements, we propose a strategy for PCR target selection (TCRD+TAL1TCRBTCRG), which probably allows reliable minimal residual disease detection in all T-ALL patients.

Introduction

Detection of low levels of malignant cells for evaluation of treatment effectiveness, known as minimal residual disease (MRD) monitoring, is becoming a routine diagnostic tool in the management of various hematopoietic malignancies.1 MRD information is particularly valuable for childhood acute lymphoblastic leukemia (ALL) patients as shown by several large multicenter prospective studies.2,3,4,5,6 Information on early response to induction treatment contributes to an improved definition of remission in ALL, and therefore MRD diagnostics is currently being incorporated into stratification of treatment protocols.7,8

In childhood ALL, detection of MRD most frequently relies on patient-specific junctional regions of immunoglobulin (Ig) and T-cell receptor (TCR) gene rearrangements (reviewed in Szczepański et al8). These leukemia-specific ‘fingerprints’ can be identified in virtually all ALL patients and they enable routine MRD detection with sensitivities of 10−4–10−5 (ie one malignant cell in the background of 104–105 normal cells). Clonal Ig and TCR gene rearrangements are easily identified with DNA techniques, including Southern blotting, polymerase chain reaction (PCR), and sequencing methods, and the results are reproducible between laboratories as proven by international standardization.9 With the advent of real-time quantitative PCR (RQ-PCR) analysis of Ig and TCR gene junctional regions, precise quantification of MRD levels is routinely achievable.10,11,12,13,14,15

One of the most important pitfalls of MRD detection in ALL using Ig/TCR gene rearrangements is clonal evolution caused by the persistent activity of the V(D)J recombinase machinery in leukemic blasts (reviewed in Szczepański et al16). Ongoing or secondary rearrangements might lead to the loss of MRD-PCR targets in ALL patients and consequently to false-negative results. Such clonal selection processes might even occur early during induction treatment hampering reliable stratification into MRD-based risk groups.17 Our extensive study in 96 precursor-B-ALL patients showed differences in Ig/TCR gene rearrangement patterns between diagnosis and relapse in 62% of patients.18 However, based on the stability of the individual Ig and TCR gene rearrangements, we proposed a stepwise strategy for selection of PCR targets enabling successful detection of MRD in the vast majority (95%) of precursor-B-ALL patients.

Although the presence of clonal evolution phenomena has also been reported in T-ALL, the studies comparing the TCR gene rearrangement patterns at diagnosis and relapse in T-ALL either did not compare junctional region sequences or were limited to TCR gamma (TCRG) and/or TCR delta (TCRD) gene loci.19,20,21,22 Therefore, we studied the stability of the TCR gene rearrangements currently used for MRD monitoring in T-ALL, that is, TCR beta (TCRB), TCRG, TCRD, and TAL1 gene rearrangements in a series of 37 relapsed T-ALL patients (28 children and nine adults). This information formed the basis for reliable selection of MRD-PCR targets in T-ALL patients with minimal chance of false-negative MRD results.

Patients, materials, and methods

Patients

Bone marrow, peripheral blood samples, or lymph node biopsy from 28 children with T-ALL were obtained at initial diagnosis and at relapse. The age distribution ranged from 2 years until 15.9 years. The diagnosis of T-ALL was made according to FAB and standard immunophenotypic criteria.23,24 Immunophenotyping of the childhood T-ALL revealed that 18 (64%) were CD3 T-ALL, eight (29%) were TCRαβ+ T-ALL, and two (7%) were TCRγδ+ T-ALL. Three children were studied at two subsequent leukemia relapses.

In addition, diagnosis and relapse samples could be obtained from nine adult T-ALL patients aged 18 until 49 years.

The rationale, methodology and pitfalls of the stepwise molecular comparison of the Ig/TCR gene rearrangements between diagnosis and relapse samples were described in detail in our earlier precursor-B-ALL study.25

Comparative Southern blot analysis

Mononuclear cells (MNC) were separated from bone marrow or peripheral blood samples by Ficoll-Paque centrifugation (density 1.077 g/cm;3 Pharmacia, Uppsala, Sweden). DNA was isolated from frozen MNC, digested with the appropriate restriction enzymes, and blotted to nylon membranes as described previously.26 TCRB gene rearrangements were detected with TCRBJ1, TCRBJ2, and TCRBC probes (DAKO Corporation, Carpinteria, CA, USA) in EcoRI and HindIII digests.27 The configuration of the TCRD genes was analyzed with the TCRDJ1 probe (DAKO) in EcoRI and HindIII digests.28 The TCRG gene configuration was studied using the TCRGJ13 probe (DAKO) in EcoRI digests together with either the TCRGJ21 probe (DAKO) in PstI digests or a combination of the Jγ2.1 probe in EcoRI digests and the Jγ1.2 probe in BglII digests.29,30 The diagnosis and relapse samples were always run in parallel lanes. The Southern blot configuration of the TCR genes in 14 patients at diagnosis and in 10 patients at relapse has been reported previously.19,27,28,31

The comparative Southern blot analyses could only be performed in the 28 childhood T-ALL cases, because no sufficient DNA could be obtained from both the diagnosis and the relapse samples in the nine adult T-ALL cases.

PCR amplification and comparative heteroduplex analysis of PCR products

PCR analysis was performed on both diagnosis and relapse samples in all childhood and adult T-ALL patients as described previously.9,32 The sequences of the oligonucleotides used for amplification of TCRG (four Vγ family-specific forward primers, six VγI member-specific forward primers, and three reverse Jγ primers), TCRD (Vδ1-Jδ1, Vδ2-Jδ1, Vδ3-Jδ1, Dδ2-Jδ1, Vδ2-Dδ3, and Dδ2-Dδ3 primer pairs) and TAL1 gene rearrangements were published before.9 In each 50 μl PCR reaction, 50 ng DNA sample, 6.3 pmol of the forward and reverse primers, and 0.5 U AmpliTaq Gold polymerase (PE Biosystems, Foster City, CA, USA) were used. PCR conditions were: initial denaturation for 10 min at 94°C, followed by 40 cycles of 45 s at 92°C, 90 s at 60°C, and 2 min at 72°C using a Perkin-Elmer 480 thermal cycler (PE Biosystems). After the last cycle, an additional extension step of 10 min at 72°C was performed. Appropriate positive and negative controls were included in all experiments.9

Identification of clonal TCRB gene rearrangements was based on multiplex strategy using 23 Vβ, two Dβ, and 13 Jβ primers as developed by BIOMED-2 Concerted Action ‘PCR-based clonality studies for early diagnosis of lymphoproliferative disorders’ (Van Dongen et al, submitted for publication).

For heteroduplex analysis, the PCR products were denatured at 94°C for 5 min after the final cycle of amplification and subsequently cooled to 4°C for 60 min to induce duplex formation.33 Afterwards, the duplexes were immediately loaded on 6% nondenaturing polyacrylamide gels in 0.5 × Tris-borate-EDTA (TBE) buffer, run at room temperature, and visualized by ethidium bromide staining.33 For identification of gene segments involved in clonal TCRB gene rearrangements, homoduplexes of appropriate size were excised from the polyacrylamide gel and eluted as described previously.34,35 The eluted PCR products were directly sequenced either with Dβ or multiplex Vβ primers (Van Dongen et al, submitted for publication).

Relapse samples were at first analyzed 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 means of mixed heteroduplex analysis, that is, mixing of the diagnosis and relapse PCR products followed by heteroduplex analysis (Figure 1).18,25 When clonal PCR products found at diagnosis were undetectable at relapse, the relapse sample was analyzed with additional primer combinations for the involved gene loci.

Figure 1
figure1

Examples of comparative heteroduplex PCR analysis. (a) Comparative heteroduplex PCR analysis of TCRD gene rearrangements. Monoclonal homoduplexes (ho) in patients 6329, 6584, and 6787 found at diagnosis and at relapse were of the same size. Mixing of the PCR products of these disease phases followed by heteroduplex PCR analysis demonstrated no heteroduplex (he) formation, proving that these gene rearrangements had identical junctional regions. (b) Comparative heteroduplex PCR analysis of TCRG gene rearrangements showed identical rearrangements at diagnosis and relapse in patients 4643 and 6329. In patients 3025 and 6953, monoclonal homoduplexes found at diagnosis and at relapse slightly differed in size. Mixing of the Vγ–Jγ PCR products followed by heteroduplex PCR analysis demonstrated clear heteroduplex formation, proving that these Vγ–Jγ gene rearrangements had different junctional regions. (c) Comparative heteroduplex PCR analysis of TCRB gene rearrangements. Patients 5370, and 5550 with mono and biallelic rearrangements, respectively, had stable Vβ–Jβ rearrangements. In contrast, in patient 5598 with presumed second T-ALL, monoclonal Dβ1–Jβ1.2 homoduplexes found at diagnosis and at relapse had different junctional regions confirmed by heteroduplex formation after mixing of the PCR products. (d) Comparative heteroduplex PCR analysis of Dβ2–Jβ2.7 gene rearrangements. Patients 3288 and 4564 with monoallelic rearrangements had stable Dβ2–Jβ2.7 rearrangements between diagnosis and relapse. In patient 5598 with presumed second T-ALL, Dβ2–Jβ2.7 joinings at diagnosis and relapse were different as shown by mixed heteroduplex analysis. Patient 3810 was negative for Dβ2–Jβ2.7 rearrangement at diagnosis but had identical Dβ2–Jβ2.7 rearrangements in two subsequent relapse samples.

Sequence analysis of Ig/TCR gene rearrangements

Clonal PCR products discordant between diagnosis and relapse were directly sequenced. Sequencing was performed using the dye-terminator cycle sequencing kit with AmpliTaq DNA polymerase FS on an ABI 377 sequencer (PE Biosystems) as described before.36 Vβ, Dβ, and Jβ segments were identified using DNAPLOT software (W Müller, H-H Althaus, University of Cologne, Germany) by searching for homology with all known human germline Vβ, Dβ, and Jβ sequences obtained from the IMGT directory of human TCR genes (http://imgt.cines.fr).36 Vγ, Vδ, Dδ, Jγ, and Jδ gene segments were identified by comparison to germline TCRG and TCRD sequences as described before.37

Results

PCR detectability of TCR gene rearrangements in relapsed T-ALL patients

In the 28 childhood T-ALL cases, a total of 120 clonal TCR and TAL1 gene rearrangements were identified at diagnosis with an average of 4.3 per patient and a range from two to six rearrangements per patient. In the nine adult T-ALL patients, we identified a total of 30 clonal TCR gene rearrangements at diagnosis with an average of 3.3 per patient and a range from two to five rearrangements per patient. Consequently, in each T-ALL patient, at least two MRD-PCR targets were available.

TCR gene rearrangement patterns in two children suspected of a second T-ALL

Within the studied T-ALL group, two patients (3025 and 5598) displayed completely different TCR gene rearrangement sequences between diagnosis and presumed relapse (three and six rearrangements lost, respectively), which we considered highly suggestive of a second ALL (Table 1). Moreover, the emergence of a new chromosome aberration, that is, t(11;14), at relapse in patient 5598 indicated the development of a second malignancy. Both patients experienced very late relapses at 6 and 10 years from initial diagnosis, respectively. This is in striking contrast to the remaining 26 patients, who relapsed within 37 months from diagnosis (mean 14 months). Therefore, we concluded that these two patients developed a second T-ALL, and consequently they were excluded from further calculations on the stability of the rearrangements.

Table 1 TCR gene rearrangement patterns in the two childhood T-ALL patients suspected of second T-ALL

Stability of gene rearrangements in childhood T-ALL patients at relapse

A total of 95 (86%) of 111 clonal TCR and TAL1 gene rearrangements identified with heteroduplex PCR analysis at diagnosis in the 26 children with truly relapsed T-ALL were preserved at relapse (Figure 1). This concerned 17 of 17 (100%) TCRD, 42 of 49 (86%) TCRG, 35 of 44 (80%) TCRB gene rearrangements, and a single TAL1 gene rearrangement (Table 2).

Table 2 Stability of MRD-PCR targets in 35 truly relapsed T-ALL patients

In 16 children (62%), all TCR gene rearrangements identified at diagnosis were preserved at relapse (Figure 2a). In nine cases, some targets (one or two) were lost during the disease course (Figure 2b). Finally, in one patient (6953), only one incomplete TCRB rearrangement was common for both the diagnosis and the relapse sample, whereas the other three TCR gene rearrangements were absent at relapse (Figure 2b). Consequently, at least one rearrangement was preserved at relapse in all 26 childhood T-ALL cases.

Figure 2
figure2

TCR gene rearrangement stability patterns in 35 T-ALL patients (26 children and nine adults). Black squares represent complete rearrangements, while gray squares reflect incomplete rearrangements. All squares above the (upper) line represent stable MRD-PCR targets, while all squares below the (lower) line represent TCR gene rearrangements lost owing to clonal evolution. β squares between the two lines have common Dβ–Jβ stems shared by different TCRB rearrangements at diagnosis and at relapse. (a) A total of 16 childhood T-ALL patients with all rearrangements identified at diagnosis preserved at relapse, (b) nine childhood T-ALL cases with some TCR gene rearrangements (one or two) lost during the disease course and one patient (6953) with the majority of clonal markers absent at relapse, (c) nine adult T-ALL patients with 29 of the 30 rearrangements identified at diagnosis preserved at relapse. β, TCRB gene rearrangements; δ, TCRD gene rearrangements; γ, TCRG gene rearrangements; T, TAL1 gene rearrangements.

Clonal evolution was observed in seven of 16 patients with CD3 T-ALL (44%), two of eight patients with TCRαβ+ T-ALL (25%), and one of two TCRγδ+ T-ALL (50%). The CD3 group can be further subdivided based on the configuration of the TCRD genes. Clonal evolution was more frequent in T-ALL with biallelic TCRD deletions (two of three cases; 67%) as compared with five of 13 T-ALL with at least one TCRD gene rearrangement (38%), but this difference did not reach statistical significance (P>0.05, χ2 test).

It should be noted that in the eight TCRαβ+ T-ALL, at least one TCRB rearrangement (in six patients, all TCRB rearrangements) remained stable, and that also in the two TCRγδ+ T-ALL, no changes were observed in the TCRG or TCRD gene rearrangements. Apparently, the expressed TCR alleles were not affected by continuing rearrangements.

Stability of TCR gene rearrangements in adult T-ALL patients at relapse

Comparative heteroduplex PCR analysis demonstrated that 29 (97%) of the 30 clonal TCR gene rearrangements were also present at relapse (Figure 2c).

All three TCRD rearrangements and all 15 TCRG gene rearrangements remained stable, whereas only one of the 12 TCRB gene rearrangements was lost at relapse. This concerned one of the two incomplete TCRB gene rearrangements in patient 7498. Interestingly, in one patient (case 6714), two different clonal Vβ–Jβ rearrangements at diagnosis shared a common Dβ–Jβ stem, which were both present at relapse albeit at different density of the PCR signals in heteroduplex analysis and GeneScanning (data not shown). This suggested the presence of two related subclones with differential outgrowth at relapse.

Patterns of clonal evolution of TCR gene rearrangements in relapsed childhood T-ALL

Based on combined Southern blot, PCR, and sequence analysis, it was possible to follow the patterns of clonal evolution leading to the disappearance of rearrangements, which were originally present at diagnosis.

TCRD gene rearrangements

A total of 17 clonal TCRD gene rearrangements (10 Vδ1–Jδ1, four Dδ2–Jδ1, one Vδ2–Jδ1, one Vδ3–Jδ1, and one Vδ2–Dδ3) were identified by PCR in 13 childhood T-ALL patients. All 17 clonal rearrangements were preserved at relapse. In four additional patients, PCR analysis did not result in identification of clonal TCRD rearrangements, while Southern blot data showed rearrangements to the Dδ3/Jδ1 region, which could not be assigned to a particular Vδ–Jδ joining based on the size of the clonal bands. Identically rearranged bands, most probably representing Vα–Jδ1 rearrangements or translocations into the TCRD locus, were also present in these four patients at relapse.

Clonal evolution in TCRG locus

A total of 49 TCRG gene rearrangements were detected at diagnosis in all 26 childhood T-ALL patients, and in 23 cases (89%) at least one TCRG gene rearrangement was preserved at relapse. Clonal evolution of TCRG gene rearrangements was observed in five patients, leading to loss of seven MRD-PCR targets. In two patients (5775, 6584), this concerned ‘regression’ of clonal rearrangements most probably to germline configuration. In one patient (4643), Southern blot data indicated the deletion of a second allele. In another patient (4564), the two new rearrangements at relapse contained upstream Vγ and downstream Jγ segments as compared to the Vγ–Jγ rearrangements at diagnosis, which is suggestive of ongoing recombination with Vγ–Jγ replacement. Finally, in the fifth patient (6953), the sequence comparison of Vγ–Jγ rearrangements at diagnosis and at relapse excluded secondary rearrangements and indicated the emergence of a clone related to the initial (pre)leukemic clone but different from the predominant clone at diagnosis.

Clonal evolution in TCRB locus

A total of 44 TCRB gene rearrangements were detected at diagnosis in 25 childhood T-ALL cases, and in 23 cases (92%) at least one MRD-PCR target was preserved at relapse. Clonal evolution of TCRB gene rearrangements was observed in six patients leading to loss of nine MRD-PCR targets. In two patients (3288 and 6329), combined Southern blot and PCR information showed that subclones found at diagnosis disappeared at relapse. In one patient (6090), the configuration at relapse reflected ongoing Vβ to Dβ2–Jβ2 joining, deleting the Vβ–Jβ1 rearrangement present at diagnosis (Figure 3). In two patients (3810 and 6953), the sequences of the Vβ–Jβ rearrangements at diagnosis and relapse contained common Dβ–Jβ stems, which confirmed their origin from a common (pre)leukemic progenitor with such Dβ–Jβ rearrangement (Figure 3). Finally, in one TCRγδ+ T-ALL patient (5158), the sequences of the TCRB gene rearrangements at diagnosis and at relapse were completely different but the TCRG and TCRD rearrangements were identical, suggesting that the presumed leukemic progenitor probably had germline TCRB genes.

Figure 3
figure3

Examples of clonal evolution in TCRB locus. (a) On one TCRB allele in patient 3810, the Vβ20.1–Jβ2.3 rearrangement at diagnosis and the Vβ4.1–Jβ2.3 rearrangement at relapse had identical Dβ–Jβ2.3 junctions. The Dβ2–Jβ2.1 rearrangement at diagnosis and the Dβ2–Jβ2.7 rearrangement at both relapses were not related. This indicates the origin of clones at diagnosis and at relapses from a common (pre)leukemic clone with a Dβ–Jβ2.3 rearrangement on one allele and a second TCRB allele in germline configuration. (b) In patient 6953, the Vβ29–Jβ1.3 rearrangement at diagnosis and the Vβ12.4–Jβ1.3 rearrangement at relapse had identical Dβ–Jβ1.3 junctions. On the same allele, the Dβ2–Jβ2.1 rearrangement identified at diagnosis was preserved at relapse. This indicates that the origin of clones at diagnosis and at relapses form a common (pre)leukemic clone with double Dβ1–Jβ1.3 and Dβ2–Jβ2.1 rearrangements on one allele. The second allele at diagnosis was in germline configuration. (c) Example of complex secondary rearrangement and ongoing Vβ to Jβ joining in patient 6090. At diagnosis, two rearrangements were found on one allele: Vβ7.4–Jβ1.6 and Dβ2–Jβ2.3. Ongoing rearrangement of the Vβ6.5 segment, located immediately upstream to the Vβ7.4 segment, to Dβ2 resulted in a new Vβ6.5–Jβ2.3 rearrangement with a preserved Dβ2–Jβ2.3 stem at relapse. The configuration on the second TCRB allele (Dβ1–Jβ2.2 rearrangement) was identical at diagnosis and relapse.

An additional example of clonal evolution in the TCRB genes was found at relapse of patient 5598. Two of the oligoclonal rearrangements at relapse (Vβ20.1–Jβ1.2 and Dβ1–Jβ1.2) contained identical Dβ1–Jβ1.2 junctions, which is consistent with ongoing Vβ to Dβ–Jβ joining.

Stability of TCR gene rearrangements between consecutive relapses

The configuration of TCR genes compared between two subsequent relapses in the three patients analyzed showed no evidence for clonal evolution, while in two of the three cases we observed some changes in gene rearrangement patterns (loss of two TCRB gene rearrangements in both cases) between diagnosis and first relapse (Figure 3).

Discussion

When compared to precursor-B-ALL, T-ALL are generally characterized by more high-risk clinical features at presentation and by a more aggressive clinical course.38,39 This is also clearly visible in the MRD kinetics during the first year of treatment in childhood T-ALL: the frequency of MRD-positive patients and the MRD levels are generally higher in T-ALL than in precursor-B-ALL, reflecting the more frequent occurrence of resistant disease in T-ALL.6 This more aggressive disease kinetics is also associated with shorter progression-free survival in T-ALL patients.6,40 In fact, the vast majority of T-ALL relapses occur within the first 2–3 years from diagnosis.40,41

Surprisingly, two children in our study with very late T-ALL relapses (6 and 10 years from initial diagnosis) showed fully changed TCR gene configurations, very much suggestive of a second leukemia (Table 1). This was in striking contrast to the other 26 childhood and nine adult T-ALL cases, which showed moderate-to-high stability of the identified MRD-PCR targets. The only alternative explanation for such different TCR gene rearrangement patterns would be the emergence of clones at diagnosis and relapse from a common progenitor with all TCR genes in germline configuration. The second character of one of the two T-ALL was further supported by the finding of a novel cytogenetic aberration at relapse. Also the early treatment response in the latter patient, as assessed by MRD kinetics, was very good with MRD negativity at 5 and 14 weeks after diagnosis (data not shown). In patients with such a good response to treatment, the risk of relapse is extremely low.6 Consequently, we have excluded these two patients from our calculations on the stability of TCR gene rearrangements.

Secondary ALL are rare, comprising only 5–10% of secondary leukemias and second T-ALL was previously described only anecdotally and never in association with a primary T-ALL.42,43,44,45,46 In fact, our hypothesis that a proportion of very late T-ALL recurrences represent second T-ALLs has never been put forward previously. In contrast, LoNigro et al22 studied TCRD and TCRG gene configurations in two relapsed T-ALL patients with remission duration of 72 and 77 months and found in both patients at least one identical clonal TCR gene rearrangement at diagnosis and at relapse. Therefore, it would be of great importance to study more patients with very late T-ALL recurrences to address the question of second T-ALL in this group. In fact, such patients might have an inherited predisposition for T-ALL development.

Our comparative Southern blot, PCR, and sequencing analyses of T-ALL at diagnosis and relapse have provided detailed insight into the stability and changes of TCR gene rearrangements during the disease course. This information is essential for application of such clonal rearrangements as PCR targets in MRD studies in T-ALL. Our previous study in a large group of childhood precursor-B-ALL patients demonstrated extreme clonal evolution in approximately 20% of patients, with all MRD-PCR targets lost in 7% of patients.18 In contrast, the loss of virtually all MRD-PCR targets was observed in only one of the 35 truly relapsed T-ALL patients (patient 6953 in Figure 2b). Also the proportion of patients with all MRD-PCR targets preserved at relapse was markedly higher in T-ALL (69%) as compared to precursor-B-ALL (40%).

DNA breakpoints of chromosome aberrations are ideal targets for MRD detection, since they are linked to the oncogenic process and are therefore stable throughout the disease course.47 However, such aberrations, for example, TAL1 gene rearrangements, can be identified in only 10–25% of T-ALL patients.48,49 Only one T-ALL case in our study group had a TAL1 gene rearrangement with an identical breakpoint sequence at diagnosis and at relapse.

When analyzing the stability of particular MRD-PCR targets, TCRD gene rearrangements were uniformly preserved at relapse in all 16 T-ALL patients. Recombination of the TCRD locus is presumed to be one of the earliest events in T-cell development in the thymus. In line with this observation, virtually all (>95%) T-ALL have rearranged or deleted TCRD genes on at least one allele.28 The TCRD gene rearrangements might be lost during the disease course via ongoing recombination in the TCRA locus, thereby deleting the TCRD genes.50 Such clonal evolution at relapse has indeed been described in two patients, associated in one case with a phenotypic shift from CD3 T-ALL to TCRαβ+ T-ALL.21,22 Nevertheless, in concordance with our study, the vast majority of previously described T-ALL patients analyzed at diagnosis and relapse showed a fully stable TCRD configuration.19,21,22

In contrast to TCRD gene rearrangements, which are detectable in only half of the T-ALL patients, TCRG gene rearrangements are present in approximately 95% of T-ALL patients31 and accordingly could be identified in 34 out of 35 patients in our study. In 85% (29 of 34) of patients, the TCRG gene configuration was fully identical between diagnosis and relapse, and in 91% (31 of 34) of patients at least one TCRG rearrangement was preserved. We could have anticipated such high stability from our earlier finding that most TCRG gene recombinations in T-ALL are end-stage rearrangements.31 Nevertheless, in a subgroup of patients, we and others20 observed clonal ‘regressions’ to a less mature configuration, which in fact represented the outgrowth of a different subclone at relapse. In these cases (patients 5775, 6584, and 6953), the oncogenic event most probably occurred before the TCRG gene rearrangement. Another potential disadvantage of using TCRG junctions as MRD-PCR targets might be their limited sensitivity owing to the limited size of TCRG junctional regions and the abundant background of polyclonal TCRG gene rearrangements in normal T cells.14,51 The currently used RQ-PCR techniques aim at sensitivities of 10−4, but in approximately one-third of TCRG gene rearrangements in T-ALL such sensitivity cannot be easily reached.14

The development of multiplex PCR approaches for detection of TCRB gene rearrangements resulted in their introduction as additional PCR targets for MRD detection (Van Dongen et al, submitted for publication). Clonal TCRB gene rearrangements were identified in 34 of the 35 patients in our series. Although their overall stability (46 of 56; 82%) was inferior to the stability of TCRG gene rearrangements (89%) and TCRD gene rearrangements (100%), at least one TCRB PCR target was preserved at relapse in 94% (32 of 34) of patients. Particularly, complete Vβ–Jβ rearrangements are attractive MRD PCR targets because of their extensive junctional regions, which guarantee good sensitivities in RQ-PCR analysis in virtually all patients (Brüggemann et al, submitted for publication). However, we observed several examples of clonal evolution phenomena owing to continuing rearrangements as well as resulting from the selection of subclones with partly related or unrelated TCRB genes (Figure 3). In patients with extreme clonal evolution, the TCRB gene rearrangements might represent postoncogenic events (see Figure 3), which might be related to the observation that TCRB gene rearrangements occur relatively late during T-cell differentiation.52,53

The overall stability of TCR gene rearrangements was higher in relapsed adult T-ALL (29 of 30; 97%) as compared to truly relapsed childhood T-ALL (95 of 111; 86%). Consequently, most adult T-ALL patients (eight of nine) preserved all potential MRD-PCR targets at relapse, while this was found in 62% (16 of 26) childhood T-ALL patients.

Based on the comparative analysis of the TCR gene configuration at diagnosis and relapse of T-ALL, we propose a stepwise strategy for MRD-PCR target selection. Although in our study at least one target was preserved in all patients, we recommend that two MRD-PCR targets should be used per patient. TCRD and TAL1 gene rearrangements are highly stable targets and should be selected as first choice (Figure 4). TCRB gene rearrangements are slightly more stable at the patient level than TCRG gene rearrangements and are characterized by better sensitivity in RQ-PCR-based MRD assays and therefore should be selected as second choice target. If possible, patient-specific oligonucleotides should be positioned on Dβ–Jβ junctions to prevent false-negative results owing to ongoing Vβ to Dβ–Jβ rearrangements. Finally, TCRG gene rearrangements have a fair stability but are less sensitive RQ-PCR targets and should therefore be selected as third-choice MRD-PCR targets. In our study group, such stepwise strategy (TCRD+TAL1TCRBTCRG) would have enabled successful detection of MRD in all T-ALL patients.

Figure 4
figure4

Flow diagram for the stepwise selection of PCR targets for MRD detection in T-ALL.

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Acknowledgements

We are grateful to Professor Dr R Benner and Professor Dr D Sońta-Jakimczyk for their continuous support, and Mrs WM Comans-Bitter for preparation of the figures.

We thank the Board and the Clinicians of the Dutch Childhood Oncology Group for kindly providing T-ALL cell samples.

This study was supported by the Dutch Cancer Foundation/Koningin Wilhelmina Fonds (Grants SNWLK 97-1567 and SNWLK 2000-2268).

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Correspondence to J J M van Dongen.

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Szczepański, T., Velden, V., Raff, T. et al. Comparative analysis of T-cell receptor gene rearrangements at diagnosis and relapse of T-cell acute lymphoblastic leukemia (T-ALL) shows high stability of clonal markers for monitoring of minimal residual disease and reveals the occurrence of second T-ALL. Leukemia 17, 2149–2156 (2003) doi:10.1038/sj.leu.2403081

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Keywords

  • TCR gene rearrangements
  • T-ALL
  • clonal evolution
  • minimal residual disease
  • second T-ALL

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