The BIOMED-2 Concerted Action BMH4-CT98-3936 on ‘Polymerase chain reaction (PCR)-based clonality studies for early diagnosis of lymphoproliferative disorders’ developed standardized PCR protocols for detection of immunoglobulin (Ig) and T-cell receptor (TCR) rearrangements, including TCR beta (TCRB). As no comparable TCRB PCR method pre-existed and only a limited number of samples was tested within the BIOMED-2 study, we initiated this study for further validation of the newly developed TCRB PCR approach by comparing PCR data with previously generated Southern blot (SB) data in a series of 66 immature (ALL) and 36 mature T-cell malignancies. In 91% of cases, concordant PCR and SB results were found. Discrepancies consisted of either failure to detect SB-detected TCRB rearrangements by PCR (6.5%) or detection of an additional non-SB defined rearrangement (2.5%). In 99% of cases (99/100), at least one clonal TCRB rearrangement was detected by PCR in the SB-positive cases. A predominance of complete Vβ-Jβ rearrangements was seen in TCRαβ+ T-cell malignancies and CD3-negative T-ALL (100 and 90%, respectively), whereas in TCRγδ+ T-ALL, more incomplete Dβ-Jβ TCRB rearrangements were detected (73%). Our results underline the reliability of this new TCRB PCR method and its strategic applicability in clonality diagnostics of lymphoproliferative disorders and MRD studies.
During early T-cell development, T-cell receptor delta (TCRD) and TCR gamma (TCRG) rearrangements precede TCR beta (TCRB) rearrangements.1, 2 Consequently, virtually all mature T-cell malignancies contain TCRB rearrangements, but also most immature T-cell malignancies, such as acute lymphoblastic leukemias (ALL), show rearranged TCRB genes.3, 4, 5
The TCRB gene cluster contains 65 Vβ gene segments (39–47 of which are qualified as functional) and two Dβ-Jβ-Cβ regions that each consist of one Dβ gene segment (TCRBD1 and TCRBD2) followed by a Jβ cluster, which comprises six (TCRBJ1S1 to TCRBJ1S6) or seven (TCRBJ2S1 to TCRBJ2S7) segments, respectively, and a Cβ gene segment (TCRBC1 and TCRBC2) (see Figure 1).2, 6, 7, 8, 9, 10 The process of rearranging the TCRB gene is generally initiated by Dβ-Jβ joining, leading to an incomplete TCRB rearrangement. In a second rearrangement step, a Vβ gene segment is added, resulting in a complete Vβ-Dβ-Jβ rearrangement.11 Rarely, other types of incomplete rearrangements can be found, such as Vβ-Dβ and Dβ-Dβ. Owing to the unique structure of the Dβ-Jβ-Cβ region with its two consecutive Dβ-Jβ clusters, more than one rearrangement per allele may be detectable: when a Vβ segment or the Dβ1 segment joins to a gene segment in the Jβ1 region, an independent, incomplete Dβ2-Jβ2 may still occur (see Figure 1).2
Molecular analysis of TCR gene rearrangements including TCRB rearrangements is a useful tool for clonality diagnostics in suspect T-cell proliferations. Detection of TCRB rearrangements can also be of major interest for minimal residual disease (MRD) studies in lymphoproliferative disorders, because the extensive combinatorial repertoire and highly diverse junctional regions make TCRB rearrangements excellent and highly stable polymerase chain reaction (PCR) targets for MRD diagnostics.12, 13, 14 TCRB rearrangements are not only detectable in most T-cell malignancies but have also been found in approximately 35% of precursor B-ALL.3, 15, 16
Analysis of TCR gene rearrangements can be performed either by Southern blot (SB) analysis, which has long been the ‘gold standard’ for clonality assessment, or by PCR. As SB is a labor-intensive and time-consuming method requiring high molecular weight DNA, the rapid and sensitive PCR technique is now broadly applied for clonality assessment. However, the extensive recombinatorial repertoire complicates the molecular analysis of TCRB rearrangements by PCR. Previously described TCRB PCR methods used highly degenerated consensus primers that limited the number of detectable rearrangements,17, 18, 19 used multiple tubes (20–25) with family-specific Vβ and multiple Jβ primers,20, 21 or used RNA in RT-PCR with a single Cβ primer to reduce the number of primers needed.22
Within the BIOMED-2 Concerted Action BMH4-CT98-3936 ‘PCR-based clonality studies for early diagnosis of lymphoproliferative disorders’, new and standardized, DNA-based PCR approaches were established for detection of immunoglobulin (Ig) and TCR gene rearrangements.23 The TCRB PCR approach was designed as a multiplex PCR for detection of complete Vβ-Jβ and incomplete Dβ-Jβ rearrangements. With 23 family-specific Vβ primers (designed to mainly recognize functional Vβ gene segments), two specific Dβ primers and 13 specific Jβ primers theoretically most complete Vβ-Jβ and all incomplete Dβ-Jβ gene rearrangements can be detected with the three BIOMED-2 multiplex tubes.23
During the last decade, ample experience with the PCR analysis of TCRG and TCRD rearrangements has been obtained, which appeared very helpful in the design and standardization of the BIOMED-2 PCR assays for these two loci. As no reliable PCR method pre-existed for the TCRB genes, PCR results obtained with the newly developed method had to be compared to classical SB analysis.4 Within Work Package 1 of the BIOMED-2 Concerted Action, only a limited number of T-cell malignancies were analyzed. Consequently, we initiated a more detailed validation study of the new TCRB multiplex PCR by direct comparison of the frequency of PCR-detected complete and incomplete TCRB rearrangements to the frequency of SB-detected rearrangements in immature (ALL) and mature T-cell malignancies.
Material and methods
Peripheral blood (PB) or bone marrow (BM) samples were selected from 66 patients with T-ALL (31 CD3 negative, 20 TCRαβ+, 15 TCRγδ+) and 36 mature TCRαβ+ malignancies, including 20 T-cell large granular lymphocyte leukemia (T-LGL), 11 T-cell prolymphocytic leukemia (T-PLL), three mycosis fungoides/Sezary's syndrome and two T-cell lymphoma cases. All samples had been analyzed previously by SB analysis.4 DNA for PCR analysis had been extracted as previously described and stored at −80°C.4 Control samples included DNA samples of patients with well-defined clonal TCRB rearrangements and polyclonal DNA derived from MNC pooled from five healthy donors.
All TCRB SB data have been published previously by Langerak et al.4 Seven TCRB probes were used for detection of TCRB rearrangements: TCRBD1U, TCRBD1, TCRBJ1, TCRBD2U, TCRBD2, TCRBJ2, TCRBC (DakoCytomation, Carpinteria, CA, USA); for sequence and position of these probes see Langerak et al.4 Using the combined information of this set of SB probes, incomplete Dβ-Jβ rearrangements, being defined via upstream Dβ (TCRBD1U, TCRBD2U) and downstream Jβ (TCRBJ1, TCRBJ2) probes, can be reliably identified. Complete Vβ-Jβ rearrangements can be identified with a considerable degree of reliability, assuming that a rearranged band with the TCRBJ1 or TCRBJ2 probe, with concomitant deletions upon TCRBD1U/TCRBD1 and/or TCRBD2U/TCRBD2 hybridization reflects a Vβ-Jβ rearrangement. Consequently, identification of complete TCRB rearrangements with Jβ probes is somewhat less reliable than identification of Dβ-Jβ rearrangements with a combination of both Dβ and Jβ probes. Therefore, it cannot be excluded that some SB-assumed Vβ-Jβ rearrangements in fact represent atypical TCRB rearrangements.4
PCR was performed according to the TCRB PCR protocol of the BIOMED-2 Concerted Action.23 In short, a multiplex PCR assay with 23 consensus Vβ, 2 specific Dβ and 13 specific Jβ primers was applied to identify clonal TCRB rearrangements. The 38 different primers were combined in three multiplex reactions, detecting complete Vβ-Jβ (tubes A and B) and incomplete Dβ-Jβ (tube C) rearrangements (InVivoScribe Technologies, Carlsbad, CA, USA; www.invivoscribe.com): (see Table 5)
Reaction conditions and cycling conditions were used according to the standardized BIOMED-2 TCRB multipex PCR protocol.23 In short, tubes A and B required 2 U AmpliTaq Gold polymerase (Applied Biosystems, Foster City, CA, USA) and 3 mM MgCl2, whereas in tube C 1 U AmpliTaq Gold polymerase and 1.5 mM MgCl2 were used.23 PCR was performed on an ABI 9600 thermal cycler (Applied Biosystems) for 35 cycles with a denaturation temperature of 94°C for 60 s, annealing at 60°C for 60 s and extension at 72°C for 60 s (initial denaturation and final extension were extended to 10 and 30 min, respectively).23 The 13 Jβ primers were labeled at their 5′end with 6′FAM (6-carboxyfluorescein, Applied Biosystems) for automated fluorescent fragment analysis (GeneScanning) of the PCR product.23 Each PCR product was analyzed for clonality by GeneScanning as well as by heteroduplex analysis, as described in the BIOMED-2 report.23
GeneScanning of PCR products
Reaction products were analysed with the GeneScanning software by exact size determination and quantitative measurement of fluorescence intensity after electrophoresis on a high-resolution polyacrylamide gel on an ABI PRISM 377 sequencer (Applied Biosystems). For exact protocol, see manufacturer's instructions and the BIOMED-2 report.23
For heteroduplex analysis, PCR products were denatured for 5 min at 94°C and renaturated for 60 min at 4°C run to induce duplex formation.23, 24 Subsequently, hetero- and homoduplexes were separated on 6% nondenaturing polyacrylamide gels and/or on commercially available polyacrylamide gels (GeneGel Excel Kit) in a GenePhor electrophoresis unit (Amersham Biosciences, Buckinghamshire, UK). Products were visualized using EtBr or by silver staining (DNA silver stain kit) in a Hoefer automated gel stainer (Amersham Biosciences).
Results and discussion
Detection of TCRB rearrangements is an important tool for clonality diagnostics in patients with suspect T-cell lymphoproliferations as well as for identification of PCR targets in MRD studies. In the past, SB analysis has been the most reliable technique for detection of clonal TCRB rearrangements. Recently, a new DNA-based multiplex TCRB PCR method has been developed within the BIOMED-2 Concerted Action BMH4-CT98-3936.23 Comparison of PCR data with SB data of a limited series of T-cell malignancies was an initial step in the validation process during Work Package 1 of the BIOMED-2 Concerted Action. The aim of the present study was the further validation of the BIOMED-2 TCRB PCR protocol23 using our earlier generated SB data of a large series of 102 T-cell malignancies: 36 mature TCRαβ+ T-cell malignancies, 31 CD3 negative T-ALL, 15 TCRγδ+ T-ALL and 20 TCRαβ+ T-ALL.4 Additionally, more information about the frequency of complete and incomplete TCRB rearrangements in mature TCRαβ+ T-cell malignancies and T-ALL was obtained.
The specific structure of the TCRB locus with the Dβ-Jβ-Cβ1 and Dβ-Jβ-Cβ2 clusters leads to the possibility of two rearrangements per allele, and a maximum number of four detectable TCRB rearrangements per sample (see Figure 1). To achieve a reliable PCR-SB comparison of single samples, we therefore decided to establish a scoring system by counting four possible loci for rearrangements on the two alleles in every sample. Unfortunately, due to technical limitations in the PCR protocol, it was impossible to design distinct multiplex tubes with Jβ1 and Jβ2 primers separated, whereas differentially labeled Jβ1 and Jβ2 primers were tested only in a late stage of the BIOMED-2 Concerted Action.23 Hence, deduction of the involved Jβ region from the PCR data was limited. For evaluation of the PCR results, three categories were applied: detection of a complete Vβ-Jβ rearrangement, detection of an incomplete Dβ-Jβ rearrangement, and no detectable PCR product. The first two categories could directly be correlated to SB results; the classification of no detectable PCR product may reflect various SB results: germline configuration, deletion of the locus or rarely occurring atypical rearrangements not detectable by PCR (Tables 1 and 2). As described by Langerak et al,4 SB analysis for example also allows to detect the rare Vβ-Dβ and Dβ-Dβ rearrangements. Scoring was performed by comparing SB information from all four loci with the PCR results so that four results were counted for every sample. The only exception was a sample from a T-LGL patient (96-019), which was proven to be biclonal and therefore counted double.25
TCRB gene rearrangement frequencies detected by PCR and SB
In 91% (179/204 plus 197/208 is 374/412) of all analyzed loci, a concordant result between PCR and SB could be found (Table 1). Direct comparison of PCR and SB data showed that 88% of all SB-defined rearrangements have also been found by TCRB PCR and that 95% concordance of SB data and PCR results was found in those loci where no PCR product could be expected from the SB results (Table 1). Similarly high proportions of 86 and 98%, respectively, were found in the general testing phase of WorkPackage 1 of the BIOMED-2 study.23 According to both PCR and SB, 28% (116/412) of all loci contained a Vβ-Jβ rearrangement and 15% (63/412) contained an incomplete Dβ-Jβ rearrangement. For 48% (197/412) of the loci, no PCR product or PCR-detectable SB result was found.
Discrepancies between PCR- and SB-based analysis of TCRB gene rearrangements
Discrepancies between PCR and SB results were found in a total of 9% (36/412) of the loci (Table 2). These findings either comprised the inability to detect a SB-defined rearrangement by PCR (25/412 loci (6%)) or the PCR detection of an additional rearrangement, not detected by SB (11/412 loci (3%): six extra Vβ-Jβ and five extra Dβ-Jβ). Of the 25 missed rearrangements, 18 concerned complete Vβ-Jβ and seven incomplete Dβ-Jβ rearrangements. In a single T-ALL case, the failure of PCR detection of one particular rearrangement lead to complete loss of information for the TCRB gene.
Failure to detect TCRB rearrangements by PCR can have various reasons: (1) atypical rearrangements, not detectable with the BIOMED-2 TCRB PCR primers;4, 26 (2) rearrangement of a nonfunctional Vβ gene segment, not recognized by the BIOMED-2 primers; (3) sequence variations (eg polymorphisms) of the rearranged gene segments leading to the impossibility of the PCR primers to anneal appropriately;8 (4) lack of PCR sensitivity for particular Vβ-Jβ rearrangements, because Vβ primers were tested with polyclonal samples and not in all cases with predefined clonal samples involving the specific Vβ segment;23 (5) lack of one of two Vβ-Jβ rearrangements due to preferential amplification of one rearrangement. Detection of additional rearrangements in PCR can be explained by (1) inability to detect a rearrangement by SB, due to for example, comigration with the germline band or another rearranged band; (2) an additional smaller clone not detectable by SB, due to the lesser sensitivity of SB; (3) selective amplification of a pseudoclone, although so far this has mainly been found in B-cell proliferations.23, 27
In an attempt to further explain the discrepant results on Vβ-Jβ rearrangements in our samples, we could exploit available data from Vβ-Cβ RT-PCR analysis in two TCRαβ+ T-ALL and 10 mature TCRαβ+ T-cell malignancies.25 Together, these 12 samples represented 7/18 missed Vβ-Jβ rearrangements in multiplex PCR and 6/6 extra Vβ-Jβ multiplex PCR products (Table 3). By comparing earlier described RT-PCR-derived Vβ-Cβ sequences25 with sequences of all detected multiplex Vβ-Jβ PCR products in these samples, we found identical results between these two PCR methods in six cases, thereby confirming the multiplex PCR data (Table 3). Interestingly, five out of the six extra PCR products in four cases (T015, 91–004, 93–067, 95–082) concerned a rearrangement to Jβ1.1; due to the position of the HindIII restriction site downstream of Jβ1.1, rearrangements involving this segment can only be detected using EcoRI digests, which is probably why they are more easily missed in SB analysis. The sixth extra Vβ-Jβ product concerned a rearrangement to Jβ2.1. In the two other cases (T069, 95–123), in which the results from the multiplex PCR and the RT-PCR were identical, the SB-assumed Vβ-Jβ2 rearrangements most probably concerned atypical rearrangements (Table 3). Another finding in this evaluation was that in four samples (96–019, 96–020, 96–042, 96–154), one of the two sequences (as known from the Vβ-Cβ RT-PCR products) was missed in the Vβ-Jβ multiplex PCR; close examination showed that competition of Vβ-Jβ targets probably played a role, since in all, except sample 96–154, the two products should have been amplified in the same multiplex tube (Table 3). Finally, one case remained unexplained through lack of reliable sequence data from the multiplex PCR products.
Interpretation problems of PCR reactions cannot always be overcome as they are caused by an inherent disadvantage of the PCR technology: its extreme sensitivity leading to false-positive results in case of subclone formation. To verify a doubtful clonal result multifold PCR and subsequent heteroduplex analysis of the mixed PCR products, or sequencing of the PCR products should therefore be considered. Also, additional information regarding the clinical, morphological and immunohistochemical status of the tested sample should always be included for final interpretation.
Comparison between GeneScanning and heteroduplex analysis
As described in the original BIOMED-2 TCRB PCR protocol,23 GeneScanning as well as heteroduplex analysis can be used to analyze the clonality of the PCR products. In our current series, no clear discrepancies in the interpretation of GeneScanning and heteroduplex results appeared.
In only 2/102 (2%) cases (both CD3 negative T-ALL), heteroduplex analysis revealed a single monoclonal band, whereas in GeneScanning an additional, but weak, monoclonal peak was detected. However, as the second peak in GeneScanning was so weak, the overall result was scored as monoallelic. In SB, biallelic rearrangements were found. Both discrepancies concerned tube A and might have been caused by preferential amplification of one Vβ-Jβ rearrangement over the other.
Overall, less discrepancies were found in this series compared to the general testing phase of Work Package 1 of the BIOMED-2 study, which is probably due to the selection of T-cell malignancies with a definite tumor load of at least 80% in the current series,4 whereas in the general testing phase of the BIOMED-2 project also many B-cell malignancies and samples with a lower tumor load were included.23
Nevertheless, the combined use of heteroduplex analysis and GeneScanning is still recommended for diagnostic application of the new TCRB PCR method.23 On the one hand, in heteroduplex analysis the polyclonal background is separated from the clonal signal thus facilitating detection, but on the other hand, the sensitivity of heteroduplex analysis is somewhat lower than for GeneScanning.23, 24 Another advantage of GeneScanning is the exact size determination that allows to distinguish PCR products outside the expected size range and to use GeneScanning for qualitative follow-up.14, 18, 23, 28
Frequency of TCRB rearrangements per disease category
In 97% (99/102) of the total group of mature TCRαβ+ T-cell malignancies and T-ALL, at least one clonal TCRB gene rearrangement was found by PCR: 100% in mature TCRαβ+ malignancies, 94% in CD3-negative T-ALL, 93% in TCRγδ+ T-ALL, 100% in TCRαβ+ T-ALL, which is in line with previously published data (Table 4 bottom).3, 4, 14, 15, 29, 30
Of the remaining three cases, two did not show a TCRB configuration in SB analysis that should lead to PCR-detectable TCRB rearrangements: one TCRγδ+ T-ALL case was germline on both alleles, whereas one CD3-negative T-ALL case showed two incomplete Vβ-Dβ rearrangements in SB. In the third case (CD3 negative T-ALL), SB-defined Vβ-Jβ and Dβ-Jβ rearrangements failed to be detected by PCR. Consequently, in 99 of the 100 samples (99%) that showed TCRB rearrangements in SB, at least one TCRB rearrangement, either complete or incomplete, was found with the BIOMED-2 TCRB multiplex PCR.
In the category of mature TCRαβ+ T-cell malignancies, 100% (36/36) of cases had at least one complete Vβ-Jβ rearrangement, whereas in 64% (23/36) of cases an incomplete Dβ-Jβ rearrangement was detected. In the total group of T-ALL, in 83% (55/66) of cases a complete Vβ-Jβ rearrangement was present, compared to 50% (33/66) of cases with incomplete Dβ-Jβ rearrangements (Table 4 top). Further division of T-ALL categories revealed differences between the subtypes: similar to mature TCRαβ+ T-cell malignancies, TCRαβ+ T-ALL and CD3-negative T-ALL also have a high frequency of complete Vβ-Jβ rearrangements and a lower frequency of incomplete Dβ-Jβ rearrangements (100 and 45% for TCRαβ+ T-ALL, and 90 and 41% for CD3-negative T-ALL, respectively). In contrast, in TCRγδ+ T-ALL the frequency of incomplete Dβ-Jβ rearrangements (73%) was essentially higher than the frequency of complete Vβ-Jβ rearrangements (46%).
TCRB rearrangement is initiated very early during T-cell differentiation. As a first step, a Dβ segment is combined with a Jβ gene segment followed by joining of a Vβ segment to the Dβ-Jβ complex. This might explain the high frequency of complete Vβ-Jβ rearrangements in cells with TCRαβ expression compared to the higher frequency of incomplete Dβ-Jβ rearrangements in TCRγδ+ T-ALL, which are thought to be derived from a separate γδ differentiation pathway, split off very early during T-cell development but after the onset of the TCRB gene rearrangement process.1, 3 This is line with a recent study by Asnafi et al31 who consider immature CD3-negative T-ALL with only Dβ-Jβ rearrangements as belonging to the γδ lineage, whereas the presence of complete Vβ-Jβ rearrangements would predispose them to the αβ lineage. Again in line with the data from Asnafi et al31 apparently a large fraction of CD3-negative T-ALL are associated with the αβ lineage, since they have a TCRαβ-like TCRB gene rearrangement pattern. The differences in TCRB gene rearrangement patterns between γδ lineage and αβ lineage T-cell malignancies become even more prominent by the frequencies of cases with two or more rearranged TCRB loci: 33% in TCRγδ+ T-ALL vs 65, 80 and 68% in CD3-negative T-ALL, TCRαβ+ T-ALL and mature TCRαβ+ T-cell malignancies, respectively (Table 4 bottom).
Irrespective of the differences in TCRB rearrangement patterns between categories, the overall detection rate of 97% (99/102) makes the TCRB rearrangement an attractive marker for follow-up of MRD, especially in T-ALL.14, 18
Strategical implications for clonality and MRD analysis of TCRB rearrangements
In the subanalysis of complete Vβ-Jβ rearrangements, a predominance of tube A-positive PCR reactions was found: 63% (57/91) of cases with a Vβ-Jβ rearrangement were positive in tube A (Jβ1 or Jβ2) and 54% (49/91) were positive in tube B (only Jβ2). A total of 16 cases were positive in both tube A and tube B (data not shown).
Knowledge about unequal distribution of complete Vβ-Jβ and incomplete Dβ-Jβ rearrangements between the disease categories could be applied for a strategic use of the PCR tubes in clonality assessment and identification of clonal markers for MRD studies. For clonality assessment in suspect mature T-cell proliferations or for identification of an MRD-PCR target in CD3-negative or TCRαβ+ T-ALL, detection of a complete Vβ-Jβ rearrangement is more likely. Therefore, analysis should be started with tubes A and B (a predominance for positivity of tube A was found in this series); for TCRγδ+ T-ALL, tube C might preferentially be applied.
In summary, our results show a good concordance of PCR results generated with the multiplex TCRB PCR approach developed by the BIOMED-2 study group compared to SB data on the same series of mature TCRαβ+ T-cell malignancies and T-ALL. In the vast majority of the cases, the combined use of GeneScanning and heteroduplex analysis for clonality assessment of the PCR products leads to conclusive interpretations. These data underline the reliability of the new BIOMED-2 TCRB PCR method and its applicability in clonality diagnostics of (T cell) lymphoproliferative disorders as well as for PCR-based MRD studies.
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We gratefully acknowledge Petra Chall, Frauke Hemken, Ellen van Gastel-Mol for their technical assistance and Mr Tar van Os for help in preparation of the figure. This work was supported by the BIOMED-2 Concerted Action BMH4-CT98-3936 ‘PCR-based clonality studies for early diagnosis of lymphoproliferative disorders’ of the European Commission.
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Cite this article
Droese, J., Langerak, A., Groenen, P. et al. Validation of BIOMED-2 multiplex PCR tubes for detection of TCRB gene rearrangements in T-cell malignancies. Leukemia 18, 1531–1538 (2004). https://doi.org/10.1038/sj.leu.2403428
- acute lymphoblastic leukemia
- minimal residual disease
- BIOMED-2 Concerted Action
- T-cell leukemia
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Refining the diagnosis of T-cell large granular lymphocytic leukemia by combining distinct patterns of antigen expression with T-cell clonality studies
Flow cytometric detection of peripheral blood involvement by mycosis fungoides and Sézary syndrome using T-cell receptor Vβ chain antibodies and its application in blood staging
Modern Pathology (2010)