T-cell receptor delta gene recombination in common acute lymphoblastic leukemia: preferential usage of V delta 2 and frequent involvement of the J alpha cluster

A high frequency (greater than 80%) of acute lymphoblastic leukemias (ALL) exhibit a recombination of the T-cell receptor (TCR) delta chain locus. Interestingly, distinct TCR delta elements are preferentially used in immunologic subtypes. In a recent series of 201 children with common ALL (cALL) we observed a TCR delta rearrangement in 162 patients, 57% of the latter showing a hybridization pattern in Southern blots suggestive of a V delta 2 to D delta 3 recombination. To verify this interpretation and to elucidate in more detail the diversity of this common type of TCR delta recombination we amplified and sequenced the junctional region of nine cALL patients and cell line REH-6 by polymerase chain reaction (PCR). A V delta 2 D delta 3 recombination was confirmed in all cases; convincing evidence for the participation of D delta 1 or D delta 2 elements was not obtained. Eight of nine patients and REH-6 showed complete 5' D delta 3 boundaries within V delta 2 D delta 3 segments, a limitation of junctional diversity also detected in 50% of peripheral blood cell clones derived from two healthy probands. Notably, sequence identity at the V delta 2 D delta 3 junction was demonstrated for a cALL and one of the control clones. Another group of 35 of 162 cALL patients was characterized by V delta 2 rearrangements and biallelic deletion of J delta and C delta sequences. Using a J alpha consensus primer, PCR-directed sequence analysis demonstrated V delta 2 D delta 3 J alpha recombinations in all four cases analyzed by this approach. The J alpha segments of these patients differed, but were identical or homologous to published J alpha elements. Our data suggest a recombination pathway of the TCR delta/alpha locus leading to chimeric TCR alpha molecules, containing V delta and, remarkably, also D delta sequences.     

Human common acute lymphoblastic leukemia-derived cell lines are competent to recombine their T-cell receptor delta/alpha regions along a hierarchically ordered pathway.

Rearrangements of the T-cell receptor (TCR) delta locus are observed in the majority of human B-cell precursor acute lymphoblastic leukemias (ALL) with a striking predominance of V delta 2(D)D delta 3 recombinations in common ALL (cALL) patients. Recently, we and others showed that almost 20% of cALL cases are characterized by further recombination of V delta 2(D)D delta 3 segments to J alpha elements, thereby deleting the TCR delta locus in analogy to the delta Rec/psi J alpha pathway in differentiating alpha/beta-positive T cells. We report here that two human cALL-derived cell lines, REH and Nalm-6, are competent to recombine the TCR delta/alpha locus under standard tissue culture conditions. Analysis of different REH subclones obtained by limiting dilution of the initial culture showed a biased recombination of V delta 2D delta 3 to distinct J alpha elements. During prolonged tissue culture, a subclone acquired growth advantage and displaced parental cells as well as other subclones. Frequently, the DJ junctions of REH subclones contained extended stretches of palindromic sequences derived from modified D delta 3 coding elements. The other cell line, Nalm-6, started the TCR delta/alpha recombination with an unusual signal joint of a cryptic recombinase signal sequence (RSS) upstream of D delta 3 to the 3' RSS of D delta 3. The RSS dimer was subsequently rearranged in all investigated subclones to an identical J alpha element. Both cell lines might become valuable tools to unravel the complex regulation of TCR delta/alpha recombination pathways in malignant and normal lymphopoiesis.     

Minimal residual disease monitoring in adult T-cell acute lymphoblastic leukemia: a molecular based approach using T-cell receptor G and D gene rearrangements

BACKGROUND AND OBJECTIVES: Minimal residual disease (MRD) is important in the measurement of response to treatment in childhood B- and T-cell acute lymphoblastic leukemia (ALL) and in adult B-cell ALL. Little is known about MRD evaluation in adult T-cell ALL. This study aimed to determine the prognostic significance of MRD measurements in adult T-cell ALL. DESIGN AND METHODS: T-cell receptor (TCR) gamma (G) and TCR delta (D) gene analyses were carried out at presentation in 49 patients with de novo T-ALL using a polymerase chain reaction (PCR) approach. In 26 of the patients bone marrow (BM) samples were collected at sequential time points (0-2, 3-5, 6-9, 10-24 months) after diagnosis for MRD investigation. The relationship between MRD status and clinical outcome was investigated and correlated with age, gender and white blood cell count at presentation. RESULTS: TCRG clonal gene rearrangements were found in 40 patients (82%). Eleven patients showed TCRD rearrangements (22%), in one of them as the sole molecular marker. V(gamma)I family rearrangements predominated (45 of 65 alleles) together with V(delta)1-J(delta)1/2 (9 of 13 alleles). Continuous clinical remission (CCR) occurred in 17 patients while nine patients relapsed. MRD analysis showed that negative tests during the first 6 months post-induction, and persisting negative MRD after induction were the best predictors of CCR. A positive test after 5 months was better at predicting relapse. In only four of seven patients was relapse preceded by a positive test the 5 months preceding relapse. INTERPRETATION AND CONCLUSIONS: Overall the ability of positive and negative tests to predict relapse or CCR was weaker in this cohort of adult T-ALL patients than in T- and B-lineage childhood ALL and B-lineage adult ALL. TCRG and TCRD gene analysis provides a clonal marker in the majority of adult T-ALL. These results suggest that caution should be taken in using MRD data based on TCR gene rearrangements to predict prognosis in adult T-ALL. Biological reasons may underlie differences between the performance of MRD tests in B- and T-lineage ALL. Further studies in a larger cohort of patients are needed to determine the exact role that MRD determination has in the management of T-ALL in adults.     

Differential usage of delta recombining element and V delta genes during T-cell ontogeny.

We analyzed the usage of the delta recombining element (delta Rec) and six V delta genes in cell samples from 15 patients with CD3- and 10 patients with CD3+ T-cell acute lymphoblastic leukemia in an attempt to define the hierarchy of genetic events that is associated with the T-cell receptor (TCR) alpha/delta gene complex during T-cell ontogeny. Based on the deletion patterns of these genes, we surmised their relative order on chromosome 14 to be as follows: 5'-V delta 4, V delta 6, V delta 1, V delta 5, delta Rec, V delta 2, D delta 1-3, J delta 1-3, C delta, V delta 3-3'. In agreement with previous reports, V delta 1 was found to be preferentially rearranged in CD3+ samples. In CD3- samples, V delta 2 and V delta 3 rearrangements were observed at a high frequency. Incomplete V delta D delta rearrangements using V delta 2 or V delta 3, which are closest to C delta, were observed in three patients with CD3- and one patient with CD3+. These results suggest that V delta 2- and V delta 3-(Dn)D delta 3 recombinations are among the earliest recombinational events. Delta Rec was observed to be rearranged to phi J alpha on one allele. In addition, delta Rec rearrangements to J delta 1 and J alpha close to phi J alpha were also demonstrated on three alleles and one allele, respectively. Delta Rec rearrangements to J delta and J alpha other than phi J alpha also inhibit expression of the TCR delta locus. Approximately half of the alleles with J delta rearrangements showed no involvement of known V delta or delta Rec, indicating the existence of other, yet-uncharacterized V delta or delta Rec-like segments.     

Detection of T cell receptor delta gene rearrangements in childhood B and T lineage acute lymphoblastic leukaemia by southern blot and PCR: technical comparison of two methods of analysis

Molecular analysis of antigen receptor genes (Ig and TCR) has been useful for clonal studies in acute lymphoblastic leukaemia (ALL) patients. Rearrangements of these genes can be used to track the persistence of the leukaemic clone during the therapy. The purpose of our study was to analyse the percentage and the pattern of the rearrangements at the TCR D locus in a series of ALL patients, comparing the results obtained by Southern blot and PCR. Genomic DNA was extracted from mononuclear BM cells of 40 paediatric ALL cases, digested with different restriction enzymes and hybridized to TCRDJ1 probe to study the TCR delta locus. Amplification of the rearranged TCR delta genes was performed by PCR to define the gene segments involved. The junctional region was deduced from the sequence to obtain patient-specific primers. Among the 31 B lineage ALL samples, one or two TCR delta alleles proved to be rearranged in 53% of cases. Two different types of rearrangements were chiefly detected: Vdelta2Ddelta3 and Ddelta2Ddelta3. In T-ALL patients, the predominant rearrangement involved the Vdelta1 and the Jdelta1 gene segments.     

Developmental process of the T-cell receptor α and δ gene assembly in B-cell precursor acute lymphoblastic leukaemia

We analysed the organization of V delta genes and delta recombining element (delta Rec) in 27 children with B-cell precursor acute lymphoblastic leukaemia. Twenty-two of 54 alleles showed rearrangements of the T-cell receptor (TCR) delta locus. These rearrangements resulted either from D2D delta 3 (2 alleles) or V delta 2(Dn)D delta 3 (20 alleles) recombinations, and the other V delta and delta Rec were not rearranged. Of 23 alleles with deletion of C delta and rearrangements of J alpha, V delta 2, V delta 4 and V delta 5 appeared to rearrange to J alpha on five alleles. With regard to the relationship between the rearranged V alpha/delta and J alpha genes, gene segments 5' to V delta 2 frequently rearranged to J alpha more proximal to C alpha, whereas V delta 2 and gene segments 3' to V delta 2 showed a tendency to rearrange to J alpha distal to C alpha. Based on these findings, we suggest that the initial recombination event of the TCR-alpha/delta gene may be D2D delta 3 joining, followed by V delta 2 recombination with the D2D delta 3 complex. It was also suggested that use of V alpha/delta and J alpha/delta may depend on the distance between the involved V alpha/delta and J alpha/delta at least in B-lineage cells. These rearrangements in B-precursor cells appear to be aberrant. However, this recombinational process may be one of the normal differentiation pathways in T-lineage cells, because cells with a V delta 2(Dn)D delta 3 rearrangement were detected in 0.1-0.01% of normal peripheral mononuclear cells by the polymerase chain reaction.     

T-cell receptor delta/alpha rearrangements in lymphoid neoplasms

Rearrangements within the T-cell receptor (TCR)delta/alpha locus were analyzed in a wide variety of lymphoid neoplasms by eight DNA probes specific for TCR J delta, J alpha and C alpha segments. In all 11 T- cell malignancies, rearrangement and/or deletion of TCR delta was detected irrespective of the stage of maturation of the tumor. The organization of TCR delta correlated with the phenotype of the tumor: In "prethymic" T-cell acute lymphocytic leukemia (ALL), TCR delta was the only TCR gene to be rearranged. More mature T cell malignancies expressing CD4 together with CD3 showed deletion of both alleles of TCR delta, suggestive of TCR V alpha-J alpha rearrangement. All 43 B-cell tumors expressing surface immunoglobulin (sIg), including two cases of adult B-cell ALL, had germline configuration of TCR delta/alpha. In contrast, all 17 B-cell precursor ALLs (null, common, and pre-B-cell ALLs) had rearrangement and/or deletion of TCR delta/alpha. A single case of "histiocytic" lymphoma also showed biallelic deletion of TCR delta. Oligoclonal rearrangements of Ig and TCR genes were observed in two cases of B-cell precursor ALL and in one case of T-cell lymphoblastic lymphoma. Patterns of such "aberrant" TCR rearrangement were similar to those observed in T-lineage malignancies. In particular, seven of eight cases of B-cell precursor ALL and the histiocytic lymphoma which demonstrated biallelic TCR delta deletion, (suggestive of a V alpha-J alpha rearrangement) had clonal TCR beta rearrangement. These data support the hypothesis that supposedly aberrant rearrangements of the TCR genes may follow the same developmental controls as found in T-cell differentiation, despite the lack of evidence for further commitment to the T-cell lineage. TCR delta rearrangement is a useful marker of clonality of immature T-cell tumors which may have only this gene rearranged but is not specific to the T-cell lineage.     

Southern blot patterns, frequencies, and junctional diversity of T-cell receptor-delta gene rearrangements in acute lymphoblastic leukemia

Southern blot analysis of T-cell receptor (TCR)-delta gene rearrangements is useful for diagnostic studies on the clonality of lymphoproliferative diseases. We have developed 18 new TCR-delta gene probes by use of the polymerase chain reaction (PCR) techniques. Application of these probes for detailed analysis of the TCR-delta genes in normal control samples, 138 T-cell acute lymphoblastic leukemias (T-ALL), and 91 precursor B-ALL allowed us to determine the TCR-delta gene restriction map for five restriction enzymes, as well as the Southern blot restriction enzyme patterns of all theoretically possible TCR-delta gene rearrangements. Based on this information, it appeared that 97% of all 213 detected TCR-delta gene rearrangements in our series of ALL could be detected by use of the TCRDJ1 probe and that the majority (76%) of the 213 rearrangements could be identified precisely. In T-ALL, we found a strong preference for the complete rearrangements V delta 1-J delta 1 (33%), V delta 2-J delta 1 (10%), and V delta 3-J delta 1 (7%) and the incomplete rearrangement D delta 2- J delta 1 (11%). In precursor B-ALL, the majority of rearrangements consisted of V delta 2-D delta 3 (72%) and D delta 2-D delta 3 (10%). The junctional diversity of these 6 preferential TCR-delta rearrangements was analyzed and showed an extensive junctional insertion (approximately 30 nucleotides) for complete V delta-J delta rearrangements, whereas incomplete rearrangements had correspondingly smaller junctional regions. The detailed TCR-delta gene restriction map and probes presented here, in combination with the Southern blot patterns of TCR-delta gene rearrangements, are important for TCR-delta gene studies in ALL; all TCR-delta gene rearrangements can be detected and the majority can be identified precisely. Identification of rearrangements is a prerequisite for subsequent PCR analysis of TCR- delta gene junctional regions, eg, for detection of minimal residual disease during follow-up of ALL patients.     

Clonal rearrangements in childhood and adult precursor B acute lymphoblastic leukemia: a comparative polymerase chain reaction study using multiple sets of primers.

Ig heavy chain (IgH) and T-cell receptor (TCR) gene rearrangements were investigated by polymerase chain reaction (PCR) amplification of diagnostic tumour samples from 91 patients (57 children and 34 adults, with cut-off at age 16) with precursor B acute lymphoblastic leukemia (ALL). Using primers directed to the framework regions (FR) 1, 2 and 3 of the IgH gene, clonal IgH rearrangements were observed in 82, 58 and 58%, respectively, whereas clonality was presented in 45 and 27% using primers hybridising to the TCR delta and gamma genes. A combination of all five primer sets used resulted in 96% positive cases (children 100%, adults 88%). The frequency of clonal IgH rearrangements correlated to patient age with a significantly lower fraction of positive cases in the adult group. The concomitant usage of more than one V(H) family gene was similar for childhood and adult ALL, and an over-representation of V(H)6 rearrangements was found in childhood ALL. Twenty-five out of 91 cases (27%) displayed an oligoclonal pattern for either IgH or TCR gene rearrangements (children 37%, adults 12%). A comparative analysis of samples from different compartments was performed in 23 patients, and differences between two or three compartments were observed in seven cases. Unexpectedly large, clonally appearing PCR products of 540-715 bp were found in three leukemias and sequence analysis verified their clonal nature. In summary, using multiple sets of primers clonal rearrangements of IgH and TCR genes can be detected in a very high frequency, including previously neglected large size PCR products. A common heterogeneity was demonstrated in different compartments reflecting ongoing clonal evolution, which can make detection of minimal residual disease (MRD) in ALL troublesome. Therefore, we suggest that a minimum of three targets should be used to minimise false-negative results.     

Rearrangement patterns of immunoglobulin heavy chain (IgH) and light chain genes in acute lymphoblastic leukemia and chronic myelocytic leukemia lymphoid crisis cells showing oligoclonal IgH gene rearrangements.

We investigated leukemic cells with multiple immunoglobulin heavy chain (IgH) gene rearrangements from nine B-precursor cell acute lymphoblastic leukemia (ALL) patients and three chronic myelocytic leukemia lymphoid crisis (CML.Ly-BC) patients in order to determine detailed recombination patterns of the variable (V), diversity (D), and joining (J) region genes. Southern blot study, using DNA probes for DQ52 and 5'D region genes, was useful to distinguish VDJ recombination from DJ recombination at the level of each allele. Leukemic cells from seven out of eight CD10-positive ALL patients showed biallelic VDJ recombinations. Rearrangements of Ig kappa genes were found in only one case. Leukemic cells from all of the CML.Ly-BC patients had a DJ/(V)DJ IgH genotype. These findings suggest that the multiple IgH gene rearrangements in B-precursor cell ALL occurred as a consequence of continuing V-(V)DJ rearrangements after neoplastic transformation, and were closely related to the stage of bone marrow B-precursor cell differentiation. Multiple IgH gene rearrangements in CML.Ly-BC might take place earlier in the process of IgH gene rearrangements than is the case in B-precursor cell ALL. In this sense, the genotypic oligoclonality observed in ALL and CML.Ly-BC should be regarded not as 'true', but as 'pseudo' oligoclonal leukemia.     

A polymerase chain reaction study of the stability of Ig heavy-chain and T-cell receptor delta gene rearrangements between presentation and relapse of childhood B-lineage acute lymphoblastic leukemia

Ig heavy-chain (IgH) and partial V delta 2-D delta 3 T-cell receptor (TCR) gene rearrangements were investigated, by polymerase chain reaction (PCR) amplification and sequence analysis, in 52 patients at presentation and first relapse and in 14 at both first and second relapse of B-lineage acute lymphoblastic leukemia. In combination, these techniques amplified one or more clonal markers at presentation in 90% of patients (IgH-PCR, 75%; V delta 2-D delta 3-PCR, 46%; both, 33%). Changes in the pattern of amplification between presentation and first relapse were seen in 31% of patients positive by IgH-PCR at presentation and in 25% of those positive by TCR delta-PCR. Only 3 patients showed complete change in their rearrangements, which is suggestive of relapse with a new clone. Furthermore, despite the high reported rates of oligoclonality and clonal evolution at the IgH locus, the results presented show that false-negative minimal residual disease (MRD) detection can be avoided by designing D-N-J probes to all presentation rearrangements. Using a PCR approach for both gene markers, false-negative testing because of clonal evolution would have only occurred in 3 (8%) of the IgH-positive patients, in contrast to 5 (21%) of V delta 2-D delta 3-positive patients. Combining these two systems increases the proportion of patients open to study to 90%, allows comparative studies of the sensitive of the two methods, and reduces the rate of false-negative assessment of MRD caused by clonal evolution to less than 10%. We conclude that large prospective PCR studies of MRD detection should examine gene rearrangements at multiple loci to maximize their applicability and to minimize false-negative relapse prediction.     

A common Vδ2—Dδ2—Dδ3 T cell receptor gene rearrangement in precursor B acute lymphoblastic leukaemia

Despite their apparent commitment to the B lymphocytic lineage, human precursor B cell acute lymphoblastic leukaemias (ALL) frequently rearrange their T cell antigen receptor (TCR) alpha, beta and gamma chain genes. Since these three genes are active sites of rearrangement in precursor B cell neoplasms, it seemed that the recently discovered fourth TCR gene, delta, might be similarly rearranged. To investigate this possibility, a series of precursor B cell leukaemias was analysed for rearrangements at the delta chain gene locus, using probes of the variable, joining, and constant regions of the delta chain gene. The majority of precursor B cell ALLs in this series (25/32, 78%) showed rearrangement or deletion of one or more TCR delta genes. This contrasts sharply with a series of 16 mature B cell neoplasms (chronic lymphocytic leukaemia) in which no TCR delta gene rearrangements were detected. An unusual TCR delta rearrangement, rarely observed in normal or neoplastic T cells, was seen in the majority (14/18) of precursor B cell ALLs with TCR delta rearrangements. In contrast to the utilization ov V delta 1 in T cell ALL, detailed restriction mapping of precursor B ALL revealed an incomplete rearrangement without involvement of J delta segments. Direct genomic sequencing was performed on one example and demonstrated a nonproductive V delta 2-D delta 2-D delta 3 recombination in this precursor B ALL. We conclude that the TCR delta chain gene is an active locus in precursor B cell neoplasia, involves an unusual type of rearrangement and provides a clonal tumour marker for diagnosis of precursor B ALL.     

Use of polymerase chain reactions to monitor minimal residual disease in acute lymphoblastic leukemia patients.

T-cell receptor (TCR) delta gene rearrangements are observed in more than 80% of acute lymphoblastic leukemia (ALL) patients. Moreover, a preferential usage of specific genetic elements has been shown in different ALL subtypes: V delta 1 DJ delta 1 rearrangements predominate in T-ALL, while most B-precursor ALLs show a recombination of V delta 2 to D delta 3. Recently we have proposed a strategy for the detection of minimal residual disease (MRD) based on the isolation of clonospecific probes following the in vitro amplification of V delta 1 DJ delta 1 junctions by polymerase chain reaction (PCR) and now have adapted this method to the preparation of specific V delta 2 D delta 3 fragments. In the present study, clonospecific probes were generated from 11 T-ALL and 16 cALL patients (21 children, 6 adults). The sensitivity of these 27 probes in detecting residual leukemia cells varied between 10(-4) to 10(-6) as determined by semiquantitative evaluation of dilution experiments. PCR analysis of 55 bone marrow (BM) and peripheral blood (PB) samples obtained from the 27 patients during complete clinical remission showed the following results: (1) Evidence for MRD was obtained in the BM of all patients (eight of eight) investigated 2 to 6 months after remission induction and also in 6 of 11 cases on maintenance therapy 7 to 19 months after diagnosis. (2) In contrast, all patients but one (10 of 11) analyzed 6 to 41 months after the termination of treatment lacked apparent evidence for leukemia DNA; the exception was a girl exhibiting 10(-4) to 10(-5) residual cells in her PB 5.5 years after diagnosis. (3) Longitudinal analysis in nine patients disclosed marked individual differences in the intervals between achievement of clinical remission and complete eradication of the leukemia cell clone. (4) Differences in the duration of MRD were not associated with distinct clinical-hematologic features. (5) Detection of residual disease by PCR proceeded clinical relapse in two cases.     

Rapid detection of clonality in patients with acute lymphoblastic leukemia

BACKGROUND AND OBJECTIVES: Polymerase chain reaction (PCR) detection of clonal T-cell receptor (TCR) gamma and delta gene rearrangements is widely used in clonality assessment of lymphoid leukemias and lymphomas and for detection of minimal residual disease of acute lymphoblastic leukemia (ALL). Standard analyses for clonality assessment include Southern blotting or PCR-based detection of clonal TCR gene rearrangements. The latter consist of heteroduplex PCR analysis by separation of PCR products on non-denaturing polyacrylamide gel (PAGE). We describe a rapid and sensitive method to identify specific clonal rearrangements in PCR fragments obtained by amplification of TCRgamma and TCRdelta genes. DESIGN AND METHODS: We applied a semi-automated electrophoretic technique (PhastSystem , Amersham Pharmacia Biotech) and compared it with standard homo-heteroduplex analysis in 21 cases of childhood acute lymphoblastic leukemia (ALL). RESULTS: The results obtained for each sample analyzed by standard homo-heteroduplex detection were completely reproduced by the PhastSystem approach. INTERPRETATION AND CONCLUSIONS: We conclude that heteroduplex analysis of TCR gene rearrangements using the semi-automated PhastSystem is a simple, rapid, cheap and highly reproducible method which can be used as an alternative to traditional analysis for detection of clonality.     

Comparative analysis of Ig and TCR gene rearrangements at diagnosis and at relapse of childhood precursor-B-ALL provides improved strategies for selection of stable PCR targets for monitoring of minimal residual disease

Immunoglobulin (Ig) and T-cell receptor (TCR) gene rearrangements are excellent patient-specific polymerase chain reaction (PCR) targets for detection of minimal residual disease (MRD) in acute lymphoblastic leukemia (ALL), but they might be unstable during the disease course. Therefore, we performed detailed molecular studies in 96 childhood precursor-B-ALL at diagnosis and at relapse (n = 91) or at presumably secondary acute myeloid leukemia (n = 5). Clonal Ig and TCR targets for MRD detection were identified in 94 patients, with 71% of these targets being preserved at relapse. The best stability was found for IGK-Kde rearrangements (90%), followed by TCRG (75%), IGH (64%), and incomplete TCRD rearrangements (63%). Combined Southern blot and PCR data for IGH, IGK-Kde, and TCRD genes showed significant differences in stability at relapse between monoclonal and oligoclonal rearrangements: 89% versus 40%, respectively. In 38% of patients all MRD-PCR targets were preserved at relapse, and in 40% most of the targets (> or = 50%) were preserved. In 22% of patients most targets (10 cases) or all targets (10 cases) were lost at relapse. The latter 10 cases included 4 patients with secondary acute myeloid leukemia with germline Ig/TCR genes. In 5 other patients additional analyses proved the clonal relationship between both disease stages. Finally, in 1 patient all Ig/TCR gene rearrangements were completely different between diagnosis and relapse, which is suggestive of secondary ALL. Based on the presented data, we propose stepwise strategies for selection of stable PCR targets for MRD monitoring, which should enable successful detection of relapse in most (95%) of childhood precursor-B-ALL.     

Immunogenotyping with antigen receptor gene probes as a diagnostic tool in childhood acute lymphoblastic leukaemia

13 cases of childhood acute lymphoblastic leukaemia (ALL) were studied combining cell surface marker analysis with immunogenotyping by Southern blot hybridisation with a panel of antigen receptor gene probes. The immunophenotypes were unequivocal: 7 patients had B-phenotype and 6 patients T-phenotype ALL. In several patients immunogenotypes were not fully consistent with the respective phenotypes. For example, 2 B-cell precursor ALL had rearranged TCR beta chain genes and 2 T-ALL rearrangement of Ig heavy-chain genes. All cases showed clonal rearrangement or deletions within the TCR delta gene locus. TCR delta gene rearrangements might, therefore, serve as markers of clonality but not of B- or T-lineage in immature lymphoid neoplasms. We conclude that in current diagnostic practice immunogenotyping is a supplement rather than an alternative to immunophenotyping by surface marker analysis.     

Molecular monitoring of residual disease using antigen receptor genes in childhood acute lymphoblastic leukaemia

Immunoglobulin (Ig) and T-cell receptor (TCR) gene rearrangements are assumed to be unique 'fingerprint-like' sequences for each acute lymphoblastic leukaemia (ALL). Various clonal Ig/TCR gene rearrangements can be identified at diagnosis in virtually all childhood ALL patients, representing molecular targets for detection of minimal residual disease (MRD) during follow-up analysis. The usage of at least two MRD-PCR targets per patient generally ensures high sensitivity (相似文献   

Detection of minimal residual disease in acute lymphoblastic leukemia by in vitro amplification of rearranged T-cell receptor delta chain sequences

Human T-cell receptor (TCR) delta-chain diversity mainly originates from high junctional variability, since only a limited number of germline elements is available. This extraordinary diversity at the V.J junction, due to the use of two D delta elements and extensive incorporation of N nucleotides, constitutes a specific clonal marker for cell populations exhibiting rearranged TCR delta genes. To this end we amplified in vitro by polymerase chain reaction (PCR) the TCR delta junctional region of five acute lymphoblastic leukemias (ALL), isolated respective DNA fragments, and used them directly as clonospecific probes. The combination of PCR technology and hybridization to clonospecific probes permitted the detection of leukemia DNA at dilution of 1:100,000 in all five cases. Moreover, we were able to investigate one of the ALL patients 11 months after achieving continuous complete remission. Conventional Southern blot analysis failed to detect rearranged TCR genes at this stage. However, residual leukemic cells could readily be detected by PCR technique. We conclude that the strategy proposed here is a very sensitive tool to detect minimal residual disease in a significant proportion of human lymphoid neoplasias.