Site-specific deletions involving the tal-1 and sil genes are restricted to cells of the T cell receptor alpha/beta lineage: T cell receptor delta gene deletion mechanism affects multiple genes.

Site-specific deletions in the tal-1 gene are reported to occur in 12-26% of T cell acute lymphoblastic leukemias (T-ALL). So far two main types of tal-1 deletions have been described. Upon analysis of 134 T-ALL we have found two new types of tal-1 deletions. These four types of deletions juxtapose the 5' part of the tal-1 gene to the sil gene promoter, thereby deleting all coding sil exons but leaving the coding tal-1 exons undamaged. The recombination signal sequences (RSS) and fusion regions of the tal-1 deletion breakpoints strongly resemble the RSS and junctional regions of immunoglobulin/T cell receptor (TCR) gene rearrangements, which implies that they are probably caused by the same V(D)J recombinase complex. Analysis of the 134 T-ALL suggested that the occurrence of tal-1 deletions is associated with the CD3 phenotype, because no tal-1 deletions were found in 25 TCR-gamma/delta + T-ALL, whereas 8 of the 69 CD3- T-ALL and 11 of the 40 TCR-alpha/beta + T-ALL contained such a deletion. Careful examination of all TCR genes revealed that tal-1 deletions exclusively occurred in CD3- or CD3+ T-ALL of the alpha/beta lineage with a frequency of 18% in T-ALL with one deleted TCR-delta allele, and a frequency of 34% in T-ALL with TCR-delta gene deletions on both alleles. Therefore, we conclude that alpha/beta lineage commitment of the T-ALL and especially the extent of TCR-delta gene deletions determines the chance of a tal-1 deletion. This suggests that tal-1 deletions are mediated via the same deletion mechanism as TCR-delta gene deletions.

Differential expression of p73 isoforms in relation to drug resistance in childhood T-lineage acute lymphoblastic leukaemia.

The T-lineage phenotype of childhood acute lymphoblastic leukaemia (ALL) is associated with an increased relapse-risk and in vitro resistance to drugs as compared to a precursor B phenotype. Antiapoptotic isoforms of p73 that lack part of the transactivation (TA) domain (DeltaTA-p73, i.e. p73Deltaex2, p73Deltaex3, p73Deltaex2/3 and DeltaN-p73) may cause resistance to anticancer agents through inhibition of p53 and/or proapoptotic p73 family members (TA-p73). We demonstrate in our study that the expression of total p73 mRNA was higher in childhood T-ALL compared to controls (P=0.004). In T-ALL, the relative contribution of antiapoptotic DeltaTA-p73 (88%) was larger than of proapoptotic TA-p73 (12%). Leukaemic cells of T-ALL patients expressing higher levels of antiapoptotic p73 were more resistant to the DNA-damaging drug daunorubicin compared to cells of patients with low or negative expression or these isoforms (P(trend)=0.045). Interestingly, p73Deltaex2 was the most abundantly expressed antiapoptotic isoform in daunorubicin-resistant patient cells (44% of total p73). No association was found between high expression of proapoptotic TA-p73 or antiapoptotic DeltaTA-p73 and relapse-risk. Our results suggest that childhood T-ALL is associated with a high expression of DeltaTA-p73. These isoforms may play a role in cellular resistance to DNA-damaging drugs in children at initial diagnosis of T-ALL.

A new recurrent 9q34 duplication in pediatric T-cell acute lymphoblastic leukemia.

Over the last decade, genetic characterization of T-cell acute lymphoblastic leukemia (T-ALL) has led to the identification of a variety of chromosomal abnormalities. In this study, we used array-comparative genome hybridization (array-CGH) and identified a novel recurrent 9q34 amplification in 33% (12/36) of pediatric T-ALL samples, which is therefore one of the most frequent cytogenetic abnormalities observed in T-ALL thus far. The exact size of the amplified region differed among patients, but the critical region encloses approximately 4 Mb and includes NOTCH1. The 9q34 amplification may lead to elevated expression of various genes, and MRLP41, SSNA1 and PHPT1 were found significantly expressed at higher levels. Fluorescence in situ hybridization (FISH) analysis revealed that this 9q34 amplification was in fact a 9q34 duplication on one chromosome and could be identified in 17-39 percent of leukemic cells at diagnosis. Although this leukemic subclone did not predict for poor outcome, leukemic cells carrying this duplication were still present at relapse, indicating that these cells survived chemotherapeutic treatment. Episomal NUP214-ABL1 amplification and activating mutations in NOTCH1, two other recently identified 9q34 abnormalities in T-ALL, were also detected in our patient cohort. We showed that both of these genetic abnormalities occur independently from this newly identified 9q34 duplication.

Incidence of additional genetic changes in the TEL and AML1 genes in DCOG and COALL-treated t(12;21)-positive pediatric ALL, and their relation with drug sensitivity and clinical outcome.

Clinical heterogeneity within t(12;21) or TEL/AML1-positive ALL (25% of childhood common/preB ALL) indicates that additional genetic changes might contribute to outcome. We studied the relation between additional genetic changes in TEL(ETV6) and AML1(RUNX1) (FISH), drug sensitivity (MTT assay) and clinical outcome in 143 DCOG and COALL-treated t(12;21)-positive ALL patients. Additional genetic changes in TEL and AML1 were present in 83% of the patients, and consisted of (partial) deletion of the second TEL gene (70%), an extra AML1 gene (23%) or an extra der(21)t(12;21) (10%). More than one additional change was observed in 20%. Disease-free survival (pDFS) of DCOG patients without additional genetic changes (4 years pDFS +/- s.e. 53 +/- 17%) and of those with an extra der(21)t(12;21) (60 +/- 22%) is poorer than that of compared to patients with other additional genetic changes in TEL or AML1 (79 +/- 6%; P-trend = 0.02). This was mainly due to the occurrence of early relapses within 2.5 years after the first diagnosis. Similar observations were found in the COALL cohort, albeit not significant owing to limited follow-up. Multivariate analysis including age, WBC and genetic abnormalities in TEL and/or AML1 showed that especially, in vitro resistance to prednisolone (hazard ratio 5.78, 95% CI 1.45-23.0; P=0.01) is an independent prognostic factor in DCOG- and COALL-treated t(12;21)-positive ALL.

Relation between genetic variants of the ataxia telangiectasia-mutated (ATM) gene, drug resistance, clinical outcome and predisposition to childhood T-lineage acute lymphoblastic leukaemia.

The T-lineage phenotype in children with acute lymphoblastic leukaemia (ALL) is associated with in vitro drug resistance and a higher relapse-risk compared to a precursor B phenotype. Our study was aimed to investigate whether mutations in the ATM gene occur in childhood T-lineage acute lymphoblastic leukaemia (T-ALL) that are linked to drug resistance and clinical outcome. In all, 20 different single nucleotide substitutions were found in 16 exons of ATM in 62/103 (60%) T-ALL children and 51/99 (52%, P = 0.21) controls. Besides the well-known polymorphism D1853N, five other alterations (S707P, F858L, P1054R, L1472W, Y1475C) in the coding part of ATM were found. These five coding alterations seem to occur more frequently in T-ALL (13%) than controls (5%, P = 0.06), but did not associate with altered expression levels of ATM or in vitro resistance to daunorubicin. However, T-ALL patients carrying these five coding alterations presented with a higher white blood cell count at diagnosis (P = 0.05) and show an increased relapse-risk (5-year probability of disease-free survival (pDFS) = 48%) compared to patients with other alterations or wild-type ATM (5-year pDFS = 76%, P = 0.05). The association between five coding ATM alterations in T-ALL, their germline presence, white blood cell count and unfavourable outcome may point to a role for ATM in the development of T-ALL in these children.

Treatment strategy and results in children treated on three Dutch Childhood Oncology Group acute myeloid leukemia trials.

This report describes the long-term follow-up data of three consecutive Dutch Childhood Oncology Group acute myeloid leukemia (AML) protocols. A total of 303 children were diagnosed with AML, of whom 209 were eligible for this report. The first study was the AML-82 protocol. Results were inferior (5-year probability of overall survival (pOS) 31%) to other available regimes. Study AML-87 was based on the BFM-87 protocol, with prophylactic cranial irradiation in high-risk patients only, and without maintenance therapy. This led to a higher cumulative incidence of relapse than that reported by the Berlin-Frankfurt-Münster (BFM), but survival was similar (5-year pOS 47%), suggesting successful retrieval at relapse. The subsequent study AML-92/94 consisted of a modified BFM-93 protocol, that is, without maintenance therapy and prophylactic cranial irradiation. However, all patients were to be transplanted (auto- or allogeneic), although compliance was poor. Antileukemic efficacy was offset by an increase in the cumulative incidence of nonrelapse mortality, especially in remission patients, and survival did not improve (5-year pOS 44%). Our results demonstrate that outcome in childhood AML is still unsatisfactory, and that further intensification of therapy carries the risk of enhanced toxicity. Our patients are currently included in the MRC AML studies, based on the results of their AML 10 trial.

Breakpoint heterogeneity in t(10;11) translocation in AML-M4/M5 resulting in fusion of AF10 and MLL is resolved by fluorescent in situ hybridization analysis.

Ten AML-M4/M5 patients' samples containing a t(10;11) translocation, but with different cytogenetic breakpoints on chromosome 11q (11q13-23), were studied by G- and R-banding and fluorescent in situ hybridization. Southern blotting analysis, studied in five patients, revealed a rearranged MLL gene. Reverse transcription-PCR analysis carried out in six patients showed a 5' MLL-3' AF-10 fusion transcript. Fluorescent in situ hybridization studies suggested that in 8 of 10 patients, the rearrangement/fusion transcript resulted from an inversion of a part of 11q (q13q23) translocated to 10p12. In the other two patients, it is assumed that an inversion/translocation has occurred of a part of 10p to the der(11). The results suggest that the orientation of the AF-10 gene on 10p is 5' telomeric and 3' centromeric. This is the first example of opposite-oriented genes being involved in translocation to yield fusion transcripts.

Fusion of the homeobox gene HLXB9 and the ETV6 gene in infant acute myeloid leukemias with the t(7;12)(q36;p13).

Recently, we and others reported a recurrent t(7;12)(q36;p13) found in myeloid malignancies in children < or =18 months of age and associated with a poor prognosis. Fluorescence in situ hybridization studies mapped the 12p13 breakpoint to the first intron of ETV6 and narrowed down the region of 7q36 involved. By using the sequences made public recently by the Human Genome Project, two candidate genes in 7q36 were identified: the homeobox gene HLXB9 and c7orf3, a gene with unknown function. Reverse transcription-PCR of two cases with t(7;12), using primers for c7orf3 and ETV6, was negative. However, reverse transcription-PCR for HLXB9-ETV6 demonstrated alternative splicing; the two major bands corresponded to fusion of exon 1 of HLXB9 to exons 2 and 3, respectively, of ETV6. The reciprocal ETV6-HLXB9 transcript was not detected. It remains to be elucidated if the leukemic phenotype is attributable to the formation of the HLXB9-ETV6 fusion protein, which includes the helix-loop-helix and E26 transformation-specific DNA binding domains of ETV6 or to the disruption of the normal ETV6 protein.

High cure rate with a moderately intensive treatment regimen in non-high-risk childhood acute lymphoblastic leukemia. Results of protocol ALL VI from the Dutch Childhood Leukemia Study Group.

PURPOSE: Here we report the results of a nationwide cooperative study in the Netherlands on acute lymphoblastic leukemia (ALL) in children. The aim of the study was to improve the cure rate and to minimize side effects in a group of non-high-risk ALL patients, especially with regard to the CNS. A second aim was to study potential prognostic factors. METHODS: Children (age 0 to 15 years) with non-high-risk ALL (WBC count < 50 x 10(9)/L, no mediastinal mass, no B-cell phenotype, and no CNS involvement) were treated with a uniform protocol, ALL VI. The treatment protocol used 6-week induction regimen with three drugs (vincristine, dexamethasone, and asparaginase), three weekly doses of intravenous (IV) medium high-dose methotrexate (2 g/m2), and 2-year maintenance therapy that consisted of alternating 5-week periods of methotrexate and mercaptopurine and 2-week periods of vincristine and dexamethasone. In the first year of maintenance, triple intrathecal therapy was administered every 7 weeks. RESULTS: From December 1, 1984 until July 1, 1988, 291 children with ALL were diagnosed; 206 were categorized as non-high-risk (71%), and 190 were treated according to protocol ALL VI. At 8 years, the event-free survival (EFS) rate was 81% (SE = 3%) and survival rate 85% (SE = 2.9%); the median follow-up time was 7.3 years (range, 36 to 117 months). The CNS relapse rate was 1.1% (two of 184 patients who achieved a complete remission [CR]). The only factor found to be of negative prognostic importance in terms of EFS (P = .05) was a positive acid phosphatase reaction. CONCLUSION: For children with non-high-risk ALL, the combination of IV medium high-dose methotrexate (2 g/m2 times three), triple intrathecal therapy in the first year of maintenance treatment, and the use of dexamethasone for induction and pulses during maintenance treatment has proved to be highly effective, especially in the prevention of CNS relapse. A high cure rate was achieved without the use of anthracyclines, alkylating agents, and cranial irradiation.

Patient stratification based on prednisolone-vincristine-asparaginase resistance profiles in children with acute lymphoblastic leukemia.

PURPOSE: To confirm the prognostic value of a drug resistance profile combining prednisolone, vincristine, and l-asparaginase (PVA) cytotoxicity in an independent group of children with acute lymphoblastic leukemia (ALL) treated with a different protocol and analyzed at longer follow-up compared with our previous study of patients treated according to the Dutch Childhood Leukemia Study Group (DCLSG) ALL VII/VIII protocol. PATIENTS AND METHODS: Drug resistance profiles were determined in 202 children (aged 1 to 18 years) with newly diagnosed ALL who were treated according to the German Cooperative Study Group for Childhood Acute Lymphoblastic Leukemia (COALL)-92 protocol. RESULTS: At a median follow-up of 6.2 years (range, 4.1 to 9.3 years), the 5-year disease-free survival probability (pDFS) rate +/- SE was 69% +/- 7.0%, 83% +/- 4.4%, and 84% +/- 6.8% for patients with resistant (PVA score 7 to 9), intermediate-sensitive (PVA score 5 to 6), and sensitive (SPVA score 3 to 4) profiles, respectively (sensitive and intermediate-sensitive v resistant, P

Differences in immunoglobulin heavy chain gene rearrangmeent patterns between bone marrow and blood samples in childhood precursor B-acute lymphoblastic leaukemia at diagnosis.

Bone marrow (BM) and corresponding peripheral blood (PB) samples from 30 patients with precursor B-acute lymphoblastic leukemia (precursor B-ALL) were analyzed for the configuration of their immunoglobulin (Ig) heavy chain (IgH) and Ig kappa chain (Ig kappa) genes. Rearrangements and/or delections of the IgH and Ig kappa genes were detected in 100 and 47% of patients in this series of precursor B-ALL, respectively. Multiple rearranged IgH gene bands, generally differing in density, were found in 10 precursor B-ALL samples. This multi-band pattern is most probably caused by subclone formation due to continuing rearrangement processes. In five of the 10 bi/oligoclonal cases (50%) differences in IgH gene rearrangement patterns between BM and PB samples were observed, which could be interpreted as the presence of an edeletections of the IgH and Ig kappa genestra subclone in two cases and differences in the size of the subclones in three cases. In the 20 monoclonal precursor B-ALL, no dissimilarities in IgH gene rearrangement patterns between BM and the corresponding PB samples were found. Differences in Ig kappa gene rearrangement patterns between BM and PB were not observed in this series of precursor B-ALL, which is in line with the finding that no multiple Ig kappa gene rearrangements were detectable. In all five cases, the edelections of the IgH and Ig kappa genestra subclones or the relatively larger sized subclones were found in the BM samples, suggesting that subclone formation in precursor B-ALL occurs in the tissue compartment from which the precursor B-ALL cells are thought to originate. This phenomenon will lead to underestimation of subclone formation, if only IgH gene analysis of PB samples is performed. In addition, it will hamper the detection of minimal residual disease by the polymerase chain reaction mediated amplification of 'leukemia-specific' IgH gene junctional regions, because it is unpredictable which subclone will cause minimal residual disease and/or relapse.

tal-1 deletions in T-cell acute lymphoblastic leukemia as PCR target for detection of minimal residual disease.

Polymerase chain reaction (PCR) techniques based on amplification and identification of leukemia-specific DNA sequences provide a sensitive diagnostic method for detection of minimal residual disease (MRD) with a detection limit of 10(-5) to 10(-6) (1-10 malignant cells in 10(6) normal cells). To date, the main leukemia-specific DNA sequences used as PCR targets in detection of MRD are breakpoint fusion regions of chromosome translocations and junctional regions of rearranged immunoglobulin (Ig) or T-cell receptor (TcR) genes. The recently identified tal-1 deletions involving the sil and tal-1 genes, provide a potential MRD-PCR target. tal-1 deletions are site-specific because they are mediated via recombination signal sequences homologous to Ig/TcR genes. In line with this homology, tal-1 deletions also show random insertion and deletion of nucleotides at their breakpoints, resulting in highly variable breakpoint fusion regions. The fusion region diversity can be applied to design patient-specific oligonucleotide probes. Our Southern blot analyses of a large series of 313 acute leukemias with a specific tal-1 deletion probe (SILDB) demonstrated that tal-1 deletions exclusively occur in T-cell acute lymphoblastic leukemia (T-ALL) and not in precursor B-ALL or acute non-lymphocytic leukemias. In addition, we did not detect tal-1 deletions in normal blood cells and normal thymocytes by PCR analysis. The diversity observed in tal-1 deletion fusion regions with an average insertion and deletion of approximately 7 and approximately 6 nucleotides, respectively, allowed us to design fusion-region-specific probes. The specificity of the fusion-region probes was proven and the detection limit of the MRD-PCR technique was tested in a series of dilution experiments. The observed detection limit of 10(-5) indicates that tal-1 deletions in T-ALL represent ideal leukemia-specific PCR targets for detection of MRD.

Cellular drug resistance profiles that might explain the prognostic value of immunophenotype and age in childhood acute lymphoblastic leukemia.

Immunophenotype and age have prognostic value in childhood acute lymphoblastic leukemia (ALL) but how this operates is not understood. In 84 children with ALL at initial diagnosis we studied the correlation between these factors and the in vitro resistance to eight drugs, determined with the 3-(4,5-dimethylthiazol-2-yl-2, 5-diphenyl tetrazolium bromide (MTT) assay. B-lineage ALL samples were classified into four differentiation stages: the CD10- proB ALL; cALL; preB ALL with cytoplasmic mu positive ALL cells; and B-ALL with surface immunoglobulin-positive (Ig+) cells. cALL and preB ALL cases have the best prognosis; proB and T-ALL cases show a worse prognosis and B-ALL the poorest prognosis. Patients aged < 18 months and > 10 years have a poor prognosis compared to patients in the intermediate age group. Our results show that cALL and preB ALL cells were the most drug-sensitive cells compared to the other phenotypes. No differences were found between cALL and preB ALL cases with the exception that preB cells were more sensitive to mustine and mafosfamide (Maf). Compared to cALL and preB ALL cases, T-ALL cases were significantly more resistant to prednisolone (Pred), daunorubicin (DNR), L-asparaginase (L-Asp), cytosine arabinoside (AraC), and Maf; proB ALL cases were more resistant to Pred, DNR, L-Asp, and 6-thioguanine. The three B-ALL cases were resistant to vincristine and DNR. Two out of three B-ALL were resistant to Pred. Compared to cells from patients aged 18 months to 10 years, cells from children < 18 months were more resistant to Pred and DNR; cells from children > 10 years were more resistant to Pred. We conclude that cellular drug-resistance patterns might at least partly explain the prognostic value of immunophenotype and age in childhood ALL.

Extensive junctional diversity of gamma delta T-cell receptors expressed by T-cell acute lymphoblastic leukemias: implications for the detection of minimal residual disease.

Rearrangements, junctional regions and expression of T-cell receptor (TcR)-gamma and TcR-delta genes were analyzed in thirteen TcR-gamma delta + T-cell acute lymphoblastic leukemias (T-ALL). All TcR-gamma genes were rearranged except for one deleted allele and one allele in germline configuration. The expressed TcR-gamma protein chains showed a preference for V gamma l (11/13), J gamma 2.3 (7/13) and C gamma 2 (8/13). Furthermore, the TcR-gamma combinatorial repertoire appears to be even more limited by the fact that particular V gamma genes are preferentially used in TcR-gamma 1 or TcR-gamma 2 derived receptors, i.e. in disulfide-linked or non-disulfide-linked TcR-gamma delta receptors. The combinatorial repertoire of the expressed TcR-delta genes was homogeneous, as all thirteen T-ALL expressed V delta 1-D delta-J delta 1-C delta protein chains. In contrast to the limited combinatorial repertoire of the TcR-gamma and TcR-delta genes, the junctional diversity of both TcR genes was extensive due to insertion of N-region, P-region, and D delta gene nucleotides in addition to deletion of nucleotides by trimming. Using polymerase chain reaction (PCR)-mediated amplification and subsequent direct sequencing, we determined the junctional region sequences of all TcR-gamma and TcR-delta rearrangements. Sequence analysis showed that in the TcR-gamma junctional regions insertion varied from 0 to 25 nucleotides (average 8.0) and deletion from 1 to 27 nucleotides (average 8.7). In TcR-delta junctional regions, nucleotide insertion varied from 5 to 47 nucleotides (average 25.5) and deletion from 0 to 29 nucleotides (average 6.2) per junctional region. In general, the N-region nucleotides were the most prominent element in the junctional regions, i.e. 97% of the TcR-gamma and 55% of the TcR-delta junctional regions. D delta genes contributed significantly (40%) to the TcR-delta junctional diversity, whereas P-regions were hardly found in both TcR genes. Altogether, the junctional regions of TcR-delta genes were far more diverse than the junctional regions of TcR-gamma genes. With respect to the new methods of detecting minimal residual disease (MRD) in lymphoid malignancies utilizing PCR-mediated amplification of the junctional regions of TcR genes, our data indicate that this MRD-PCR analysis will generally be more sensitive if TcR-delta instead of TcR-gamma junctional-region-specific probes are used.

Limited combinatorial repertoire of gamma delta T-cell receptors expressed by T-cell acute lymphoblastic leukemias.

Detailed analysis of the rearrangement and expression of T-cell receptor gamma (TcR-gamma) and TcR-delta genes was performed in ten TcR-gamma delta+ T-cell acute lymphoblastic leukemias (T-ALL). In nine T-ALL the TcR-gamma genes were rearranged on both alleles, whereas in the tenth leukemia one allele was rearranged and the other was in germline configuration. Twelve out of the 19 rearranged alleles contained rearrangements of the J gamma 2.3 gene segment, five of which were to the V gamma 8 gene segment and three to the V gamma 3 gene segment. This implies that the combinatorial repertoire of the rearranged TcR-gamma gene is restricted due to the preferential usage of several V gamma and J gamma gene segments. The TcR-delta genes were rearranged on both alleles in nine T-ALL, whereas in the tenth leukemia one allele was rearranged and the other deleted. The combinatorial repertoire of the TcR-delta genes was homogeneous, as in all ten T-ALL at least one allele contained a V delta 1-J delta 1 rearrangement. In at least nine of the ten T-ALL the V delta 1-J delta 1 allele coded for the expressed TcR-delta chain, as was supported by reactivity with the anti-V delta 1-J delta 1 (delta TCS1) antibody in all T-ALL tested. As the total repertoire of the TcR molecules is not only dependent on combinations of gene segments, but also on the size and diversity of the junctional regions, we studied the V delta 1-J delta 1 junctional regions using the polymerase chain reaction (PCR) technique. These PCR analyses showed that the size of the V delta 1-J delta 1 junctional regions differed markedly (up to approximately 30 bases or more) between the leukemias. Therefore we conclude that the combinatorial repertoire of TcR-delta S+ T-ALL is limited, especially due to the homogeneous TcR-gamma gene rearrangements, but that the junctional repertoire of the TcR-delta genes seems to be extensive.

Multiple rearranged immunoglobulin genes in childhood acute lymphoblastic leukemia of precursor B-cell origin.

Sixty precursor B-cell acute lymphoblastic leukemia (ALL) patients were analyzed for the configuration of their immunoglobulin (Ig) genes. Rearrangements and/or deletions of the Ig heavy chain (IgH), Ig kappa chain (Ig kappa), and Ig lambda chain (Ig lambda) genes were detected in 98, 48, and 23% of cases, respectively. Although these percentages suggest the presence of a hierarchical order in IgH and Ig light chain (IgL) gene rearrangements during B-cell differentiation, no correlation was found between the immunophenotype of the precursor B-ALL and the arrangement patterns of their IgH and IgL genes. Multiple rearranged IgH gene bands, generally differing in density, were found in 27 (45%) of the precursor B-ALL in various restriction enzyme digests. Cytogenetic data were used to determine whether the presence of more than two rearranged IgH gene bands was caused by hyperdiploidy of chromosome 14 or other chromosome 14 aberrations. The combined cytogenetic and IgH gene data allowed the precursor B-ALL to be divided into three groups: a monoclonal group (n = 36; 60%), a biclonal group (n = 16; 27%), and an oligoclonal group (n = 8; 13%). In five biclonal ALL biclonality at the Ig kappa gene level was also found. Such subclone formation was not detected at the Ig lambda gene level. As the detection limit of the Southern blot technique is 2-5%, it might well be that small subclones remained undetected, implying that the frequency of subclone formation at the IgH gene level in precursor B-ALL is probably higher than 40%. It has been suggested that precursor B-ALL with multiple IgH gene rearrangements have a higher tendency to relapse. Although higher relapse rates were found in the oligoclonal group (53%) and in the combined bi-oligoclonal group (33%) compared with the monoclonal group (20%), the log rank trend test showed no significance. The occurrence of multiple subclones in precursor B-ALL as found by IgH gene analyses will severely hamper the detection of minimal residual disease using the polymerase chain reaction (PCR) mediated amplification of 'tumor-specific' IgH gene junctional regions, because it cannot be predicted which detectable (or undetectable) subclone will cause minimal residual disease and/or relapse. Therefore it can be expected that the PCR technique will frequently produce false negative results during the follow-up of precursor B-ALL.

Immunoglobulin kappa deleting element rearrangements in precursor-B acute lymphoblastic leukemia are stable targets for detection of minimal residual disease by real-time quantitative PCR.

Immunoglobulin gene rearrangements are used as PCR targets for detection of minimal residual disease (MRD) in acute lymphoblastic leukemia (ALL). We investigated the occurrence of monoclonal immunoglobulin kappa-deleting element (IGK-Kde) rearrangements by Southern blotting and PCR/heteroduplex analysis at diagnosis, their stability at relapse, and their applicability in real-time quantitative PCR (RQ-PCR) analysis. In 77 selected children with precursor-B-ALL, Southern blotting detected 122 IGK-Kde rearrangements, 12 of which were derived from subclones in six patients (8%). PCR/heteroduplex analysis with BIOMED-1 Concerted Action primers identified 100 of the 110 major IGK-Kde rearrangements (91%). Comparison between diagnosis and relapse samples from 21 patients with PCR-detectable IGK-Kde rearrangements (using Southern blotting, PCR/heteroduplex analysis, and sequencing) demonstrated that 27 of the 32 rearrangements remained stable at relapse. When patients with oligoclonal IGK-Kde rearrangements were excluded, 25 of the 27 rearrangements remained stable at relapse and at least one stable rearrangement was present in 17 of the 18 patients. Subsequently, RQ-PCR analysis with allele-specific forward primers, a germline Kde TaqMan-probe, and a germline Kde reverse primer was evaluated for 18 IGK-Kde rearrangements. In 16 of the 18 targets (89%) a sensitivity of < or =10(-4) was reached. Analysis of MRD during follow-up of eight patients with IGK-Kde rearrangements showed comparable results between RQ-PCR data and classical dot-blot data. We conclude that the frequently occurring IGK-Kde rearrangements are generally detectable by PCR (90%) and are highly stable MRD-PCR targets, particularly where monoclonal rearrangements at diagnosis (95%) are concerned. Furthermore, most IGK-Kde rearrangements (90%) can be used for sensitive detection of MRD (< or =10(-4)) by RQ-PCR analysis.

T cell receptor gamma gene rearrangements as targets for detection of minimal residual disease in acute lymphoblastic leukemia by real-time quantitative PCR analysis.

Several studies have shown that quantitative detection of minimal residual disease (MRD) predicts clinical outcome in childhood acute lymphoblastic leukemia (ALL). In this report we investigated the applicablility of T cell receptor gamma (TCRG) gene rearrangements as targets for MRD detection by real-time quantitative PCR analysis. Seventeen children with precursor-B-ALL and 15 children with T-ALL were included in this study. Using an allele-specific (ASO) forward primer in combination with germline Jgamma reverse primers and Jgamma TaqMan probes, a reproducible sensitivity of < or =10(-4) (defined by strict criteria) was obtained in only four out of 19 (21%) TCRG gene rearrangements in precursor-B-ALL patients and in 10 out of 15 (67%) TCRG gene rearrangements in T-ALL patients. The main reason for not obtaining a reproducible sensitivity of < or =10(-4) in approximately 60% of cases was the non-specific amplification of TCRG gene rearrangements in normal T-lymphocytes. A maximal sensitivity of < or =10(-4) (defined by less strict criteria) was obtained in 42% of TCRG gene rearrangements in precursor-B-ALL patients. The number of inserted nucleotides was significantly higher in T-ALL (mean: 8.5) as compared to precursor-B-ALL (mean: 6.8) and appeared to be the most important predictor for reaching a reproducible sensitivity < or =10(-4). The usage of a touchdown PCR or the usage of an ASO reverse primer in combination with Vgamma member forward primers and TaqMan probes did not clearly improve the overall results. Nevertheless, RQ-PCR analysis of TCRG gene rearrangements in follow-up samples obtained from 12 ALL patients showed the applicability of this method for MRD detection. We conclude that RQ-PCR analysis of TCRG gene rearrangements can be used for the detection of MRD, but that sensitivities might be limited due to non-specific amplification. This method is applicable in the majority of T-ALL patients and in almost half of precursor-B-ALL patients, particularly when used as second-choice target for confirmation of the MRD results obtained via the first-choice target.

Immunoglobulin light chain gene rearrangements display hierarchy in absence of selection for functionality in precursor-B-ALL.

The general order of the immunoglobulin (Ig) gene rearrangement process in human precursor-B cells is largely known. However, the exact Ig rearrangement patterns reflecting this process, especially those of the Ig light chain genes, are not well established. This requires detailed analysis of the gene configuration of all six IGH, IGK and IGL alleles at the single cell level. As such extensive analyses are difficult to perform in a reliable way within a single normal precursor-B cell, we used 169 precursor-B-ALL (ie six pro-B-ALL, 112 common ALL, and 51 pre-B-ALL) as clonal 'single cell' model system. The Ig gene recombinations show hierarchy starting with IGH gene rearrangements in all cases, followed by IGK rearrangements, IGK deletions and/or IGL rearrangements in 71% of cases. IGK deletions were found in the absence of IGL rearrangements in 34% of cases, which might be explained by the continuous recombinase activity in precursor-B-ALL, resulting in 'end-stage' IGK rearrangements, together with an apparently limited accessibility of the IGL locus. Remarkably, in 5% of cases IGL rearrangements took place in the absence of IGK rearrangements. In addition we found that in-frame IGH rearrangements are not necessarily required for the induction of Ig light chain gene rearrangements and that IGL rearrangements can be induced irrespective of the frame of the accompanying IGK rearrangements. In conclusion, precursor-B-ALL constitute a model system for studying Ig gene rearrangement processes without selection for functionality of the rearrangements or the influence of somatic hypermutations. Nevertheless, the hierarchy of IGH, IGK and IGL rearrrangements is apparent in precursor-B-ALL.

Immunophenotypic changes between diagnosis and relapse in childhood acute lymphoblastic leukemia.

To get more insight into the phenotypic changes of childhood acute lymphoblastic leukemia (ALL) at relapse, a detailed morphological and immunophenotypic study in 40 childhood ALL cases (32 precursor B-ALL and 8 T-ALL) was performed. Expression patterns of non-lineage specific markers (terminal deoxynucleotidyl transferase (TdT), CD34, and HLA-DR), B-lineage markers (CD10, CD19, CD20, and CD22), T-lineage markers (CD1, CD2, CD3, CD4, CD5, CD7, and CD8), and cross-lineage myeloid markers (CD14, CD15, and CD33) were compared at diagnosis and relapse. In case of low blast counts (< or = 70%) at relapse, double labeling for membrane markers and TdT was used in order to define the precise immunophenotype of the TdT+ leukemic cells. An immunological marker-shift was defined as either a conversion from positive to negative and vice versa or a difference in positivity of > or = 50%. Morphological differences between diagnosis and relapse were detected in 34% of precursor B-ALL and 14% of T-ALL. Differences in immunological marker expression were found in 72% of precursor B-ALL and in 75% of T-ALL, and generally concerned minor shifts with loss or acquisition of a few markers. The morphological shifts and immunophenotypic shifts were not correlated. Immunophenotypic shifts were found for all markers tested in precursor B-ALL, except for HLA-DR. Shifts in CD10 expression (16% of cases) were only observed in relapses occurring 30 months or more after diagnosis. In four precursor B-ALL an intra-lineage shift was found at relapse (one common ALL to null ALL and three pre-B-ALL to common ALL or null ALL) and two precursor B-ALL cases were diagnosed as acute non-lymphocytic leukemia at relapse based on morphology and immunophenotype. In T-ALL, neither intra-lineage nor inter-lineage shifts were observed, although shifts were detected in all T cell markers tested, except for the lineage specific CD3 and T cell receptor (TcR) markers. In conclusion, immunophenotypic shifts at relapse frequently occur in precursor B-ALL and T-ALL, in a small percentage leading to an intra-lineage shift (10%) or inter-lineage shift (5%). Therefore immunophenotypic monitoring of minimal residual disease in ALL patients should be based on multiple marker combinations, preferably together with polymerase chain reaction analysis of rearranged immunoglobulin and/or TcR genes or chromosome aberrations.