ETV6/RUNX1 abrogates mitotic checkpoint function and targets its key player MAD2L1.

Approximately 25% of childhood B-cell precursor acute lymphoblastic leukemia have an ETV6/RUNX1 (E/R) gene fusion that results from a t(12;21). This genetic subgroup of leukemia is associated with near-triploidy, near-tetraploidy, and trisomy 21 as rather specific types of secondary changes. Here, we show that, unlike various controls, E/R-expressing Ba/F3 clones acquire a tetraploid karyotype on prolonged culture, corroborating the assumption that E/R may attenuate the mitotic checkpoint (MC). Consistent with this notion, E/R-expressing diploid murine and human cell lines have decreased proportions of cells with 4N DNA content and a lower mitotic index when treated with spindle toxins. Moreover, both RUNX1 and E/R regulate mitotic arrest-deficient 2 L1 (MAD2L1), an essential MC component, by binding to promoter-inherent RUNX1 sites, which results in down-regulation of MAD2L1 mRNA and protein in E/R-expressing cells. Forced expression of E/R also abolishes RUNX1-induced reporter activation, whereas E/R with a mutant DNA-binding site leads to only minor effects. Our data link for the first time E/R, MC, and MAD2L1 and provide new insights into the function of the E/R fusion gene product. Although tetraploidy is an almost exclusive feature of E/R-positive leukemias, its rarity within this particular subgroup implies that further yet unknown factors are required for its manifestation.

Prognostic significance of minimal residual disease in infants with acute lymphoblastic leukemia treated within the Interfant-99 protocol.

Acute lymphoblastic leukemia (ALL) in infants younger than 1 year is a rare but relatively homogeneous disease ( approximately 80% MLL gene rearranged, approximately 70% CD10-negative) when compared with childhood and adult ALL. Several studies in children and adults with ALL have shown that minimal residual disease (MRD) status is a strong and independent prognostic factor. We therefore evaluated the prognostic significance of MRD in infant ALL. Ninety-nine infant patients treated according to the Interfant-99 protocol were included in this study. MRD was analyzed by real-time quantitative PCR analysis of rearranged immunoglobulin genes, T-cell receptor genes and MLL genes at various time points (TP) during therapy. Higher MRD levels at the end of induction (TP2) and consolidation (TP3) were significantly associated with lower disease-free survival. Combined MRD information at TP2 and TP3 allowed recognition of three patients groups that significantly differed in outcome. All MRD-high-risk patients (MRD levels > or =10(-4) at TP3; 26% of patients) relapsed. MRD-low-risk patients (MRD level <10(-4) at both TP2 and TP3) constituted 44% of patients and showed a relapse-rate of only 13%, whereas remaining patients (MRD-medium-risk patients; 30% of patients) had a relapse rate of 31%. Comparison between the current Interfant-06 stratification at diagnosis and the here presented MRD-based stratification showed that both stratifications recognized different subgroups of patients. These data indicate that MRD diagnostics has added value for recognition of risk groups in infant ALL and that MRD diagnostics can be used for treatment intervention in infant ALL as well.

Chromosome 14 copy number-dependent IGH gene rearrangement patterns in high hyperdiploid childhood B-cell precursor ALL: implications for leukemia biology and minimal residual disease analysis.

Childhood B-cell precursor acute lymphoblastic leukemia (BCP ALL) is generally a clonal disease in which the number of IGH rearrangements per cell does not exceed the number of the IGH alleles on chromosome 14. Consequently, monoclonal high hyperdiploid (HeH) cases with a trisomy 14 can harbor three rearrangements, a pattern that otherwise may be misinterpreted to be oligoclonal. Oligoclonal IGH rearrangements, on the other hand, may be instable at relapse and should therefore not be used for minimal residual disease analysis. We thus investigated the association between IGH allele copy numbers and the IGH rearrangement patterns in 90 HeH BCP ALL with either two (13%) or three copies (87%) of chromosome 14. HeH cases (44%) had an oligoclonal IGH rearrangement pattern, but true oligoclonality--after correction for the respective copy number of IGH alleles--was only 16%. Monoclonal and oligoclonal HeH cases had predominantly V(H) to preexisting DJ(H) recombinations, a finding that contrasts with oligoclonal cases of other major genetic BCP ALL subgroups in which V(H) replacements prevail. We conclude that for the precise assessment and correct interpretation of clonality patterns in BCP ALL, the IGH allele copy number has to be taken into consideration.

Cluster analysis of genomic ETV6-RUNX1 (TEL-AML1) fusion sites in childhood acute lymphoblastic leukemia.

Fusion between ETV6 and RUNX1 defines the largest genetic subgroup in childhood ALL. The genomic fusion site, unique to individual patients and specific for the malignant clone, represents an ideal molecular marker for quantification of minimal residual disease. Sequencing of DNA breakpoints has been difficult due to the extended size of the respective breakpoint cluster regions. We therefore evaluated a specially designed multiplex long-range PCR assay in 65 diagnostic bone marrow samples for its suitability in routine use. Resulting fusion sites and breakpoints derived from previous studies were subject to cluster analysis to identify potential sequence motifs involved in translocation initiation.

Analysis of minimal residual disease by Ig/TCR gene rearrangements: guidelines for interpretation of real-time quantitative PCR data.

Most modern treatment protocols for acute lymphoblastic leukaemia (ALL) include the analysis of minimal residual disease (MRD). To ensure comparable MRD results between different MRD-polymerase chain reaction (PCR) laboratories, standardization and quality control are essential. The European Study Group on MRD detection in ALL (ESG-MRD-ALL), consisting of 30 MRD-PCR laboratories worldwide, has developed guidelines for the interpretation of real-time quantitative PCR-based MRD data. The application of these guidelines ensures identical interpretation of MRD data between different laboratories of the same MRD-based clinical protocol. Furthermore, the ESG-MRD-ALL guidelines will facilitate the comparison of MRD data obtained in different treatment protocols, including those with new drugs.

Acute lymphoblastic leukemia with t(4;11) in children 1 year and older: The 'big sister' of the infant disease?

The t(4;11)-positive acute lymphoblastic leukemia (ALL) is a rare disease in children above the age of 1 year. We studied the clinical and biological characteristics in 32 consecutively diagnosed childhood cases (median age 10.0 years, range 1.0-17.1 years). Immunophenotyping revealed a pro-B and a pre-B stage in 24 and eight cases, respectively. IGH genes were rearranged in 84% of leukemias with a predominance of incomplete DJ(H) joints. Whereas IGK-Kde and TCRD rearrangements were rare, TCRG rearrangements were present in 50% of cases and involved mainly Vgamma11 or Vgamma9 together with a Jgamma1.3./2.3 gene segment, an unusual combination among t(4;11)-negative B-cell precursor ALL. Oligoclonality was found in about 30% as assessed by heterogeneous IGH and TCRG rearrangements. Our data are in line with transformation of a precursor cell at an early stage of B-cell development but retaining the potential to differentiate to the pre-B cell stage in vivo. Although a distinct difference between infant and older childhood cases with t(4;11) became evident, no age-related biological features were found within the childhood age group. In contrast to infants with t(4;11)-positive ALL, childhood cases had a relatively low cumulative incidence of relapse of 25% at 3.5 years with BFM-based high-risk protocols.

Optimization of PCR-based minimal residual disease diagnostics for childhood acute lymphoblastic leukemia in a multi-center setting.

Minimal residual disease (MRD) diagnostics is used for treatment stratification in childhood acute lymphoblastic leukemia. We aimed to identify and solve potential problems in multicenter MRD studies to achieve and maintain consistent results between the AIEOP/BFM ALL-2000 MRD laboratories. As the dot-blot hybridization method was replaced by the real-time quantitative polymerase chain reaction (RQ-PCR) method during the treatment protocol, special attention was given to the comparison of MRD data obtained by both methods and to the reproducibility of RQ-PCR data. Evaluation of all key steps in molecular MRD diagnostics identified several pitfalls that resulted in discordant MRD results. In particular, guidelines for RQ-PCR data interpretation appeared to be crucial for obtaining concordant MRD results. The experimental variation of the RQ-PCR was generally less than three-fold, but logically became larger at low MRD levels below the reproducible sensitivity of the assay (<10(-4)). Finally, MRD data obtained by dot-blot hybridization were comparable to those obtained by RQ-PCR analysis (r(2)=0.74). In conclusion, MRD diagnostics using RQ-PCR analysis of immunoglobulin/T-cell receptor gene rearrangements is feasible in multicenter studies but requires standardization; particularly strict guidelines for interpretation of RQ-PCR data are required. We further recommend regular quality control for laboratories performing MRD diagnostics in international treatment protocols.

High incidence and unique features of antigen receptor gene rearrangements in TEL-AML1-positive leukemias.

The t(12;21) translocation resulting in the TEL-AML1 gene fusion is found in 25% of childhood B-cell precursor (BCP) acute lymphoblastic leukemias (ALL). Since TEL-AML1 has been reported to induce cell cycle retardation and thus may influence somatic recombination, we analyzed 214 TEL-AML1-positive ALL by PCR for rearrangements of the immunoglobulin (Ig) and T-cell receptor (TCR) genes. As a control group, 174 childhood BCP ALL without a TEL-AML1 were used. The majority of TEL-AML1-positive leukemias had a higher number of Ig/TCR rearrangements than control ALL. They also had a more mature immunogenotype characterized by their high frequency of complete IGH, IGK-Kde, and TCRG rearrangements. While IGK-Kde and TCRG were more frequently rearranged on both alleles at higher age, IGH and TCRD rearrangements decreased in their incidence along with a decrease in biallelic IGH rearrangements. This suggests that the recombination process continues in these leukemias leading to ongoing rearrangements and possibly also deletions of antigen receptor genes. We here provide first evidence that somatic recombination of antigen receptor genes is affected by the TEL-AML1 fusion, and that further age-related differences are probably caused by the longer latency period of the prenatally initiated TEL-AML1-positive leukemias in older children.

Presence of N regions in the clonotypic DJ rearrangements of the immunoglobulin heavy-chain genes indicates an exquisitely short latency in t(4;11)-positive infant acute lymphoblastic leukemia.

Childhood acute lymphoblastic leukemia (ALL) is frequently initiated in utero at a time of developmentally regulated insertion of N regions into the DJ(H) rearrangements of immunoglobulin heavy-chain (Ig(H)) genes. Here it is shown that N regions are present in the clonotypic DJ(H) rearrangements in 11 of 12 infant ALLs with t(4;11). These data are compared with the 122 previously published DJ(H) sequences and were found to have a pattern similar to that of ALL in children older than 3 years at diagnosis but were unlike that in children younger than 3 years who predominantly lack N regions. These findings, therefore, indicate that t(4;11)-positive infant ALL is initiated later in fetal development than most B-cell precursor ALL from children younger than 3 years and that they have a shorter latency period already in utero.

Origins of "late" relapse in childhood acute lymphoblastic leukemia with TEL-AML1 fusion genes.

Approximately 20% of childhood B-precursor acute lymphoblastic leukemia (ALL) has a TEL-AML1 fusion gene, often in association with deletions of the nonrearranged TEL allele. TEL-AML1 gene fusion appears to be an initiating event and usually occurs before birth, in utero. This subgroup of ALL generally presents with low- or medium-risk features and overall has a very good prognosis. Some patients, however, do have relapses late or after the cessation of treatment, at least on some therapeutic protocols. They usually achieve sustained second remissions. Posttreatment relapses, or even very late relapses (5-20 years after diagnosis), in childhood ALL are clonally related to the leukemic cells at diagnosis (by IGH or T-cell receptor [TCR] gene sequencing) and are considered, therefore, to represent a slow re-emergence or escape of the initial clone seen at diagnosis. Microsatellite markers and fluorescence in situ hybridization identified deletions of the unrearranged TEL allele and IGH/TCR gene rearrangements were analyzed; the results show that posttreatment relapse cells in 2 patients with TEL-AML1-positive ALL were not derived from the dominant clone present at diagnosis but were from a sibling clone. In contrast, a patient who had a relapse while on treatment with TEL-AML1 fusion had essentially the same TEL deletion, though with evidence for microsatellite instability 5(') of TEL gene deletion at diagnosis, leading to extended 5(') deletion at relapse. It is speculated that, in some patients, combination chemotherapy for childhood ALL may fail to eliminate a fetal preleukemic clone with TEL-AML1 and that a second, independent transformation event within this clone after treatment gives rise to a new leukemia masquerading as relapse. (Blood. 2001;98:558-564)

Biased distribution of chromosomal breakpoints involving the MLL gene in infants versus children and adults with t(4;11) ALL.

Derivative chromosomes of 40 patients diagnosed with t(4;11) acute lymphoblastic leukemia (ALL) were analysed on the genomic DNA level. Chromosomal breakpoints were identified in most cases within the known breakpoint cluster regions of the involved MLL and AF4 genes. Due to our current knowledge of the primary DNA sequences of both breakpoint cluster regions, specific features were identified at the chromosomal fusion sites, including deletions, inversions and duplications of parental DNA sequences. After separation of all t(4;11) leukemia patients into two age classes (below and above 1 year of age), the analysis of chromosomal fusion sites revealed significant differences in the distribution of chromosomal breakpoints and led to the definition of two hotspot areas within the MLL breakpoint cluster region. This may point to the possibility of different age-linked mechanisms that were leading to t(4;11) chromosomal translocations.

Influence of transplantation regimen on prognostic significance of high-level minimal residual disease before allogeneic stem cell transplantation in children with ALL.

High MRD levels before transplantation in children receiving T cell-depleted unrelated grafts for relapsed ALL were associated with a 100% relapse risk. We report on two children with relapsed ALL who underwent non T cell-depleted BMT from unrelated donors. Despite a high residual tumor load pre- transplant and the occurrence of only aGVHD grade I, they are still in second complete remission 2.7 and 5.2 years after transplantation, respectively. Thus, we propose that the transplant regimens influence the prognostic significance of high MRD levels pre-BMT.

Molecular detection of minimal residual disease is a strong predictive factor of relapse in childhood B-lineage acute lymphoblastic leukemia with medium risk features. A case control study of the International BFM study group.

The medium-risk B cell precursor acute lymphoblastic leukemia (ALL) accounts for 50-60% of total childhood ALL and comprises the largest number of relapses still unpredictable with diagnostic criteria. To evaluate the prognostic impact of minimal residual disease (MRD) in this specific group, a case control study was performed in patients classified and treated as medium (or intermediate)-risk according to the criteria of national studies (ALL-BFM 90, DCLSG protocol ALL-8, AIEOP-ALL 91), which includes a good day 7 treatment response. Standardized polymerase chain reaction (PCR) analysis of patient-specific immunoglobulin and T cell receptor gene (TCR) rearrangements were used as targets for semi-quantitative estimation of MRD levels: > or =10(-2), 10(-3), < or =10(-4). Twenty-nine relapsing ALL patients were matched with the same number of controls by using white blood cell count (WBC), age, sex, and time in first complete remission, as matching factors. MRD was evaluated at time-point 1 (end of protocol Ia of induction treatment, ie 6 weeks from diagnosis) and time-point 2 (before consolidation treatment, ie 3 months from diagnosis). MRD-based high risk patients (> or =10(-3) at both time-points) were more frequently present in the relapsed cases than in controls (14 vs 2), while MRD-based low risk patients (MRD negative at both time-points) (1 vs 18) showed the opposite distribution. MRD-based high risk cases experienced a significantly higher relapse rate than all other patients, according to the estimated seven-fold increase in the odds of failure, and a much higher rate than MRD-based low risk patients (OR = 35.7; P= 0.003). Using the Cox model, the prediction of the relapse-free interval at 4 years was 44.7%, 76.4% and 97.7% according to the different MRD categories. MRD-based risk group classification demonstrate their clinical relevance within the medium-risk B cell precursor ALL which account for the largest number of unpredictable relapses, despite the current knowledge about clinical and biological characteristics at diagnosis. Therefore, MRD detection during the first 3 months of follow-up can provide the tools to target more intensive therapy to those patients at true risk of relapse.

Detection of residual disease in pediatric B-cell precursor acute lymphoblastic leukemia by comparative phenotype mapping: method and significance.

The present review summarizes our efforts in developing a novel immunologic approach ("Comparative Phenotype Mapping") targeted at assessing minimal residual disease (MRD) in B-cell precursor (BCP) acute lymphoblastic leukemia (ALL) patients. The method relies on quantitatively aberrant, leukemia-associated antigen expression patterns which allow to discriminate leukemic from normal BCP using a limited panel of antibody combinations and multidimensional flow cytometry. In an analysis of 63 follow up bone marrow samples of patients with BCP-ALL we show that this approach enables to efficiently detect MRD. Further clinical observation revealed that the patients which were MRD-positive by flow cytometry (although in morphological remission) had a very high probability of early disease recurrence compared to the good chances of a relapse-free survival (RFS) in the MRD-negative cohort (RFS 0.0 vs. 0.76 at 3 years). Comparative Phenotype Mapping thus proves to be a reliable method for MRD detection in BCP-ALL. Concluding remarks relate to the optional applications of the method as well as to future perspectives. An ongoing large prospective study which we are now conducting on the basis of Comparative Phenotype Mapping will clarify the clinical significance of MRD detection in ALL patients by this method, and will determine its value compared to related as well as molecular-genetic techniques.

Requirement of residual thymus to restore normal T-cell subsets after human allogeneic bone marrow transplantation.

BACKGROUND: To determine the effect of residual thymic activity in reconstituting the T-cell system after T cell-depleting therapy, we monitored T-cell subsets of a unique thymectomized cancer patient in comparison to thymus-bearing patients after allogeneic bone marrow transplantation (BMT). METHODS: T cells and T-cell subsets previously shown in murine studies to be regulated by the thymus were analyzed by FACS from 6 to >48 months after BMT. The investigation of thymus-bearing patients included 32 examinations of 9 children and 14 adults. None of the investigated cases had severe graft-versus-host disease or severe infections when examined. RESULTS: In the thymectomized host, T-cell regeneration occurred by donor cell expansion and was characterized by two prominent features: (i) a persistent failure to regenerate naive (CD45RA+) T-helper cells (14%, median), consistent with the recently developed concept of a thymus-dependency; and (ii) persistently elevated proportions of CD3+CD4-CD8- cells (double-negative cells, median 29%), which were identified in T cell receptor (TCR)gamma delta+ (22%, median of CD3+ cells, 88% double negatives) but also TCRalpha beta+ T-cell populations (78%, median of CD3+ cells, 17% double negatives). In thymus-bearing patients, 10 of 12 and 6 of 14 examinations of children and adults, respectively, performed later than 12 months after BMT showed the proportion of CD4+CD45RA+ cells appropriate for age (>52% and >28% in children and adults, respectively). Elevated double-negative cells (>10%) were found in only three patients, but none had elevated double-negative cells with a TCRalpha beta+ phenotype. CONCLUSION: Residual thymic activity might, in addition to its well-established role for regenerating naive T-helper (CD4+CD45RA+) cells, control the expansion of double-negative cells. A normal T-cell subset regeneration in a proportion of thymus-bearing adult hosts indicates the potential of an effective residual thymic activity even beyond childhood.

Rapid molecular response during early induction chemotherapy predicts a good outcome in childhood acute lymphoblastic leukemia.

Early response to therapy is an independent prognostic factor in childhood acute lymphoblastic leukemia. Although most patients have rapid early responses, as detected by morphology, 15% to 20% of patients have relapses. The authors evaluated residual disease by molecular methods on day 15 of minimal residual disease (MRD) therapy and compared these data with their recently established MRD-based risk stratification, defined by MRD levels 5 weeks after induction treatment and before consolidation. All 68 children treated according to current Berlin-Frankfurt-Münster (BFM) protocols went into morphologically complete remission after induction. There was a significant difference in outcome between children with rapid disease clearance and those with high levels of day-15 MRD (P =.035). Among patients with high levels of day-15 MRD, only the MRD-based risk stratification was predictive of the outcome. All patients with negative or low day-15 MRD had excellent prognoses and were in the MRD-based low-risk group. Thus, after only 2 weeks of treatment, the authors were able to identify a patient population of 20% who may benefit from the least intensive treatment.

Presence of clone-specific antigen receptor gene rearrangements at birth indicates an in utero origin of diverse types of early childhood acute lymphoblastic leukemia.

There is strong evidence that infant leukemias with a t(4;11) translocation originate in utero. To test whether other subtypes of childhood leukemias are also initiated during fetal life, we used clone-specific genetic markers for the analysis of neonatal blood spots from 5 children aged 6 months to 4 years 8 months at diagnosis of pro-B, common acute lymphoblastic leukemia (ALL), and T-ALL. In all children, the clonotypic antigen receptor gene rearrangements were already present at birth. The estimated amount of clonotypic cells was in the range of 10 to 100 cells per blood spot. In 2 infants with a t(4;11) positive ALL, we detected similar amounts of the fusion gene sequences compared with the clonal antigen receptor gene rearrangements, suggesting the presence of both markers in the same cells. Our data indicate that the first leukemogenic event of diverse types of childhood ALL may already occur in utero. (Blood. 2000;95:2722-2724)

Heterogeneous TCR delta Vdelta2-Ddelta3 rearrangements and their relation to IgH and TCR gamma gene status in childhood B cell precursor leukaemias.

In this study we aimed to test the hypothesis that leukaemias with oligoclonal Vdelta2-Ddelta3 rearrangements are clonal at the IgH and TCRG gene status and that the oligoclonality is a poor prognostic marker. In ten leukaemias the individual Vdelta2-Ddelta3 rearrangements characterised different populations as deduced from single cell analysis and/or from densitometric differences of PCR products. Five of these leukaemias were clonal by the IgH and/or TCRG gene status. We therefore conclude that ALL is a clonal disease despite the presence of heterogeneous TCRD rearrangements. Our clinical data show that oligoclonality at the TCRD level does not represent an adverse prognostic factor.

Multiplex PCR reaction for the detection and identification of immunoglobulin kappa deleting element rearrangements in B-lineage leukaemias.

Immunoglobulin kappa (Igkappa) gene recombinations can be used - similarly to IgH rearrangements - as clonal markers in B-lineage leukaemias. Based on the extensive junctional diversity, these rearrangements represent valuable targets for the analysis of minimal residual disease (MRD). In order to provide a simple method for the rapid detection of leukaemia clone-specific kappa deleting element (Kde) mediated rearrangements, we developed a multiplex PCR reaction that is able to amplify the five most frequent rearrangements in one tube. Position of the amplimers were chosen to enable identification of the involved segments according to the size of the PCR product. This method was tested on 101 B-lineage leukaemias (71 childhood B-cell precursor acute lymphoblastic leukaemias (BCP ALL) and 30 chronic lymphocytic leukaemias (CLL)). 39 and 22 Kde rearrangements could be readily detected in 30 (44%) BCP ALL and 22 (56%) CLL, respectively. 36% of the Kde rearrangements in BCP ALL and 45% in CLL were intron recombination signal sequence (RSS)-Kde rearrangements. The other Kde rearrangements involved the Vkappa families: VkappaI in 36% and 50%, VkappaII in 32% and 16.7%, VkappaIII in 24% and 25%, and VkappaIV in 8% and 8.3% in BCP ALL and CLL, respectively. The sensitivity of the multiplex system was 10-2-10-3. We compared this multiplex PCR assay with multiple single PCR reactions using different sets of primer combinations. Thereby the number and types of rearrangements were confirmed in all cases. Clonality of rearrangements was proven by sequence analysis. Our data show that by this method clonal Kde rearrangements were rapidly detected and precisely identified.