Stability and prognostic influence of FLT3 mutations in paired initial and relapsed AML samples.

In acute myeloid leukemia (AML), activating mutations in the fms-like tyrosine kinase 3 (FLT3) gene predict poor prognosis. We determined FLT3 internal tandem duplications (FLT3/ITD) and D835 point mutations in paired initial and relapse samples from 80 pediatric and adult AML patients. One D835 point mutation was found in an initial pediatric AML sample. Fms-like tyrosine kinase 3/ITDs were present in 21 initial and 22 relapse samples (26.3 and 27.5%, respectively). Interestingly, FLT3/ITD positivity was related to a significantly shorter time to relapse, most pronounced when the ITD-positive status was found at relapse (P<0.001). However, FLT3/ITD status changed between diagnosis and relapse in 14 cases. In four patients, the FLT3/ITD became undetectable at relapse in five patients FLT3/ITDs were only detected at relapse, and in five patients the length or number of FLT3/ITDs changed. Gain of FLT3/ITDs may suggest oligoclonality with selective outgrowth of the FLT3/ITD-positive clone, whereas losses may reflect ITDs in the more mature leukemic cells rather than in the leukemic stem cell, or, alternatively, that other genetic aberrations provided a greater selective advantage. Studying FLT3/ITD kinetics in minimal residual disease setting may provide some answers for the changes we observed. Fms-like tyrosine kinase 3/ITD is a relevant marker for prognosis, and remains an important target for therapeutic inhibition.

A comparison of the in vitro cytotoxicity of daunorubicin and liposomal daunorubicin in pediatric acute leukemia.

Anthracyclines are effective in the treatment of leukemia, but their use is limited because of cardiotoxicity. Liposomal daunorubicin (L-DNR) is potentially less cardiotoxic than daunorubicin (DNR). We compared in vitro cytotoxicity in pediatric acute leukemia samples and found no significant differences between cytotoxicity of DNR and L-DNR.

Causes of death--other than progressive leukemia--in childhood acute lymphoblastic (ALL) and myeloid leukemia (AML): the Dutch Childhood Oncology Group experience.

We analyzed causes of death, other than resistant disease or relapse, in 875 children with acute lymphoblastic leukemia (ALL) and 229 with acute myeloid leukemia (AML), treated on three different Dutch Childhood Oncology Group (DCOG) ALL and three AML protocols. Overall, 23 (2.6%) ALL and 44 (19.2%) AML patients died. Early death (ED, before remission was reached) occurred in nine ALL (1%) and thirty AML (13.1%) patients, including three and ten deaths before treatment was initiated. Chemotherapy-related mortality in remission (CRM) occurred in nine ALL (1.1%) and eight AML (4.4%) patients. For ALL, both ED and CRM declined over time, although this was not statistically significant. For AML a decrease in ED was observed (from 26% to approximately 10%), but counter-balanced by an increase in CRM (from 3 to 8%), maybe related to the scheduling of intensification blocks in AML-92/94. Including transplant-related mortality, death in CR rates in AML increased from 3 to 15% in the last study. The main cause of ED was hemorrhage, often associated with hyperleucocytosis, and infection for CRM. We conclude that mortality dropped favorably in ALL, but not in AML. Especially for AML, effective but less toxic therapy and better supportive care guidelines need to be developed.

Mutations in KIT and RAS are frequent events in pediatric core-binding factor acute myeloid leukemia.

Activating mutations in RAS and receptor tyrosine kinases such as KIT and FLT3 are hypothesized to cooperate with chimeric transcription factors in the pathogenesis of acute myeloid leukemia (AML). To test this hypothesis, we genotyped 150 pediatric AML samples for mutations in KIT (exons 8, 17), NRAS and KRAS (exons 1, 2) and FLT3/ITD. This is the largest cohort of pediatric AML patients reported thus far screened for all four mutations. Of the children with AML, 40% had a mutation in KIT (11.3%), RAS (18%) or FLT3/ITD (11.1%), and 70% of cases of core-binding factor (CBF) leukemia were associated with a mutation of KIT or RAS. Mutations in RAS or FLT3/ITD were frequently found in association with a normal karyotype. Patients with a FLT3/ITD mutation had a significantly worse clinical outcome. However, the presence of a KIT or RAS mutation did not significantly influence clinical outcome. We demonstrate that KIT exon 8 mutations result in constitutive ligand-independent kinase activation that can be inhibited by clinically relevant concentrations of imatinib. Our results demonstrate that abnormalities of signal transduction pathways are frequent in pediatric AML. Future clinical studies are needed to determine whether selective targeting of these abnormalities will improve treatment results.

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.

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.

Prognostic significance of karyotype at diagnosis in childhood acute lymphoblastic leukemia [corrected].

Acute lymphoblastic leukemia (ALL) is the most frequent childhood cancer. The disease is heterogeneous. The leukemic cells in childhood ALL patients show various karyotypic abnormalities, express diverse phenotypes, and respond variably to treatment. Identification of prognostic factors will make it possible to predict treatment outcome and to identify the patients that require different therapeutic approaches. The important prognostic implications of chromosome number and of the presence of several well defined structural chromosomal aberrations have been established by several groups. We analyzed 145 children with ALL at diagnosis for cytogenetic features. In 135 cases cytogenetic analysis was successful. Of these, 101 showed abnormal karyotype (70%). Structural chromosomal rearrangements were detected in 71 out of 135 cases investigated (53%), i.e. 71% of the cytogenetically abnormal cases. These cytogenetic findings were correlated with clinical outcome. In our series the ploidy of the karyotype appeared to be of prognostic importance. Our findings concerning karyotype, phenotype, and treatment outcome of this subgroup were in most cases in agreement with earlier reports from other investigators. We observed an increased risk for central nervous system relapse in childhood ALL patients with abnormalities involving the short arm of chromosome 12 in combination with pre-B or common ALL phenotype.

Recombinant hematopoietic growth factors fail to induce a proliferative response in precursor B acute lymphoblastic leukemia.

We have tested a panel of recombinant hematopoietic growth factors (HGF) including the interleukins (IL) 1, 2, 3, 4, and 6 and the colony stimulating factors GM-CSF, G-CSF, M-CSF for their ability to induce proliferation of precursor B acute lymphoblastic leukemia cells (ALL) from 19 patients. In Ficoll-Isopaque isolated and T cell-depleted ALL bone marrow samples, IL2 (two cases), IL3 (four cases), and GM-CSF (one case) infrequently stimulated DNA synthesis measured by 3H-thymidine (TdR) uptake, and the other recombinant growth factors completely failed to do so. In repeat experiments with ALL blasts purified by fluorescence activated cell sorting (FACS), IL2, IL3, and GM-CSF responses could not be reproduced, suggesting that nonleukemic contaminant cells, and not the ALL blasts, had been stimulated by these factors. Cocktails containing combinations of IL1-IL4 and IL6 also lacked proliferation inducing potency. Depending on the purity of the incubated ALL cell samples, an impure preparation of B cell growth factors that has been reported to contain a highly effective stimulatory activity for precursor B ALL cells induced proliferation of residual normal cells as well as the ALL cells, as was evident from combined analysis of DNA synthesis and karyotyping. Exposure of the ALL blasts to artificial activators of protein kinase C and Ca2+ mobilization resulted in significant rises in 3H-TdR uptake, suggesting that these intracellular compounds are involved in transducing signals that upregulate proliferation. Although it remains possible that some of the human recombinant growth factors promote the growth of precursor B ALL cells in combination with other stimuli, a dominant role in the regulation of proliferation of these cells cannot be attributed to any of these cytokines at the present time.

Cytogenetic clonality analysis in myelodysplastic syndrome: monosomy 7 can be demonstrated in the myeloid and in the lymphoid lineage.

Bone marrow and blood from three patients with myelodysplastic syndrome (MDS) and monosomy 7 were studied for cell lineage involvement of the chromosomal abnormality. Cytogenetic involvement of the myeloid and erythroid cell lineages in MDS with monosomy 7 has been shown before. Lymphoid subpopulations have also been investigated but generally with negative results. A combined technique of May-Grünwald-Giemsa (MGG) for cell cytology and interphase fluorescence in situ hybridization (FISH) using a chromosome 7 specific DNA probe was applied. Further, immunophenotype and genotype of the cells were simultaneously examined with alkaline phosphatase anti-alkaline phosphatase (APAAP) immunostaining and FISH. The monosomy 7 was found in the blasts and in all or in subpopulations of myeloid and erythroid cells. T cells (CD3+, CD5+) did not appear to be involved. B cells (CD19+, CD22+) showed a normal distribution of FISH spots in two patients. In one patient however the loss of a chromosome 7 was found in approximately 70% of the cells positive for B cell markers including CD79a. The results of this study show that in some cases MDS is a disease arising in a progenitor cell with repopulative abilities restricted to myelopoiesis and erythropoiesis. In other cases, the pluripotent progenitor cells in MDS may show the capacities to differentiate into B lineage lymphoid cells, as well as suggesting that in those instances MDS represents a condition of more primitive transformed hematopoietic ancestor cells.

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.

12p chromosomal aberrations in precursor B childhood acute lymphoblastic leukemia predict an increased risk of relapse in the central nervous system and are associated with typical blast cell morphology.

Recently we reported cytogenetic, clinical, and immunologic data of 135 childhood ALL patients, who were diagnosed and treated in The Sophia Children's Hospital between January 1, 1980 and November 1, 1990. An increased risk for a first relapse in the central nervous system (CNS) was detected in a subgroup of childhood ALL patients with common ALL or pre-B ALL phenotype and chromosomal aberrations of the short arm of chromosome 12. In this paper we report clinical, cytogenetic, immunologic, morphologic and cytochemical data on these eight childhood ALL patients with aberrations of the short arm of chromosome 12 and of an additional four cases that were diagnosed and treated between November 1, 1990 and February 1, 1992. We found that three out of six common ALL, two out of three pre-B ALL and one out of three T-ALL patients with 12p chromosomal rearrangements developed a first relapse in the CNS. On the contrary, the frequency of CNS relapse in our childhood ALL patients without 12p aberrations was 10%. Furthermore, morphologic and cytochemical analysis of the bone marrow smears of these 12 patients with aberrations of the short arm of chromosome 12 revealed that the nine cases with pre-B or common ALL phenotype had typical morphologic characteristics that are unusual for newly diagnosed childhood ALL. Typical for this subtype is the presence of large polymorphic blast cells without nucleoli. The nuclei are irregularly shaped showing folds and clefts and a stripy pattern. The nucleus and cytoplasm are often abundantly vacuolated. The cytoplasm has a foamy light-blue appearance.

Prognostic implication of hyperdiploidy as based on DNA flow cytometric measurement in childhood acute lymphocytic leukemia--a multicenter study.

The pretreatment distribution of DNA content was determined in the leukemic blasts of 114 children with standard risk acute lymphocytic leukemia. The patients were admitted to four different centers for pediatric oncology, participating in a national study ALL-V. In 39 of 107 evaluable patients (36.4%), a single aneuploid leukemic line was detected with a median DNA Index of 1.22 (range 1.10-1.40). These hyperdiploid patients did not differ from those with diploid disease for the presenting features of age, sex, FAB classification, immunophenotype, or white blood cell count. However, patients with hyperdiploid acute lymphocytic leukemia had a significantly longer (p = 0.021) disease-free survival after a median observation period of 52 months. These observations indicate that routinely applied flow cytometry of DNA content can identify a fairly large subgroup of children with standard risk acute lymphocytic leukemia who have a low probability of relapse. It may be considered to exempt these patients from more intensive treatment regimens.

Translocation (6;9) may be associated with a specific TdT-positive immunological phenotype in ANLL.

Two patients with acute nonlymphocytic leukemia (ANLL) (FAB-M4) and t(6;9)(p23;q34) are described. Immunological marker analysis revealed a phenotype of HLA-DR+/partly terminal deoxynucleotidyl transferase (TdT)+/CD13+ in both cases and CD33 positivity in one. The expression of CD13 and CD33 by TdT-positive cells was demonstrated by double immunofluorescence staining. Although it has been postulated that TdT plays a role in gene rearrangement, Southern blot analysis performed in one leukemia revealed that both the T cell receptor beta chain genes and the immunoglobulin heavy chain genes were in germ line configuration. Since we could not detect CD13+/TdT+ cells and CD33+/TdT+ cells in control bone marrow samples, double marker analysis was used to detect low numbers of residual leukemic cells during follow-up of one patient. A gradually increasing percentage of CD33+/TdT+ cells was detected in the bone marrow in a period of 6 months before hematological relapse. Although the t(6;9) may not be correlated to a specific French-American-British subtype, it may be associated with TdT-positive ANLL. Since TdT-positive ANLL seems to have a poor outcome, detection of TdT expression in ANLL patients is particularly important for diagnostic purposes. In addition, our results indicate that double immunological marker analysis for a myeloid marker and TdT allows detection of residual disease during follow-up.

In vitro cytotoxicity of mitoxantrone, daunorubicin and doxorubicin in untreated childhood acute leukemia.

Mitoxantrone (MIT) has not been studied as a single agent in children with untreated leukemia. The antileukemic activity of MIT in these patients and its activity in relation to clinical and cell biological features is unknown. We studied the in vitro cytotoxicity of MIT, daunorubicin (DNR) and doxorubicin (DOX) in untreated childhood acute lymphoblastic leukemia (ALL, n = 131) and acute nonlymphoblastic leukemia (ANLL, n = 20) samples, using the MTT assay. There were marked interindividual differences in resistance to all three drugs. A strong, significant cross-resistance was found in ALL between MIT, DNR and DOX. All samples of the T-lineage, a prognostically unfavorable immunophenotype, however, were significantly more resistant to DNR and DOX, but not to MIT, than common or pre-B ALL samples. ALL cells from children with a prognostically unfavorable age at diagnosis, especially those < 2 years, showed a relative resistance to all three drugs compared to the intermediate age-group. This was found within all patients, but also within the common or pre-B ALL cases only. Sex, white blood cell count, or FAB type was not related to in vitro drug resistance. None of the three drugs showed an overall preferential activity in ALL or ANLL. We conclude that the in vitro antileukemic activity of MIT, DNR and DOX is related to certain clinical and cell biological features. There were no major differences between the three drugs in antileukemic activity, except that T-ALL samples were more resistant than common or pre-B ALL samples to DNR and DOX, while MIT was equally active in these two immunophenotypes.

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.

Detection of residual disease in AML patients by use of double immunological marker analysis for terminal deoxynucleotidyl transferase and myeloid markers.

In the majority of patients with acute myeloid leukemia (AML) immature leukemic subpopulations expressing myeloid markers and terminal deoxynucleotidyl transferase (TdT) are present. The normal counterparts of these double-positive cells are rare in bone marrow (BM) (< 0.03%; if they occur at all) and are not detectable in peripheral blood (PB). In 14 patients with TdT+ AML at diagnosis, we have performed a prospective follow-up study to monitor the myeloid-marker+, TdT+ cells during and after chemotherapy. One patient did not obtain complete remission (CR), a second patient relapsed under therapy, whereas the other 12 patients were in cytomorphological CR at the end of chemotherapy. During subsequent follow-up, seven of these 12 patients developed one or two relapses (total of ten relapses). Nine of these ten relapses were preceded by a gradual increase of myeloid-marker+, TdT+ cells in BM and PB samples over a period of 14-38 weeks. Based on comparable results in BM and PB samples and doubling times of 15-20 days, we propose that monitoring of AML patients should include PB sampling each 4-6 weeks. In one patient the relapse was not preceded by a gradual increase of double-positive cells. This false negative result was caused by a phenotypic shift, since at relapse the AML cells did not express TdT. In the five AML patients who still are in continuous cytomorphological CR for 32-46 months we repeatedly detected relatively high percentages of myeloid-marker+, TdT+ cells in BM (up to 0.1%) and PB (up to 0.02%). Although we could not prove the leukemic origin of these double-positive cells, they might represent residual dysplastic AML cells which survived chemotherapy but which are not capable of causing leukemia regrowth as yet. This would be in line with recent polymerase chain reaction studies, which could demonstrate the persistence of leukemic clones in the majority of AML patients in continuous CR. It is concluded that double immunofluorescence labeling for myeloid markers and TdT is useful for detection of residual disease in TdT+ AML patients. A gradual increase of double-positive cells is suggestive for leukemic cell growth and can be used to predict relapse.

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.

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.

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.

Characterization of the blast cells in acute leukemia with translocation (4;11): report of eight additional cases and of one case with a variant translocation.

Eight new cases (five adults and three children) of acute leukemia characterized by the presence in bone marrow cells of a t(4;11)(q21;q23)and one similar case, a child, with a t(1;11)(p32;q23) are reported. All patients were diagnosed as having acute lymphoblastic leukemia (ALL) with high-risk features. Immunologically the blast cells of the nine cases showed a strikingly consistent immature lymphoid phenotype, i.e., TdT+, HLA-DR+, B4(CD19)+, CALLA(CD10)-, Smlg-, cmu-, BA-2(CD9)+ corresponding to a "null ALL." In addition, six of nine cases expressed the myeloid marker VIM-D5(CD15). By double immunofluorescence staining it was determined that the VIM-D5(CD15) antigen was expressed by terminal deoxynucleotidyl transferase-positive blast cells, excluding the possibility of double leukemia. In five cases investigation of the Ig heavy chain genes by Southern blot analysis showed clonal rearrangement of both Ig heavy chain gene alleles. These data suggest that the blast cells involved in t(4;11) leukemia represent early B-cell progenitors with "aberrant" expression of myelomonocytic features, possibly related to the 11q23 breakpoint.