Search

Background and objective: The characteristic chromosomal translocation t(9;22)(q34;q11) in chronic myeloid leukaemia (CML) mainly results in the two different BCR/ABL fusion transcripts b2a2 or b3a2. Both transcript variants can occur simultaneously due to alternative splicing of the b3a2 transcript. Conflicting results have been reported on the influence of the transcripts on haematological findings at diagnosis and the course of the disease in adults while data concerning these topics on childhood CML are still missing. This paper reports on a correlation of BCR/ABL transcript variants with patients’ characteristics in childhood CML. Design and methods: Transcript types were determined in 146 paediatric patients with CML enrolled in trial CML-paed-I. Fifty-five patients (38%) expressed b2a2, 53 patients (36%) b3a2 and 38 patients (26%) both transcripts, respectively. These findings were correlated with patients’ characteristics (sex, age, WBC, Hb, platelet count, hepatosplenomegaly, etc.) assessed at diagnosis. Results: While the co-expression of both transcripts was evenly distributed among genders [b2a2 + b3a2: 22 females (28%), 16 males (24%)] a highly significant difference (P = 0.007) was found concerning the expression of the b2a2 transcript [34 male (51%) vs. 21 female (27%)] and vice versa of the b3a2 transcript [17 male (25%) vs. 36 female (45%)]. High platelet counts and the combination of high platelet counts in conjunction with pronounced leukocytosis were observed more often in patients expressing the b3a2 transcript. Conclusions: These findings demonstrate that in children like in adults specific BCR/ABL transcript types present at diagnosis are associated with distinct haematological alterations (e.g. a high platelet count with the transcript b3a2). However, the sex-dependent skewed distribution of the BCR/ABL transcript types observed so far in this paediatric cohort only deserves further investigation.

Clinical and histopathological characteristics have limited prognostic value for children with anaplastic large-cell lymphoma (ALCL). We evaluated the presence, extent, and prognostic impact of circulating tumor cells in bone marrow (BM) and peripheral blood (PB) of children and adolescents with NPM-ALK–positive ALCL at diagnosis using qualitative and quantitative polymerase chain reaction (PCR) for NPM-ALK. Numbers of NPM-ALK transcripts were normalized to 104 copies ABL (NCNs). BM was analyzed from 80 patients and PB from 52. BM was positive for NPM-ALK in 47.5% of patients, and positivity was significantly correlated with clinical stage, mediastinal or visceral involvement, microscopic BM involvement, and histologic subtype. Qualitative and quantitative PCR results in BM and PB strongly correlated. BM PCR was associated with the cumulative incidence of relapses (CI-Rs): CI-R was 50% ± 10% for 38 PCR-positive and 15% ± 7% for 42 PCR-negative patients (P < .001). Sixteen patients with more than 10 NCNs NPM-ALK in BM had a CI-R of 71% ± 14% compared with a CI-R of 18% ± 6% for 59 patients with 10 or fewer NCNs (P < .001). PB PCR results led to a similar grouping. Thus, quantitative PCR in BM or PB allows identification of 20% of patients experiencing 60% of all relapses with an event-free survival of 20%.

We obtained a global view of gene expression in both cell lines and pediatric acute lymphoblastic leukemia (ALL) samples that harbor one of several selected chromosomal abnormalities. When the cell lines were studied alone, we found that these chromosomal abnormalities were associated with the predominant variation in transcriptional programs across the set of cell lines studied. When cell lines and clinical samples were studied together, we found that each chromosomal abnormality (TEL/AML1, BCR/ABL, or MLL abnormalities) was associated with a characteristic gene expression signature that was shared by both cell lines and clinical samples. However, BCR/ABL was associated with a much more heterogeneous pattern of expression than were TEL/AML1 and MLL abnormalities. This observation has important implications for the study of BCR/ABL ALL. In addition, we systematically identified genes whose expression was associated with TEL/AML1, BCR/ABL, or MLL abnormalities in both clinical samples and cell lines. Although some of these genes have previously been described, many have not previously been reported to be associated with one of these chromosomal abnormalities. Notably, we found that the erythropoietin receptor (EPOR) is consistently highly expressed in TEL/AML1 ALL compared with BCR/ABL or MLL. (Blood. 2004;103:1043-1049)

The family of multidrug resistance-associated proteins (MRPs) belongs to the superfamily of adenosine triphosphate-binding-cassette (ABC) transporters, which have the ability to function as outward pumps for chemotherapeutic drugs and therefore might be involved in drug resistance. In this study the expression of the MRP2, MRP3, MRP4, MRP5, and SMRP genes was measured using TaqMan real-time polymerase chain reaction (PCR) in 103 children with previously untreated acute lymphoblastic leukemia (ALL) (precursor B-cell ALL [B-ALL], n = 71; T-cell ALL [T-ALL], n = 32). All 5 genes were expressed with a great variability. Only MRP3 expression was associated with a significantly worse prognosis (P = .008). The median expression of MRP3 was 10-fold higher in T-ALL than in precursor B-ALL (P < .001) and 4-fold higher in male patients than in female patients (P < .001). The prognostic impact of MRP3 was independent of immunophenotype or sex. Higher levels of MRP3 were found in patients with a poor in vivo response to prednisone, but this could not be confirmed in an independent case-control study (40 patients) for prednisone response. In healthy donors, the median expression of MRP4 was 4-fold higher in bone marrow and 8-fold higher in CD34+ stem cells compared with peripheral blood (P = .002). Our results suggest that MRP3 is involved in drug resistance in childhood ALL. It therefore represents an interesting target to overcome multidrug resistance. High levels of MRP3 could possibly be the reason for the poorer prognosis of male patients or patients who have T-ALL. Similar to other members of the family of ABC transporters, MRP4 seems to be a marker for immature stem cells. (Blood. 2003;102:4493-4498)

Chromosomal translocations are a characteristic feature of leukaemia and other malignant diseases. As clonal markers, they can be applied to identify and quantify the number of malignant cells by polymerase chain reaction (PCR) methods. The translocation t(4;11) is present in >60% of infant leukaemia. In order to facilitate the sequencing of chromosomal breakpoints, we developed an optimized set of 30 PCR primers and a new approach, designated as asymmetric multiplex PCR (am-PCR). Due to the high number of primers, small breakpoint-spanning DNA fragments are obtained in one nested multiplex PCR reaction. All PCR products contain an identical binding site for the initiation of direct sequencing. By using am-PCR, the translocation t(4;11) was examined in bone marrow and blood samples from children with acute leukaemia. Compared with previously described methods for the determination of genomic breakpoints, am-PCR may be advantageous with regard to its simplicity and rapidity. Breakpoint-spanning sequences were also evaluated with regard to their applicability as unique clonal markers to design primers and probes for minimal residual disease quantification by real-time PCR. This approach can easily be adapted to other chromosomal translocations in malignant diseases for the detection and analysis of clone-specific DNA markers.

The fusion transcript AML1/ETO corresponding to translocation t(8;21)(q22;q22) can be found in approximately 7-12% of childhood de novo AML. Despite the favorable prognosis, some of these patients relapse. Most of MRD studies so far were performed on adults treated not uniformly. Therefore, we analyzed the follow-up of 15 AML1/ETO-positive children using real-time quantitative reverse transcription PCR (RQ-RT-PCR), all enrolled in the multicenter therapy trial AML-BFM 98. AML1/ETO copy numbers were normalized to the control gene ABL and the results were expressed in copy numbers AML1/ETO per 10 000 copies ABL. At diagnosis, a median of 10 789 copies AML1/ETO was found. A linear decrease to about 10 copies (2-4 log) could be seen in most of the children by the start of consolidation. In the majority of cases they remained positive at this low level during the ongoing therapy. Four children relapsed and two of them had a decrease of less than 2 log before starting consolidation. Three of the relapsed children showed, prior to relapse, an increase of the AML1/ETO fusion transcript at 6, 9, and 11 weeks, respectively. These results suggest that monitoring of minimal residual disease using RQ-RT-PCR could be helpful in detecting patients with a higher risk of relapse.

The translocation t(9;11)(p22;q23) is a recurring chromosomal abnormality in acute myeloid leukemia (AML) fusing two genes designated as MLL and AF9. Within MLL, almost all rearrangements cluster in an 8.3-kb restricted region and fuse 5' portions of MLL to a variety of heterologous genes in various 11q23 translocations. AF9 is one of the most common fusion partners of MLL. It spans more than 100 kb, and two breakpoint cluster regions (BCRs) have been identified in a telomeric region of intron 4 (BCR1) and within introns 7 and 8 (BCR2). We investigated 11 children's bone marrow or peripheral blood samples (3 AML, 5 t-AML, 2 ALL, 1 ALL relapse) and two cell lines (THP-1 and Mono-Mac-6) with cytogenetically diagnosed translocations t(9;11). By use of an optimized multiplex nested long-range PCR assay, a breakpoint-spanning DNA fragment from each sample was amplified and directly sequenced. In four patients and two cell lines, the AF9 breakpoints were located within BCR1 and in two patients within BCR2, respectively. However, in five patients the AF9 breakpoints were found outside the previously described BCRs within the centromeric region of intron 4 and even within intron 3 in one case. All five patients with a secondary AML, who had not received etoposides during treatment of the primary malignant disease, revealed almost identical MLL breakpoints very close to a breakage hot spot inducible by topoisomerase II inhibitors or apoptotic triggers in vitro. Sequence patterns around the breakpoints indicated involvement of a "damage-repair mechanism" in the development of t(9;11) similar to t(4;11) in infants' acute leukemia.

The translocation t(8;21)(q22;q22), which results in the fusion of the AML1 (RUNX1) and ETO (CBFA2T1) genes, is a recurrent aberration in acute myeloid leukemia (AML), preferentially correlated with FAB M2, and has the highest incidence in childhood AML. Because of the favorable prognosis, the evidence of the t(8;21) or the AML1/ETO fusion gene is mandatory in most of the therapy trials, allowing the stratification of the patients to the correct risk group in terms of treatment. Here we present six out of 59 children with AML who were positive for AML1/ETO by RT-PCR, but showed no evidence of the classical t(8;21)(q22;q22) by conventional cytogenetics. Because of the discrepancies between molecular and cytogenetic analyses, these six patients were further investigated by fluorescence in situ hybridization analysis. Small hidden interstitial insertions resulting in an AML1/ETO rearrangement were detected in five (8.5%) of the 59 patients, whereas the sixth patient showed a cryptic three-way translocation. The insertions could be characterized as ins(21;8) in three patients and ins(8;21) in the remaining two. Additionally, three of the patients showed secondary chromosome aberrations leading to a higher complexity of the karyotype. In conclusion, the combination of more than one standard technique in the analysis of AML1/ETO is useful to reveal the overall frequency of cryptic chromosome rearrangements and permits a better understanding of the mechanisms involved in the generation of this fusion gene. © 2003 Wiley-Liss, Inc.

We used karyotyping, fluorescence in situ hybridization (FISH), Southern blotting, and RT-PCR in order to analyze prospectively 77 infants (less than 1 year of age) with acute lymphoblastic leukemia for the occurrence of 11q23/MLL rearrangements and/or other cytogenetic abnormalities. Out of the 69 informative samples we found an 11q23/MLL rearrangement in 42 cases (61%). Regarding only pro-B ALL cases, the incidence of 11q23/MLL rearranged cases, however, reached more than 90% The infants were treated within the therapy studies ALL-BFM90, ALL-BFM95 and CoALL-05-92. For patients with an adequate follow-up of 4 years the event-free survival of the 11q23/MLL-positive and 11q23/MLL-negative group was 0.2 or 0.64, respectively (P = 0.024). The monoclonal antibody 7.1. (moab 7.1) does not react with normal hematopoetic precursors or mature blood cells but was shown to specifically react with leukemic cells bearing a rearrangement of chromosome 11q23 or the MLL gene, respectively. We, therefore, specifically addressed the question whether the reactivity of moab 7.1, as determined by flow cytometry, may substitute for molecular testing of an 11q23/MLL rearrangement in this cohort of infant ALLs. Reactivity of moab 7.1 indicated a 11q23/MLL rearrangement with a specificity of 100%. However, five of the 11q23/MLL-positive cases did not react with moab 7.1 indicating a sensitivity of 84% only. Three of these five moab 7.1-negative but 11q23/MLL-positive cases could be identified by their unique expression pattern of CD65s and/or CD15. Thus, 95% of all 11q23/MLL-positive ALL cases in infancy may be identified by flow cytometry based on their expression of CD15, CD65s and/or moab 7.1.

The expression of the PRAME gene (preferentially expressed antigen of melanoma) was measured by quantitative reverse transcriptase polymerase chain reaction in 50 children with newly diagnosed acute myeloid leukemia (AML), three samples of CD34(+) stem cells, six bone marrow samples, and 10 peripheral blood samples of healthy donors, as well as three AML cell-lines (KG-1, U937, and HL-60). Eight patients were also analyzed in relapse. Contrary to previous reports, we could show that the PRAME gene is expressed by CD34(+) stem cells. This might constitute a problem in using PRAME for tumor immunotherapy. Overexpression of PRAME was found in 62% (n=31) of our patients. The rates of overall and disease-free survival in this group were higher than in patients with no or low expression (P0.05). PRAME expression was negatively correlated to the white blood cell count at diagnosis (P0.05) and significantly higher in patients with t(8;21). The levels of expression at diagnosis corresponded with those at relapse (P0.001) and increased levels could be found prior to the relapse in one patient who was regularly monitored. Our results suggest that the expression of PRAME is an indicator of favorable prognosis and could be a useful tool for monitoring minimal residual disease in childhood AML.

We have isolated the human GRAF gene (for GTPase regulator associated with the focal adhesion kinase pp125 FAK ). This gene was fused with MLL in a unique t(5;11)(q31;q23) that occurred in an infant with juvenile myelomonocytic leukemia. GRAF encodes a member of the Rho family of the GTPase-activating protein (GAP) family. On the protein level, it is 90% homologous to the recently described chicken GRAF gene that functions as a GAP of RhoA in vivo and is thus a critical component of the integrin signaling transduction pathway. The particular position of the human GRAF gene at 5q31 and the proposed antiproliferative and tumor suppressor properties of its avian homologue suggest that it also might be pathogenetically relevant for hematologic malignancies with deletions of 5q. To investigate this possibility, we sequenced 4–5 individual cDNA clones from 13 cases in which one allele of GRAF was deleted. We found point mutations within the GAP domain of the second GRAF allele in one patient. In two additional patients we found an insertion of 52 or 74 bp within the GRAF cDNA that generates a reading frame shift followed by a premature stop codon. GRAF maps outside the previously defined commonly deleted 5q31 region. Nevertheless, inactivation of both alleles in at least some cases suggests that deletions and mutations of the GRAF gene may be instrumental in the development and progression of hematopoeitic disorders with a del(5q).

Chromosomal rearrangements in childhood acute lymphoblastic leukemia (ALL) play an important role in the identification of clinical relevant subgroups. For rapid and easy detection of the clinically most important gene rearrangements, a nested multiplex reverse transcriptase polymerase chain reaction (multiplex PCR) was developed. This multiplex PCR enables the detection of M-BCR/ABL, m-BCR/ABL, TEL/ AML1, and MLL/AF4 fusion transcripts in one PCR reaction. However, the existence of splicing variants and different breakpoints on the DNA level hampers the discrimination of the rearrangements by their fragment size on an agarose gel. Therefore, one of the internal primers of each translocation (ABL-2, TEL-2, AF4-2) was labeled with a characteristic fluorescent dye, and an automatic fluorescence-based DNA fragment analysis was performed. The sensitivity of this multiplex PCR is in the same range as that of the corresponding single PCR reaction and allows a fast screening for the detection of therapy-relevant rearrangements, with a high turnover of samples.

TEL/AML1 gene fusion is the most frequent genetic lesion in pediatric acute lymphoblastic leukemia (ALL). It occurs as a consequence of the cryptic chromosomal translocation t(12;21)(p13;q22). In a cohort of 50 RT-PCR-positive TEL/AML1 patients, karyotype examination by GTG banding and fluorescence in situ hybridization (FISH) allowed us to identify chromosome anomalies in addition to the already existing t(12;21). Secondary aberrations were found in 29 out of 41 patients (71%) at initial diagnosis and in all 9 patients with relapse. Structural rearrangements affected chromosome arms 2p, 2q, 5q, 9p, 12p (n = 2), 6q, 11p (n = 3), and 21q (n = 4). An extra chromosome 21 was found to be the most frequent anomaly. It was detected in 6 out of 41 patients at initial diagnosis (15%) and in 7 out of the 9 patients at relapse. No karyotype with trisomy 21 exceeded 47 chromosomes. Gain of chromosome 21 was the sole anomaly in GTG-banding analysis in 2/41 patients at initial diagnosis and in 4/9 at relapse. Notably, chromosome painting analysis performed in 11 out of the 13 patients with an extra chromosome 21 revealed duplication of the normal chromosome 21 in 8, and duplication of der(21)t(12;21) in 3 patients. Furthermore, gain of der(21)t(12;21) chromosome was confined exclusively to the relapse patients.

Treatment response and residual-disease monitoring in initial and relapsed TEL-AML1 positive childhood ALL

Quantification of minimal residual disease (MRD) based on clonotypic immunoglobulin/ T-cell receptor (Ig/TCR) gene rearrangements is widely used as an independent prognostic parameter in childhood acute lymphoblastic leukemia (ALL). In this study we compared MRD by quantification of Ig/TCR targets and genomic ETV6-RUNX1 specific sequences. In ten of twelve patients with t(12;21)+ ALL we observed concordance with rapid blast reduction in nine, and high-level persistence in one case. The two remaining patients showed low-level persistence of the genomic breakpoint specific sequence. These patients have remained in complete remission for 38 and 41 months, so far, indicating that a small ETV6-RUNX1-positive clone is not detrimental to the short-term prognosis of affected children.

NAD(P)H:quinone oxidoreductase 1 (NQO1) is an enzyme that protects cells against mutagenicity from free radicals and toxic oxygen metabolites. The gene coding for NQO1 is subject to a genetic polymorphism at nucleotide position 609 (C--T) of the human NQO1 cDNA. Heterozygous individuals (C/T) have intermediate activity and homozygotes for the variant allele (T/T) are deficient in NQO1 activity. In previous studies, genotypes conferring lower NQO1 activity have been associated with an increased risk of acute leukemia, particularly infant leukemia carrying MLL/AF4 fusion genes. In the present study, we investigated this association in our population and extended the analysis to other subgroups of pediatric hematologic neoplasms characterized by specific fusion genes.We genotyped 138 patients with childhood acute lymphoblastic leukemia (ALL) carrying distinct fusion genes (MLL/AF4=35; BCR/ABL=31; TEL/AML1=72), 71 cases of pediatric sporadic Burkitt's lymphoma and 190 healthy control individuals for the NQO1 C609T polymorphism.When compared to the healthy control group, only children with Burkitt's lymphoma significantly more often had NQO1 genotypes associated with lower NQO1 activity (odds ratio, 1.81; p=0.036), predominantly at a younger age (9 years at diagnosis: odds ratio, 3.02; p=0.003).Our results suggest that in our population the NQO1 C609T polymorphism does not confer an increased risk of the investigated entities of childhood ALL. However, there may be a modulating role for NQO1 in the pathogenesis of pediatric sporadic Burkitt's lymphoma.

Detection of minimal residual disease (MRD) has proven to provide independent prognostic information for treatment stratification in several types of leukemias such as childhood acute lymphoblastic leukemia (ALL), chronic myeloid leukemia (CML) and acute promyelocytc leukemia. This report focuses on the accurate quantitative measurement of fusion gene (FG) transcripts as can be applied in 35-45% of ALL and acute myeloid leukemia, and in more than 90% of CML. A total of 26 European university laboratories from 10 countries have collaborated to establish a standardized protocol for TaqMan-based real-time quantitative PCR (RQ-PCR) analysis of the main leukemia-associated FGs within the Europe Against Cancer EAC) program. Four phases were scheduled: (1) training, (2) optimization, (3) sensitivity testing and (4) patient sample testing. During our program, three quality control rounds on a large series of coded RNA samples were performed including a balanced randomized assay, which enabled final validation of the EAC primer and probe sets. The expression level of the nine major FG transcripts in a large series of stored diagnostic leukemia samples (n = 278) was evaluated. After normalization, no statistically significant difference in expression level was observed between bone marrow and peripheral blood on paired samples at diagnosis. However, RQ-PCR revealed marked differences in FG expression between transcripts in leukemic samples at diagnosis that could account for differential assay sensitivity. The development of standardized protocols for RQ-PCR analysis of FG transcripts provides a milestone for molecular determination of MRD levels. This is likely to prove invaluable to the management of patients entered into multicenter therapeutic trials.

To define prognostic factors in infant acute lymphoblastic leukemia (ALL), the outcome of 106 infants (age ≤12 months) during 3 consecutive multicenter trials of the Berlin-Frankfurt-Münster group (ALL-BFM 83, 86, and 90) was retrospectively analyzed according to presenting features and early in vivo response to prednisone. The prednisone response was defined as the cytoreduction (number of blood blasts per microliter at day 8) to a 7-day prednisone prephase and 1 intrathecal dose of methotrexate on day 1. Prednisone good responder (PGR

The purpose of this review is to present an account of our current understanding of the structure, organization and evolution of mitochondrial genomes, and to discuss the origin and evolution of mitochondria from the perspective recently provided by the extensive sequencing of various mitochondrial genomes. Mitochondrial-en-coded protein phylogenies are congruent with nuclear phylogenies and strongly support a monophyletic origin of mitochondria. The newly available data from red-algal mitochondrial genomes, in particular, show that the structural and functional diversity of mitochondrial genomes can be accounted for by paralogous evolution. We also discuss the influence of other constraints, such as uniparental inheritance, on the evolution of genome organization in mitochondria.

Treatment of relapsed acute myelogeneous leukaemia with MLL/ AF6 fusion after stem cell transplantation by intensive reinduction followed by adoptive immunotherapy

Mitochondrial DNA from the unicellular rhodophyte Cyanidium caldarium RK-1 and the multicellular Chondrus crispus were isolated, cloned, and sequenced. Two genes, sdhB and sdhC, that encode subunits of the succinate dehydrogenase, were identified by similarity. These genes form a cluster (sdhCB) in both red algae.

Granulocyte colony-stimulating factor receptor expression and 11q23/ MLL genotype in childhood acute lymphoblastic leukemia developing during the first 18 months of life

Acute lymphoblastic leukemia (ALL) is the most common malignancy in childhood. In vivo response to initial therapy, as assessed by determination of minimal residual disease (MRD) at 5 and 12 weeks of treatment, has evolved as the strongest prognostic factor in pediatric ALL patients treated according to the BFM regime. In this study, ten patients with a poor treatment response (MRD load ≥ 10−3 at week 12, MRD-high risk, HR) and five treatment-sensitive (no measurable MRD at weeks 5 and 12, MRD-standard risk, SR) patients were investigated by means of high-resolution bacterial artificial chromosome (BAC) array-based comparative genomic hybridization (array-CGH). To ensure homogeneity with regard to prognostic factors, the following inclusion criteria were used: B cell precursor or common ALL, DNA index of 1.0, no BCR/ABL, no MLL/AF4, no TEL/AML1 rearrangements. A gain for all chromosome 21-related BAC clones was observed in all SR patients. None of the ten HR patients showed a gain of any region of chromosome 21. This result could be confirmed by means of fluorescence in situ hybridization using the LSI AML1/ETO DC probe set. Besides the basic level of chromosome 21 gain, higher levels of gain or amplification observed for individual regions were found in three SR patients. Recurrent genomic alterations in the group of HR patients were loss of chromosomal region 2p11.22 (9/10), a gain of 8q24.13 (7/10) and loss of 14q32 (8/10). To see whether these findings may help to further subdivide the intermediate risk group patients (any MRD positivity at week 5, MRD load < 10−3 at week 12) we additionally analyzed three patients of this group: two patients in long-term complete remission and one patient who relapsed 29 months after diagnosis. Again, a gain for all chromosome 21-related clones was detected in both patients in complete remission. In contrast, the leukemic sample of the relapsed patient demonstrated no gain of chromosome 21. We conclude that array CGH analysis in childhood ALL may lead to the identification of additional prognostic markers. The relevance of the observed gain of chromosome 21 has to be validated in a larger set of patients.

The ALK gene which is typically rearranged in anaplastic large cell lymphoma (ALCL) and inflammatory myofibroblastic tumor (IMT) is located in the band 2p23. An involvement of this band was detected in 6 patients (0.35%) when we checked the cytogenetic database of our laboratory with approximately 1500 successfully analyzed karyotypes of childhood AML and MDS. Patient 1 had a t(2;4)(p23;q27) and patient 2 showed a del(2)(p23). In both cases, the 2p23 abnormality was part of a complex karyotype that included several non-specific alterations. Among the remaining four children, three (pts. 3–5) showed an inv(2)(p23q13) and patient 6 a t(2;2)(p23;q13). We performed a hybridization using the LSI ALK Dual Color Break Apart Rearrangement Probe (Vysis, Downers Grove, IL) onto all six patients. Whereas in the nuclei of pts 1, 2, 3 two normal fusion signals were found, a split hybridization signal showed the ALK involvement in the inv(2) and the t(2;2) of patients 4, 5 and 6. The latter case was further analyzed using chromosome arm-specific painting probes and this approach also confirmed the translocation. For the identification of the partner gene we used the primers for RANBP2/ALK which were described for IMT. In all patients with ALK involvement a PCR product was found and the sequencing identified identical breakpoints within the ALK and RANBP2 gene. In addition, a FISH and RT-PCR screening was performed in 20 children with AML or MDS, but no ALK involvement could be detected. All 3 ALK rearranged patients were originally diagnosed as myeloid disease (AML/MDS), but showed different unspecific morphological features. To the best of our knowledge this is the first description of an inv(2)(p23q13) and a t(2;2)(p23;q13) and the first report of an RANBP2/ALK rearrangement in childhood AML/MDS. Further studies will help to elucidate the role of this rearrangement in pediatric leukemia.

The adaptor protein SLP-65 plays an essential role during B cell differentiation. A crucial consequence of SLP-65 deficiency in mice is a high incidence of pre-B-cell leukemia, suggesting a tumor suppressor role for SLP-65 in pre-B-cells. While the link between SLP-65 deficiency and leukemia development is established in mice, experiments mainly using microarrays for gene expression profiling suggested normal expression of SLP-65 in human precursor B-cell ALL. This analysis however does not discriminate between normal and aberrant SLP-65 transcripts with the latter being unable to generate functional protein. To examine the correlation between SLP-65 deficiency and childhood precursor B-cell ALL, we determined SLP-65 expression in 119 precursor B-cell ALL samples by both RNA and protein methods. The expression of SLP-65 was compared to clinical and laboratory findings, cytogenetics as well as to the outcome data within this uniformly treated cohort of patients. Loss of slp-65 protein was significantly associated with the occurrence of the TEL/AML1 rearrangement (p=0.026) but not with any other clinical or cytogenetic feature. We found a profound disconnection between slp-65 mRNA and protein expression in 38 out of the 119 leukemic samples pointing to a posttranscriptional regulation of slp-65 (Table). To confirm that SLP-65 transcript expression does not automatically correlate with its protein expression, we analyzed a panel of human cell lines derived from precursor B-cell ALL patients. The cell lines HPB-NULL and BV-173 showed a deficiency in SLP-65 protein expression, although SLP-65 transcripts can easily be detected in both lines. Together, the data suggest that SLP-65 expression might be regulated at the posttranscriptional level and that the presence of SLP-65 transcripts does not necessarily lead to SLP-65 protein and function. In one particular patient, we found a truncated slp-65 transcript and the predicted slp-65 protein lacks its SH2 domain. We tested whether this SLP-65 protein lacking the SH2 domain is functional in pre-B cells. To this end, we transfected murine SLP-65 −/− pre-B cells with retroviral constructs for either wild-type (wt SLP-65) or truncated SLP-65 (SLP-65delSH2) and analysed pre-BCR downregulation, Ca2+ release and pre-B cell differentiation. The results showed that, in contrast to wt SLP-65, SLP-65delSH2 failed to induce any effects in the performed experiments. Together with previous findings showing that SLP-65-deficient mice develop pre-B cell leukemia, the data suggest that SLP-65 acts as a tumor suppressor that limits pre-B cell proliferation by inducing differentiation. Disconnection between slp-65 transcripts and protein expression total slp-65 protein+ (51 patients) slp-65 protein weak (19 patients) slp-65 protein- (49 patients) PCR+ 108 51(9 TEL/AML+, 42 TEL/AML-) 19 (9 TEL/AML+, 10 TEL/AML-) 38 (15 TEL/AML+, 23 TEL/AML-) PCR- 11 0 0 11 (T-ALL)

The hallmark of chronic myeloid leukemia (CML) is the reciprocal translocation t(9;22)(q34;q11) which creates a hybrid BCR/ABL gene that is transcribed into a chimeric mRNA. In the 8.5-kb mRNA either exon b2 or exon b3 of the major breakpoint cluster region (M-bcr) is coupled to C-ABL exon 2. These two junction variants (b2a2 or b3a2) differ in size by 75 nucleotides and can be selectively amplified by semi-nested PCR. A controversy exists whether or not the type of transcript and/or the breakpoint site exerts an influence on the clinical course of CML in adult patients (Mills KI 1991; Foiretos T 1993; Melo JV 1996). CML is a rare disease in childhood contributing only 2 – 3 % of all pediatric leukemia cases. This may be the reason why there are only scant data from small series of children with CML giving information on the transcript variant present at diagnosis. During an 11 year period (1995 – 2005) 203 pts younger than 19 years (median age: 11.4 yrs; 101 boys, 102 girls) with Philadelphia-chromosome positive CML were treated within the multicenter GPOH-trial “CML-paed”. 85% of the pts were diagnosed in chronic phase (CP). Treatment was performed by hydroxyurea +/− interferon and pts were scheduled for stem cell transplantation (SCT) from an HLA-matched family donor within 6 months after diagnosis and from an unrelated donor within 12 months. Bone marrow and/or peripheral blood specimen for molecular analysis were collected at diagnosis and during follow-up examinations at 3 – 6 months intervals. RT-PCR with analysis of the breakpoint and transcript type was performed in 154 pts (77.4 % of the total study population). 133 pts exhibited a Major-BCR rearrangement, 5 pts a minor-BCR rearrangement and in 16 pts both rearrangements were present. In pts with M-BCR either 55 pts expressed the b2a2 transcript (37 %) or the b3a2 transcript (37 %), respectively. Both types of transcripts were found either simultaneously or alternately during follow-up examinations in 38 pts (25 %). One pt showed an atypical rearrangement which is awaiting further classification. No statistical significant differences (p > 0.05) could be found with respect to hematological parameters at diagnosis (white cell count, platelets count, percentage of basophils in peripheral blood), clinical findings (liver size, spleen size) and disease status (CP versus advanced phase). The impact of either transcript upon the course of the disease (e.g. duration of chronic phase) was not analyzed because 70 % of the study population was transplanted early within 1 year after diagnosis. We conclude that in contrast to some reports in adults the type of BCR breakpoint seems to have no impact in pediatric pts with CML.

The overall unfavorable prognosis of CD10 negative (CD10−) precursor B-cell acute lymphoblastic leukemia (BCP-ALL) is well known. We analyzed 4473 pediatric patients (pts) n* 5y-pEFS* (%) SE (%) p (log-rank) *5 pts w/o reinduction were excluded sex female 109 55 5 0.022 male 119 40 5 age Out of a number of immunophenotypic markers, analyzed at different expression cut-off points, CD24 at missing or weak expression of age Including age, WBC, PR and MLL/AF4 status as covariables, out of the analyzed markers only CD65 proved to be an independent prognostic factor in CD10− BCP-ALL (Cox regression analysis: RR 1.5, 95% CI 1.1–2.9, p=0.018). The identification of additional prognosis associated immunophenotypic markers may contribute to further refinement of treatment strategies for CD10− BCP-ALL pts.

Objective: The adaptor protein SLP-65 (also known as BLNK or BASH) plays an essential role in B cell differentiation. A crucial consequence of SLP-65 deficiency in mice is the high incidence of pre-B cell leukemia, suggesting a tumor suppressor role for SLP-65 in pre-B cells. We recently demonstrated deficiency slp-65 in 16 out of 34 pre-B ALL samples from children (Jumaa et al., Nature, 2003). These data were recently questioned by another study in which slp-65 levels were found to be as high as in normal B cells (Imai, et al., Leukemia, 2004). To resolve this issue we investigated another series of 148 primary ALL-samples by quantitative RT-PCR, Western-blotting and sequencing of aberrant slp-65 transcripts. We provide evidence that a newly identified slp-65 aberration, with inclusion of a newly identified exon 11a, leads to a functionless slp-65 protein. Materials: Samples were collected at diagnosis and contained at least 80 % marrow blasts. Screening for the presence of the fusion genes BCR/ABL, MLL/AF4, or TEL/AML1 was done by standard RT-PCR. For immunophenotyping and classification into pro-B, pre-B, c-, B- or T-cell ALL we used the standard criteria. Slp-65 mRNA was measured by taqman technology, protein expression by Western blotting with the B-211 antibody. To assess the functional consequences of a truncated form of slp-65, we measured Ca-responsiveness of the pre-B cell receptor. Pre-B cell differentiation properties of the wildtype and the truncated isoform of slp-65 were functionally tested as described previously (Flemming et al., Nat. Immunol, 2003). Results: The slp-65 mRNA expression ranged widely among the samples but was not correlated to any of the clinical or genetically parameters tested, such as sex, Age, WBC, or event-free survival. With respect to the immunophenotype, slp-65 mRNA was extremely low in T-ALL and but highly expressed in pro-B-ALL (p

The molecular approach for the analysis of leukemia associated chromosomal translocations has led to the identification of prognostic relevant subgroups. In pediatric acute lymphoblastic leukemia (ALL), the most common translocations, t(9; 22) and t(4; 11), have been associated with a poorer clinical outcome. Recently the TEL gene at chromosome 12p13 and the AML1 gene at chromosome 21q22 were found to be involved in the translocation t(12; 21)(p13; q22). By conventional cytogenetics, however, this chromosomal abnormality is barely detectable and occurs in less than 0.05% of childhood ALL. To investigate the frequency of the molecular equivalent of the t(12; 21), the TEL/AML1 gene fusion, we have undertaken a prospective screening in the running German Berlin-Frankfurt-Münster (BFM) and Italian Associazione Italiana Ematologia Oncologia Pediatrica (AIEOP) multicenter ALL therapy trials. We have analyzed 334 unselected cases of pediatric ALL patients consecutively referred over a period of 5 and 9 months, respectively. The overall incidence of the t(12; 21) in pediatric ALL is 18.9%. The 63 cases positive for the TEL/AML1 chimeric products ranged in age between 1 and 12 years, and all but one showed CD10 and pre-B immunophenotype. Interestingly, one case displayed a pre-pre–B immunophenotype. Among the B-lineage subgroup, the t(12; 21) occurs in 22.0% of the cases. Fifteen of 61 (24.6%) cases coexpressed at least two myeloid antigens (CD13, CD33, or CDw65) in more than 20% of the gated blast cells. DNA index was available for 59 of the 63 TEL/AML1 positive cases; a hyperdiploid DNA content (≥1.16) was detected in only four patients, being nonhyperdiploid in the remaining 55. Based on this prospective analysis, we retrospectively evaluated the impact of TEL/AML1 in prognosis by identifying the subset of B-lineage ALL children enrolled in the closed German ALL-BFM-90 and Italian ALL-AIEOP-91 protocols who had sufficient material for analysis. A total of 342 children were investigated for the presence of TEL/AML1 fusion gene and 99 cases (28.9%) were positive. The patients expressing the TEL/AML1 fusion mRNA appeared to have a better event-free survival (EFS) than the patients who lacked this chimeric product. Whereas three of the TEL/AML1 positive cases (3.0%) have relapsed to date, 27 patients without TEL/AML1 rearrangement (11.1%) suffered from relapse. To date, the only subset of B-lineage ALL with a favorable prognosis has been the hyperdiploid group (DNA index ≥1.16