p21 Downregulation is an important component of PAX3/FKHR oncogenicity and its reactivation by HDAC inhibitors enhances combination treatment.

A number of drugs developed against cancer-specific molecular targets have been shown to offer survival benefits alone or in combination with standard treatments, especially for those cases in which tumor pathogenesis is dominated by a single molecular abnormality. One example for such a tumor type is alveolar rhabdomyosarcoma (aRMS), which is characterized by a specific translocation creating the oncogenic PAX3/FKHR transcription factor, believed to be the molecular basis of the disease. Recently, we were able to show that the small molecule inhibitor PKC412 (midostaurin) shows strong antitumor activity against aRMS by reducing the transcriptional activity of PAX3/FKHR. In this study, we screened for combination strategies that are superior to PKC412-only treatment and found that the combination of PKC412 with histone deacetylase inhibitors like valproic acid (VPA) synergistically induced apoptosis resulting in suppressed aRMS tumor growth in vivo. We provide evidence that the antitumor effect on combination treatment is achieved by VPA-induced reactivation of p21, which is downregulated in aRMS cells by destabilization of the transcriptional regulator EGR1 by PAX3/FKHR. Our study highlights a possible mechanism behind the increased efficacy and indicates that different arms of PAX3/FKHR oncogenicity can be exploited therapeutically by the specific combination of drugs to increase their therapeutic potential.

The Wnt receptor FZD1 mediates chemoresistance in neuroblastoma through activation of the Wnt/beta-catenin pathway.

The development of chemoresistance represents a major obstacle in the successful treatment of cancers such as neuroblastoma (NB), a particularly aggressive childhood solid tumour. The mechanisms underlying the chemoresistant phenotype in NB were addressed by gene expression profiling of two doxorubicin (DoxR)-resistant vs sensitive parental cell lines. Not surprisingly, the MDR1 gene was included in the identified upregulated genes, although the highest overexpressed transcript in both cell lines was the frizzled-1 Wnt receptor (FZD1) gene, an essential component of the Wnt/beta-catenin pathway. FZD1 upregulation in resistant variants was shown to mediate sustained activation of the Wnt/beta-catenin pathway as revealed by nuclear beta-catenin translocation and target genes transactivation. Interestingly, specific micro-adapted short hairpin RNA (shRNAmir)-mediated FZD1 silencing induced parallel strong decrease in the expression of MDR1, another beta-catenin target gene, revealing a complex, Wnt/beta-catenin-mediated implication of FZD1 in chemoresistance. The significant restoration of drug sensitivity in FZD1-silenced cells confirmed the FZD1-associated chemoresistance. RNA samples from 21 patient tumours (diagnosis and postchemotherapy), showed a highly significant FZD1 and/or MDR1 overexpression after treatment, underlining a role for FZD1-mediated Wnt/beta-catenin pathway in clinical chemoresistance. Our data represent the first implication of the Wnt/beta-catenin pathway in NB chemoresistance and identify potential new targets to treat aggressive and resistant NB.

Minimal residual disease-directed risk stratification using real-time quantitative PCR analysis of immunoglobulin and T-cell receptor gene rearrangements in the international multicenter trial AIEOP-BFM ALL 2000 for childhood acute lymphoblastic leukemia.

Detection of minimal residual disease (MRD) is the most sensitive method to evaluate treatment response and one of the strongest predictors of outcome in childhood acute lymphoblastic leukemia (ALL). The 10-year update on the I-BFM-SG MRD study 91 demonstrates stable results (event-free survival), that is, standard risk group (MRD-SR) 93%, intermediate risk group (MRD-IR) 74%, and high risk group (MRD-HR) 16%. In multicenter trial AIEOP-BFM ALL 2000, patients were stratified by MRD detection using quantitative PCR after induction (TP1) and consolidation treatment (TP2). From 1 July 2000 to 31 October 2004, PCR target identification was performed in 3341 patients: 2365 (71%) patients had two or more sensitive targets (< or =10(-4)), 671 (20%) patients revealed only one sensitive target, 217 (6%) patients had targets with lower sensitivity, and 88 (3%) patients had no targets. MRD-based risk group assignment was feasible in 2594 (78%) patients: 40% were classified as MRD-SR (two sensitive targets, MRD negativity at both time points), 8% as MRD-HR (MRD > or =10(-3) at TP2), and 52% as MRD-IR. The remaining 823 patients were stratified according to clinical risk features: HR (n=108) and IR (n=715). In conclusion, MRD-PCR-based stratification using stringent criteria is feasible in almost 80% of patients in an international multicenter trial.

Comparative expression profiling identifies an in vivo target gene signature with TFAP2B as a mediator of the survival function of PAX3/FKHR.

The chromosomal translocation t(2;13), characteristic for the aggressive childhood cancer alveolar rhabdomyosarcoma (aRMS), generates the chimeric transcription factor PAX3/FKHR with a well known oncogenic role. However, the molecular mechanisms mediating essential pathophysiological functions remain poorly defined. Here, we used comparative expression profiling of PAX3/FKHR silencing in vitro and PAX3/FKHR-specific gene signatures in vivo to identify physiologically important target genes. Hereby, 51 activated genes, both novel and known, were identified. We also found repression of skeletal muscle-specific genes suggesting that PAX3/FKHR blocks further differentiation of aRMS cells. Importantly, TFAP2B was validated as direct target gene mediating the anti-apoptotic function of PAX3/FKHR. Hence, we developed a pathophysiologically relevant transcriptional profile of PAX3/FKHR and identified a critical target gene for aRMS development.

The MLL recombinome of acute leukemias.

Chromosomal rearrangements of the human MLL gene are a hallmark for aggressive (high-risk) pediatric, adult and therapy-associated acute leukemias. These patients need to be identified in order to subject these patients to appropriate therapy regimen. A recently developed long-distance inverse PCR method was applied to genomic DNA isolated from individual acute leukemia patients in order to identify chromosomal rearrangements of the human MLL gene. We present data of the molecular characterization of 414 samples obtained from 272 pediatric and 142 adult leukemia patients. The precise localization of genomic breakpoints within the MLL gene and the involved translocation partner genes (TPGs) was determined and several new TPGs were identified. The combined data of our study and published data revealed a total of 87 different MLL rearrangements of which 51 TPGs are now characterized at the molecular level. Interestingly, the four most frequently found TPGs (AF4, AF9, ENL and AF10) encode nuclear proteins that are part of a protein network involved in histone H3K79 methylation. Thus, translocations of the MLL gene, by itself coding for a histone H3K4 methyltransferase, are presumably not randomly chosen, rather functionally selected.

Disseminated Fusarium oxysporum infection in hemophagocytic lymphohistiocytosis.

The portal of entry of disseminated Fusarium spp. infections is still not clearly defined. We report on a disseminated Fusarium oxysporum infection occurring during a long period of severe neutropenia in a child with hemophagocytic lymphohistiocytosis. A nasogastric feeding tube was the possible source of entry of the fungus.

Port-A-Cath infections in children with cancer.

Implanted subcutaneous (s.c.) central venous port accesses including Port-A-Cath (PAC) facilitate the administration of chemotherapy or blood products and are frequently used in children with cancer. The incidence of PAC-related infections was determined in 155 consecutive paediatric cancer patients with PAC followed for a total of 134,773 days (median, 738; range, 25-2080). Overall, 48 bloodstream infections occurred in 26 patients. 12 (25%) of these infections and 3 local infections at the insertion site were treatment-resistant and demanded removal of the PAC. Coagulase-negative staphylococci were involved in 12 of these 15 episodes. The rate of clearly PAC-related infections in this so far largest reported series was 0.11 episodes per 1000 PAC days, one of the lowest in the literature. Although catheter-related infections demanded PAC removal in 8% of our patients, the long periods PAC were in use and their benefits argue for continued PAC use in the paediatric cancer population.

Prenatal origin of separate evolution of leukemia in identical twins.

Several studies involving identical twins with concordant leukemia and retrospective scrutiny of archived neonatal blood spots have shown that the TEL-AML1 fusion gene in childhood acute lymphoblastic leukemia (ALL) frequently arises before birth. A prenatal origin of childhood leukemia was further supported by the detection of clonotypic immunoglobulin gene rearrangements on neonatal blood spots of children with various other subtypes of ALL. However, no comprehensive study is available linking these clonotypic events. We describe a pair of 5-year-old monozygotic twins with concordant TEL-AML1-positive ALL. Separate leukemic clones were identified in the diagnostic samples since distinct IGH and IGK-Kde gene rearrangements could be detected. Additional differences characterizing the leukemic clones included an aberration of the second, nonrearranged TEL allele observed in one twin only. Interestingly, both the identical TEL-AML1 fusion sequence and distinct immunoglobulin gene rearrangements were identified on the neonatal blood spots indicating that separate preleukemic clones evolved already before birth. Finally, we compared the reported twins with an additional 31 children with ALL by using the microarray technology. Gene expression profiling provided evidence that leukemia in twins harbours the same subtype-typical feature as TEL-AML1-positive leukemia in singletons suggesting that the leukemogenesis model might also be applicable generally.

Prognostic impact of chromosomal aberrations in Ewing tumours.

Although greater than 50% of Ewing tumours contain non-random cytogenetic aberrations in addition to the pathognomonic 22q12 rearrangements, little is known about their prognostic significance. To address this question, tumour samples from 134 Ewing tumour patients were analysed using a combination of classical cytogenetics, comparative genomic and fluorescence in situ hybridisation. The evaluation of the compiled data revealed that gain of chromosome 8 occurred in 52% of Ewing tumours but was not a predictive factor for outcome. Gain of 1q was associated with adverse overall survival and event-free survival in all patients, irrespective of whether the tumour was localised or disseminated (overall survival: P=0.002 and P=0.029; event-free survival: P=0.018 and P=0.010). Loss of 16q was a significant predictive factor for adverse overall survival in all patients (P=0.008) and was associated with disseminated disease at diagnosis (P=0.039). Gain of chromosome 12 was associated with adverse event-free survival (P=0.009) in patients with localised disease. These results indicate that in addition to a 22q12 rearrangement confirmation in Ewing tumours it is important to assess the copy number of 1q and 16q to identify patients with a higher probability of adverse outcome.

The investigation of karyotypic instability in the high-hyperdiploidy subgroup of acute lymphoblastic leukemia.

Acute lymphoblastic leukemia (ALL) of childhood has been cytogenetically well characterized, and approximately 25% of cases will have a high-hyperdiploid (51-68) chromosome complement. In a 5 year period a consecutive series of 152 presentation ALL's were karyotyped. In all cases a result was obtained and 138 (91%) had a detectable abnormal clone of which 44 (29%) were high-hyperdiploid. Within the high-hyperdiploidy group karyotypic cell to cell variation was observed in many cases. To provide further evidence of this phenomenon a dual-color fluorescence in-situ hybridization (FISH) experiment was performed on stored fixed suspension from 14 ALL's with such a karyotype. In each case 4-6 probes were investigated, employing probes to centromeres of chromosomes X, 4, 6, 8, and 10 and a locus specific probe to chromosome 21q22. It was found that the FISH produced results that were generally in good agreement with the G-banding findings and supported the notion of karyotypic cell to cell variation. FISH further showed that most of cases would have two extra copies of chromosome 21 in the majority of leukemic cells and a single extra copy in the minority. A further finding was that fewer cells contained extra copies of chromosomes 6, 8 and 10 than was expected based on the comparison of the signal number of the other probes investigated. In contrast chromosomes X, 4, and 21 seldom displayed this feature. We have demonstrated that karyotypic instability as defined by karyotypic cell to cell variation is a feature of the high-hyperdiploid subgroup in childhood ALL. It is questioned whether the underlying defect resulting in the observed karyotypic instability of this subgroup is one of the primary causative events in the formation of the leukemia.

Detection of unidentified chromosome abnormalities in human neuroblastoma by spectral karyotyping (SKY).

Spectral karyotyping (SKY) is a novel technique based on the simultaneous hybridization of 24 fluorescently labeled chromosome painting probes. It provides a valuable addition to the investigation of many tumors that can be difficult to define by conventional banding techniques. One such tumor is neuroblastoma, which is often characterized by poor chromosome morphology and complex karyotypes. Ten primary neuroblastoma tumor samples initially analyzed by G-banding were analyzed by SKY. In 8/10 tumors, we were able to obtain additional cytogenetic information. This included the identification of complex rearrangements and material of previously unknown origin. Structurally rearranged chromosomes can be identified even in highly condensed metaphase chromosomes. Following the SKY results, the G-banding findings were reevaluated, and the combination of the two techniques resulted in a more accurate karyotype. This combination allows identification not only of material gained and lost, but also of breakpoints and chromosomal associations. The use of SKY is therefore a powerful tool in the genetic characterization of neuroblastoma and can contribute to a better understanding of the molecular events associated with this tumor.

Loss of X chromosome in childhood acute lymphoblastic leukemia.

We present six cases of childhood acute lymphoblastic leukemia (ALL) in which an acquired loss of the X chromosome was detected. The cases derive from a consecutive series of 178 childhood ALL, consisting of 80 girls and 98 boys. In five cases the presence of the TEL-AML1, t(12;21), fusion product was detected by FISH. The single negative case had an unusual t(1;19)(p13;q13). In addition, this was the only case that did not have a cytogenetically visible rearrangement involving one of the chromosome regions 6q, 9p, or 12p. The six cases showed the typical presentation features of an ALL of FAB type L1, a common ALL immunophenotype with myeloid marker co-expression, and a median presenting age of 7 years. We, therefore, conclude that loss of chromosome X may be a secondary event in older girls with TEL-AML1-positive ALL.

Constitutional balanced chromosomal rearrangements and neoplasm in children.

A predisposition to tumor development is currently associated with some, but not all, constitutional chromosomal abnormalities. In a series of 578 children, in which conventional cytogenetic investigation was performed on material from various benign and malignant tumors, four boys and one girl were also found to have constitutional balanced chromosomal rearrangements. The figure of 5 in 578 is notable because the reported incidence of balanced rearrangements in newborns is approximately 1 in 450. Thereby suggesting that some, if not all, children with balanced constitutional chromosomal rearrangements have an increased predisposition for neoplasms developing.

Long-term follow-up and residual sequelae after treatment for intracerebral germ-cell tumour in children and adolescents.

BACKGROUND: Information on long-term follow-up of children and adolescents treated for intracerebral germ-cell tumour is scant. We report on the results of a small series of patients treated at a single institution. PATIENTS AND METHODS: Hospital records from 15 patients treated between 1980 and 1998 were reviewed. An attempt was made to correlate sequelae to tumour location and treatment modalities. RESULTS: This cohort constitutes 5.5% of all brain tumours diagnosed at our institution. HISTOLOGY: 10 germinomas, 2 benign teratomas, 2 malignant teratomas, and one mixed germ-cell tumour. Overall survival was 87%, with a mean follow-up time of 7 years and 8 months. The majority of patients have long-term sequelae involving one or several organ systems. In 66% endocrine, in 47% ophthalmologic, in 60% neuropsychological defects were observed. Endocrine and ophthalmologic sequelae show a correlation to tumour location. Neuropsychological long-term abnormalities are frequent and are associated with cranial irradiation in particular at young age, but less with tumour location, irradiation dose or surgery. CONCLUSIONS: Our preliminary data suggest that today intracerebral germinomas and mature teratomas have a good prognosis even when a relapse occurs. The outcome for mixed germ-cell tumours and malignant teratomas is less favourable. Although long-term sequelae are present in the majority of patients, there is some evidence that patients treated after 1990 suffer fewer severe long-term defects, thereby indicating that recent treatment protocols may result in a reduction of sequelae.

Molecular features of a human rhabdomyosarcoma cell line with spontaneous metastatic progression.

A novel human cell line was established from a primary botryoid rhabdomyosarcoma. Reverse transcription polymerase chain reaction investigations of this cell line, called RUCH-2, demonstrated expression of the regulatory factors PAX3, Myf3 and Myf5. After 3.5 months in culture, cells underwent a crisis after which Myf3 and Myf5 could no longer be detected, whereas PAX3 expression remained constant over the entire period. Karyotype analysis revealed breakpoints in regions similar to previously described alterations in primary rhabdomyosarcoma tumour samples. Interestingly, cells progressed to a metastatic phenotype, as observed by enhanced invasiveness in vitro and tumour growth in nude mice in vivo. On the molecular level, microarray analysis before and after progression identified extensive changes in the composition of the extracellular matrix. As expected, down-regulation of tissue inhibitors of metalloproteinases and up-regulation of matrix metalloproteinases were observed. Extensive down-regulation of several death receptors of the tumour necrosis factor family suggests that these cells might have an altered response to appropriate apoptotic stimuli. The RUCH-2 cell line represents a cellular model to study multistep tumorigenesis in human rhabdomyosarcoma, allowing molecular comparison of tumorigenic versus metastatic cancer cells.

Trisomy 1q generating translocations in Wilms tumor.

Unbalanced translocations generating trisomy of 1q are common in Wilms tumor (WT). We present eight unbalanced 1q translocations from seven tumors and a review of the literature. An unbalanced translocation that results in a der(16)t(1q;16q) chromosome represents more than half of the published +1q generating translocations in WT. This translocation is also common to many other tumor types. Four of the tumors presented here contained this chromosome and,in two cases, it was the primary acquired cytogenetic abnormality within the tumor. The other four translocations involved 9q31, 9q34, 17p1?, and 21p11 as the partner to 1q. The chromosome 17 and 21 translocations occurred within the same tumor as apparently independent events. In contrast with the 16q translocations, these other translocations were secondary cytogenetic events, thereby indicating a role in tumor progression rather than initiation. Probes mapping to 1q12 and 1q21 were employed for fluorescence in situ hybridization and it was demonstrated that different 1q breakpoints are possible. In this series, the majority of breakpoints either mapped to 1q12 or were centromeric to this region.

Cytogenetic abnormalities in Langerhans cell histiocytosis.

We present the cytogenetic investigations of five histiocytic tumour lesions from children. In four cases there was a confirmed diagnosis of Langerhans cell histiocytosis (LCH) and one case of histiocytosis that did not fulfil all the criteria for true LCH. All five cases showed cytogenetic abnormalities, including the first report of an abnormal clone in LCH. The clone showed a t(7;12)(q11.2;p13) translocation and was detected in only a small percentage of cells. This case and a further three also contained non-clonal abnormalities and an increase in chromosome breakage. The fifth case, the only one in which no acquired abnormalities were seen, had a constitutional paracentric inversion of chromosome 13q.

A further case of a t(11;20)(p15;q11.2) translocation in an acute myeloid leukemia (FAB M2).

PURPOSE: The clinical implications of many cytogenetic abnormalities in acute myeloid leukemia (AML) are now well established. However, questions about the significance of rarer abnormalities still exist, particularly in childhood disease. PATIENTS AND METHODS: We report a case of a 9 1/2-year-old girl who had AML of the FAB M2 subtype. A diagnostic bone marrow aspirate and subsequent bone marrow aspirates were investigated using conventional cytogenetic methods. RESULTS: Cytogenetic analysis of the diagnostic bone marrow aspirate showed a t(1;20)(p15;q11.2) translocation as the sole acquired abnormality. After one course of chemotherapy, the patient achieved hematopoietic and cytogenetic remission which has been sustained for 1 year after presentation. CONCLUSION: This report demonstrates that rare non-random cytogenetic abnormalities are still to be described in AML, and that care should be taken when ascribing clinical significance to cytogenetic findings in childhood disease based on those of older patients.

Clustered organization of S100 genes in human and mouse.

S100 Ca2+-binding proteins became of major interest because of their differential expression in tissues and their association with human diseases. Earlier studies showed that 13 S100 genes are located as a cluster on human chromosome 1q21. Since a number of mouse S 100 genes, such as S100A4 and S100A6, have been localized to a syntenic region on mouse chromosome 3, we investigated if the S100 gene cluster exists in mouse and is structurally conserved during evolution. First we identified the cDNA sequences of mouse S100A1, S100A3 and S100A5. Then we isolated a 490 kb mouse YAC clone which gives a specific signal by FISH most likely on chromosome 3. Hybridization studies with different mouse S100 cDNAs revealed that eight mouse S100 genes are arranged in a clustered organization similar to that in human. The linkage relationships between the genes S100A8-S100A9 and S100A3-S100A4-S100A5-S100A6 were conserved during divergence of human and mouse about 70 million years ago. However, the separation of the mouse S100 genes S100A1 and S100A13 in comparison to the human linkage group suggests rearrangement processes between human and mouse. Our data demonstrate that the S100 gene cluster is structurally conserved during evolution. Further studies on the genomic organization of the S100 genes including various species could generate new insights into gene regulatory processes and phylogenetic relationships.

Routine karyotyping in Wilms tumor.

We describe the karyotypes of nine Wilms tumors (WT). Four tumors were initially karyotyped from diagnostic needle core biopsies, 3 after postchemotherapy tumor resection and the remainder from xenografts grown in nude mice. The 9 nephroblastomas were composed of 7 with favorable histology (intermediate-grade malignancy) and 2 with unfavorable histology (anaplastic or high-grade malignancy). The 7 tumors with favorable histology had karyotypes typical of WT, with the previously described nonrandom abnormalities +1q, +6, +7, +8, +12, +13, +18 and structural abnormalities of 1p and 16q present in at least 1 case. The most common abnormalities were trisomy 18 (4 cases) and +1q (3 cases). The 2 tumors with unfavorable histology both had complex karyotypes atypical for WT. We suggest that cytogenetics can act as a marker when histologic grade is in doubt. Karyotypic analysis from needle core biopsies was attempted in 6 samples, including 1 from a nephrogenic rest (NR) of the nonaffected kidney and provided a result on 5 occasions. The NR were present in the sole case with a constitutional abnormality, a mosaic partial duplication of 8q. However, both the tumor and the NR were apparently derived from the normal cell line. Here we demonstrate that a cytogenetic result can be routinely obtained from needle core biopsies and will thus facilitate true diagnostic tumor karyotypes in both WT and other tumors.