F. Speleman

Emerging patterns of cryptic chromosomal imbalance in patients with idiopathic mental retardation and multiple congenital anomalies: a new series of 140 patients and review of published reports

Background: Chromosomal abnormalities are a major cause of mental retardation and multiple congenital anomalies (MCA/MR). Screening for these chromosomal imbalances has mainly been done by standard karyotyping. Previous array CGH studies on selected patients with chromosomal phenotypes and normal karyotypes suggested an incidence of 10–15% of previously unnoticed de novo chromosomal imbalances. Objective: To report array CGH screening of a series of 140 patients (the largest published so far) with idiopathic MCA/MR but normal karyotype. Results: Submicroscopic chromosomal imbalances were detected in 28 of the 140 patients (20%) and included 18 deletions, seven duplications, and three unbalanced translocations. Seventeen of 24 imbalances were confirmed de novo and 19 were assumed to be causal. Excluding subtelomeric imbalances, our study identified 11 clinically relevant interstitial submicroscopic imbalances (8%). Taking this and previously reported studies into consideration, array CGH screening with a resolution of at least 1 Mb has been undertaken on 432 patients with MCA/MR. Most imbalances are non-recurrent and spread across the genome. In at least 8.8% of these patients (38 of 432) de novo intrachromosomal alterations have been identified. Conclusions: Array CGH should be considered an essential aspect of the genetic analysis of patients with MCA/MR. In addition, in the present study three patients were mosaic for a structural chromosome rearrangement. One of these patients had monosomy 7 in as few as 8% of the cells, showing that array CGH allows detection of low grade mosaicisims.

Quality Assessment of Genetic Markers Used for Therapy Stratification

PURPOSEnTherapy stratification based on genetic markers is becoming increasingly important, which makes commitment to the highest possible reliability of the involved markers mandatory. In neuroblastic tumors, amplification of the MYCN gene is an unequivocal marker that indicates aggressive tumor behavior and is consequently used for therapy stratification. To guarantee reliable and standardized quality of genetic features, a quality-assessment study was initiated by the European Neuroblastoma Quality Assessment (ENQUA; connected to International Society of Pediatric Oncology) Group.nnnMATERIALS AND METHODSnOne hundred thirty-seven coded specimens from 17 tumors were analyzed in 11 European national/regional reference laboratories using molecular techniques, in situ hybridization, and flow and image cytometry. Tumor samples with divergent results were re-evaluated.nnnRESULTSnThree hundred fifty-two investigations were performed, which resulted in 23 divergent findings, 17 of which were judged as errors after re-evaluation. MYCN analyses determined by Southern blot and in situ hybridization led to 3.7% and 4% of errors, respectively. Tumor cell content was not indicated in 32% of the samples, and 11% of seemingly correct MYCN results were based on the investigation of normal cells (eg, Schwann cells). Thirty-eight investigations were considered nonassessable.nnnCONCLUSIONnThis study demonstrated the importance of revealing the difficulties and limitations for each technique and problems in interpreting results, which are crucial for therapeutic decisions. Moreover, it led to the formulation of guidelines that are applicable to all kinds of tumors and that contain the standardization of techniques, including the exact determination of the tumor cell content. Finally, the group has developed a common terminology for molecular-genetic results.

A new recurrent inversion, inv(7)(p15q34), leads to transcriptional activation of HOXA10 and HOXA11 in a subset of T-cell acute lymphoblastic leukemias

Chromosomal translocations with breakpoints in T-cell receptor (TCR) genes are recurrent in T-cell malignancies. These translocations involve the TCRαδ gene (14q11), the TCRβ gene (7q34) and to a lesser extent the TCRγ gene at chromosomal band 7p14 and juxtapose T-cell oncogenes next to TCR regulatory sequences leading to deregulated expression of those oncogenes. Here, we describe a new recurrent chromosomal inversion of chromosome 7, inv(7)(p15q34), in a subset of patients with T-cell acute lymphoblastic leukemia characterized by CD2 negative and CD4 positive, CD8 negative blasts. This rearrangement juxtaposes the distal part of the HOXA gene cluster on 7p15 to the TCRβ locus on 7q34. Real time quantitative PCR analysis for all HOXA genes revealed high levels of HOXA10 and HOXA11 expression in all inv(7) positive cases. This is the first report of a recurrent chromosome rearrangement targeting the HOXA gene cluster in T-cell malignancies resulting in deregulated HOXA gene expression (particularly HOXA10 and HOXA11) and is in keeping with a previous report suggesting HOXA deregulation in MLL-rearranged T- and B cell lymphoblastic leukemia as the key factor in leukaemic transformation. Finally, our observation also supports the previous suggested role of HOXA10 and HOXA11 in normal thymocyte development.

NOTCH1 and FBXW7 mutations have a favorable impact on early response to treatment, but not on outcome, in children with T-cell acute lymphoblastic leukemia (T-ALL) treated on EORTC trials 58881 and 58951

Risk-adjusted treatment stratification in T-cell acute lymphoblastic leukemias (T-ALLs) is currently based only on early response to chemotherapy. We investigated the prognostic implication of hyperactivation of NOTCH pathway resulting from mutations of NOTCH1 or FBXW7 in children with T-ALL enrolled in EORTC-CLG trials. Overall, 80 out of 134 (60%) patients were NOTCH+ (NOTCH1 and/or FBXW7 mutated). Although clinical presentations were not significantly associated with NOTCH status, NOTCH+ patients showed a better early response to chemotherapy as compared with NOTCH− patients, according to the rate of poor pre-phase ‘responders’ (25% versus 44%; P=0.02) and the incidence of high minimal residual disease (MRD) levels (11% (7/62) versus 32% (10/31); P=0.01) at completion of induction. However, the outcome of NOTCH+ patients was similar to that of NOTCH− patients, with a 5-year event-free survival (EFS) of 73% and 70% (P=0.82), and 5-year overall survival of 82% and 79% (P=0.62), respectively. In patients with high MRD levels, the 5-year EFS rate was 0% (NOTCH+) versus 42% (NOTCH−), whereas in those with low MRD levels, the outcome was similar: 76% (NOTCH+) versus 78% (NOTCH−). The incidence of isolated central nervous system (CNS) relapses was relatively high in NOTCH1+ patients (8.3%), which could be related to a higher propensity of NOTCH+ leukemic blasts to target the CNS.

Molecular cytogenetic study of 126 unselected T-ALL cases reveals high incidence of TCR|[beta]| locus rearrangements and putative new T-cell oncogenes

Chromosomal aberrations of T-cell receptor (TCR) gene loci often involve the TCRαδ (14q11) locus and affect various known T-cell oncogenes. A systematic fluorescent in situ hybridization (FISH) screening for the detection of chromosomal aberrations involving the TCR loci, TCRαδ (14q11), TCRβ (7q34) and TCRγ (7p14), has not been conducted so far. Therefore, we initiated a screening of 126 T-cell acute lymphoblastic leukemia (T-ALL) and T-cell lymphoblastic lymphoma cases and 19 T-ALL cell lines using FISH break-apart assays for the different TCR loci. Genomic rearrangements of the TCRβ locus were detected in 24/126 cases (19%), most of which (58.3%) were not detected upon banding analysis. Breakpoints in the TCRαδ locus were detected in 22/126 cases (17.4%), whereas standard cytogenetics only detected 14 of these 22 cases. Cryptic TCRαδ/TCRβ chromosome aberrations were thus observed in 22 of 126 cases (17.4%). Some of these chromosome aberrations target new putative T-cell oncogenes at chromosome 11q24, 20p12 and 6q22. Five patients and one cell line carried chromosomal rearrangements affecting both TCRβ and TCRαδ loci. In conclusion, this study presents the first inventory of chromosomal rearrangements of TCR loci in T-ALL, revealing an unexpected high number of cryptic chromosomal rearrangements of the TCRβ locus and further broadening the spectrum of genes putatively implicated in T-cell oncogenesis.

MYCN and ALKF1174L are sufficient to drive neuroblastoma development from neural crest progenitor cells

Neuroblastoma is an embryonal tumor with a heterogeneous clinical course. The tumor is presumed to be derived from the neural crest, but the cells of origin remain to be determined. To date, few recurrent genetic changes contributing to neuroblastoma formation, such as amplification of the MYCN oncogene and activating mutations of the ALK oncogene, have been identified. The possibility to model neuroblastoma in mice allows investigation of the cell of origin hypothesis in further detail. Here we present the evidence that murine neural crest progenitor cells can give rise to neuroblastoma upon transformation with MYCN or ALKF1174L. For this purpose we used JoMa1, a multipotent neural crest progenitor cell line, which is kept in a viable and undifferentiated state by a tamoxifen-activated c-Myc transgene (c-MycERT). Expression of MYCN or ALKF1174L, one of the oncogenic ALK variants identified in primary neuroblastomas, enabled these cells to grow independently of c-MycERT activity in vitro and caused formation of neuroblastoma-like tumors in vivo in contrast to parental JoMa1 cells and JoMa1 cells-expressing TrkA or GFP. Tumorigenicity was enhanced upon serial transplantation of tumor-derived cells, and tumor cells remained susceptible to the MYC-inhibitor, NBT-272, indicating that cell growth depended on functional MYCN. Our findings support neural crest progenitor cells as the precursor cells of neuroblastoma, and indicate that neuroblastomas arise as their malignant progeny.

MicroRNA-193b-3p acts as a tumor suppressor by targeting the MYB oncogene in T-cell acute lymphoblastic leukemia

The MYB oncogene is a leucine zipper transcription factor essential for normal and malignant hematopoiesis. In T-cell acute lymphoblastic leukemia (T-ALL), elevated MYB levels can arise directly through T-cell receptor-mediated MYB translocations, genomic MYB duplications or enhanced TAL1 complex binding at the MYB locus or indirectly through the TAL1/miR-223/FBXW7 regulatory axis. In this study, we used an unbiased MYB 3′untranslated region–microRNA (miRNA) library screen and identified 33 putative MYB-targeting miRNAs. Subsequently, transcriptome data from two independent T-ALL cohorts and different subsets of normal T-cells were used to select miRNAs with relevance in the context of normal and malignant T-cell transformation. Hereby, miR-193b-3p was identified as a novel bona fide tumor-suppressor miRNA that targets MYB during malignant T-cell transformation thereby offering an entry point for efficient MYB targeting-oriented therapies for human T-ALL.

Amplification units and translocation at chromosome 17q and c-erbB-2 overexpression in the pathogenesis of breast cancer

Abstractu2002Hyperplasia without and with atypia is considered to be a precursor lesion for certain breast carcinomas. The cytogenetic events and the molecular pathology involved in the multistep process from normal to invasive carcinoma are unknown. To characterise the sequence of early genetic abnormalities of chromosome 17q and their biological consequences in the pathogenesis of breast cancer, we performed immunohistochemistry on 451 breast tissues including 180 normal breast specimens, 28 hyperplastic lesions without atypia and 44 with atypia, 100 cases of ductal carcinoma in situ (DCIS) and 99 cases of invasive ductal carcinoma. We correlated the overexpression of the c-ErbB-2 protein, the histological and the recently proposed differentiation classification of DCIS with the extent of DCIS. For fluorescence in situ hybridisation (FISH) analysis, different probes spanning the 17q region including the c-erbB-2 gene locus and those which are found adjacent, were used. Reverse painting and comparative genomic hybridisation (CGH) were performed on several breast cancer cell lines. c-ErbB-2 overexpression was observed in only 29% of DCIS and 23% of invasive carcinomas, but not in hyperplastic and normal tissue. c-ErbB-2 overexpression is correlated with poor differentiation in DCIS but not in invasive carcinoma. In DCIS, there was no correlation with the histological subtype classification. The average extent of DCIS is significantly increased from 13.81u2005mm in c-ErbB-2 negative cases to 29.37u2005mm in c-ErbB-2 positive cases. The increase was considered to be a possible consequence of the overexpression and is probably due to the previously described motility enhancing effect of the c-ErbB-2 protein. The histological and differentiation classification of DCIS did not correlate with the extent of disease. Using FISH, amplified genes at 17q12, always including the c-erbB-2 gene, were detected in all cases of DCIS and invasive carcinoma with c-ErbB-2 overexpression. The centromeric region and the NF1 locus, which is located between the centromere and c-erbB-2, were not amplified in any of the DCIS and invasive breast carcinomas, but co-amplification of the myeloperoxidase gene was detected in 3/5 DCIS and 1/5 invasive carcinomas with c-ErbB-2 overexpression. In contrast to c-erbB-2, immunohistochemical overexpression of their respective gene products was not observed. FISH, reverse painting and CGH show similar amplified genes with amplified c-erbB-2 in c-ErbB-2 overexpressing SK-BR-3 and BT474 human breast cancer cells. The amplified genes are part of two different amplicons. Extensive modifications of the 17q chromosomal region, caused by translocation, were also observed in these cell lines. It is concluded that the modifications of chromosome 17q, inducing overexpression of c-ErbB-2 protein, occur at the level of transition from hyperplasia to DCIS. They are preserved in invasive carcinoma with overexpression of c-ErbB-2 protein. This had led to the hypothesis that these modifications at 17q may lead to a larger extent of DCIS.

Molecular analysis of 1p36 breakpoints in two Merkel cell carcinomas

Merkel cell carcinoma (MCC) is a rare aggressive neuroendocrine tumor of the skin. Only little information is available on the genetic alterations occurring in this tumor. Cytogenetic studies thus far have not shown recurrent chromosomal changes, although various structural chromosome 1 rearrangements, including deletions, often leading to loss of distal 1p material appear to be frequent. We report on fluorescence in situ hybridization and loss of heterozygosity analyses of an MCC tumor and MCC cell line UISO. The present study has shown that two distinct regions in the most distal band 1p36 on the short arm of chromosome 1 can be implicated in MCC. One region at 1p36.3 was delineated by a distal deletion in the MCC tumor as a result of an unbalanced translocation, resulting in loss of all markers distal to ENO1. This region was previously shown to be deleted in different tumor types including neuroblastoma. In cell line UISO an insertion in 1p36.2 was identified. The insertion breakpoint indicates a second, more proximal, region on 1p involved in MCC. The insertion breakpoint was mapped within a cluster of repetitive tRNA and snRNA genes and thus could coincide with the constitutional 1p36 breakpoint previously reported in a patient with neuroblastoma. Genes Chromosomes Cancer 23:67–71, 1998.

Escape from p53-mediated tumor surveillance in neuroblastoma: switching off the p14 ARF -MDM2-p53 axis

A primary failsafe program against unrestrained proliferation and oncogenesis is provided by the p53 tumor suppressor protein, inactivation of which is considered as a hallmark of cancer. Intriguingly, mutations of the TP53 gene are rarely encountered in neuroblastoma tumors, suggesting that alternative p53-inactivating lesions account for escape from p53 control in this childhood malignancy. Several recent studies have shed light on the mechanisms by which neuroblastoma cells circumvent the p53-driven antitumor barrier. We review here these mechanisms for evasion of p53-mediated growth control and conclude that deregulation of the p14ARF-MDM2-p53 axis seems to be the principal mode of p53 inactivation in neuroblastoma, opening new perspectives for targeted therapeutic intervention.