Marije IJszenga

Novel and highly recurrent chromosomal alterations in Sezary syndrome

This study was designed to identify highly recurrent genetic alterations typical of Sézary syndrome (Sz), an aggressive cutaneous T-cell lymphoma/leukemia, possibly revealing pathogenetic mechanisms and novel therapeutic targets. High-resolution array-based comparative genomic hybridization was done on malignant T cells from 20 patients. Expression levels of selected biologically relevant genes residing within loci with frequent copy number alteration were measured using quantitative PCR. Combined binary ratio labeling-fluorescence in situ hybridization karyotyping was done on malignant cells from five patients. Minimal common regions with copy number alteration occurring in at least 35% of patients harbored 15 bona fide oncogenes and 3 tumor suppressor genes. Based on the function of the identified oncogenes and tumor suppressor genes, at least three molecular mechanisms are relevant in the pathogenesis of Sz. First, gain of cMYC and loss of cMYC antagonists (MXI1 and MNT) were observed in 75% and 40% to 55% of patients, respectively, which were frequently associated with deregulated gene expression. The presence of cMYC/MAX protein heterodimers in Sézary cells was confirmed using a proximity ligation assay. Second, a region containing TP53 and genome maintenance genes (RPA1/HIC1) was lost in the majority of patients. Third, the interleukin 2 (IL-2) pathway was affected by gain of STAT3/STAT5 and IL-2 (receptor) genes in 75% and 30%, respectively, and loss of TCF8 and DUSP5 in at least 45% of patients. In sum, the Sz genome is characterized by gross chromosomal instability with highly recurrent gains and losses. Prominent among deregulated genes are those encoding cMYC, cMYC-regulating proteins, mediators of MYC-induced apoptosis, and IL-2 signaling pathway components.

EWSR1-CREB1 and EWSR1-ATF1 Fusion Genes in Angiomatoid Fibrous Histiocytoma

Purpose: Angiomatoid fibrous histiocytoma (AFH) is a low-grade mesenchymal neoplasm which usually occurs in children and adolescents. Either FUS-ATF1 or EWSR1-ATF1 have been detected in the few cases published, pointing to the interchangeable role of FUS and EWSR1 in this entity. EWSR1-ATF1 also represents the most frequent genetic alteration in clear cell sarcoma, suggesting the existence of a molecular homology between these two histotypes. We investigated the presence of EWSR1-CREB1, recently found in gastrointestinal clear cell sarcoma, and FUS-CREB1, as well as the already reported FUS-ATF1 and EWSR1-ATF1 in a series of AFH. Experimental Design: Fourteen cases were analyzed by fluorescence in situ hybridization (FISH) on paraffin-embedded tissue sections, using a commercial EWSR1 probe and custom-designed probes for FUS, ATF1, and CREB1. In two cases, four-color FISH was also done. Reverse transcription-PCR for the four hypothetical fusion genes was done in one case, for which frozen material was available. Results: Thirteen cases showed rearrangements of both EWSR1 and CREB1, whereas one case showed the rearrangement of both EWSR1 and ATF1. Four-color FISH confirmed the results in two selected cases. Reverse transcription-PCR showed EWSR1-CREB1 transcript in the case analyzed. Conclusion: We identified the presence of either EWSR1-CREB1 or EWSR1-ATF1 in all the cases, strengthening the concept of chromosomal promiscuity between AFH and clear cell sarcoma. Either the occurrence of a second unknown tumor-specific molecular event or, perhaps more likely, divergent differentiation programs of the putatively distinct precursor cells of AFH and clear cell sarcoma might be invoked in order to explain the two different phenotypes.

The NFATc2 Gene Is Involved in a Novel Cloned Translocation in a Ewing Sarcoma Variant That Couples Its Function in Immunology to Oncology

Purpose: Ewing sarcoma is an aggressive sarcoma and is the second most common bone sarcoma in childhood. Disease-specific t(11;22) (∼85-90%), t(21;22) (∼5-10%), or rarer variant translocations with the involvement of chromosome 22 (∼5%) are present. At the gene level, the EWSR1 gene fuses with FLI1, ERG, or other ETS transcription factor family members. Thus far, no Ewing sarcoma has been identified with a fusion to transcription factors other than ETS. Experimental Design: Using molecular tools such as multicolor fluorescence in situ hybridization and array comparative genomic hybridization, a ring chromosome containing chromosomes 20 and 22 was identified in four Ewing sarcoma cases. The breakpoint was mapped with (fiber-) fluorescence in situ hybridization and reverse transcription-PCR followed by sequencing of the fusion partners. Results: Molecular karyotyping showed the translocation and amplification of regions of chromosomes 20q13 and 22q12. Cloning of the breakpoint showed an in-frame fusion between the EWSR1 and NFATc2 genes, resulting in loss of the NH2-terminal, calcineurin-dependent control region and an intact active domain of NFATc2 controlled by the transactivation domains of EWSR1. Conclusion: A new translocation involving EWSRI and NFATc2 was cloned. NFATc2 is a transcription factor that is not a member of the ETS family and functions in T-cell differentiation and immune response. Direct involvement of NFATc2 has not yet been observed in oncogenesis. We show that due to the shared sequence recognition of NFATc2 and the ETS family, shared transcriptional control is possible using activating protein complex 1.

Frequent deletion of the CDKN2A locus in chordoma: analysis of chromosomal imbalances using array comparative genomic hybridisation

The initiating somatic genetic events in chordoma development have not yet been identified. Most cytogenetically investigated chordomas have displayed near-diploid or moderately hypodiploid karyotypes, with several numerical and structural rearrangements. However, no consistent structural chromosome aberration has been reported. This is the first array-based study characterising DNA copy number changes in chordoma. Array comparative genomic hybridisation (aCGH) identified copy number alterations in all samples and imbalances affecting 5 or more out of the 21 investigated tumours were seen on all chromosomes. In general, deletions were more common than gains and no high-level amplification was found, supporting previous findings of primarily losses of large chromosomal regions as an important mechanism in chordoma development. Although small imbalances were commonly found, the vast majority of these were detected in single cases; no small deletion affecting all tumours could be discerned. However, the CDKN2A and CDKN2B loci in 9p21 were homo- or heterozygously lost in 70% of the tumours, a finding corroborated by fluorescence in situ hybridisation, suggesting that inactivation of these genes constitute an important step in chordoma development.

Spectrum of genetic changes in gastro-esophageal cancer cell lines determined by an integrated molecular cytogenetic approach

Adenocarcinomas arising around the gastro-esophageal junction (GEJ) are highly malignant, and their incidence has risen rapidly in the last decades. Cell lines are the basic in vitro system for functional and therapeutic studies in GEJ tumors, but only a small number of cell lines are currently available, and none of them has been fully karyotyped. We analyzed 5 GEJ tumor cell lines using a combination of 24-color fluorescence in situ hybridization (FISH), comparative genomic hybridization (CGH) and genomic microarrays. Using CGH we demonstrated that these cell lines present imbalances similar to those we had previously observed in primary GEJ tumors, namely gains on 1q, 7q, 8q, 17q, 19q, 20, and X, and losses on 3p, 4, 5q, 9p, 18q, and 21. Multicolor FISH karyotyping revealed multiple structural rearrangements involving chromosomes 1, 5, 6, 7, 8, 9, 13, 17, 18, and 22. Rearrangements of chromosome 8 involved 10 different chromosomes, while rearrangements of chromosome 17 involved 5. Different rearrangements resulted in imbalances of similar chromosome regions, suggesting that similar genomic imbalances are constitutively important but are achieved through different pathways. The use of a commercially available genomic array excluded TOP2A (17q), and MYBL2, PTPT1, CSE1L, and ZNF217 (20q) as candidate genes for frequently amplified areas on these chromosomes, and contributed to refining the limits of chromosome regions involved in genomic imbalances.

A balanced t(5;17) (p15;q22-23) in chondroblastoma: frequency of the re-arrangement and analysis of the candidate genes

BackgroundChondroblastoma is a benign cartilaginous tumour of bone that predominantly affects the epiphysis of long bones in young males. No recurrent chromosomal re-arrangements have so far been observed. Methods: We identified an index case with a balanced translocation by Combined Binary Ratio-Fluorescent in situ Hybridisation (COBRA-FISH) karyotyping followed by breakpoint FISH mapping and array-Comparative Genomic Hybridisation (aCGH). Candidate region re-arrangement and candidate gene expression were subsequently investigated by interphase FISH and immunohistochemistry in another 14 cases.ResultsA balanced t(5;17)(p15;q22-23) was identified. In the index case, interphase FISH showed that the translocation was present only in mononucleated cells and was absent in the characteristic multinucleated giant cells. The t(5;17) translocation was not observed in the other cases studied. The breakpoint in 5p15 occurred close to the steroid reductase 5α1 (SRD5A1) gene. Expression of the protein was found in all cases tested. Similar expression was found for the sex steroid signalling-related molecules oestrogen receptor alpha and aromatase, while androgen receptors were only found in isolated cells in a few cases. The breakpoint in 17q22-23 was upstream of the carbonic anhydrase × (CA10) gene region and possibly involved gene-regulatory elements, which was indicated by the lack of CA10 protein expression in the index case. All other cases showed variable levels of CA10 expression, with low expression in three cases.ConclusionWe report a novel t(5;17)(p15;q22-23) translocation in chondroblastoma without involvement of any of the two chromosomal regions in other cases studied. Our results indicate that the characteristic multinucleated giant cells in chondroblastoma do not have the same clonal origin as the mononuclear population, as they do not harbour the same translocation. We therefore hypothesise that they might be either reactive or originate from a distinct neoplastic clone, although the occurrence of two distinct clones is unlikely. Impairment of the CA10 gene might be pathogenetically relevant, as low expression was found in four cases. Diffuse expression of SRD5A1 and sex steroid signalling-related molecules confirms their role in neoplastic chondrogenesis.