Identification of tumor-specific molecular signatures in intracranial ependymoma and association with clinical characteristics.

PURPOSE: To delineate clinically relevant molecular signatures of intracranial ependymoma. MATERIALS AND METHODS: We analyzed 24 primary intracranial ependymomas. For genomic profiling, microarray-based comparative genomic hybridization (CGH) was used and results were validated by fluorescent in situ hybridization and loss of heterozygosity mapping. We performed gene expression profiling using microarrays, real-time quantitative reverse transcriptase polymerase chain reaction, and methylation analysis of selected genes. We applied class comparison analyses to compare both genomic and expression profiling data with clinical characteristics. RESULTS: A variable number of genomic imbalances were detected by array CGH, revealing multiple regions of recurrent gain (including 2q23, 7p21, 12p, 13q21.1, and 20p12) and loss (including 5q31, 6q26, 7q36, 15q21.1, 16q24, 17p13.3, 19p13.2, and 22q13.3). An ependymoma-specific gene expression signature was characterized by the concurrent abnormal expression of developmental and differentiation pathways, including NOTCH and sonic hedgehog signaling. We identified specific differentially imbalanced genomic clones and gene expression signatures significantly associated with tumor location, patient age at disease onset, and retrospective risk for relapse. Integrated genomic and expression profiling allowed us to identify genes of which the expression is deregulated in intracranial ependymoma, such as overexpression of the putative proto-oncogene YAP1 (located at 11q22) and downregulation of the SULT4A1 gene (at 22q13.3). CONCLUSION: The present exploratory molecular profiling study allowed us to refine previously reported intervals of genomic imbalance, to identify novel restricted regions of gain and loss, and to identify molecular signatures correlating with various clinical variables. Validation of these results on independent data sets represents the next step before translation into the clinical setting.

Expression studies in gliomas and glial cells do not support a tumor suppressor role for LGI1.

Disruptions of LGI1 in glioblastoma (GBM) cell lines and LGI1 mutations in families with autosomal dominant epilepsy imply a role for LGI1 in glial cells as well as in neurons. Although we and others could not find LGI1 mutations in malignant gliomas, our initial studies appeared to support the idea that LGI1 is poorly expressed or absent in these tumors. Microarray data suggested that LGI1 could be involved in the control of matrix metalloproteinases, and we found that tumors derived from U87 glioblastoma cells overexpressing LGI1 were less aggressive than U87 control tumors. To our surprise, we observed that LGI1 expression after differentiation of murine neural stem cells was robust in neurons but negligible in glial cells, in agreement with immunohistochemistry studies on rodent brain. This observation could suggest that the variable levels of LGI1 expression in gliomas reflect the presence of neurons entrapped within the tumor. To test this hypothesis, we investigated LGI1 expression in parallel with expression of the neuronal marker NEF3 by real-time PCR on 30 malignant gliomas. Results showed a strong, positive correlation between the expression levels of these two genes (P < 0.0001). Thus, our data confirm that LGI1 is involved in cell-matrix interactions but suggest that its expression is not relevant in glial cells, implying that its role as a tumor suppressor in gliomas should be reconsidered.

Wilms tumor in monozygous twins: clinical, pathological, cytogenetic and molecular case report.

The concomitant occurrence of Wilms tumor (WT) was observed in two monozygotic twin sisters without evidence of congenital malformations. Twin 1 was diagnosed with a stage I WT at 11 months of age, whereas twin 2 developed a bilateral (stage V) WT at 13 months of age. In both cases pathologic examination showed a nonanaplastic stromal type WT, with marked rhabdomyomatous elements. Cytogenetic analyses performed on blood samples and on tumor specimens revealed no karyotypic abnormality. No alteration of the WT1 and POU6F2 genes was identified in constitutional and tumor DNA of both sisters, and no anomaly in WT1 expression was evidenced in the normal kidney of one of them. However, loss of heterozygosity on chromosome 11p, involving the alleles of maternal origin, was detected both in the single tumor of twin 1 and in the two distinct tumors of twin 2, thus suggesting a common etiology of the diseases. To the authors' knowledge, this is the first report describing at both the clinical and genetic level a couple of monozygotic twins concordant for WT development.

SMARCB1/INI1 tumor suppressor gene is frequently inactivated in epithelioid sarcomas.

Epithelioid sarcoma is a rare soft tissue neoplasm of uncertain lineage that usually arises in the distal extremities of adults, presents a high rate of recurrences and metastases and frequently poses diagnostic dilemmas. The recently reported large-cell "proximal-type" variant is characterized by increased aggressiveness, deep location, preferential occurrence in proximal/axial regions of older patients, and rhabdoid features. Previous cytogenetic studies indicated that the most frequent alterations associated with this tumor entity affect chromosome 22. In this study, combined spectral karyotyping, fluorescence in situ hybridization, and array-based comparative genomic hybridization analyses of two proximal-type cases harboring a rearrangement involving 10q26 and 22q11 revealed that the 22q11 breakpoints were located in a 150-kb region containing the SMARCB1/INI1 gene, and that homozygous deletion of the gene was present in the tumor tissue. The SMARCB1/INI1 gene encodes for an invariant subunit of SWI/SNF chromatin remodeling complex and has been previously reported to act as a tumor suppressor gene frequently inactivated in infantile malignant rhabdoid tumors. We analyzed SMARCB1/INI1 gene status in nine additional epithelioid sarcoma cases (four proximal types and five conventional types) and altogether we identified deletions of SMARCB1/INI1 gene in 5 of 11 cases, all proximal types. We confirmed and further extended the number of cases with SMARCB1/INI1 inactivation to 6 of 11 cases, by real-time quantitative PCR analysis of mRNA expression and by SMARCB1/INI1 immunohistochemistry. Overall, these results point to SMARCB1/INI1 gene involvement in the genesis and/or progression of epithelioid sarcomas. Analysis of larger series of epithelioid sarcomas will be necessary to highlight putative clinically relevant features related to SMARCB1/INI1 inactivation.

Genetic alterations and in vivo tumorigenicity of neurospheres derived from an adult glioblastoma.

Pediatric brain tumors may originate from cells endowed with neural stem/precursor cell properties, growing in vitro as neurospheres. We have found that these cells can also be present in adult brain tumors and form highly infiltrating gliomas in the brain of immunodeficient mice. Neurospheres were grown from three adult brain tumors and two pediatric gliomas. Differentiation of the neurospheres from one adult glioblastoma decreased nestin expression and increased that of glial and neuronal markers. Loss of heterozygosity of 10q and 9p was present in the original glioblastoma, in the neurospheres and in tumors grown into mice, suggesting that PTEN and CDKN2A alterations are key genetic events in tumor initiating cells with neural precursor properties.

Germline mutations of the POU6F2 gene in Wilms tumors with loss of heterozygosity on chromosome 7p14.

Wilms tumor (WT) is a kidney malignancy of childhood characterized by highly heterogeneous genetic alterations. We previously reported the molecular and cytogenetic characterization of a WT (Case 30) carrying an interstitial deletion in chromosome 7p14 between markers D7S555 and D7S668. Loss of heterozygosity (LOH) analyses had revealed that this same region was lost in 8 out of 38 examined WTs, suggesting that the identified interval contains a putative tumor suppressor gene. To confirm this hypothesis, in this work, we analyzed an additional 35 WTs, four of which showed LOH in the region of interest. Furthermore, we were able to more accurately define the extension of the deletion in Case 30, mapping it within an interval not exceeding 390 kb, proximally to D7S555. To date, only a single expressed gene, POU6F2 (the POU domain, class 6, transcription factor 2; also known as RPF1), has been recognized in this interval. Sequencing of the gene in the 12 WTs showing LOH and in a corresponding numbers of WT cases without LOH, led to the identification of two germline nucleotide substitutions. The first occurred in the 5'-untranslated region, while the second caused an amino acid change in a glutamine repeat domain. These mutations, whose occurrence was not observed in more than 100 control subjects, were detected in two patients showing the loss of the constitutionally wild-type allele in tumor DNA. Together with the finding of the expression of the POU6F2 mouse homolog in both fetal and adult kidney, our observations suggest that the gene is a tumor suppressor and is involved in hereditary predisposition to WT.

Molecular cytogenetic characterization of proximal-type epithelioid sarcoma.

Proximal-type epithelioid sarcoma is a recently described soft-tissue tumor that is distinguished from conventional-type epithelioid sarcoma by a far more aggressive clinical course, frequent location in the proximal anatomic regions, and variable rhabdoid morphology. Because of their rarity and peculiar morphology, proximal-type epithelioid sarcomas frequently pose serious diagnostic dilemmas, being easily misdiagnosed as a variety of other malignant neoplasms. To date, the information available on the genetic alterations associated with this tumor entity has been confined to single conventional cytogenetic reports. In this article, we present the results of a conventional and molecular cytogenetic analysis of six proximal-type epithelioid sarcomas. Spectral karyotyping analysis of these cases deciphered the characteristics of several marker chromosomes and complex translocations, leading to the recognition of recurrent rearrangements. The most frequently involved chromosome arm was 22q, and the identification of two cases with a similar translocation, t(10;22), suggests a role for one or more genes on chromosome 22 in the pathogenesis of this tumor and provides an opportunity for finely mapping the translocation-associated breakpoints. Chromosome arm 8q gain was also a frequent event and correlated with gain of MYC gene copy number, as demonstrated by fluorescence in situ hybridization. A review of both cases reported in the literature and those presented in this study reinforced the involvement of chromosomes 8 and 22 and also indicated frequent rearrangements of chromosomes 7, 14, 18, and 20.