1019

CDK4

CMM3|PSK-J3;cyclin-dependent kinase 4;CDK4;cyclin-dependent kinase 4

cell division protein kinase 4

12q14

protein-coding

Rhabdomyosarcomas are a heterogeneous group of malignant tumors and are the mostcommon soft-tissue sarcoma of childhood. Rhabdomyosarcomas resemble developing skeletal muscle, notably in their expression of the MRF family of transcription factors and the PAX3 and PAX7 genes. These PAX genes are also involved through specific translocations, t(2;13)(q35;q14) and variant t(1;13)(p36;q14) in the alveolar subtype, which result in PAX3-FKHR and PAX7-FKHR fusion genes, respectively. The fusion genes are thought critically to affect downstream targets of PAX3 and PAX7 or possibly have novel targets. Similar downstream changes may also be involved in embryonal and fusion gene negative cases. Genomic amplification of such genes as MYCN, MDM2, CDK4, and PAX7-FKHR is a feature mainly of the alveolar subtype, while specific chromosomal gains, including chromosomes 2, 8, 12, and 13, are associated with the embryonal subtype. Loss ofalleles and imprinting at 11p15.5 and disruption of genes such as IGF2, ATR, PTC, P16, and TP53 have also been implicated in rhabdomyosarcoma development. Whereasthere is now a realistic possibility of cure in the majority of cases, there remains a subset that is resistant to multimodality therapy, including high-dosechemotherapy. Characterization of the defining molecular features of tumors thatare likely to behave aggressively represents a particular challenge. Current research is leading toward a better understanding of rhabdomyosarcoma tumorigenesis, which may ultimately result in novel therapeutic strategies that increase the overall cure. Genes Chromosomes cancer 26:275-285, 1999.#CI- Copyright 1999 Wiley-Liss, Inc.

Although common among adult intracranial neoplasms, pediatric malignant astrocytomas (PMAs) comprise a relatively small proportion of the brain tumors that occur in children. The scarcity of such cases generally requires that molecular analyses of PMAs are based on the utilization of paraffin-embedded material, and here we have used 39 such specimens to examine the incidence and prognostic significance of oncogene and tumor suppressor gene alterations (including amplifications of EGFR, CDK4, and MDM2 as well as inactivating mutations of CDKN2A, TP53, and PTEN) in these tumors. In general, the frequency of alteration for the genes we have studied fell within ranges that have been reported for adult astrocytomas. However, EGFR amplification, which is usually observed in approximately 40% and 15% of adult grade 4 and grade 3 astrocytomas,respectively, was not detected in any member of this series. With regard to prognosis, PTEN mutations were significantly associated with decreased survival among grade 3 and grade 4 PMA patients, a potentially important observation because neither patient age nor tumor malignancy grade was correlated with outcome for these individuals. In total, our data suggest at least one significant distinction between the genetic etiology of pediatric and adult astrocytomas and additionally reveal that analysis of PTEN mutations in PMA patients may be useful in the differential diagnosis of these tumors.

Rhabdomyosarcoma (RMS) is a family of soft tissue tumors that are associated with the skeletal muscle lineage and generally occur in the pediatric population. Based on histopathologic features, two subtypes, embryonal (ERMS) and alveolar (ARMS), were identified and associated with distinct clinical characteristics and genetic alterations. ARMS is associated with 2;13 or 1;13 chromosomal translocations, which generate PAX3-FKHR and PAX7-FKHR fusion products, respectively. These translocations result in altered expression, function, and subcellular localization of the fusion products relative to the wild-type proteins, and ultimately contribute to oncogenic behavior by modifying growth, differentiation, and apoptosis pathways. In contrast to the specific translocations found in ARMS, most ERMS cases have allelic loss at chromosome 11p15.5. Chromosome fragment transfer studies demonstrated that this region represses tumor cell growth, suggesting the presence of tumor suppressor gene(s)in this region. In both ERMS and ARMS, there is evidence of collaborating alterations that affect common targets, such as the p53 and RB pathways. One mechanism for perturbing these pathways involves amplification of genes such as MDM2 and CDK4; these amplification events occur frequently in ARMS but only rarely in ERMS. Therefore, despite similarities in the downstream targets of these genetic alterations, the striking cytogenetic and molecular differences between ARMS and ERMS indicate distinct molecular etiologies in these two subtypes.

Amplification of the EGFR, mdm2, CDK4 and PDGFR A genes has been widely demonstrated in adult malignant gliomas, almost exclusively glioblastomas. To determine the role of these mutational events in pediatric astrocytic gliomas weinvestigated the presence of EGFR, mdm2, CDK4 and PDGFR A gene amplification in 38 childhood brain tumor biopsies, including 24 low-grade astrocytomas and 14 malignant tumors. We used differential PCR assay on DNA extracted either from paraffin embedded or frozen tissues. EGFR gene amplification was detected in 4 out of 14 malignant tumors; no low-grade astrocytoma showed amplification. tumors with EGFR gene amplification were negative for the presence of p53 mutations, asobserved in a previous study. One glioblastoma showed PDGFR A amplification, while no amplifications were observed for mdm2 and CDK4 genes. These data are inline with those obtained from studies on gliomas of adults and suggest the existence of two different subsets of malignant gliomas also in pediatric brain tumors: one carrying EGFR gene amplification, the other showing p53 mutations.

BACKGROUND: childhood ependymoma remains a major therapeutic challenge despite surgery, chemotherapy, and irradiation. We hypothesized that p53 function might be abrogated in ependymomas and implicated in their resistance to anti-cancer therapy. PROCEDURE: Primary ependymomas at diagnosis or relapse from 24 childrenwere analyzed for p53 pathway, using a functional assay in yeast, RT-PCR, Western blot analysis, and/or immunohistochemistry for TP53 mutation, p14(ARF) deletion and promoter hypermethylation, MDM2 and PAX5 expression, respectively. p53-mediated response to radiation-induced DNA damage was evaluated using Western blot and flow cytometry analysis in two ependymoma xenograft models, IGREP37 andIGREP83, derived from primary anaplastic childhood ependymomas. RESULTS: No TP53, MDM2, p14(ARF), PAX5 gene abnormalities were detected in the primary ependymomastumors and xenografts tested. Interestingly, despite the lack of these abnormalities, p53 induced p21-mediated G(1) growth arrest in response to irradiation was altered in the IGREP37 xenograft tumors. Although irradiation induced necrosis and apoptotic cell death, IGREP37 tumors were moderately sensitive to radiation therapy in vivo. In contrast, irradiation yielded significant tumor growth delays and tumor regressions in the p53 functional IGREP83 xenografts. CONCLUSION: Alterations in p53-mediated growth arrest in ependymomas might be implicated in the radio-resistance of these tumors and demand further evaluation.

Rhabdomyosarcomas (RMS) are the most common pediatric soft tissue sarcomas. Theyresemble developing skeletal muscle and are histologically divided into two mainsubtypes; alveolar and embryonal RMS. Characteristic genomic aberrations, including the PAX3- and PAX7-FOXO1 fusion genes in alveolar cases, have led to increased understanding of their molecular biology. Here, we determined the effect of genomic copy number on gene expression levels through array comparative genomic hybridization (CGH) analysis of 13 RMS cell lines, confirmed by multiplex ligation-dependent probe amplification copy number analyses, combined with theircorresponding expression profiles. Genes altered at the transcriptional level bygenomic imbalances were identified and the effect on expression was proportionalto the level of genomic imbalance. Extrapolating to a public expression profiling dataset for 132 primary RMS identified features common to the cell lines and primary samples and associations with subtypes and fusion gene status. Genes identified such as CDK4 and MYCN are known to be amplified, overexpressed, and involved in RMS tumorigenesis. Of the many genes identified, those with likely functional relevance included CENPF, DTL, MYC, EYA2, and FGFR1. Copy number and expression of FGFR1 was validated in additional primary material and found amplified in 6 out of 196 cases and overexpressed relative to skeletal muscle and myoblasts, with significantly higher expression levels in the embryonal comparedwith alveolar subtypes. This illustrates the ability to identify genes of potential significance in tumor development through combining genomic and transcriptomic profiles from representative cell lines with publicly available expression profiling data from primary tumors.

Cytogenetic and molecular genetic studies were performed on a pleomorphic sarcoma removed from the left atrium of a 15-year-old girl. Histologic analysis was consistent with a storiform-pleomorphic malignant fibrous histiocytoma (MFH). Although MFH is the most common soft-tissue sarcoma of late adulthood. It is extremely rare in childhood and its existence in the pediatric population remains controversial. Cytogenetic analysis revealed several alterations previously associated with adult MFH, including abnormalities of chromosomal bands 11p11 and 19p13. Moreover, the tumor demonstrated homogeneously staining regions (HSR) anddouble minute chromosomes (dmin) suggestive of gene amplification. We therefore screened the case for amplification of genes localized to chromosomal bands 12q13-14, including the putative protooncogenes MDM2, CDK4, SAS, CHOP, and CLI, which are frequently amplified and overexpressed in adult MFH. Southern and Northern blot analysis confirmed the coamplification of MDM2, CDK4, SAS, and CHOP. To our knowledge, such coamplification studies of the 12q13-14 amplicon have not been previously detected in pediatric MFH. Our results provide cytogenetic and molecular genetic evidence that pediatric and adult MFH are histogenetically related entities.

Abnormality of the cyclin D1/cdk4/p16INK4a/pRb pathway during tumorigenesis has recently been reported. Hepatoblastoma is a rare malignant liver tumor of childhood, but underlying abnormalities of cell-cycle regulating protein remain to be elucidated. The expression of cyclin D1, cdk4, p16 and retinoblastoma geneproduct (pRb) was studied by immunohistochemistry in 17 paraffin-embedded tissues consisting of both tumor and corresponding non-neoplastic tissues. tumor tissuesshowed overexpression of cyclin D1 (13/17, 76%) and cdk4 (15/17, 88%). Eleven cases showed co-overexpression of both cyclin D1 and cdk4. No abnormal p16 or pRb expression was noted. In the group with a high score (+4) for cyclin D1 expression, a positive correlation with tumor recurrence was noted (P = 0.043). These data suggest that overexpressed cyclin D1 and cdk4 protein might play an important role in the tumorigenesis of hepatoblastoma and that in the group withhigh cyclin D1 expression, tumor recurrence may be more frequent.