Molecular subgroups of atypical teratoid rhabdoid tumours in children: an integrated genomic and clinicopathological analysis.

BACKGROUND: Rhabdoid brain tumours, also called atypical teratoid rhabdoid tumours, are lethal childhood cancers with characteristic genetic alterations of SMARCB1/hSNF5. Lack of biological understanding of the substantial clinical heterogeneity of these tumours restricts therapeutic advances. We integrated genomic and clinicopathological analyses of a cohort of patients with atypical teratoid rhabdoid tumours to find out the molecular basis for clinical heterogeneity in these tumours. METHODS: We obtained 259 rhabdoid tumours from 37 international institutions and assessed transcriptional profiles in 43 primary tumours and copy number profiles in 38 primary tumours to discover molecular subgroups of atypical teratoid rhabdoid tumours. We used gene and pathway enrichment analyses to discover group-specific molecular markers and did immunohistochemical analyses on 125 primary tumours to evaluate clinicopathological significance of molecular subgroup and ASCL1-NOTCH signalling. FINDINGS: Transcriptional analyses identified two atypical teratoid rhabdoid tumour subgroups with differential enrichment of genetic pathways, and distinct clinicopathological and survival features. Expression of ASCL1, a regulator of NOTCH signalling, correlated with supratentorial location (p=0·004) and superior 5-year overall survival (35%, 95% CI 13-57, and 20%, 6-34, for ASCL1-positive and ASCL1-negative tumours, respectively; p=0·033) in 70 patients who received multimodal treatment. ASCL1 expression also correlated with superior 5-year overall survival (34%, 7-61, and 9%, 0-21, for ASCL1-positive and ASCL1-negative tumours, respectively; p=0·001) in 39 patients who received only chemotherapy without radiation. Cox hazard ratios for overall survival in patients with differential ASCL1 enrichment treated with chemotherapy with or without radiation were 2·02 (95% CI 1·04-3·85; p=0·038) and 3·98 (1·71-9·26; p=0·001). Integrated analyses of molecular subgroupings with clinical prognostic factors showed three distinct clinical risk groups of tumours with different therapeutic outcomes. INTERPRETATION: An integration of clinical risk factors and tumour molecular groups can be used to identify patients who are likely to have improved long-term radiation-free survival and might help therapeutic stratification of patients with atypical teratoid rhabdoid tumours. FUNDING: C17 Research Network, Genome Canada, b.r.a.i.n.child, Mitchell Duckman, Tal Doron and Suri Boon foundations.

Digital expression profiling identifies RUNX2, CDC5L, MDM2, RECQL4, and CDK4 as potential predictive biomarkers for neo-adjuvant chemotherapy response in paediatric osteosarcoma.

Osteosarcoma is the most common malignancy of bone, and occurs most frequently in children and adolescents. Currently, the most reliable technique for determining a patients' prognosis is measurement of histopathologic tumor necrosis following pre-operative neo-adjuvant chemotherapy. Unfavourable prognosis is indicated by less than 90% estimated necrosis of the tumor. Neither genetic testing nor molecular biomarkers for diagnosis and prognosis have been described for osteosarcomas. We used the novel nanoString mRNA digital expression analysis system to analyse gene expression in 32 patients with sporadic paediatric osteosarcoma. This system used specific molecular barcodes to quantify expression of a set of 17 genes associated with osteosarcoma tumorigenesis. Five genes, from this panel, which encoded the bone differentiation regulator RUNX2, the cell cycle regulator CDC5L, the TP53 transcriptional inactivator MDM2, the DNA helicase RECQL4, and the cyclin-dependent kinase gene CDK4, were differentially expressed in tumors that responded poorly to neo-adjuvant chemotherapy. Analysis of the signalling relationships of these genes, as well as other expression markers of osteosarcoma, indicated that gene networks linked to RB1, TP53, PI3K, PTEN/Akt, myc and RECQL4 are associated with osteosarcoma. The discovery of these networks provides a basis for further experimental studies of role of the five genes (RUNX2, CDC5L, MDM2, RECQL4, and CDK4) in differential response to chemotherapy.

MicroRNA-34c inversely couples the biological functions of the runt-related transcription factor RUNX2 and the tumor suppressor p53 in osteosarcoma.

Osteosarcoma (OS) is a primary bone tumor that is most prevalent during adolescence. RUNX2, which stimulates differentiation and suppresses proliferation of osteoblasts, is deregulated in OS. Here, we define pathological roles of RUNX2 in the etiology of OS and mechanisms by which RUNX2 expression is stimulated. RUNX2 is often highly expressed in human OS biopsies and cell lines. Small interference RNA-mediated depletion of RUNX2 inhibits growth of U2OS OS cells. RUNX2 levels are inversely linked to loss of p53 (which predisposes to OS) in distinct OS cell lines and osteoblasts. RUNX2 protein levels decrease upon stabilization of p53 with the MDM2 inhibitor Nutlin-3. Elevated RUNX2 protein expression is post-transcriptionally regulated and directly linked to diminished expression of several validated RUNX2 targeting microRNAs in human OS cells compared with mesenchymal progenitor cells. The p53-dependent miR-34c is the most significantly down-regulated RUNX2 targeting microRNAs in OS. Exogenous supplementation of miR-34c markedly decreases RUNX2 protein levels, whereas 3'-UTR reporter assays establish RUNX2 as a direct target of miR-34c in OS cells. Importantly, Nutlin-3-mediated stabilization of p53 increases expression of miR-34c and decreases RUNX2. Thus, a novel p53-miR-34c-RUNX2 network controls cell growth of osseous cells and is compromised in OS.

Array comparative genomic hybridization in osteosarcoma.

Osteosarcoma, the most frequent primary bone tumor, is a malignant mesenchymal sarcoma with a peak incidence in young children and adolescents. Left untreated, it progresses relentlessly to local and systemic disease, ultimately leading to death within months. Genomically, osteosarcomas are aneuploid with chaotic karyotypes, lacking the pathognomonic genetic rearrangements characteristic of most sarcomas. The familial genetics of osteosarcoma helped in elucidating some of the etiological molecular disruptions, such as the tumor suppressor genes RB1 in retinoblastoma and TP53 in Li-Fraumeni, and RECQL4 involved in DNA repair/replication in Rothmund-Thomson syndrome. Genomic profiling approaches such as array comparative genomic hybridization (aCGH) have provided additional insights concerning the mechanisms responsible for generating complex osteosarcoma genomes. This chapter provides a brief introduction to the clinical features of conventional osteosarcoma, the predominant subtypes, and a general overview of materials and analytical methods of osteosarcoma aCGH, followed by a more detailed literature overview of aCGH studies and a discussion of emerging genes, molecular mechanisms, and their clinical implications, as well as more recent application of integrative genomics in osteosarcoma. aCHG is helping elucidate genomic events leading to tumor development and evolution as well as identification of prognostic markers and therapeutic targets in osteosarcoma.

The genetics of osteosarcoma.

Osteosarcoma is a primary bone malignancy with a particularly high incidence rate in children and adolescents relative to other age groups. The etiology of this often aggressive cancer is currently unknown, because complicated structural and numeric genomic rearrangements in cancer cells preclude understanding of tumour development. In addition, few consistent genetic changes that may indicate effective molecular therapeutic targets have been reported. However, high-resolution techniques continue to improve knowledge of distinct areas of the genome that are more commonly associated with osteosarcomas. Copy number gains at chromosomes 1p, 1q, 6p, 8q, and 17p as well as copy number losses at chromosomes 3q, 6q, 9, 10, 13, 17p, and 18q have been detected by numerous groups, but definitive oncogenes or tumour suppressor genes remain elusive with respect to many loci. In this paper, we examine studies of the genetics of osteosarcoma to comprehensively describe the heterogeneity and complexity of this cancer.

Multiple CDK/CYCLIND genes are amplified in medulloblastoma and supratentorial primitive neuroectodermal brain tumor.

Embryonal brain tumors, which include medulloblastoma and the more aggressive supratentorial primitive neuroectodermal tumor (sPNET), comprise one of the largest group of malignant pediatric brain tumors. We observed in high resolution array comparative genomic hybridization and polymerase chain reaction analyses that several different components of the CDK/CYCLIND/pRB regulatory complex, including the CDK4/6 and CCND1/2 loci, are targets of gene amplification in medulloblastoma and sPNET. CDK6 and CCND1 gene amplification were respectively most common and robust, and overall CDK/CYCLIND gene amplification was more commonly observed in sPNET (25%) than medulloblastoma (1-5%). CDK6 overexpression enhanced in vitro and in vivo oncogenicity and endogenous CDK6 or CCND1 knockdown decreased pRB phosphorylation and impaired cell cycle progression in both medulloblastoma and sPNET cell lines. Although animal models implicate the pRB tumor suppressor pathway in medulloblastoma and sPNET, mutations of RB1 or the related INK4 tumor suppressor loci are rare in primary human tumors. Our data suggest that CDK/CYCLIND gene amplification may represent important mechanisms for functional inactivation of pRB in medulloblastoma and sPNET.

The CIC-DUX4 fusion transcript is present in a subgroup of pediatric primitive round cell sarcomas.

Pediatric undifferentiated soft tissue sarcomas are a group of diagnostically challenging tumors. Recent studies have identified a subgroup of undifferentiated soft tissue sarcomas with primitive round to plump spindle cell morphology and a t(4;19)(q35;q13.1) translocation resulting in the expression of a CIC-DUX4 fusion transcript, including 2 tumors previously reported by our laboratory (Cancer Genet Cytogenet 2009;195:1). In the present study, reverse transcriptase polymerase chain reaction assays developed for both frozen and paraffin-based tissues were applied to a series of 19 pediatric undifferentiated soft tissue sarcomas using a combination of primer sets covering the CIC-DUX4 fusion transcript. Of the 19 undifferentiated soft tissue sarcomas, 16 had primitive round to plump spindle cell morphology, and 3 had pure spindle cell morphology. Three of the 16 undifferentiated soft tissue sarcomas with primitive round cell morphology were found to harbor the CIC-DUX4 fusion transcript by reverse transcriptase polymerase chain reaction. Automated DNA sequencing of the polymerase chain reaction products identified 2 distinct transcript variants. One CIC-DUX4-positive tumor showed membranous CD99 positivity, 2 showed focal S100 positivity, and 1 showed focal CD57 positivity. With the 2 previously reported cases, the total number of CIC-DUX4-positive primitive round cell sarcomas identified at our institution has been brought to 5 (28%) of 18. Given the consistent involvement of the CIC-DUX4 fusion in a subset of primitive round cell undifferentiated soft tissue sarcomas, these findings suggest a central role for the fusion transcript in such tumors. The current findings further define a novel genetic subset of pediatric primitive round cell sarcomas and provide an additional diagnostic tool for their characterization and diagnosis.

Analysis of miRNA-gene expression-genomic profiles reveals complex mechanisms of microRNA deregulation in osteosarcoma.

Osteosarcoma is an aggressive sarcoma of the bone characterized by a high level of genetic instability and recurrent DNA deletions and amplifications. This study assesses whether deregulation of microRNA (miRNA) expression is a post-transcriptional mechanism leading to gene expression changes in osteosarcoma. miRNA expression profiling was performed for 723 human miRNAs in 7 osteosarcoma tumors, and 38 miRNAs differentially expressed ≥10-fold (28 under- and 10 overexpressed) were identified. In most cases, observed changes in miRNA expression were DNA copy number-correlated. However, various mechanisms of alteration, including positional and/or epigenetic modifications, may have contributed to the expression change of 23 closely linked miRNAs in cytoband 14q32. To develop a comprehensive molecular genetic map of osteosarcoma, the miRNA profiles were integrated with previously published array comparative genomic hybridization DNA imbalance and mRNA gene expression profiles from a set of partially overlapping osteosarcoma tumor samples. Many of the predicted gene targets of differentially expressed miRNA are involved in intracellular signaling pathways important in osteosarcoma, including Notch, RAS/p21, MAPK, Wnt, and the Jun/FOS pathways. By integrating data on copy number variation with mRNA and miRNA expression profiles, we identified osteosarcoma-associated gene expression changes that are DNA copy number-correlated, DNA copy number-independent, mRNA-driven, and/or modulated by miRNA expression. These data collectively suggest that miRNAs provide a novel post-transcriptional mechanism for fine-tuning the expression of specific genes and pathways relevant to osteosarcoma. Thus, the miRNA identified in this manner may provide a starting point for experimentally modulating therapeutically relevant pathways in this tumor.

Immunohistochemical expression and cluster analysis of mesenchymal and neural stem cell-associated proteins in pediatric soft tissue sarcomas.

Pediatric undifferentiated soft tissue sarcomas (USTSs) are a group of malignancies composed predominantly of primitive round cell sarcomas, the histogenesis of which is uncertain. Thus, diagnosis and therapy remain a challenge. The aims of the current study were to determine whether differential expression of stem cell-associated proteins could be used to aid in determining the histogenesis of pediatric USTSs and to determine whether pediatric USTSs expressed a unique panel of stem cell-associated proteins to aid diagnosis. Tumors included 28 Ewing sarcoma/primitive neuroectodermal tumors (ESs), 22 embryonal rhabdomyosarcomas (ERMSs), 8 alveolar rhabdomyosarcomas (ARMSs), 5 synovial sarcomas (SSs), 5 malignant peripheral nerve sheath tumors (MPNSTs), and 13 USTSs. Stem cell antibodies included 3 mesenchymal stem cell markers (CD44, CD105, and CD166) and 5 neural stem cell markers (CD15, CD29, CD56, CD133, and nestin). Sections were scored followed by statistical analysis, clustering analysis, and visualizations using Partek Genomic Suite Software. The Euclidean clustering divided the tumors into 2 major groups. ESs and USTSs formed the majority of the 1st group, whereas ERMSs, ARMSs, MPNSTs, and SSs formed the 2nd group. Reduced expression of CD56 was strongly associated with the ES/USTS cluster (P < 0.0001). ESs and USTSs were further separated by CD166 staining, wherein increased expression was associated with ES (P < 0.0001). The 2nd group included the majority of other sarcomas, with no consistent separation between subtypes. The current study demonstrates the usefulness of applying stem cell markers to pediatric sarcomas and indicates that USTSs and ESs are closely related and may share a common histogenesis.

Increased detection of metastatic melanoma in pediatric sentinel lymph node biopsies using RT-PCR on paraffin-embedded tissue.

Sentinel lymph node (SLN) biopsy has become integral in the staging of patients with melanoma, and entails detailed histologic examination with immunohistochemistry. Reverse transcriptase-polymerase chain reaction (RT-PCR) for tyrosinase transcripts has been used to increase sensitivity but requires a dedicated piece of tissue that does not undergo histologic examination. We developed a nested RT-PCR assay for tyrosinase applicable on paraffin-embedded tissue and applied this to a series of SLNs from pediatric patients with melanoma. Thirty-six SLNs from 4 females and 4 males were included in the study. Eight lymph nodes with reactive changes were included as controls. SLNs were examined histologically and immunohistochemically for S100, tyrosinase, and MART1. Seven patients had between 1 and 4 morphologically-positive SLNs and one patient had negative SLNs (HISTO+; 12/36, 33%). Three lymph nodes were excluded from molecular analysis owing to inadequate RNA, and 29 of the remaining 33 nodes were positive (MOL+; 88%). All patients had at least 1 SLN positive by RT-PCR. Twelve were HISTO+/MOL+; 17 were HISTO-/MOL+; and 4 were HISTO-/MOL-. All control lymph nodes were negative for tyrosinase transcripts. The application of RT-PCR for tyrosinase to paraffin-embedded tissue significantly increased the number of positive SLNs and upstaged one patient from negative to positive. The prognostic implications of such findings require further investigation, especially in the pediatric age group. Nonetheless, this technique provides a useful tool to determine the clinical significance of RT-PCR positivity in melanoma SLNs.

Expression analysis of genes associated with human osteosarcoma tumors shows correlation of RUNX2 overexpression with poor response to chemotherapy.

BACKGROUND: Human osteosarcoma is the most common pediatric bone tumor. There is limited understanding of the molecular mechanisms underlying osteosarcoma oncogenesis, and a lack of good diagnostic as well as prognostic clinical markers for this disease. Recent discoveries have highlighted a potential role of a number of genes including: RECQL4, DOCK5, SPP1, RUNX2, RB1, CDKN1A, P53, IBSP, LSAMP, MYC, TNFRSF1B, BMP2, HISTH2BE, FOS, CCNB1, and CDC5L. METHODS: Our objective was to assess relative expression levels of these 16 genes as potential biomarkers of osteosarcoma oncogenesis and chemotherapy response in human tumors. We performed quantitative expression analysis in a panel of 22 human osteosarcoma tumors with differential response to chemotherapy, and 5 normal human osteoblasts. RESULTS: RECQL4, SPP1, RUNX2, and IBSP were significantly overexpressed, and DOCK5, CDKN1A, RB1, P53, and LSAMP showed significant loss of expression relative to normal osteoblasts. In addition to being overexpressed in osteosarcoma tumor samples relative to normal osteoblasts, RUNX2 was the only gene of the 16 to show significant overexpression in tumors that had a poor response to chemotherapy relative to good responders. CONCLUSION: These data underscore the loss of tumor suppressive pathways and activation of specific oncogenic mechanisms associated with osteosarcoma oncogenesis, while drawing attention to the role of RUNX2 expression as a potential biomarker of chemotherapy failure in osteosarcoma.

IGF2 is highly expressed in pediatric undifferentiated sarcomas and reveals two distinct cytoplasmic trafficking patterns.

Pediatric undifferentiated soft tissue sarcomas (USTSs) are tumors composed of primitive mesenchymal cells. As such, they form an attractive model for studying the early events in sarcoma development. In an effort to better understand the critical molecular aberrations leading to sarcoma development, gene expression array analysis and post-array validation techniques were applied to several USTSs; the results were consistent with upregulation of the excitatory components of the insulin-like growth factor (IGF) pathway. Particularly high expression of the insulin-like growth factor 2 (IGF2) ligand was seen and confirmed by real-time reverse transcriptase-polymerase chain reaction. Immunohistochemistry performed using antibodies against IGF2 revealed overexpression of the IGF2 protein in 19 of 21 (90%) USTSs and revealed 2 distinct staining patterns, 1 in which there was diffuse cytoplasmic staining (16/19) and 1 in which there was punctate perinuclear positivity (3/19). Using ultrastructural immunogold localization of IGF2, it was determined that IGF2 was primarily localized to Golgi-derived vesicles and multivesicular bodies in tumor cells showing the punctate pattern, and to both the cytoplasm and plasma membrane of tumor cells showing the diffuse pattern. The results suggest that upregulation of the IGF pathway in pediatric USTSs is a critical early event in the development of sarcomas. Furthermore, findings from the immunocytochemical and immunogold analyses confirm the presence of 2 different cytoplasmic trafficking patterns and storage motifs of IGF2 within this type of tumor. Given that in one subcellular pattern the IGF2 protein does not appear to reach the membrane, these findings may have functional significance.

Detailed cytogenetic and array analysis of pediatric primitive sarcomas reveals a recurrent CIC-DUX4 fusion gene event.

Pediatric undifferentiated soft tissue sarcomas (USTS) are a diagnostically challenging group of neoplasms. Recently, a subcategory of USTS with primitive round cell morphology and a t(4;19)(q35;q13) rearrangement has been defined. The present study applied high-throughput array comparative genomic hybridization together with spectral karyotyping, four-color fluorescence in situ hybridization (FISH), and reverse transcriptase-polymerase chain reaction (RT-PCR) to a series of three pediatric USTS. Two of these had primitive round cell morphology with CD99 positivity; the third had a spindled and myxoid appearance. By genomic analyses, both primitive round cell sarcomas had t(4;19)(q35;q13) [corrected] rearrangements in addition to several imbalances throughout the genome. Four-color FISH and in silico analyses of the breakpoint region at 19q13 identified the potential involvement of the candidate oncogene CIC. By RT-PCR, fusion transcripts involving CIC (19q13) and DUX4 (4q35) were confirmed to be present in both primitive round cell sarcomas, further defining the breakpoints seen by genomic analysis. Described here are two tumors belonging to the rare category of CIC-DUX4-positive primitive sarcomas, with detailed cytogenetic and genomic information regarding this novel subclass of pediatric malignancy. Molecular and cytogenetic techniques for the detection of the CIC-DUX4 fusion gene are described, to aid in recognition and diagnosis.

Gene expression using microarrays in transplant recipients at risk of EBV lymphoproliferation after organ transplantation: preliminary proof-of-concept.

We hypothesized that aspects of the virus-host interaction could be measured to help predict progression to EBV-PTLD. We examined the spectrum of host genes differentially expressed and any relevant clustering in children at risk of EBV lymphoproliferation after organ transplantation. We compared the genes expressed among patients with different levels of viral loads. Gene expression was measured by microarray analysis of RNA from CD19+ B lymphocytes using the Human Genome U133 Plus 2.0 GeneChip. Among 27 samples from 26 transplant recipients, the viral load categories were: low or undetectable loads (LU), n = 14; high or intermediate loads (HI), n = 13. There were seven healthy EBV-seropositive (P) and -seronegative controls (N). Median time of post-transplantation was 0.5 yr (range 0.1-3.8). We identified 24-54 differentially expressed genes in each of four comparisons of HI vs. P, LU vs. P, HI vs. LU, and P vs. N. We identified patterns of 563 gene expressions, creating five clusters aligned with levels of viral load. PTLD occurred in four of five clusters. In summary, we demonstrated varying degrees of alignment between levels of VL and gene clusters. Analyses for differential expression of genes showed genes that could be implicated in the pathogenesis of EBV-PTLD.

Identification of interactive networks of gene expression associated with osteosarcoma oncogenesis by integrated molecular profiling.

Altered gene expression in tumors can be caused by copy number alterations to DNA or mutation affecting coding or regulatory regions of genes. However, epigenetic events may also influence gene expression. Malignant cells can show major disruptions in DNA methylation profiles, which are manifested as aberrant hypermethylation or as hypomethylation of gene promoters, as well as global genomic hypomethylation. In this study we performed integrative whole-genome analysis of DNA copy number, promoter methylation and gene expression using 10 osteosarcomas. We identified significant changes including: hypomethylation, gain, and overexpression of histone cluster 2 genes at chromosome 1q21.1-q21.3; loss of chromosome 8p21.2-p21.3 and underexpression of DOCK5 and TNFRSF10A/D genes; and amplification-related overexpression of RUNX2 at chromosome 6p12.3-p21.1. Amplification and overexpression of RUNX2 could disrupt G2/M cell cycle checkpoints, and downstream osteosarcoma-specific changes, such as failure of bone differentiation and genomic polyploidization. Failure of DOCK5-signaling, together with p53 and TNFRSF10A/D-related cell cycle and death pathways, may play a critical role in abrogating apoptosis. Our analyses show that the RUNX2 interactome may be constitutively activated in osteosarcoma, and that the downstream intracellular pathways are strongly associated with the regulation of osteoblast differentiation and control of cell cycle and apoptosis in osteosarcoma.

Recurrent RECQL4 imbalance and increased gene expression levels are associated with structural chromosomal instability in sporadic osteosarcoma.

Osteosarcoma (OS) is an aggressive bone tumor with complex abnormal karyotypes and a highly unstable genome, exhibiting both numerical- and structural-chromosomal instability (N- and S-CIN). Chromosomal rearrangements and genomic imbalances affecting 8q24 are frequent in OS. RECQL4 gene maps to this cytoband and encodes a putative helicase involved in the fidelity of DNA replication and repair. This protective genomic function of the protein is relevant because often patients with Rothmund-Thomson syndrome have constitutional mutations of RECQL4 and carry a very high risk of developing OS. To determine the relative level of expression of RECQL4 in OS, 18 sporadic tumors were studied by reverse transcription-polymerase chain reaction. All tumors overexpressed RECQL4 in comparison to control osteoblasts, and fluorescence in situ hybridization analysis of tumor DNA showed that expression levels were strongly copy number-dependent. Relative N- and S-CIN levels were determined by classifying copy number transitions within array comparative genomic hybridization profiles and by enumerating the frequency of break-apart fluorescence in situ hybridization within 8q24 using region-specific and control probes. Although there was no evidence that disruption of 8q24 in OS led to an elevated expression of RECQL4, there was a marked association between increased overall levels of S-CIN, determined by copy number transition frequency and higher levels of RECQL4.

Mixed embryonal/alveolar rhabdomyosarcoma of the prostate: report of a case with molecular genetic studies and literature review.

Alveolar rhabdomyosarcoma (RMS) is 1 of 2 main subtypes of RMS in the pediatric age group and tends to occur in the extremities. The urogenital tract is another common site for RMS, but this typically involves the embryonal subtype including sarcoma botryoides. We report a 28-year-old male with a prostatic tumor that was excised en bloc and showed a RMS with separate areas of embryonal and solid alveolar morphologies at the light microscopic level. Both areas showed diffuse nuclear expression for myogenin, and both areas expressed the PAX3-FKHR fusion gene, a genetic change associated with alveolar but not embryonal RMS. A review of the literature documented only 5 cases of RMS primary to the prostate showing alveolar or mixed histology. Ours is the 6th case and the 1st with molecular findings. Although the diagnostic category of mixed embryonal/alveolar RMS remains in use, the nature of this type of RMS is incompletely understood. In our case, although the morphology was mixed embryonal/alveolar, at the genetic level this tumor was alveolar in nature.

Distinct patterns of structural and numerical chromosomal instability characterize sporadic ovarian cancer.

Sporadic ovarian cancer is a particularly aggressive tumor characterized by highly abnormal karyotypes exhibiting many features of genomic instability. More complex genomic changes in tumors arise as a consequence of chromosomal instability (CIN), which can generate both numerical [(N)-CIN] and structural chromosomal instability [(S)-CIN]. In this study, molecular cytogenetic analysis was used to evaluate the relative levels of both (N)-CIN and (S)-CIN. Six tumors had a near-diploid chromosome number, two were near-tetraploid, and two were near-triploid. (N)-CIN levels increased as a function of overall tumor genomic content, with near-diploid tumors exhibiting numerical instability indices ranging from 7.0 to 21.0 and near-tetraploid and triploid tumors exhibiting instability indices ranging from 24.9 to 54.9. In contrast, the extent of (S)-CIN was generally more evident in the diploid tumors compared with the near-tetraploid tumors. To determine whether the associated chromosomal constitution and/or ploidy changes were influenced by mitotic segregation errors, centrosome analyses were performed on all 10 tumors. The near-diploid tumors, with the lowest numerical change, were observed to possess fewer cells with centrosome abnormalities (5.5% to 14.0%), whereas the near-tetraploid tumors possessed much higher levels of (N)-CIN and were characterized by a trend of elevating percentages of cells with abnormal centrosomes (16.0% to 20.5%). These observations suggest that two distinct processes governing genome stability may be disrupted in ovarian cancer: those that impact on numerical segregation and ploidy of chromosomes and those that affect the fidelity of DNA repair and lead to structural aberrations.

A new molecular variant of desmoplastic small round cell tumor: significance of WT1 immunostaining in this entity.

Desmoplastic small round cell tumor is a rare aggressive neoplasm, often presenting in young adult males. Although the classic features are well described, considerable clinical, pathologic, and immunohistochemical variation has been reported. The defining feature is a reciprocal translocation, t(11;22)(p13;q12), which fuses EWS on chromosome 22 to WT1 on chromosome 11. WT1 immunohistochemistry is reportedly useful in distinguishing desmoplastic small round cell tumor from other tumors. Herein, we describe a desmoplastic small round cell tumor of soft tissue with an unusual pattern of WT1 expression associated with a novel variant EWS-WT1 fusion transcript. We compare WT1 expression pattern with 5 intra-abdominal desmoplastic small round cell tumors and review the literature on WT1 expression and variant transcripts in desmoplastic small round cell tumor. Immunohistochemistry for the N- and C-terminals of WT1 was performed in 6 desmoplastic small round cell tumors. The EWS-WT1 fusion transcript was confirmed in all cases by reverse transcriptase polymerase chain reaction. Sequencing of fusion transcripts and reverse transcriptase polymerase chain reaction for wild-type WT1 was performed in 4 cases. The soft tissue desmoplastic small round cell tumor was negative for the WT1 C-terminal and showed nuclear staining with the N-terminal antibody. This case demonstrated 2 novel fusion transcripts, both lacking WT1 exons 9 and 10 and one containing additional exons of WT1 (exons 3-7). This tumor also strongly expressed full-length WT1. Five intra-abdominal desmoplastic small round cell tumors showed nuclear staining for WT1 C-terminal, but not for the N-terminal antibody. Although WT1 immunohistochemistry reflects the EWS-WT1 fusion transcript in most desmoplastic small round cell tumors, some cases express full-length WT1 or have variant transcripts, resulting in atypical staining patterns. Hence, interpretation of WT1 immunostaining requires knowledge of antibody target epitopes and correlation with clinical, morphological, and molecular genetic findings for establishing a diagnosis of desmoplastic small round cell tumor.

In vitro analysis of integrated global high-resolution DNA methylation profiling with genomic imbalance and gene expression in osteosarcoma.

Genetic and epigenetic changes contribute to deregulation of gene expression and development of human cancer. Changes in DNA methylation are key epigenetic factors regulating gene expression and genomic stability. Recent progress in microarray technologies resulted in developments of high resolution platforms for profiling of genetic, epigenetic and gene expression changes. OS is a pediatric bone tumor with characteristically high level of numerical and structural chromosomal changes. Furthermore, little is known about DNA methylation changes in OS. Our objective was to develop an integrative approach for analysis of high-resolution epigenomic, genomic, and gene expression profiles in order to identify functional epi/genomic differences between OS cell lines and normal human osteoblasts. A combination of Affymetrix Promoter Tilling Arrays for DNA methylation, Agilent array-CGH platform for genomic imbalance and Affymetrix Gene 1.0 platform for gene expression analysis was used. As a result, an integrative high-resolution approach for interrogation of genome-wide tumour-specific changes in DNA methylation was developed. This approach was used to provide the first genomic DNA methylation maps, and to identify and validate genes with aberrant DNA methylation in OS cell lines. This first integrative analysis of global cancer-related changes in DNA methylation, genomic imbalance, and gene expression has provided comprehensive evidence of the cumulative roles of epigenetic and genetic mechanisms in deregulation of gene expression networks.