Maria Zielenska

Absence of TMPRSS2:ERG fusions and PTEN losses in prostate cancer is associated with a favorable outcome

TMPRSS2:ERG gene fusions and PTEN deletions are the most common genomic aberrations in prostate cancer. Recent work has suggested that the TMPRSS2:ERG fusion is associated with a more aggressive phenotype. Similarly, PTEN deletion has been associated with biochemical recurrence and lymph node metastasis. To date, there has been no systematic analysis of the combined influence of genomic PTEN deletion with TMPRSS2:ERG gene fusions on clinical parameters of prostate cancer progression. We carried out a retrospective analysis of 125 prostate cancers with known clinical outcome using interphase fluorescence in situ hybridization to detect the relative prevalence of TMPRSS2:ERG rearrangements and/or PTEN genomic deletions. TMPRSS2:ERG rearrangement was found in 60 of 125 (48%) prostate cancers. Duplication of TMPRSS2:ERG fusion was observed in seven (6%) tumors. Gleason grade (P=0.0002)/score (P=0.001), median tumor volume (P=0.0024), preoperative PSA (P=0.001) and perineural invasion (P=0.0304) were significantly associated with biochemical recurrence by univariate analysis with TMPRSS2:ERG approaching significance (P=0.0523). By multivariate analysis, relevant factors associated with recurrence were Gleason scores 7 (P=0.001) and 8–10 (P=0.015), PTEN homozygous deletion (P=0.013) and concurrent TMPRSS2:ERG fusion and PTEN deletion (P=0.036). Kaplan–Meier analysis indicated that the presence of TMPRSS2:ERG fusion was marginally less favorable in comparison to no fusion. Duplication of fusion gene showed worse prognosis. It was possible to determine the relative frequencies of PTEN deletion and/or TMPRSS2:ERG fusions in 82 of 125 prostate cancers. With biochemical recurrence as an endpoint, the genomic biomarkers identified three patient groups: (1) ‘poor genomic grade’ characterized by both PTEN deletion and TMPRSS2:ERG fusions (23/82, 28%); (2) ‘intermediate genomic grade’ with either PTEN deletion or TMPRSS2:ERG fusion (35/82, 43%) and (3) ‘favorable genomic grade’ in which neither rearrangement was present (24/82, 29%). Kaplan–Meier and multivariate analysis indicate that TMPRSS2:ERG fusion and PTEN loss together are a predictor of earlier biochemical recurrence of disease.

High‐resolution mapping of amplifications and deletions in pediatric osteosarcoma by use of CGH analysis of cDNA microarrays

Conventional cytogenetic and comparative genomic hybridization (CGH) studies have shown that osteosarcomas (OSs) are characterized by complex structural and numerical chromosomal alterations and gene amplification. In this study, we used high‐resolution CGH to investigate recurrent patterns of genomic imbalance by use of DNA derived from nine OS tumors hybridized to a 19,200‐clone cDNA microarray. In six OSs, there was copy number gain or amplification of 6p, with a minimal region of gain centering on segment 6p12.1. In seven OSs, the pattern of amplification affecting chromosome arm 8q showed high‐level gains of 8q12–21.3 and 8q22–q23, with amplification of the MYC oncogene at 8q24.2. Seven OSs showed copy number gain or amplification of 17p between the loci bounded by GAS7 and PMI (17p11.2–17p12), and three of these tumors also showed small losses at 17p13, including the region containing TP53. An in silico analysis of the distribution of segmental duplications (duplicons) in this region identified a large number of tracts consisting of paralogous sequences mapping to the 17p region, encompassing the region of deletions and amplifications in OS. Interestingly, within this same region there were clusters of duplicons and several genes that are expressed during bone morphogenesis and in OS. In summary, microarray CGH analysis of the chromosomal imbalances of OS confirm the overall pattern observed by use of metaphase CGH and provides a more precise refinement of the boundaries of genomic gains and losses that characterize this tumor.

Cause and Consequences of Genetic and Epigenetic Alterations in Human Cancer

Both genetic and epigenetic changes contribute to development of human cancer. Oncogenomics has primarily focused on understanding the genetic basis of neoplasia, with less emphasis being placed on the role of epigenetics in tumourigenesis. Genomic alterations in cancer vary between the different types and stages, tissues and individuals. Moreover, genomic change ranges from single nucleotide mutations to gross chromosomal aneuploidy; which may or may not be associated with underlying genomic instability. Collectively, genomic alterations result in widespread deregulation of gene expression profiles and the disruption of signalling networks that control proliferation and cellular functions. In addition to changes in DNA and chromosomes, it has become evident that oncogenomic processes can be profoundly influenced by epigenetic mechanisms. DNA methylation is one of the key epigenetic factors involved in regulation of gene expression and genomic stability, and is biologically necessary for the maintenance of many cellular functions. While there has been considerable progress in understanding the impact of genetic and epigenetic mechanisms in tumourigenesis, there has been little consideration of the importance of the interplay between these two processes. In this review we summarize current understanding of the role of genetic and epigenetic alterations in human cancer. In addition we consider the associated interactions of genetic and epigenetic processes in tumour onset and progression. Furthermore, we provide a model of tumourigenesis that addresses the combined impact of both epigenetic and genetic alterations in cancer cells.

Pediatric renal cell carcinoma: clinical, pathologic, and molecular abnormalities associated with the members of the mit transcription factor family.

We describe the clinical features, outcome, pathology, cytogenetics, and molecular aspects of 13 pediatric papillary renal cell carcinomas during a 19-year period. Seven cases (54%) had translocations involving Xp11.2 (TFE3). They were identified by cytogenetic, molecular, and/or immunohistochemical analyses. All Xp11.2+ translocations were TFE3+ by immunostaining. Cytogenetic and/or polymerase chain reaction analyses identified 3 cases with t(X17) and 1 case with t(1;17), and all had additional translocations. Histologic features in common in TFE3+ tumors also were present in some TFE3- tumors. One TFE3- tumor had complex cytogenetic abnormalities, 55XY,+2,del(3)(p14),+7,+8,+12,+13,+16,+17,+20[11 ], and 2 cases had normal karyotypes. None had t(6;11)/TFEB+ immunostaining. Five cases had focal, weak MITF tumor immunostaining. The key clinical findings were as follows: (1) The presence of an Xp11.2 (TFE3) translocation frequently is associated with advanced stage at initial examination. (2) All patients who underwent complete, partial nephrectomy with clear margins (adequate only for stage 1) and resection of metastases were alive and relapse-free at last follow-up. (3) The mean +/- SD event-free survival and overall survival rates at 5 years were both 92% +/- 7.4%. (4) One patients with a TFE3+ and MITF+ tumor and 66-87,XXY,der(1)t(1;8)del(4)(q?) der(11)t(11;15)der17t(X;17 abnormalities died 9 months after diagnosis.

Spectral karyotyping identifies recurrent complex rearrangements of chromosomes 8, 17, and 20 in osteosarcomas.

Conventional cytogenetic studies have shown that osteosarcomas (OSs) are often highly aneuploid, with a large number of both structural and numerical chromosomal alterations. To investigate the complexity of OS karyotypes in detail, we applied spectral karyotyping (SKY) to a series of 14 primary OS tumors and four established OS cell lines. A total of 531 rearrangements were identified by SKY, of which 300 breakpoints could be assigned to a specific chromosome band. There was an average of 38.5 breakpoints identified by SKY per primary tumor. Chromosome 20 was involved in a disproportionately high number of structural rearrangements, with 38 different aberrations being detected. Chromosomal rearrangements between chromosomes 20 and 8 were evident in four tumors. FISH analysis using a 20q13 subtelomeric probe identified frequent involvement of 20q in complex structural rearrangements of OS cell lines. Characterization of the structural aberrations of chromosomes 8 and 17 by use of SKY demonstrated frequent duplication or partial gains of chromosome bands 8q23–24 and 17p11–13. Other chromosomes frequently involved in structural alteration were chromosomes 1 (47 rearrangements) and 6 (38 rearrangements). Centromeric rearrangements often involving chromosomes 1, 6, 13, 14, 17, and 20 were present. Four of the 14 primary OS tumors were characterized by nonclonal changes that included both structural and numerical alterations. In summary, OS tumors have a very high frequency of structural and numerical alterations, compounded by gross changes in ploidy. This intrinsic karyotype instability leads to a diversity of rearrangements and the acquisition of composite chromosomal rearrangements, with the highest frequency of alteration leading to gain of 8q23–24 and 17p11–13 and rearrangement of 20q. These findings suggest that specific sequences mapping to these chromosomal regions will likely have a role in the development and progression of OS.

Chromosomal localization of DNA amplifications in neuroblastoma tumors using cDNA microarray comparative genomic hybridization.

Conventional comparative genomic hybridization (CGH) profiling of neuroblastomas has identified many genomic aberrations, although the limited resolution has precluded a precise localization of sequences of interest within amplicons. To map high copy number genomic gains in clinically matched stage IV neuroblastomas, CGH analysis using a 19,200-feature cDNA microarray was used. A dedicated (freely available) algorithm was developed for rapid in silico determination of chromosomal localizations of microarray cDNA targets, and for generation of an ideogram-type profile of copy number changes. Using these methodologies, novel gene amplifications undetectable by chromosome CGH were identified, and larger MYCN amplicon sizes (in one tumor up to 6 Mb) than those previously reported in neuroblastoma were identified. The genes HPCAL1, LPIN1/KIAA0188, NAG, and NSE1/LOC151354 were found to be coamplified with MYCN. To determine whether stage IV primary tumors could be further subclassified based on their genomic copy number profiles, hierarchical clustering was performed. Cluster analysis of microarray CGH data identified three groups: 1) no amplifications evident, 2) a small MYCN amplicon as the only detectable imbalance, and 3) a large MYCN amplicon with additional gene amplifications. Application of CGH to cDNA microarray targets will help to determine both the variation of amplicon size and help better define amplification-dependent and independent pathways of progression in neuroblastoma.

Human Telomere Reverse Transcriptase Expression Predicts Progression and Survival in Pediatric Intracranial Ependymoma

PURPOSE Pediatric intracranial ependymomas are a heterogeneous group of neoplasms with unpredictable clinical and biologic behavior. As part of ongoing studies to identify potential biologic and therapeutic markers, we analyzed the role of human telomere reverse transcriptase (hTERT; the catalytic subunit of telomerase) expression as a prognostic marker for this disease. PATIENTS AND METHODS Primary intracranial ependymomas that were resected at our institution between 1986 and 2004 were identified through the pathology and oncology databases. A tissue array was constructed from the patient samples and hTERT expression was evaluated by immunohistochemistry. Twenty-one samples were also analyzed for telomerase activity (telomerase repeat amplification protocol assay). RESULTS Eighty-seven tumors from 65 patients were analyzed. Five-year progression-free survival was 57% (SEM, 12%) and 21% (SEM, 8%) for hTERT-negative and hTERT-positive tumors, respectively (P = .002). Five-year overall survival was 84% (SEM, 7%) and 41% (SEM, 7%) for hTERT-negative and hTERT-positive tumors, respectively (P = .001). There was good correlation between telomerase activity and hTERT expression (kappa = 0.637). Multivariate analysis revealed hTERT expression to be the single most important predictor of survival of all known pathologic, clinical, and treatment factors (hazard ratio, 60.4; 95% CI, 6.4 to 561). All four patients with hTERT-negative tumors at relapse are still alive, with median follow-up of 11.2 years. CONCLUSION In this study, hTERT expression was the strongest predictor of outcome and was independent of other clinical and pathologic prognostic markers. It represents a simple and reliable biologic prognostic factor for intracranial ependymomas. These results should be confirmed in larger prospective trials.

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.

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.

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.