Cassandra Graham

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.

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.

Genomic instability and copy-number heterogeneity of chromosome 19q, including the kallikrein locus, in ovarian carcinomas

Many tissue kallikrein (KLK) genes and proteins are candidate diagnostic, prognostic and predictive biomarkers for ovarian cancer (OCa). We previously demonstrated that the KLK locus (19q13.3/4) is subject to copy‐number gains and structural rearrangements in a pilot study of cell lines and ovarian cancer primary tissues, shown to overexpress KLK gene family members. To determine the overall frequency of genomic instability and copy‐number changes, a retrospective study was conducted using formalin‐fixed paraffin embedded (FFPE) tissues. Eighty‐one chemotherapy naïve serous OCas were examined using 3‐colour fluorescence in situ hybridization (FISH) to identify structural and numerical changes on 19q, including the KLK locus; in addition to immunohistochemistry (IHC) for KLK6, which has been shown to be overexpressed in OCa. The KLK locus was subject to copy‐number changes in ∼83% of cases: net gain in 51%, net loss in 30% and amplified in 2%; and found to be chromosomally unstable (p < 0.001). All cases showed a wide range of immuoreactivity for KLK6 by IHC. Although no strong correlation could be found with copy‐number, the latter was contributing factor to the observed KLK6 protein overexpression. Moreover, univariate and multivariate analyses showed an association between the net loss of the KLK locus and longer disease‐free survival. Interestingly, FISH analyses indicated that chromosome 19q was subjected to structural rearrangement in 62% of cases and was significantly correlated to tumor grade (p < 0.001). We conclude that numerical and structural aberrations of chromosome 19q, affect genes including the KLK gene members, may contribute to ovarian carcinoma progression and aggressiveness.

Impact of cytogenetic and genomic aberrations of the kallikrein locus in ovarian cancer

The tissue kallikrein (KLK) genes are a new source for biomarkers in ovarian cancer. However, there has been no systematic analysis of copy number and structural rearrangements related to their protein expression. Chromosomal rearrangements and copy number changes of the KLK region were studied by FISH with protein levels measured by ELISA. Ovarian cancer and cell lines revealed the KLK region was subject to copy number imbalances or involved in unbalanced translocations and were associated with increased protein expression of KLKs 5, 6, 7, 8, 9, 10 and 11. In this initial study, we introduce the potential for long‐range chromosomal effects and copy number as a mechanism for the previously reported aberrant expression of many KLK genes in ovarian cancers.

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.

Integrated Cytogenetic and High-Resolution Array CGH Analysis of Genomic Alterations Associated with MYCN Amplification

Amplification of oncogenes and closely linked flanking genes is common in some types of cancer and can be associated with complex chromosome rearrangements and/or co-amplification of non-syntenic chromosomal regions. To better understand the etiology and structural complexity of focal MYCN amplicons in human neuronal cancer, we investigated the precise chromosomal locations of high copy number genomic regions in MYCN amplified cell lines. An integrated cytogenetic map of the MYCN amplicon was created using high-resolution array CGH, spectral karyotyping (SKY), multi-color banding (mBAND), and fluorescence in situ hybridization (FISH) in 4 human neuronal tumor cell lines. The evidence of complex intra- and inter-chromosomal events, providing clues concerning the nature of the genomic mechanisms that contributed to the process of MYCN amplification, was observed. The presence of multiple co-amplified syntenic or non-syntenic sequences in the MYCN amplicon is quite intriguing. MYCN is usually centrally located in the amplicon; however, the structure and complexity of the amplicons were highly variable. It is noteworthy that clusters of unstable repetitive regions characterized by CNV sequences were present throughout the regions encompassed by MYCN gene amplification, and these sequences could provide a mechanism to destabilize this region of the genome. Complex structural rearrangements involving genomic losses and gains in the 2p24 region lead to MYCN amplification and that these rearrangements can trigger amplification events.

t(4;19)(q35;q13) in pediatric undifferentiated soft tissue sarcomas

Review on t(4;19)(q35;q13) in pediatric undifferentiated soft tissue sarcomas, with data on clinics, and the genes involved.

Abstract 1168: Integrated genomic, microRNA (miRNA) and proteomic profiling by stable isotope labeling with amino acids in cell culture (SILAC) of ovarian carcinoma for biomarker discovery

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Ovarian cancer (OCa) is the fifth leading cause of cancer-related deaths in North American women, and the first for gynecologic malignancies. The long-term effectiveness of standard therapy is poor. Thus, there is a need for developing markers for diagnosis, prognosis, and for predicting therapeutic response. Transformation, malignancy and therapy resistance are consequences of changes at the DNA, RNA and protein levels, requiring an integrative approach for biomarker discovery. We, and others, have previously demonstrated that elevated protein levels of Kallikrein 6 (KLK6) in OCa are clinically relevant; and that copy-number gains of the KLK locus (19q13.3/13.4) is associated with elevated levels of KLKs, increasing grade and genomic instability. Recent work has indicated KLK6 protein expression is also regulated by microRNAs (miRNAs). Our profiling of OCa cell lines and primary tumours showed the differential expression of miRNAs, consistent with other published studies. Moreover, miRNAs predicted to target KLK6 were shown to be decreased in a KLK6-overexpressing OCa cell line (OVCAR-3), in comparison to a KLK6-non-expressing cell line (TOV21G) or to miRNAs derived from normal ovarian tissue. Among these are members of the hsa-let-7 family of miRNAs, found also to be in regions of copy-number loss in OVCAR-3. Since miRNAs can affect the protein expression of many genes, the identification of differentially expressed proteins, in addition to KLK6 not only suggests putative biomarkers, but may help elucidate pathways for therapeutic interventions. To identify differentially expressed proteins upon the transient transfection of hsa-let-7 family members into the OVCAR-3 cell line, we utilized Stable Isotope Labelling with Amino Acids in Cell Culture (SILAC) coupled to mass spectrometry. OVCAR-3 cultures were labeled separately in light-Arg/Lys and heavy-Arg/Lys isotopes, such that “light” and “heavy” peptides of the same proteins will generate spectra that are different due to a mass shift, thus enabling relative quantification. In control experiments, equal Light/OVCAR3 and Heavy/OVCAR3 total protein mixtures were profiled with 2,800 proteins identified, and 2,465 quantified. Over 94% of these quantified proteins showed a heavy:light ratio between 0.8 and 1.2, making this a robust system for distinguishing differentially expressed proteins. Light/OVCAR-3 was transfected with hsa-let-7a or hsa-let-7e while heavy/OVCAR-3 was transfected with a scrambled miRNA control. KLK6-specific ELISA confirmed its decrease by 50% in transfected cultures over controls. Preliminary SILAC profiling has identified a number of differentially expressed proteins upon transfection with the miRNAs, which will be discussed in the context of OCa pathogenesis and implications for novel biomarker panels and proteomic signatures. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1168. doi:10.1158/1538-7445.AM2011-1168

Abstract 3423: Immunohistochemical expression and cluster analysis of mesenchymal and neural stem cell-associated proteins in pediatric undifferentiated soft tissue sarcomas

Pediatric undifferentiated soft tissue sarcomas (USTS) are a challenging group of tumors to diagnose and accurately categorize. By definition, they do not exhibit consistent immunohistochemical profiles or harbor recurrent chromosomal rearrangements to aid in their recognition. Traditionally, they have been classified with poorly differentiated forms of embryonal rhabdomyosarcoma. More recently, however, this notion has been challenged as such tumors share characteristics with the Ewing family of tumors. Thus, the histogenesis of pediatric USTS remains unclear and optimum therapy remains a challenging decision. The aims were twofold: firstly, to determine whether differential expression of stem cell-associated proteins could be used to aid in determining the histogenesis of pediatric USTSs; and secondly, to determine whether pediatric USTS expressed a unique panel of stem cell-associated proteins and form a distinct sarcoma subcategory. Tumors included 32 Ewing sarcoma/PNETs (EWS), 22 embryonal rhabdomyosarcomas (ERMSs), 8 alveolar rhabdomyosarcomas (ARMSs), 5 synovial sarcomas (SSs), 5 malignant peripheral nerve sheath tumours (MPNSTs) and 16 USTSs. Stem cell antibodies used included a series of three mesenchymal stem cell markers (CD44, CD105 and CD166) and five neural stem cell markers (CD15, CD29, CD56, CD133 and nestin). Antibodies were applied to the sections using standard immunohistochemical techniques and the sections were scored on a six-tiered scoring system using both intensity and distribution components. The scores were then imported into Partek Genomic Suite Software, for statistical analyses, cluster analysis, and visualizations. The datasets were analyzed by comparing the expression scores from the groups using the 1-way ANOVA. Each group was compared to each individual group, as well as the remaining groups combined. The resultant Euclidean clustering divided the tumors into two major groups. EWSs and USTSs formed the majority of the first group, whereas ERMSs, ARMSs, MPNSTs and SSs formed the second group. Negativity for CD56 was strongly associated with the EWS/USTS cluster (p Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3423.

Abstract 3037: Integrated Genomic, MicroRNA (miRNA) and Proteomic Profiling of Ovarian Carcinoma for Biomarker Discovery

Ovarian cancer (OCa) is the fifth leading cause of cancer-related deaths in North American women and the first due to a gynecologic malignancy. The long-term effectiveness of standard therapy is generally poor and is accompanied by serious side effects. Thus there is a need for developing markers not only for diagnosis and prognosis, but also for predicting therapeutic response. Tumour progression and resistance to therapy is a consequence of the complexity of DNA, RNA and proteins. The search for effective and specific biomarkers should integrate aspects of all these factors. We have previously demonstrated that KLK6 is a promising biomarker for OCa and its observed over-expression is linked to copy-number gains of the 19q13.3/4 locus. Here, we demonstrate by multi-colour FISH analyses that the KLK locus in 81 OCas is subject to high-level of genomic instability (p 2 -PCR showed the differential expression of miRNAs, consistent with other published studies in OCa. Since miRNAs can potentially affect the protein expression of hundreds of genes, the identification of such differentially expressed proteins not only provides putative biomarkers, but may also elucidate pathways for therapeutic intervention. Using Stable Isotope Labelling with Amino Acids in Cell Culture (SILAC) coupled to mass spectrometry for the OVCAR-3 cell line, cultures were labeled separately in light-Arg/Lys and heavy-Arg/Lys. In this control experiment, over 2,800 proteins were identified with 2,465 quantified. Over 94% of these quantified proteins showed a heavy:light ratio between 0.8 and 1.2, making this a robust system for quantitatively distinguishing differentially expressed proteins in the presence of miRNA precursors or inhibitors. Our profiling, as well as others, has shown the loss of expression of let-7 family members and hsa-125a-5p in OCas, and both miRNAs show decreased expression in OVCAR-3. Interestingly, these miRNAs are also predicted to target KLK6. Thus, we hypothesize that these miRNAs will not only affect the expression of KLK6, but also the expression of other target genes. In the future, using SILAC, we will identify the differentially expressed proteins affected upon re-introduction of these miRNAs through the differential labeling of miRNA-transfected OVCAR-3 vs. non-transfected OVCAR-3; thus revealing novel biomarkers for OCa. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3037.