Chromosome-breakage genomic instability and chromothripsis in breast cancer

Background: Chromosomal breakage followed by faulty DNA repair leads to gene amplifications and deletions in cancers. However, the mere assessment of the extent of genomic changes, amplifications and deletions may reduce the complexity of genomic data observed by array comparative genomic hybridization (array CGH). We present here a novel approach to array CGH data analysis, which focuses on putative breakpoints responsible for rearrangements within the genome. Results: We performed array comparative genomic hybridization in 29 primary tumors from high risk patients with breast cancer. The specimens were flow sorted according to ploidy to increase tumor cell purity prior to array CGH. We describe the number of chromosomal breaks as well as the patterns of breaks on individual chromosomes in each tumor. There were differences in chromosomal breakage patterns between the 3 clinical subtypes of breast cancers, although the highest density of breaks occurred at chromosome 17 in all subtypes, suggesting a particular proclivity of this chromosome for breaks. We also observed chromothripsis affecting various chromosomes in 41% of high risk breast cancers. Conclusions: Our results provide a new insight into the genomic complexity of breast cancer. Genomic instability dependent on chromosomal breakage events is not stochastic, targeting some chromosomes clearly more than others. We report a much higher percentage of chromothripsis than described previously in other cancers and this suggests that massive genomic rearrangements occurring in a single catastrophic event may shape many breast cancer genomes

Defining a link with asthma in mice congenitally deficient in eosinophils

Eosinophils are often dominant inflammatory cells present in the lungs of asthma patients. Nonetheless, the role of these leukocytes remains poorly understood. We have created a transgenic line of mice (PHIL) that are specifically devoid of eosinophils, but otherwise have a full complement of hematopoietically derived cells. Allergen challenge of PHIL mice demonstrated that eosinophils were required for pulmonary mucus accumulation and the airway hyperresponsiveness associated with asthma. The development of an eosinophi-less mouse now permits an unambiguous assessment of a number of human diseases that have been linked to this granulocyte, including allergic diseases, parasite infections, and tumorigenesis

STAG2 is a clinically relevant tumor suppressor in pancreatic ductal adenocarcinoma

Background Pancreatic ductal adenocarcinoma (PDA) is a highly lethal cancer characterized by complex aberrant genomes. A fundamental goal of current studies is to identify those somatic events arising in the variable landscape of PDA genomes that can be exploited for improved clinical outcomes. Methods We used DNA content flow sorting to identify and purify tumor nuclei of PDA samples from 50 patients. The genome of each sorted sample was profiled by oligonucleotide comparative genomic hybridization and targeted resequencing of STAG2. Transposon insertions within STAG2 in a KRASG12D-driven genetically engineered mouse model of PDA were screened by RT-PCR. We then used a tissue microarray to survey STAG2 protein expression levels in 344 human PDA tumor samples and adjacent tissues. Univariate Kaplan Meier analysis and multivariate Cox Regression analysis were used to assess the association of STAG2 expression relative to overall survival and response to adjuvant therapy. Finally, RNAi-based assays with PDA cell lines were used to assess the potential therapeutic consequence of STAG2 expression in response to 18 therapeutic agents. Results STAG2 is targeted by somatic aberrations in a subset (4%) of human PDAs. Transposon-mediated disruption of STAG2 in a KRASG12D genetically engineered mouse model promotes the development of PDA and its progression to metastatic disease. There was a statistically significant loss of STAG2 protein expression in human tumor tissue (Wilcoxon-Rank test) with complete absence of STAG2 staining observed in 15 (4.3%) patients. In univariate Kaplan Meier analysis nearly complete STAG2 positive staining (>95% of nuclei positive) was associated with a median survival benefit of 6.41 months (P = 0.031). The survival benefit of adjuvant chemotherapy was only seen in patients with a STAG2 staining of less than 95% (median survival benefit 7.65 months; P = 0.028). Multivariate Cox Regression analysis showed that STAG2 is an independent prognostic factor for survival in pancreatic cancer patients. Finally, we show that RNAi-mediated knockdown of STAG2 selectively sensitizes human PDA cell lines to platinum-based therapy. Conclusions Based on these iterative findings we propose that STAG2 is a clinically significant tumor suppressor in PDA

Glioblastomas are composed of genetically divergent clones with distinct tumourigenic potential and variable stem cell-associated phenotypes

Glioblastoma (GBM) is known to be a heterogeneous disease; however, the genetic composition of the cells within a given tumour is only poorly explored. In the advent of personalised medicine the understanding of intra-tumoural heterogeneity at the cellular and the genetic level is mandatory to improve treatment and clinical outcome. By combining ploidy-based flow sorting with array-comparative genomic hybridization we show that primary GBMs present as either mono- or polygenomic tumours (64 versus 36 %, respectively). Monogenomic tumours were limited to a pseudodiploid tumour clone admixed with normal stromal cells, whereas polygenomic tumours contained multiple tumour clones, yet always including a pseudodiploid population. Interestingly, pseudodiploid and aneuploid fractions carried the same aberrations as defined by identical chromosomal breakpoints, suggesting that evolution towards aneuploidy is a late event in GBM development. Interestingly, while clonal heterogeneity could be recapitulated in spheroid-based xenografts, we find that genetically distinct clones displayed different tumourigenic potential. Moreover, we show that putative cancer stem cell markers including CD133, CD15, A2B5 and CD44 were present on genetically distinct tumour cell populations. These data reveal the clonal heterogeneity of GBMs at the level of DNA content, tumourigenic potential and stem cell marker expression, which is likely to impact glioma progression and treatment response. The combined knowledge of intra-tumour heterogeneity at the genetic, cellular and functional level is crucial to assess treatment responses and to design personalized treatment strategies for primary GBM

Abstract 69: Genomic deletions clustered on chromosomes 8 and 9 are associated with pancreatic cancer progression

Abstract Pancreatic cancer cells that have mesenchymal traits can be particularly migratory and resistant to chemotherapeutics. These cells are presumed to represent a more advanced and deadly population of cells relative to cancer cells with more differentiated, epithelial traits. The factors leading to the acquisition of a mesenchymal phenotype in pancreatic cancer are not well characterized. In this study, we tested the hypothesis that the development of a mesenchymal phenotype occurs selectively in tumor cells that harbor specific enabling genomic alterations. As a model system, we used pancreatic cancer cell lines that had either an epithelial-like or a mesenchymal-like phenotype when cultured in vitro, as defined by morphology and key molecular indicators. We used whole-genome comparative genomic hybridization to compare the genomic amplifications and deletions between 17 epithelial-like cell lines and 9 mesenchymal-like cell lines. An unbiased search revealed that 18 genes (each comprising at least three contiguous probes in a coding region) had alterations in a significantly greater percentage of the mesenchymal-like cells than the epithelial-like cells, whereas no genes had more frequent alterations in the epithelial-like cells. Sixteen of the 18 alterations were deletions, and 14 of the deletions were clustered in specific regions on chromosomes 8 and 9, suggesting the importance of those regions for suppressing de-differentiation. Some of the 18 genes were previously identified in studies of pancreatic cancer, such as SGCZ, KIAA1797 and SMAD4, while others were not. Certain genes showed a good correlation between DNA copy number and mRNA expression, providing evidence for the functional importance of those alterations. The known functions of the 18 genes include cell cycle control, cell membrane homeostasis, immune system regulation, and TGF-β pathway transduction, the deletion of which might provide improved survival advantage and cell structure modification required for metastasis. These findings support the concept that mesenchymal-like cancer cells are genetically more progressed than their epithelial-like counterparts and provide leads on the genomic hits that enable such progression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 69. doi:1538-7445.AM2012-69</jats:p

Elucidating potentially significant genomic regions involved in the initiation and progression of undifferentiated pleomorphic sarcoma

Sarcomas are cancers that arise in soft tissues or bone and make up a small percentage of malignancies. In an effort to identify potential genetic targets for therapy, this study explores the genomic landscape of a metastatic undifferentiated pleomorphic sarcoma (UPS) with spindle cell morphology. Thick sections (50 µm) of formalin-fixed, paraffin-embedded tissue from a primary, recurrent, and metastatic tumor were collected and processed from a single patient for DNA content-based flow-sorting and analyses. Nuclei of diploid and aneuploid populations were sorted from the malignant tissues and their genomes interrogated with array comparative genomic hybridization. The third sample was highly degraded and did not contain any intact ploidy peaks in our flow assays. A 2.5N aneuploid population was identified in the primary and recurrent sample. We detected a series of shared and unique genomic aberrations in the sorted aneuploid populations. The patterns of aberrations suggest that two similar but independent clonal populations arose during the clinical history of this rare tumor. None of these aberrations were detected in the matching sorted diploid samples. The targeted regions of interest might play a role in UPS and may lead to clinical significance with further investigation. </jats:p

Unique genomic and neoepitope landscapes across tumors: a study across time, tissues, and space within a single lynch syndrome patient

AbstractLynch syndrome (LS) arises in patients with pathogenic germline variants in DNA mismatch repair genes. LS is the most common inherited cancer predisposition condition and confers an elevated lifetime risk of multiple cancers notably colorectal and endometrial carcinomas. A distinguishing feature of LS associated tumors is accumulation of variants targeting microsatellite repeats and the potential for high tumor specific neoepitope levels. Recurrent somatic variants targeting a small subset of genes have been identified in tumors with microsatellite instability. Notably these include frameshifts that can activate immune responses and provide vaccine targets to affect the lifetime cancer risk associated with LS. However the presence and persistence of targeted neoepitopes across multiple tumors in single LS patients has not been rigorously studied. Here we profiled the genomic landscapes of five distinct treatment naïve tumors, a papillary transitional cell renal cell carcinoma, a duodenal carcinoma, two metachronous colorectal carcinomas, and multi-regional sampling in a triple-negative breast tumor, arising in a LS patient over 10 years. Our analyses suggest each tumor evolves a unique complement of variants and that vaccines based on potential neoepitopes from one tissue may not be effective across all tumors that can arise during the lifetime of LS patients.</jats:p

Abstract 969: Amplifications, gene fusions, and therapeutic targets in triple negative and refractory ER+ and HER2+ breast cancers

Abstract Breast cancer is known to be heterogeneous and choices of targeted therapy are still limited for refractory estrogen receptor positive (ER+), refractory HER2+, and triple negative (TNBC) breast cancer. In order to identify novel drug targets that are either focally amplified or involved in fusion or translocation events in such disease segments, we applied high resolution DNA copy number profiling and whole genome sequencing to a set of DNA content sorted breast cancer samples. We first applied DNA-content based flow cytometry to purify diploid and aneuploid cells in each of 54 samples. Purified populations of both aneuploid and diploid cells were then subjected to high resolution aCGH profiling. Our initial analysis of high resolution copy number profiling data identified a list of genes focally amplified and harboring break points. We then selected 8 of these samples (four ER+ and four triple negative) for whole genome sequencing to identify their fusion partners. Paired-end sequencing data from HiSeq for each flow sorted sample was aligned against the human genome. A custom analysis workflow based on published software tools and custom developed scripts for computationally predicting structural variation events was implemented. Paired ends that mapped to discontinuous regions in the genome were merged with our aCGH data to identify candidate gene fusions, inversions, and translocations in each sample. Automatic scan and custom examination for pair-ends mapped to discontinuous genomic regions identified a list of novel gene fusion and translocation candidates. A subset of such fusion candidates showed precise break point position concordance across the NGS and aCGH data and equal copy number amplification of two fusion partners, further strengthening our confidence that such genes are indeed involved in fusion and translocation events. These candidates include transcription factors (e.g. ZNF492), kinases (e.g. DYRK1A), and phosphatases (e.g. PTPRG). Some of these fusion genes are known to be involved in fusion in other diseases or tumor types but have not been reported in breast cancer, such as AUTS2, which is reported as translocation partner in autism and mental retardation patients and B-cell ALL, and MECOM (aka EVI1/MDS1), which is fused with AML1 in AML patients. Others are completely novel such as BICD1-ZNF492. In conclusion, we have successfully identified a list of fusions genes in ER+ refractory and triple negative breast cancer patients by combining high resolution aCGH profiling of DNA copy number analysis and whole genome sequencing. The next step is to validate the expression of these fusion proteins and their functional relevance to breast cancer. The driver fusion genes identified can provide potential therapeutic targets for ER+ refractory and TN breast cancer patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 969. doi:1538-7445.AM2012-969</jats:p