Genome-wide Chromosomal Instability Research Articles

EPEN-09. SUPER ENHANCER GENES AS MOLECULAR TARGETS IN C11ORF95-RELA FUSION EPENDYMOMA

Genomic sequencing has driven precision-based oncology therapy; however, genetic drivers remain unknown or non-targetable for many malignancies, demanding alternative approaches to identify therapeutic leads. Ependymomas comprise histologically similar tumor entities driven by distinct molecular mechanisms, such as fusion oncoproteins, genome-wide chromosomal instability, or disruption of DNA methylation patterns. Despite these differences, ependymomas resist chemotherapy and lack available targeted agents for clinical trial development. Based on our previous findings, we hypothesized that mapping chromatin landscapes and super enhancers (SE) could uncover transcriptional dependencies as targets for therapy (Mack, Pajtler, Chavez et al., Nature, 2018). To functionally test the requirement of these SE genes for ependymoma cellular growth, we designed a pooled RNA interference screen against 267 SE associated genes. Our screen was performed in one C11ORF95-RELA-fusion model and two PF-EPN-A models as controls in biological triplicates. As an indication that our screen was successful, positive controls scored among lead hits including KIF11, BUB1B, PHF5A and MYC. Importantly, we identified many subtype specific dependencies in both C11ORF95-RELA-fusion and PF-EPN-A models, thus revealing novel pathways that potentially govern subgroup-specific ependymoma cell growth. Further, several candidates detected across all ependymoma lines were also identified as pan-cancer dependencies or glioma/glioblastoma specific essential genes from the DepMap Cancer Dependency Gene Resource. Our findings reveal novel targets and pathways that are essential for ependymoma cell growth, which may provide new insight into therapeutic strategies against these aggressive brain tumors.

EPEN-16. TRANSCRIPTIONAL REGULATORY CIRCUITRIES AS MOLECULAR TARGETS IN EPENDYMOMA

Genomic sequencing has driven precision-based oncology therapy; however, genetic drivers remain unknown or non-targetable for many malignancies, demanding alternative approaches to identify therapeutic leads. Ependymomas comprise histologically similar tumor entities driven by distinct molecular mechanisms, such as fusion oncoproteins, genome-wide chromosomal instability, or disruption of DNA methylation patterns. Despite these differences, ependymomas resist chemotherapy and lack available targeted agents for clinical trial development. Based on our previous findings, we hypothesized that mapping chromatin landscapes and super enhancers (SE) could uncover transcriptional dependencies as targets for therapy (Mack, Pajtler, Chavez et al., Nature, 2018). To functionally test the requirement of these SE genes for ependymoma cellular growth, we designed a pooled RNA interference screen against 267 SE associated genes. Our screen was performed in one C11ORF95-RELA-fusion model and two PF-EPN-A models as controls in biological triplicates. As an indication that our screen was successful, positive controls scored among lead hits including KIF11, BUB1B, PHF5A and MYC. Importantly, we identified many subtype specific dependencies in both C11ORF95-RELA-fusion and PF-EPN-A models, thus revealing novel pathways that potentially govern subgroup-specific ependymoma cell growth. Further, several candidates detected across all ependymoma lines were also identified as pan-cancer dependencies or glioma/glioblastoma specific essential genes from the DepMap Cancer Dependency Gene Resource. Our findings reveal novel targets and pathways that are essential for ependymoma cell growth, which may provide new insight into therapeutic strategies against these aggressive brain tumors.

Transcriptome analysis of a ring chromosome 20 patient cohort.

Ring chromosomes occur when the ends of normally rod-shaped chromosomes fuse. In ring chromosome 20 (ring 20), intellectual disability and epilepsy are usually present, even if there is no deleted coding material; the mechanism by which individuals with complete ring chromosomes develop seizures and other phenotypic abnormalities is not understood. We investigated altered gene transcription as a contributing factor by performing RNA-sequencing (RNA-seq) analysis on blood from seven patients with ring 20, and 11 first-degree relatives (all parents). Geographic analysis did not identify altered expression in peritelomeric or other specific chromosome 20 regions. RNA-seq analysis revealed 97 genes potentially differentially expressed in ring 20 patients. These included one epilepsy gene, NPRL3, but this finding was not confirmed on reverse transcription Droplet Digital polymerase chain reaction analysis. Molecular studies of structural chromosomal anomalies such as ring chromosome are challenging and often difficult to interpret because many patients are mosaic, and there may be genome-wide chromosomal instability affecting gene expression. Our findings nevertheless suggest that peritelomeric altered transcription is not the likely pathogenic mechanism in ring 20. Underlying genetic mechanisms are likely complex and may involve differential expression of many genes, the majority of which may not be located on chromosome 20.

Genome-wide chromosomal instability by cell-free DNA sequencing predicts survival in patients with metastatic breast cancer

BackgroundGenome-wide chromosomal instability, instead of specific somatic mutations or copy-number alterations in selected genes, is a significant property of cancer and may suggest a new strategy for treatment. Here we utilized cell-free DNA (cfDNA) sequencing to display the whole picture of chromosomal instability in patients with metastatic breast cancer (MBC), and evaluate its predictive value for patient survival. MethodsThe clinical data of 65 patients who had frozen plasma and planned to change the therapeutic regimen were retrospectively enrolled. Low-coverage whole-genome sequencing of cfDNA was performed to generate the chromosomal instability represented by chromosomal instability (CIN) score. ResultsTumors with diverse status of hormone receptor and HER2 represented diverse chromosomal instability across the whole genome. According to the receiver operating characteristic curve and the statistical distribution, CIN score exceed 3881 was defined as “High”. 32 (53.3%) patients with high CIN score had similar clinicopathologic characteristics compared with low CIN score patients. The median overall survival of patients with high CIN score was 21.2 months (95% CI 14.1–28.3), which was significantly inferior to those with low CIN score (not reached, P = 0.006). Regardless of various treatment regimens, the median progression free survival in patients with high CIN score was 7.3 months, which was significantly worse than those in the low CIN score population (11.0 months, P = 0.034). Multivariate analysis revealed that CIN score was an independent prognostic factor, with hazard ratio of 3.563 (P = 0.005). ConclusionsTo our knowledge, this is the first study illustrating the prognostic value of chromosomal instability derived from cfDNA in MBC.

Genome-Wide Chromosomal Instability by Cell-Free DNA Sequencing Predicts Patient Survival in Metastatic Breast Cancer

Background: Cell-free DNA (cfDNA) could well recapitulate the genomic features of tumor with minimal invasion. We aimed to evaluate the prognostic value of genome-wide chromosomal instability derived from cfDNA in patients with metastatic breast cancer (MBC). Methods: We retrospectively identified 65 patients with MBC who were intended to change line of therapy and had blood draw for cfDNA. We performed low-coverage whole-genome sequencing of cfDNA from plasma, and chromosomal instability was analyzed and represented by chromosomal instability (CIN) score. Findings: 32 (53.3%) samples resulted to have high CIN score, with similar clinicopathologic characteristics relative to patients with low CIN. Different subtypes of MBC exhibit remarkably diverse copy number profiles. High CIN score was associated with significantly shorter overall survival after blood draw, median 21.2 months (95% CI 14.1-28.3) versus not reached (P=0.006). Besides, patients with high CIN had worse progression free survival compared with the low CIN group (median 7.3 vs 11.0 months, P=0.034). CIN score remained an independent prognostic factor in multivariate analysis (hazard ratio, 3.563; 95%CI, 1.481 to 8.572; P=0.005). Interpretation: Genome-wide chromosomal instability tracking by cfDNA is reliable and predicts survival in patients with MBC. Funding Statement: This study was supported by National Natural Science Foundation of China (81874122, 81772490), National Key R&D Program of China (2018YFC0115204), the Chinese Academy of Medical Sciences (CAMS) Innovation Fund for Medical Sciences (CIFMS) (Grants 2017-I2M-3-004 and 2019-I2M-1-003), and Capital’s Funds for Health Improvement and Research (CFH2018-4-4024). Declaration of Interests: Ziliang Qian is a salaried employee of Prophet Genomics Inc., a provider of cancer genome-based diagnostic testing. The remaining authors declare no conflict of interest. Ethics Approval Statement: Use of patients’ clinicopathologic data and cfDNA draws were approved by institutional review board, and written informed consent was obtained from all patients.

Genome-wide chromosomal instability by cell-free DNA sequencing to predict patient survival in metastatic breast cancer.

e12591 Background: Cell-free DNA (cfDNA) could well recapitulate the genomic features of tumor with minimal invasion. For metastatic breast cancer (MBC), prior applications of cfDNA have focused on tracking specific gene locus. We aimed to evaluate the prognostic value of genome-wide chromosomal instability derived from cfDNA in patients with MBC. Methods: We retrospectively identified 65 patients with MBC who were intended to change line of therapy and had blood draw for cfDNA. We performed low-coverage whole-genome sequencing of cfDNA from plasma, and chromosomal instability was analyzed and represented by chromosomal instability (CIN) score. Results: 32 (53.3%) samples resulted to have high CIN score, with similar clinicopathologic characteristics relative to patients with low CIN. None of patient’s age (P = 0.073), receptor status (P = 0.136), disease stage at primary diagnosis (P = 0.353), or previous lines of therapy (P = 0.067) was correlated with genome-wide chromosomal instability. Different subtypes of MBC exhibit remarkably diverse copy number profiles. High CIN score was associated with significantly shorter overall survival after blood draw, median 21.2 months (95% CI 14.1-28.3) versus not reached (P = 0.006). Besides, patients with high CIN had worse progression free survival compared with the low CIN group (median 7.3 vs 11.0 months, P = 0.034). CIN score remained an independent prognostic factor in multivariate analysis (hazard ratio, 3.563; 95%CI, 1.481 to 8.572; P = 0.005). Conclusions: Here we present a framework for minimally invasive genomic characterization of different subtypes of MBC, and subsequent integration with clinicopathologic data and patient outcomes. Genome-wide chromosomal instability tracking by cfDNA is reliable and predicts survival in patients with MBC.

Abstract 2069: Therapeutic targeting of ependymoma as informed by oncogenic enhancers

Abstract Genomic sequencing has driven precision-based oncology therapy; however, genetic drivers remain unknown or non-targetable for many malignancies, demanding alternative approaches to identify therapeutic leads. Ependymomas comprise histologically similar tumor entities driven by distinct molecular mechanisms, such as fusion oncoproteins, genome-wide chromosomal instability, or disruption of DNA methylation patterns. Despite these differences, ependymomas commonly resist chemotherapy and lack available targeted agents for clinical trial development. In the case of genomically balanced ependymomas, and those driven by fusion oncoproteins, we hypothesized that the chromatin landscapes could uncover oncogenes that would inform actionable targets for therapy and reveal specific transcriptional circuitries to identify molecular origins of the disease. We therefore interrogated the oncogenic drivers of ependymoma by characterizing the active regulatory landscapes of 42 primary tumors through an integrative analysis of H3K27ac ChIP-seq, RNA-seq, whole-exome sequencing (WES), whole genome sequencing (WGS), copy number profiling, and DNA methylation profiling. Across the landscape of ependymal tumors, enhancer mapping revealed putative oncogenes, including NFIX, MIRLET7BHG, FGFR1, WEE1, and MTSS1L, and delineated subgroup specific enhancer lesions. Reconstruction of enhancer networks permitted the identification of core transcription factors (TFs) that establish ependymoma cell state, and lineage-specifying TFs that dictate molecular subgroup identity. Lineage-associated TFs point to distinct spatio-temporal origins of ependymoma subgroups such as FOXJ1 TF activity and expression observed preferentially in subsets of hindbrain ependymomas. To translate our results into a potential clinical paradigm, we demonstrate cancer dependencies on super enhancer associated genes and lineage TFs, and establish the utility of chromatin landscape analysis to predict novel targets for cancer therapy. Citation Format: Stephen C. Mack, Global Ependymoma Network of Excellence. Therapeutic targeting of ependymoma as informed by oncogenic enhancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2069.

EPND-01. THE ACTIVE REGULATORY LANDSCAPE OF EPENDYMAL TUMORS

AbstractGenomic sequencing has driven precision-based oncology therapy; however, genetic drivers remain unknown or non-targetable for many malignancies, demanding alternative approaches to identify therapeutic leads. Ependymomas comprise histologically similar tumour entities driven by distinct molecular mechanisms, such as fusion oncoproteins, genome-wide chromosomal instability, or disruption of DNA methylation patterns. Despite these differences, ependymomas commonly resist chemotherapy and lack available targeted agents for clinical trial development. In the case of genomically balanced ependymomas, and those driven by fusion oncoproteins, we hypothesized that the chromatin landscapes could uncover oncogenes that would inform actionable targets for therapy and reveal specific transcriptional circuitries to identify the molecular origins of the disease. To this end, we mapped active chromatin landscapes in 42 primary ependymomas in discovery and validation cohorts using H3K27 acetylation ChIP-seq. Enhancer regions revealed novel oncogenes, molecular targets, and pathways, which when subjected to small molecule inhibitor or shRNA treatment, increased survival and halted proliferation in mouse and neurosphere patient-derived models of ependymomas. Reconstruction of enhancer networks permitted the identification of core transcription factors (TFs) that establish ependymoma cell state, and lineage-specifying TFs that dictate molecular subgroup identity. Lineage-associated TFs point to distinct spatio-temporal origins of ependymoma subgroups such as FOXJ1 TF activity and expression observed preferentially in subsets of hindbrain ependymomas. To translate our results into a potential clinical paradigm, we demonstrate cancer dependencies on super enhancer associated genes and lineage TFs, and establish the utility of chromatin landscape analysis to predict novel targets for cancer therapy.

Abstract 4839: Lung cancer patients exhibit a genomewide chromosomal instability and DNA methylation correlation which varies by expression subtype

Abstract Introduction: Lung cancer is a deadly disease, which manifests gross DNA alterations. Chromosome instability (CIN) and DNA methylation (DM) instability are important DNA alteration processes that contribute to the initiation and progression of lung cancer. Considering that DNA replication and methylation maintenance are coordinated, we hypothesized that CIN and DM may be coordinated in the etiology of lung cancer patients. Methods: A cohort of 63 patients with surgically-resected lung tumors was collected. DNA copy number was measured by Affymetrix 250K_StyI microarrays. DNA methylation was measured by the MSNP assay, which involves comparing the copy number of HpaII (methylation sensitive) digested DNA to undigested DNA (Yuan, 2006). The sample genomewide CIN score was the median of chromosome arm absolute copy numbers. The sample genomewide DNA methylation score was the median of the percent methylation across all probes. mRNA expression was measured by Agilent 44K microarrays. Results: CIN and DM varied substantially over lung cancers. Histological classes did not differ in CIN or DM. Lung cancer mRNA expression subtypes (described in Hayes et al., J Clin Oncol, 2006 and Wilkerson et al., Clin Cancer Res 2010) were significantly different in CIN (P<0.05) with the magnoid adenocarcinoma, classical squamous cell carcinoma (SCC), and basal SCC subtypes having the largest CIN scores. DM was also significantly different between expression subtypes (P<0.05), with the magnoid adenocarcinoma and the classical SCC having the largest DM scores. CIN and DM were significantly positively correlated (0.37; P<0.01), indicating that the most copy number disrupted genomes were also the most DNA hypermethylated. To evaluate the combined effect of these alterations, a combined CIN and DM (CIN+DM) standardized score was evaluated. Histological classes did not differ in CIN+DM. Expression subtypes were significantly different in CIN+DM (P<0.01) with the magnoid adenocarcinoma subtype and the classical SCC subtype exhibiting the largest CIN+DM scores. Patients with distant recurrences had a trend of larger CIN+DM scores than patients with local recurrences or no observed recurrences (P<0.21). An mRNA expression signature based on the CIN+DM score was positively correlated (GSEA, FDR < 0.05) with overexpression of DNA repair pathways, providing corroborating evidence for this DNA alteration phenotype. Conclusions: Genomewide chromosomal instability and DNA methylation are significantly correlated in lung cancer. These co-occurring DNA alterations are the most extreme in the magnoid adenocarcinoma and classical squamous cell carcinoma subtypes, suggesting the subtypes have evolved differently. These results elucidate new lung cancer etiology and future therapies could be focused on exploiting these DNA alterations. 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 4839. doi:10.1158/1538-7445.AM2011-4839

Single nucleotide polymorphism-based genome-wide chromosome copy change, loss of heterozygosity, and aneuploidy in Barrett's esophagus neoplastic progression.

Chromosome copy gain, loss, and loss of heterozygosity (LOH) involving most chromosomes have been reported in many cancers; however, less is known about chromosome instability in premalignant conditions. 17p LOH and DNA content abnormalities have been previously reported to predict progression from Barrett's esophagus (BE) to esophageal adenocarcinoma (EA). Here, we evaluated genome-wide chromosomal instability in multiple stages of BE and EA in whole biopsies. Forty-two patients were selected to represent different stages of progression from BE to EA. Whole BE or EA biopsies were minced, and aliquots were processed for flow cytometry and genotyped with a paired constitutive control for each patient using 33,423 single nucleotide polymorphisms (SNP). Copy gains, losses, and LOH increased in frequency and size between early- and late-stage BE (P < 0.001), with SNP abnormalities increasing from <2% to >30% in early and late stages, respectively. A set of statistically significant events was unique to either early or late, or both, stages, including previously reported and novel abnormalities. The total number of SNP alterations was highly correlated with DNA content aneuploidy and was sensitive and specific to identify patients with concurrent EA (empirical receiver operating characteristic area under the curve = 0.91). With the exception of 9p LOH, most copy gains, losses, and LOH detected in early stages of BE were smaller than those detected in later stages, and few chromosomal events were common in all stages of progression. Measures of chromosomal instability can be quantified in whole biopsies using SNP-based genotyping and have potential to be an integrated platform for cancer risk stratification in BE.

Single nucleotide polymorphism array analysis of chromosomal instability patterns discriminates rectal adenomas from carcinomas

Total mesorectal excision (TME) is the standard treatment for rectal cancer, while transanal endoscopic microsurgery (TEM) is a recently introduced surgical approach for the treatment of rectal adenomas. Incorrect preoperative staging before TEM is a problem. To identify genetic changes that might correlate with tumour stage and could lead to optimized treatment selection we performed a genome-wide chromosomal instability search in a homogeneous, clinical cohort of rectal tumours. 78 rectal tumours during different clinical stages were analysed with 10K single nucleotide polymorphism (SNP) arrays. Logistic regression was performed to build a quantitative model of specific chromosomal aberrations. Overall, most cases (95%) had one or more chromosomal aberrations. We observed a clear correlation between the total number of aberrations and the different tumour stages. Specifically, the chromosomal events: gain of 8q22-24, 13q and 20q, and loss of 17p and 18q12-22, were far more abundant in carcinoma than in adenoma. In adenoma fractions from cases with a carcinoma (infiltrating at least in the submucosa), twice the amount of such 'malignant aberrations' was observed, compared to pure adenomas. Furthermore, combined aberrations such as gain of 13q and loss of 18q were only found in adenomatous fractions of carcinomas and not in benign lesions. Based on these five genomic events associated with carcinoma, a clear distinction between adenoma and carcinoma tissue could be made. These data should be validated further in order that they may be used in preoperative staging of rectal tumours.

A Breakage-Fusion-Bridge Cycle Generated by Telomeric Loss May Underlie the dup(21q) of Acute Lymphoblastic Leukemia.

A highly complex, rearranged chromosome 21, arising from duplication of 21q and associated with amplification of RUNX1 [dup(21q)], is linked to a poor prognosis in acute lymphoblastic leukemia (ALL). Using BAC array-based CGH (Spectral Genomics 1Mb, USA) (aCGH), we originally identified a characteristic pattern of imbalance, with common regions of amplification (CRA) and deletion (CRD) along 21q, in a series of 10 patients. The extent of these regions was refined to 6.6Mb (between 33.192 and 39.796Mb) and 3.3Mb (between 43.7 and 47Mb, which included sub-telomeric sequences), respectively, by tiling-path oligonucleotide-based aCGH (NimbleGen Inc., USA) (n=15). Six BAC clones (including a sub-telomeric one) from the 1Mb arrays, corresponding to the variable regions of amplification along 21q were used as probes for interphase FISH (iFISH) in 48 patients. The same CRA was confirmed in all, while the CRD was observed in 34/46 (77%) of them. iFISH showed that the degree of amplification corresponding to each chromosomal region differed between patients. Although these techniques, which measure genomic copy number changes, showed consistent patterns of amplification and deletion along 21q, it was intriguing as to why, at the cytogenetic level, the abnormal chromosome 21 had many different forms. The same locus-specific probes, applied to metaphases from 10 patients, revealed complex signal patterns, unique to each patient, in which the signals from each probe were distributed along 21q in unexpected positions relative to each other. Often signals from the same probe were located to more than one region of the abnormal chromosome 21. These findings were indicative of duplications of the chromosomal regions to which the probes were located, in combination with intricate intrachromosomal rearrangements, including inversions. We were able to illustrate these unique rearrangements by multicolor FISH banding (XCyte21, Metasystems, Germany) in two cases. Supervised gene expression analysis (HG-U133A arrays, Affymetrix, USA) showed a distinct signature for eight patients with dup(21q). Genomic copy number correlated with overall gene expression levels within areas of gain or loss. However, there was considerable inter-genic differences and variation between individuals. Of the 40 genes contained within the amplicon, no promising targets were upregulated when compared to patients with high hyperdiploidy, comprising at least one additional copy of chromosome 21. Collectively, our results have provided evidence for extensive intrachromosomal rearrangements and instabilityn of 21q in these patients. Although there was no evidence of other established chromosomal changes, the clonal heterogeneity we observed in the karyotypes was indicative of genome-wide chromosomal instability. These types of genomic alterations, arising from a series of chromatid breaks and reunions, which lead to intrachromosomal amplifications and deletions, are the hallmark of the breakage-fusion-bridge (BFB) cycle in solid tumors. This is the first time that cytogenetic features linked to the BFB mechanism have been described in ALL. We hypothesize that the loss or abnormal functioning of telomeric sequences may be the causal event behind this poor-risk 21q abnormality.