High Degree Of Chromosomal Instability Research Articles

Abstract 5360: Relief of chromosomal instability-induced cGAS-STING signaling sensitizes STK11-mutant non-small cell lung cancer to immune checkpoint blockade

Abstract Non-small cell lung cancers (NSCLCs) harboring deletions or inactivating mutations in STK11 (encoding LKB1) are associated with poor prognosis and immune evasion, but the underlying mechanisms are poorly understood. Through integrative analyses of >10,000 molecular profiles from public data sources and an additional ~15,000 new patient data profiles, we find identify STK11-deficient NSCLC as an archetypical chromosomally unstable tumor. Using high-content imaging, we show that both human and murine STK11-mutant models have a higher rate of micronuclei and chromosome mis-segregation events, confirming their high degree of chromosomal instability (CIN). We show that CIN-induced tonic activation of the cGAS-STING pathway results in impaired type I interferon expression, thus, turning the physiological function of this pathway on its head. We show that temporary relief of tonic CIN-induced signaling reprogrammed cancer cells towards production of type I interferons. We achieved this through orthogonal routes, including genetic suppression of CIN by overexpression of mitotic centromere-associated kinesin (MCAK), rescue of wild-type STK11 (but not its kinase-dead) allele, genetic deletion or pharmacological inhibition of cGAS. Relief through any of these approaches re-sensitized cells to acute STING agonism with its endogenous ligand (cGAMP) or pharmacological stimulation. Using otherwise isogenic, syngeneic KRAS-mutant Stk11/Lkb1-/- (KL) and KRAS-mutant p53 mutant (KP) murine models, we show that cGAS KO sensitized otherwise highly resistant KL models to immune checkpoint blockade (ICB), while an increase in CIN (through overexpression of dominant-negative MCAK) in KP conferred resistance in this otherwise ICB-sensitive model. In summary, we define STK11-mutant as archetypical chromosomally unstable form of NSCLC, provide mechanistic basis that correlates with poor clinical outcomes, and demonstrate how relief of tonic CIN-induced changes may be therapeutically leveraged. Citation Format: Lindsay A. Caprio, Christy Hong, Amit D. Amin, Somnath Tagore, Mercedes Duran, Meri Rogava, Luke Cai, Leon Ebel, Johannes Melms, Hanina Hibshoosh, Samuel Bakhoum, Benjamin Izar. Relief of chromosomal instability-induced cGAS-STING signaling sensitizes STK11-mutant non-small cell lung cancer to immune checkpoint blockade [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5360.

Abstract 5671: Profiling intra-tumor heterogeneity and chromosomal instability in malignant peripheral nerve sheath tumors

Abstract Malignant peripheral nerve sheath tumors (MPNSTs) are rare soft-tissue sarcomas that manifest on peripheral nerves, constituting 2% of all sarcomas. While previous genomic analyses of MPNSTs have been conducted on small cohorts, often limited to single samples per patient and lacking comprehensive resolution, the extent of intra-tumor heterogeneity and the contribution of chromosomal instability to MPNST development remains unclear. To address these limitations, we developed a multi-omics integration pipeline and applied it to bulk Whole Genome Sequencing (WGS), single-cell RNA sequencing(scRNA-seq), single-cell DNA sequencing (scDNA-seq), spatial transcriptomics (ST), and Laser Capture Microsection (LCM) sequencing data. 60 tumor samples were collected from 17 patients with MPNST, and the above five techniques were applied to each sample. Overall, tumors harbor a high degree of chromosomal instability and evidence of whole-genome doubling (WGD). We reconstructed tumor phylogenetic trees from WGS data using Dirichlet Process-based methods. Intriguingly, our findings indicated that for patient 2.3, primary tumor samples PT_1 and PT_2 displayed a close phylogenetic relationship, while local recurrences LR_2 and LR_3 represented a different branch of the phylogenetic tree. Moreover, scRNA-seq analysis of patient 2.16 highlighted an enrichment of immune cells, including macrophages, B cells, and T cells. To infer copy number alterations (CNA) of tumor cells from scRNA-seq data, we leveraged inferCNV, with normal cells identified within the same sample as controls. Significant copy number gains were identified on chromosomes 5, 7, 8, and 15 in patient 2.16. The inferred copy number changes derived from scRNA-seq were validated against copy number profiles obtained through WGS using Battenberg, revealing a remarkable congruence. In the scDNA analysis of patient 2.12, we observed that all tumor cells exhibited biallelic loss of CDKN2A, with a majority showcasing WGD and chromosome 7 amplification. This finding underscored the potential significance of CDKN2A in the pathogenesis of this tumor, in keeping with the observation that CDKN2A is recurrently mutated and lost in MPNSTs. Our research profiled the tumors' evolutionary histories and intra-tumor heterogeneity down to single-cell resolution, highlighting the genotype and phenotype differences between tumor subclones. By shedding light on the genomic landscape of MPNSTs, we aspire to make profound contributions to the field of sarcoma research, ultimately improving patient outcomes. Citation Format: Yixiao Cheng, Haixi Yan, Maxime Tarabichi, Zhihui Zhang, Chunxu Gao, Tom Lesluyes, Adrienne Flanagan, Peter Van Loo. Profiling intra-tumor heterogeneity and chromosomal instability in malignant peripheral nerve sheath tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5671.

Genomic analysis in patients with soft tissue sarcoma to examine gene mutations involved in chromosome segregation, mitotic spindle assembly, and degradation.

e23523 Background: Soft tissue sarcoma (STS) is a rare cancer with few drug discovery. Recent genomic analysis of STS revealed a high degree of chromosomal instability (CIN) including genome-wide copy number alterations, aneuploidy, whole genome doubling and chromothripsis. In STS patients, we have reported extensive somatic LOH in tumors, a hallmark of CIN, represented by haploid of germline mutations/variants in cancer-related genes. CIN of STS implicates abnormalities in chromosome segregation, spindle assembly and degradation in mitosis but few studies have examined the impact of gene mutations involved in the pathological mitotic processes in STS. Methods: We recruited 155 patients with metastatic/recurrent STS (135 female and 20 male, mean age 51 at analysis, 100 LMS, 19 LPS, 4 ESS, 3 UPS, 3 AS, 3 MPT and others) with information of familial cancer burden. Whole exome sequencing and analysis were performed in both blood and tumor samples as described in 2018ASCO. Results: STS Patients with tumors harboring less than 33% somatic LOH (n = 54) in a total of somatic and LOH mutations/variants in 595 COSMIC genes have a better prognosis than those (34-66%, n = 49, 67-100%, n = 52) with more LOH genes (5-year OS; 72% vs 53% or 40%, p = 0.0499, p = 0.0023, respectively). LOH was associated with neither TMB nor MSI status but with a total number of somatic and germline mutations (R = 0.874). Of the genes involved in chromosome segregation and mitosis, we found that a family of ARHGAP genes which play a role in the spindle degradation and Aurora A kinase activation was most frequently mutated in tumor and germline genomes (n = 102 in a total of 155, n = 71 in 100 LMS). Damaging ARHGAP mutations/variants are correlated with higher LOH values (54.1±2.8%, n = 102 vs. 33.1±3.8%, n = 53, mean±SE, p < 0.001, for LMS, 63.3±2.9%, n = 71 vs. 36.7±5.4%, n = 29, mean±SE, p < 0.001) and poor prognosis of patients (5-year OS; 48% n = 102 vs. 71% n = 53, p = 0.0107). We also found damaging mutations/variants in a family of CAPN (calpain) genes involved in cleavage of the cohesion ring for chromosome separation (n = 73 in 155) and CENPT gene essential for bridging between kinetochore and microtubules via NDC80 (n = 43 in 155). For CENPT, loss of wild type allele at codons encoding functional Ser121 and Arg122 was found in 10 patients with LMS and DSRCT. Damaging CAPN mutations/variants are correlated with higher LOH values (60.6±3.0%, n = 73 vs. 34.8±3.2%, n = 82, mean±SE, p < 0.001). Conclusions: This study demonstrates extensive genomic abnormalities involved in the pathways of chromosome segregation and mitosis in STS. Recent studies, including ourselves, confirmed overexpression of KIF18A, NDC80 and NUF2 in STS, lethal targets for KIF18A and CHD (Calponin Homology Domain) inhibitors in CIN tumors. Together, our results will open a new avenue evolving treatment landscape of STS.

Abstract 5944: Relief of chromosomal instability-induced cGAS-STING signaling sensitizes STK11-mutant non-small cell lung cancer to immune checkpoint blockade

Abstract Non-small cell lung cancers (NSCLCs) harboring deletions or inactivating mutations in STK11 (encoding LKB1) are associated with treatment-resistance, including to immune checkpoint blockade, and poor survival, yet the underlying mechanisms are poorly understood. Here, we combined multi-modal single-cell transcriptomics, whole-genome sequencing (WGS), and analysis of public data bases totaling >10,000 NSCLC whole-exome sequencing (WES) and when available RNA (RNA-seq) profiles of AACR GENIE, MSK IMPACT, TCGA and CPTAC, high-content imaging, and functional assays, to determine genomic and mechanistic features of STK11-mutant NSCLC. Across human WES/RNA-seq data, we find that STK11-mutant NSCLC have a significantly higher fraction of genome altered (FGA) and score strongly for the mRNA-based CIN70 signature compared to other NSCLC genotypes, overall indicating that these tumors have a higher degree of chromosomal instability (CIN). Using high-content imaging, we show that both human and murine STK11-mutant models have a higher rate of micronuclei and chromosome-mis-segregation events, confirming their CINhigh status in dynamic assays. Next, we show that tonic CIN-induced activation of cGAS-STING signaling, whose activation is typically thought of inducing type I interferon expression, results in strong suppression of anti-tumor immune signaling. Remarkably, genetic or pharmacologic suppression of cGAS, and thus depletion of the STING ligand cGAMP, results in reprogramming and re-sensitization of STK11-mutant models to cGAS-STING mediated type I interferon responses. Furthermore, we show that CIN promotes excessive production cGAMP in STK11-mutant models. We find upregulation of two ectonucleotidases (ENPP1 and CD73/NT5E) that hydrolyze exported cGAMP to adenosine, which may further contribute to the highly dysfunctional tumor-microenvironment observed in STK11-mutant tumors. Lastly, suppression of CIN through overexpression of mitotic-centromere associated kinesin (MCAK) in murine STK11-mutant models results in decreased tumor growth and sensitization to anti-PD1 therapy. In summary, we define STK11-mutant as archetypical chromosomally form of NSCLC, provide mechanistic basis that corelates with poor clinical outcomes, and demonstrate how relief of tonic CIN-induced changes may be therapeutically leveraged. Citation Format: Lindsay A. Caprio, Christy Hong, Amit Dipak Amin, Somnath Tagore, Johannes Melms, Luke Cai, Yiping Wang, Patricia Ho, Michael Mu, Hanina Hibshoosh, Brian Henick, Kwok K. Wong, Samuel F. Bakhoum, Benjamin Izar. Relief of chromosomal instability-induced cGAS-STING signaling sensitizes STK11-mutant non-small cell lung cancer to immune checkpoint blockade [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5944.

Genetic abnormalities and aberrant expression of genes involved in chromosome segregation and mitosis in patients with chromosomally unstable malignant soft tissue tumors haboring extensive somatic loss-of-heterozygosity (LOH).

11576 Background: Malignant soft tissue tumor is a rare cancer with few therapeutic options. Recent genomic analysis of soft tissue sarcoma (STS) revealed a high degree of chromosomal instability (CIN) including genome-wide copy number alteration, aneuploidy, whole genome doubling and chromothripsis. In STS patients, we observed extensive somatic LOH, a hallmark of CIN, which is haploid of germline mutations/variants in cancer-related genes. CIN of STS genome implicates abnormalities in chromosome segregation and mitosis. So far, studies on this issue, however, have not been reported in STS patients. Methods: We recruited 155 patients with metastatic/recurrent malignant soft tissue tumors (135 female and 20 male, mean age 51 at analysis, 100 LMS, 19 LPS, 4 ESS, 3 UPS, 3 AS, 3 MPT, 3 GIST and others) with information of familial cancer burden. Whole exome sequencing and analysis were performed in both blood and tumor samples as described in 2018ASCO. KIF18A expression was assessed by immunohistochemistry. Results: Patients with tumors harboring less than 33% somatic LOH in a total of somatic and LOH mutations/variants in 595 COSMIC genes (n = 54) have a better prognosis than those (34-66%, n = 49, 67-100%, n = 52) with tumors harboring more LOH genes (5-year OS; 71% vs 52% or 46%, p = 0.0299, p = 0.0117, respectively). Neither TMB nor MSI status was associated with LOH. Of the genes involved in chromosome segregation and mitosis, we found that a family of ARHGAP genes which play a role in the spindle assembly and Aurora A kinase activation was frequently mutated in both tumor and germline genomes (n = 81 in a total of 155). Damaging ARHGAP mutations/variants in tumors are correlated with higher LOH values (54±3.4%, n = 81 vs. 39±3.3%, n = 74. mean±SE, p = 0.0021) and poor prognosis of patients (5-year OS; 58% n = 81 vs. 41% n = 74, p = 0.0098). We also found that elevated and robust expression of a mitotic kinesin KIF18A in tumors harboring higher LOH and/or damaging ARHGAP mutations/variants (n = 12) but not in lower LOH tumors without ARHGAP mutations/variants (n = 4). Conclusions: This study, for the first time, demonstrates that genetic abnormalities and aberrant expression of genes involved in chromosome segregation and mitosis in patients with malignant soft tissue tumors. The results reveal a novel target of drug discovery for incurable STS because CIN tumor cells are shown to be particularly vulnerable to KIF18A inhibition, while somatic, diploid cells are not.

P–207 Heterogoneic cell division proven to occur in bovine zygotes

Abstract Study question We hypothesize that the zygote can segregate parental genomes via a non-canonical pathway. We coined this heterogoneic cell division. Can we proof the existence of this new segregational pathway? Summary answer We confirmed the existence of this non-canonical segregation mechanism leading to mixoploidy and provide a catalogue of abnormal zygotic divisions. What is known already Embryos show a high degree of chromosomal instability leading to chromosomal mosaicism. Chromosomal aberrations affect the developmental potential. We developed haplarithmisis which determines the single-cell genome-wide haplotype and copy number and allows to deduce the parental and segregational origin. Analysis of cleavage-stage bovine embryos by haplarithmisis, discovered the presence of uniparental and biparental blastomere lineages in individual embryos. We hypothesized that whole genome segregations can occur via a non-canonical zygotic division, a process termed “heterogoneic” division. Abnormal zygotic division has been observed in bovine and human in vitro produced zygotes using time-lapse, but parental genome segregation has never been disclosed. Study design, size, duration We hypothesized that abnormal cytokinetics and spindle mechanics may underlie the segregation of parental genomes in a separate blastomere line. In vitro produced zygotes were monitored by time-lapse microscopy. Zygotes cleaving 3 or 4 cells were disaggregated, picked and analysed by haplarithmisis. Participants/materials, setting, methods Blastomeres from bovine in vitro produced zygotes cleaving directly into 3 or 4 blastomeres, identified by time-lapse monitoring, were tubed following zona removal and blastomere dissociation and whole-genome amplified. Samples were subsequently hybridized on Illumina Bovine HD BeadChip SNP arrays. Data was analyzed by haplarithmisis, using a the siCHILD-bovine algorithm, to infer the haplotypes and the copy number of the parental genomes. Blastomeres showing failed haplarithmisis plots were low-coverage whole-genome sequenced on a HiSeq4000 sequencer. Main results and the role of chance: We obtained 25 bovine embryos, comprising 82 blastomeres, derived from 12 families (12 cows and 2 bulls) that cleaved directly into 3 or 4 blastomeres. Sixteen, 7 and 2 out of 25 zygotes cleaved respectively in 3 cells, 4 cells and 3 cells and a fragment. In at least 20 embryos, more than one paternal haplotype was identified, showing that a polyspermic fertilization resulted in an abnormal division. All embryos contained a whole-genome abnormality in at least one blastomere, resulting in mixoploid (5), mixed diploid biparental and androgenetic (12), polyploid (4), mixed gynogenetic and androgenetic (2) and androgenetic (2) embryos. Twenty-one embryos had at least one blastomere containing a uniparental signature. Based on the blastomere haplotype profiles we classified the embryos in six segregation categories. In twelve blastomeres haplarithmisis failed. Massive parallel sequencing of the amplified DNA showed the presence of mitochondrial DNA, indicating the blastomere did not contain any genomic DNA. This observation confirms that heterogoneic cell division does occur via different non-canonical zygotic segregations, which result in a variety of chimeric and mixoploid embryos constitutions. Limitations, reasons for caution These findings apply to a small set of bovine in vitro produced abnormally cleaving embryos. Segregation patterns may be incomplete and their true in vitro and in vivo prevalence remains unknown. Based on the haplotypes the non-canonical divisions have been reconstructed. Those patterns can now be evaluated and tested. Wider implications of the findings: This study shows that heterogoneic cell division occurs. We hypothesize this non-canonical division occurs frequently in both in vitro and in vivo, not only in cattle but also in man and hypothesize that persistence of such cell lines might explain the development of androgenetic tumorous outgrowths and mosaic uniparental individuals. Trial registration number Not applicable

Abstract 2498: miFISH single cell analysis as a potential tool to stratify the progression risk in patients with Atypical Ductal Hyperplasia (ADH)

Abstract We previously studied synchronous ductal carcinomas in situ (DCIS) and invasive ductal carcinomas (IDC) using our novel approach of multiplex interphase Fluorescence in situ Hybridization (miFISH) that allows simultaneous enumeration of copy numbers of up to 20 gene loci per single cell, providing new insights into tumor clonality and intratumor heterogeneity (ITH). A high degree of chromosomal instability was present in DCIS, and we frequently observed a direct clonal evolution from DCIS to IDC. We now ask whether this degree of instability is also present in even earlier potential precursor lesions like atypical ductal hyperplasias (ADH). We are therefore conducting a retrospective study analyzing ADH lesions from patients without synchronous DCIS or IDC (pure ADH) and from patients with ADH adjacent to higher-grade lesions. Using our miFISH assay, we simultaneously assess copy number changes of six oncogenes (COX2 (1q), PIK3CA (3q), MET (7q), MYC (8q), CCND1 (11q), HER2 (17q), ZNF217 (20q)) and six tumor suppressor genes (FHIT (3p), DBC2 (8p), RB1 (13q), CDH1 (16q), TP53 (17q), NF2 (22q), typical for breast tumorigenesis. So far, we have analyzed fifteen pure ADH and five ADH with synchronous DCIS or IDC. The miFISH analysis showed that the majority of all pure ADH displayed a stable diploid genome without any copy number changes observed for the genes tested while only 3/15 (20%) showed changes, namely (i) a COX2 and HER2 gain in a diploid genome, (ii) a CDH1and MET loss in a tetraploid genome and (iii) a COX2 gain in a diploid genome. In the group of ADH with adjacent higher-grade lesions (n=5), all DCIS and IDC lesions analyzed displayed breast cancer specific copy number changes corroborating our previous results. Of note, two of the ADH lesions adjacent to these aberrant DCIS or IDC lesions had normal diploid genomes. Of the remaining three synchronous ADH lesions, one showed a complex gain and loss pattern similar to its adjacent DCIS, the second one was characterized by a simple COX2 gain which was maintained in the adjacent DCIS in addition to a DBC2 loss and MYC gain. The third ADH displayed a tetraploid clone without any gains or losses, while the adjacent IDC revealed a complex gain and loss pattern in its tetraploid genome. Our preliminary data indicate that the majority of pure ADH does not harbor copy number changes. However, there is a small subset of pure ADH revealing gain and loss patterns typical for DCIS and IDC often involving COX2 gain indicating it as an early event. Of note, ADH lesions adjacent to DCIS or IDC tend to display similar changes as their adjacent higher-grade lesions suggesting them as direct precursor lesions. We are currently analyzing more ADH samples and plan to add material from an ADH patient cohort with long-term follow-up to explore whether miFISH can stratify ADH patients into different risk groups. Citation Format: Kiara Whitaker, Jausheng Tzeng, Daniela Hirsch, Irianna Torres, Steven Brower, Gert Auer, Miguel Sanchez, Thomas Ried, Kerstin Heselmeyer-Haddad. miFISH single cell analysis as a potential tool to stratify the progression risk in patients with Atypical Ductal Hyperplasia (ADH) [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2498.

Abstract 5123: Proteogenomic characterization reveals mitotic kinase and replication stress implicated in ovarian high-grade serous cancer

Abstract High-grade serous cancer (HGSC), the most prevalent histotype of ovarian cancer, has the lowest survival rates and is the leading cause of gynecological cancer-related deaths in the developed world. HGSC is characterized by the presence of nearly universal TP53 mutations, diverse and widespread chromosomal instability, and a general shortage of targetable driving mutations. The major clinical needs in HGSC include the identification of alternative therapeutic targets and an improved understanding of the mechanisms driving chromosomal heterogeneity. We prospectively collected 83 new ovarian HGSC samples and 20 normal precursor tissue samples (Fallopian tube, FT) for deep characterization at the genomic, transcriptomic, proteomic, and phosphoproteomic levels. We were also able to obtain cytobrush samples of Fallopian tube epithelium and provide the first extensive comparison of HGSC and FT proteomes and phosphoproteomes that used surgical specimens rather than cell lines. The ability to compare tumor and normal precursor tissues provided new biological insights regarding the role of increased proliferation in promoting replication stress in a DNA repair-deficient background, potentially explaining the high degree of chromosomal instability associated with HGSC. Our collection protocols were specifically designed to minimize the effect of ischemia on protein phosphorylation, which has been identified as a significant confounding variable. Thus, we were able to identify novel signaling interactions that would have been lost in the background of ischemic stress, most notably the activation of CDK4 and CDK7. Widespread increases in protein phosphorylation and pathway activation in tumors compared to normal tissues, particularly in the proliferation-associated CDK/RB and AURKA pathways that are targetable by FDA-approved inhibitors, provide a rational basis for the use of these therapeutics in ovarian cancer. Phosphosite-specific analysis of CDKs and FANCD was consistent with an elevated response to replication stress, potentially induced by proliferative signals. Additionally, pathway-level observations from the retrospective TCGA cohort were conserved in the prospective cohort, including histone acetylation marks associated with homologous repair deficiency. In addition to confirming key features of HGSC from previous proteogenomic study, analysis of prospectively collected HGSC samples provided significant new insights stemming from direct comparisons to normal precursor tissues; deep phosphoproteomics revealed new insights regarding the potential role of proliferation-induced replication stress in promoting the characteristic chromosomal instability of HGSC and suggested novel therapeutic targets for use in precision medicine trials. Citation Format: Jason McDermott, Osama Arshad, Vladislav Petyuk, Yi Fu, Marina Gritsenko, Chia-Feng Tsai, Samuel Payne, Mathangi Thiagarajan, Christopher Kinsinger, Ana Robles, Emily Boja, Henry Rodriguez, Daniel Chan, Bing Zhang, Zhen Zhang, Li Ding, Richard Smith, Tao Liu, Karin Rodland. Proteogenomic characterization reveals mitotic kinase and replication stress implicated in ovarian high-grade serous cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5123.

Abstract 2189: Genomic dynamics in breast cancer progression analyzed by miFISH analysis

Abstract Our previous studies of synchronous Ductal Carcinomas in Situ (DCIS) and Invasive Ductal Carcinomas (IDC) often showed a high degree of chromosomal instability already in DCIS, and frequently a direct clonal evolution from DCIS to IDC. We now asked the question whether this degree of instability is also present in DCIS that did not progress to IDC. In collaboration with Englewood Hospital and Medical Center, we are therefore conducting a retrospective study analyzing FFPE material from 20 DCIS patients with low-grade or high-grade DCIS who did not present with invasive breast carcinoma during their follow-up (5-10 years) and compared the findings to 10 DCIS patients who presented with synchronous IDC. Using our novel multiplex interphase FISH (miFISH) assay, we are able to determine copy number changes of five oncogenes (COX2, MYC, CCND1, HER2, ZNF217) and three tumor suppressor genes (DBC2, CDH1, TP53) and assessed intratumor heterogeneity (ITH) and clonal evolution during disease progression. The miFISH analysis showed that low-grade DCIS displayed mostly stable patterns of gene copy numbers in a diploid genome, while high-grade DCIS frequently had increased nuclear DNA content (ploidy) associated with a higher degree of ITH. However, some low-grade DCIS exhibited complex and unstable patterns with higher ploidy. Also, a few high-grade DCIS were defined by few copy number changes, e.g., the gain of a whole chromosome 8 (DBC and MYC), resulting in a clonal population with a low instability index. DCIS and their synchronous IDC often displayed very similar miFISH patterns indicating direct expansion of dominant clones. Of note, the DCIS cells of one case were defined by the well described 1q gain/16q loss pattern and acquired subsequently a chromosome 8 gain in the IDC, confirming our previous observation that the gain of MYC is a pivotal step in breast cancer progression. Our data also show that DCIS with or without synchronous IDC exhibited similar patterns. This could indicate that DCIS in general, regardless of grading, should be treated as lesions with progressive potential. Phylogenetic tree modeling with our custom FISHtrees software will further elucidate the dynamics of DCIS lesions. In summary, miFISH analysis and phylogenetic tree modeling allow an in-depth characterization of precursor lesions and their evolutionary trajectory. We anticipate that this approach will ultimately help to stratify progression risk in patients with DCIS. Citation Format: Irianna Marie Torres, Leanora Hernandez, Jausheng Tzeng, Russell Schwartz, Alejandro Schaffer, Edward Michael Gertz, Daniela Hirsch, Steven Brower, Miguel Sanchez, Gert Auer, Kerstin Heselmeyer-Haddad, Thomas Ried. Genomic dynamics in breast cancer progression analyzed by miFISH analysis [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 2189.

Abstract 2908: Single-cell genetic analysis of ductal carcinoma in situ with and without synchronous invasive breast cancer by multiplex FISH delineates specific patterns of tumor clonality and heterogeneity

Abstract We previously studied synchronous Ductal Carcinomas in Situ (DCIS) and Invasive Ductal Carcinomas (IDC) using a novel approach of multiplexing FISH probes that allows us to simultaneously assess the copy numbers of up to 20 loci within intact nuclei providing new insights into tumor clonality and heterogeneity. A high degree of chromosomal instability already in DCIS, and frequently, but not always, a direct clonal evolution from DCIS to IDC was detected. We now ask whether this degree of instability is also present in DCIS that did not progress to IDC and are therefore analyzing FFPE material from 20 patients with either low-grade and high-grade DCIS who did not present with invasive breast carcinoma during their follow-up (5-10 years), in addition to 10 patients who presented with synchronous IDC. The multiplex FISH assay used targets five oncogenes (COX2, MYC, CCND1, HER2, ZNF217) and three tumor suppressor genes (DBC2, CDH1, TP53) frequently altered in breast carcinomas. To date, we have analyzed two paired cases of synchronous DCIS and IDC, DCIS-IDC 3 and 4, and three cases of DCIS without IDC, OP-DCIS 1, 4 and 5. The paired cases exhibited very similar aberration patterns for synchronous DCIS and IDC indicating the invasive carcinoma is closely related to the DCIS lesion. Specifically, the major clones in DCIS 3 (low-grade) and IDC 3 showed a diploid tumor cell population with a gain of COX2, and losses of DBC2, MYC, TP53, and HER2. Of note, the only difference observed was a gain of ZNF217 in the DCIS which was not seen in the major clone of the invasive carcinoma. The other paired case, DCIS 4 (high-grade) and IDC 4, showed major clones of triploid tumor cell populations with gains of COX2, CCND1 and MYC and losses of DBC2, CDH1, TP53 and ZNF217. The three DCIS cases without synchronous or subsequent IDC exhibited varying degrees of aberration and complexity patterns in their clonal populations. Case OP-DCIS 1, a high-grade DCIS, revealed a tetraploid cell population which showed a major clone with an amplification of CCND1 and a MYC gain combined with losses of DBC2, CDH1, TP53 and HER2. A low-grade DCIS, OP-DCIS 4, exhibited a diploid cell population with a major clone showing loss of CDH1 as the only aberration. The third case OP-DCIS 5 (low-grade DCIS) exhibited a diploid cell population with one major clone showing losses of CDH1, MYC, DBC2, TP53, HER2 and CCND1. Our preliminary observations show a tendency of diploid lesions with a predominance of loss patterns for low-grade DCIS, while high-grade DCIS seem to reveal higher ploidy with more complex gain and loss patterns. However, also low-grade DCIS with mainly loss patterns progress to invasive cancers as seen in case DCIS-IDC3. We expect that the analysis of the remaining cases will further elucidate the dynamics of DCIS lesions which will hopefully help to assess and stratify progression risk in patients with DCIS. Citation Format: Irianna Marie Torres, Leanora Hernandez, Jausheng Tzeng, Russell Schwartz, Alejandro Schaffer, Edward Gertz, Stephen Brower, Miguel Sanchez, Gert Auer, Kerstin Heselmeyer-Haddad, Ried Thomas. Single-cell genetic analysis of ductal carcinoma in situ with and without synchronous invasive breast cancer by multiplex FISH delineates specific patterns of tumor clonality and heterogeneity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2908. doi:10.1158/1538-7445.AM2017-2908

Copy number analysis identifies tumor suppressive lncRNAs in human osteosarcoma.

Osteosarcoma (OS) has a high degree of chromosomal instability and total copy number (CN) changes. We examined 58 human OS samples including 40 primary tumors, 11 explants, and 7 cell lines using single nucleotide polymorphism (SNP) arrays, and revealed that 70% of the samples had one or more recurrent CN-neutral loss of heterozygosity (CNN‑LOH) also known as uniparental disomy(UPD). Importantly, 17% of the samples showed prominent homozygous deletion of 3q13.31, suggesting its role in tumorigenesis. We identified and characterized two novel lncRNAs, LOC285194 and BC040587, within this genomic locus, strongly suggesting their tumor suppressor activity. Frequent deletions and UPD suggest that OS often has mutant or non-expressed tumor suppressor genes including two lncRNAs.

Implication of genomic characterization in synchronous endometrial and ovarian cancers of endometrioid histology

ObjectivesSynchronous endometrial and ovarian carcinomas (SEOCs) present gynecologic oncologists with a challenging diagnostic puzzle: discriminating between double primary cancers and single primary cancer with metastasis. We aimed to determine the clonal relationship between simultaneously diagnosed endometrial and ovarian carcinomas. MethodsFourteen pairs of SEOCs of endometrioid type and two pairs of SEOCs with disparate histologic types (control for dual primary tumors) were subjected to massively parallel sequencing (MPS) and molecular inversion probe microarrays. ResultsThirteen of the 14 pairs of SEOCs harbored somatic mutations shared by both uterine and ovarian lesions, indicative of clonality. High degree of chromosomal instability in the tumors from 10 patients who received adjuvant chemotherapy, of whom 9 had synchronous carcinomas with significantly overlapping copy number alterations (CNAs), suggestive of single primary tumors with metastasis. The clonal relationship determined by genomic analyses did not agree with clinicopathological criteria in 11 of 14 cases. Minimal CNAs were identified in both ovarian and endometrial carcinomas in 4 patients, who did not receive adjuvant chemotherapy and experienced no recurrent diseases. In contrast, two of the 10 patients with chromosomally unstable cancers developed recurrent tumors. ConclusionOur findings support a recent paradigm-shifting concept that most SEOCs originate from a single tumor. It also casts doubt on the clinicopathological criteria used to distinguish between dual primary tumors and single primary tumor with metastasis. Testing of CNAs on SEOCs may help determining the need of adjuvant therapy.

Abstract 3745: Comprehensive integrative characterization of hepatocellular carcinoma: The TCGA HCC project

Abstract Background: Hepatocellular carcinomas (HCCs) are heterogenous primary liver cancers with pleomorphic histologic features. We present a comprehensive genetic, genomic, transcriptomic and proteomic analysis of 203 HCCs in The Cancer Genome Atlas Program (TCGA) on behalf of the TCGA Liver analysis working group to provide a deeper understanding of the molecular features of HCCs, to classify them in a clinically-relevant manner, and to provide a public resource that identifies potential targets for emerging therapies.Results: Mutation analyses confirmed the presence of TERT promoter mutations in 40% of HCCs. The analysis also revealed the first reported germline mutation in the TERT promoter in a 30 year old male, possibly reflecting an inherited predisposition to HCC. TERT promoter mutations were enriched in hepatitis C virus infected subjects. Analyses of hepatitis B virus (HBV)-induced HCCs demonstrated recurrent integrations of HBV in the TERT locus. Thus, HBV integration may substitute for TERT promoter mutations as an alternate mechanism of telomerase activation. Other tumor suppressor genes inactivated by mutation included the cell cycle genes TP53 (32% of cases), RB1 (8%), and PTEN (3%), the Wnt/beta catenin pathway gene AXIN1 (4%), and the chromatin remodeling genes ARID1A (9%), BAP1 (6%), ARID1B (4%), ARID2 (4%), and PBRM1 (3%). The oncogene CTNNB1 was mutated in 27% of HCCs. Mutations were also found in the MTOR pathway genes, MTOR (4%), TSC2 (4%) and TSC1 (3%). The patterns of mutational signatures observed included signatures characteristic of the known carcinogens aristolochic acid and aflatoxin B1. Clustering of mRNA, miRNA, or protein expression, DNA hypermethylation and DNA copy number profiles identified five, four, two, five and three clusters, respectively. Analysis of mRNA expression data identified five groups. In particular, Group 1 was associated with Asian race, higher histologic grade, and micro- and macrovascular invasion. Integrated clustering showed a grouping of HCCs into three superclusters: iCluster 1 had a low frequency of CDKN2A silencing, lack of CTNNB1 mutation, a normal-like methylation pattern, and overexpression of proliferation marker genes; iClusters 2 and 3 had a high frequency of CDKN2A silencing, CTNNB1 mutation, and overexpression of oxidative response genes; iCluster 3 tumors had a higher degree of chromosomal instability, high frequency of 17p loss, and a highly altered methylation profile. Copy number aberrations included gains in the MYC, VEGFA, CCND1, TERT, and MET loci and losses in the TP53, RB1, ERFFI1, CDKN2A, ARID1A and PTEN loci. Discussion: Based on integrated analysis of genome, transcriptome, methylome and proteome we showed that HCCs naturally separate into three to five distinct groups with associated molecular and clinical characteristics. Importantly, we identified a novel germline mutation in the TERT promoter that may be associated with an inherited risk for HCC. Note: This abstract was not presented at the meeting. Citation Format: Lewis R. Roberts, David A. Wheeler. Comprehensive integrative characterization of hepatocellular carcinoma: The TCGA HCC project. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3745. doi:10.1158/1538-7445.AM2015-3745

Profiling of GEPNETs

The tumorigenesis of gastrointestinal and pancreatic tumors (GEPNETs) is poorly understood. We need a better understanding of the molecular alterations in GEPNETs to obtain an accurate classification, and it may also provide targets for therapeutic intervention. Purpose of Paper. The purpose of this paper was to critically examine recent advances in the molecular understanding of GEPNETs gained from genome-wide and transcriptome-wide profiling studies. Special emphasis was put on diagnostic, predictive, and therapeutic implications of profiling studies. Results. Pancreatic neuroendocrine tumours (PNETs) were characterised by a distinct pattern of chromosomal alterations and a higher degree of chromosomal instability (CIN) than ileal carcinoids. Subgroups of PNETs and ileal carcinoids were identified on the basis of specific chromosomal alterations. Exome sequencing identified mutations in MEN1, ATRX/DAXX, and mTOR pathway genes as being frequent events in sporadic PNETs. Expression profiles of PNETs and ileal carcinoids were found to be different, and allowed identification of subgroups of tumors, as well discrimination between benign and malignant tumors. The molecular data provided a number of candidate genes and pathways suitable for targeted therapy. For PNETs, candidate targets include BRAF, KRAS, TERT, EGFR, RET, MDM2, IGF, MET/HGF, ANG2, LCK, PDGFRB, AKT-mTOR, and SSTR2. Some of these targets have already been evaluated in clinical trials (mTOR and SSTR2). For ileal carcinoids, significantly fewer candidate targets were provided, including ERBB2 (HER2), RET, APLP1, and Notch. Conclusion. Profiling of GEPNETs is a powerful tool for discovery of novel targets for therapeutic intervention. Further studies, combining genome, epigenome, transcriptome, and proteome data are needed to enable us to identify clinically relevant targets in GEPNETs.

A study of adrenocortical tumors in children and adolescents by a comparative genomic hybridization technique

Adrenocortical tumors (ACT) are rare neoplasms of the adrenal glands accounting for 0.2% of all pediatric cancers. However, the incidence of ACT in South Brazilian children is 10 to 15 times greater than the worldwide incidence. Comparative genomic hybridization studies have revealed the presence of a high degree of chromosomal instability in ACT. We evaluated 16 ACT, 8 of them carcinomas and 8 adenomas. The presence of changes in DNA copy numbers was determined by comparative genomic hybridization, and the findings were validated by real-time polymerase chain reaction on the basis of IGF-II gene expression. The adenomas showed a mean of 19.7 imbalances per case, with the most frequent gain and loss being 4p15.1-p15.3 and 20p11.2-p13.2, respectively. The carcinomas presented with a mean of 35.5 imbalances per case, with the more frequent gain being 2q14.1-q24.3 and the more frequent losses being 3q21-q26.2, 20q12-qter, and 22q11.2-q13.3. The most frequent imbalances in both adenomas and carcinomas were gains of 1p21-p31.2, 2p12-p21 and loss of 20p11.2-p12. The expression of IGF-II mRNA (11p15.5) was higher in samples that presented with a gain of this region. It has been established that great genomic instability exists in pediatric ACT.

The cohesin-interacting protein, precocious dissociation of sisters 5A/sister chromatid cohesion protein 112, is up-regulated in human astrocytic tumors

Glioblastoma multiforme (GBM) is the most prevalent, highly malignant, invasive and difficult-to-treat primary brain tumor in adults. At the genetic level, it is characterized by a high degree of chromosomal instability and aneuploidy. It has been shown that defects in the mitotic spindle checkpoint could lead to the development of aneuploidy as well as tumorigenesis. Additional proteins regulating sister chromatid cohesion could also be involved in maintaining the fidelity of chromosome segregation. One such protein is the precocious dissociation of sisters 5A (Pds5A), also known as sister chromatid cohesion protein 112. It is a nuclear protein, expressed from the S right through to the mitotic phase. It is highly conserved from yeast to man and plays a role in the establishment, maintenance and dissolution of sister chromatid cohesion. The mutation of Pds5A orthologs in lower organisms results in chromosome missegregation, aneuploidy and DNA repair defects. It is considered that such defects can cause either cell death or contribute to the development of cancer cells. Indeed, altered expression levels of Pds5A have been observed in tumors of the breast, kidney, oesophagus, stomach, liver and colon. Malignant gliomas, however, have not been analysed so far. Herein, we report on the cloning of Rattus norvegicus Pds5A and on the analysis of its expression pattern in rat tissue. We also show that Pds5A is significantly overexpressed at both the mRNA and protein level and that this overexpression correlates positively with the WHO grade of human gliomas. However, functional assays show that the siRNA-mediated knockdown of Pds5A affects sister chromatid cohesion but does not influence mitotic checkpoint function or the proliferation and survival of GBM cells. Although the mechanism by which Pds5A functions in GBM cells remains unclear, its overexpression in high grade gliomas implies that it could play a pivotal role during the development and progression of astrocytic tumors.

Abstract 2135: Genetic relationships between ovarian and breast cancers

Abstract Background: Ovarian cancer is the most fatal gynecological cancer, and breast cancer is the most common malignant disease in women. The existence of a common etiology to these diseases is suggested by the fact that BRCA1 or BRCA2 germline mutation-associated tumors are primarily restricted to the breast and the ovary. While in the ovary, BRCA mutations are associated with high-grade serous carcinomas (HGSC), and in the breast, BRCA1 mutation carriers can develop tumors that are phenotypic copies of sporadic basal-like breast cancer (BLBC). Hypothesis/objective: We hypothesize that HGSC and BLBC's are similar with respect to genetic instability and chromosomal aberrations and that these similarities may reflect their common pathogenesis. The objective of our proposed project is to identify the common patterns of loss of heterozygosity (LOH) and regions of gain or amplification shared by the two diseases. Materials and Methods: Fifty two HGSC were collected and tumor cells enriched by needle microdissection from frozen tissue sections if available to remove stroma before DNA isolation. DNA was subjected to Affymetrix 250K SNP array analysis. SNP array data from a DFCI-BWH cohort of breast cancers were analyzed for comparison. dChip software was used for LOH and copy number analysis. Results: LOH pattern-based hierarchical clustering defined two subgroups in HGSC, the major subgroup (∼60% of the cases) with high-level LOH [mean fraction of LOH/genome (FLOH) 36.6%] and the minor subgroup (∼40% of the cases) with lower levels of LOH (mean FLOH 13%). These data suggest the differences in chromosomal instability in the two subgroups. The major group of ovarian tumors shared similar high levels of LOH with BRCA1-associated or sporadic BLBCs. In contrast, the lower frequency of LOH in the minor subgroup of serous tumors is similar to that in HER2 positive or estrogen receptor positive luminal breast tumors. LOH on chromosome 17 is the most common alteration in ∼78% of the cases. However, frequent LOH on chromosomes 4, 5, 9, 13, 22, and X is characteristic of the major ovarian cancer subgroup defined by this analysis. These features are also found in BLBC, but not in non-basal-like breast tumors. Chromosomal copy gain/amplification is most common on chromosome 8q (>40%), and frequent on 3q, 19q12, 19q13, and 20q (>15%) in two subgroups of serous ovarian cancer. These changes on 8q and 20q are shared by a proportion of breast cancers. Conclusion: A major subset of serous ovarian cancer, like breast BLBCs, reveals a high degree of chromosomal instability with high levels of LOH. This suggests a defect(s) in maintenance of genome stability. Another subset of serous cancer, like non-basal-like breast tumors, has a lower degree of genome instability with low levels of LOH. We propose that these features may reflect a different pathogenesis of the two subgroups of serous ovarian tumors, and possibly are associated with differences in response to chemotherapy. 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 2135.

Are Keratoacanthomas Variants of Squamous Cell Carcinomas? A Comparison of Chromosomal Aberrations by Comparative Genomic Hybridization

Keratoacanthoma (KA) is a benign keratinocytic neoplasm that usually presents as a solitary nodule on sun-exposed areas, develops within 6-8 weeks and spontaneously regresses after 3-6 months. KAs share features such as infiltration and cytological atypia with squamous cell carcinomas (SCCs). Furthermore, there are reports of KAs that have metastasized, invoking the question of whether or not KA is a variant of SCC. To date no reported criteria are sensitive enough to discriminate reliably between KA and SCC, and consequently there is a clinical need for discriminating markers. We screened fresh frozen material from 132 KAs and 37 SCCs for gross chromosomal aberrations by using comparative genomic hybridization (CGH). Forty-nine KAs (37.1%) and 31 SCCs (83.7%) showed genomic aberrations, indicating a higher degree of chromosomal instability in SCCs. Gains of chromosomal material from 1p, 14q, 16q, 20q, and losses from 4p were seen significantly more frequently in SCCs compared with KAs (P-values 0.0033, 0.0198, 0.0301, 0.0017, and 0.0070), whereas loss from 9p was seen significantly more frequently in KAs (P-value 0.0434). The patterns of recurrent aberrations were also different in the two types of neoplasms, pointing to different genetic mechanisms involved in their developments.

P.2.102 Aripiprazole treatment for psychosis in patients with Alzheimer's disease

Adrenocortical tumors (ACT) are rare neoplasms of the adrenal glands accounting for 0.2% of all pediatric cancers. However, the incidence of ACT in South Brazilian children is 10 to 15 times greater than the worldwide incidence. Comparative genomic hybridization studies have revealed the presence of a high degree of chromosomal instability in ACT. We evaluated 16 ACT, 8 of them carcinomas and 8 adenomas. The presence of changes in DNA copy numbers was determined by comparative genomic hybridization, and the findings were validated by real-time polymerase chain reaction on the basis of IGF-II gene expression. The adenomas showed a mean of 19.7 imbalances per case, with the most frequent gain and loss being 4p15.1–p15.3 and 20p11.2–p13.2, respectively. The carcinomas presented with a mean of 35.5 imbalances per case, with the more frequent gain being 2q14.1–q24.3 and the more frequent losses being 3q21–q26.2, 20q12–qter, and 22q11.2–q13.3. The most frequent imbalances in both adenomas and carcinomas were gains of 1p21–p31.2, 2p12–p21 and loss of 20p11.2–p12. The expression of IGF-II mRNA (11p15.5) was higher in samples that presented with a gain of this region. It has been established that great genomic instability exists in pediatric ACT.

A high degree of chromosomal instability at 13q14 in cutaneous squamous cell carcinomas: indication for a role of a tumour suppressor gene other than Rb

Background/Aims—Loss of function of the retinoblastoma (Rb) tumour suppressor gene, located on chromosome 13, is common in many inherited and sporadic forms of cancer. Inactivation of its gene product by...