Degree Of Genomic Instability Research Articles

Ergodicity Breaking and Self-Destruction of Cancer Cells by Induced Genome Chaos.

During the progression of some cancer cells, the degree of genome instability may increase, leading to genome chaos in populations of malignant cells. While normally chaos is associated with ergodicity, i.e., the state when the time averages of relevant parameters are equal to their phase space averages, the situation with cancer propagation is more complex. Chromothripsis, a catastrophic massive genomic rearrangement, is observed in many types of cancer, leading to increased mutation rates. We present an entropic model of genome chaos and ergodicity and experimental evidence that increasing the degree of chaos beyond the non-ergodic threshold may lead to the self-destruction of some tumor cells. We study time and population averages of chromothripsis frequency in cloned rhabdomyosarcomas from rat stem cells. Clones with frequency above 10% result in cell apoptosis, possibly due to mutations in the BCL2 gene. Potentially, this can be used for suppressing cancer cells by shifting them into a non-ergodic proliferation regime.

Abstract 314: Intratumor genetic heterogeneity dictates metastatic subclones in stage II colon cancer

Abstract The high incidence of colon cancer (CC) worldwide is a major health concern. Although surgery and adjuvant chemotherapy are effective to a high extent in lymph-node negative colon cancers, there are still some 10-15% of patients that show disease relapse within the next 5-year period after intended curative surgery. We hypothesize that genomic instability and intratumor heterogeneity fuel the formation of subclonal populations in the primary tumor that might promote metastasis. In the current study, we assessed the presence of intratumor heterogeneity in CC using multiplex interphase FISH (miFISH) and whole-exome sequencing (WES) to evaluate samples obtained from primary stage II colon tumors and their patient-matched associated liver metastases using multi-region sampling (N=9). In combination with genome-wide copy-number changes obtained from WES analysis, miFISH allowed the simultaneous quantification of copy-numbers for nine CC relevant genes and a centromeric ploidy control probe in intact tumor nuclei derived from archival patient material. Our preliminary data on the four cases analyzed so far, revealed high similarities regarding copy-number changes and ploidy between primary tumor and metastatic samples for three of the cases. While two of these cases showed either triploid or highly aneuploid genomes with different degrees of genomic instability and subclonality among them, the third case displayed a diploid content for both matched lesions. Intriguingly, as seen by miFISH and WES, loss of CDX2 in the primary tumor might have originated the main clonal population giving rise to metastasis in this latter case. Conversely, the fourth case analyzed exhibited a triploid baseline for the primary tumor while the metastasis revealed a diploid population. Interestingly, despite higher absolute copy numbers in the primary tumor, there was an overlap of five of the gains and losses observed for the major clone populations of the tumor and metastasis by miFISH. However, two of the highly clonal gains observed in the tumor, namely the EGFR and MYC gain, were not present in the metastasis. Altogether, in addition to the ploidy change, results indicated major discrete copy-number changes from primary tumor to metastasis in this patient. In summary, we show here that the combined analysis of WES and miFISH is able to describe the subclonal composition of copy-number alterations in primary tumors and their patientmatched liver metastases. Future perspectives also include the assessment of subclonality affecting single-nucleotide variants to complete the genomic landscape of tumor evolution and clonal development in this patient cohort with the goal to better understand the nature of these early metastases. Citation Format: Sanam Yaghoubi, Kerstin Heselmeyer-Haddad, Ivan Archilla, Carolina Parra, Darawalee Wangsa, Giancarlo Castellano, Sara Lahoz, Veronica Pablo-Fontecha, Daniela Hirsch, Wei-Dong Chen, Thomas Ried, Miriam Cuatrecasas, Paul S. Meltzer, Jordi Camps. Intratumor genetic heterogeneity dictates metastatic subclones in stage II colon cancer [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 314.

Abstract 2590: Analysis of homologous recombination deficiency (HRD) scores in osteosarcoma

Abstract Objective: Outcomes for patients with metastatic or relapsed osteosarcoma (OS) remain poor. Many OS samples have demonstrated a high proportion of mutational signature 3 (ms3), suggestive of homologous recombination DNA repair deficiency (HRD) or a BRCAness molecular phenotype. The HRD score, which quantifies chromosomal structural rearrangements by combining loss of heterozygosity (LOH), large state transitions (LST), and telomere allelic imbalances (TAI), has been used to assess HRD status in patients with breast and ovarian cancer, with high scores found to be predictive of PARP inhibitor sensitivity. While high HRD scores have been described in OS, little is known about how HRD scores vary by stage, site, pre vs post-therapy, presence of homologous recombination pathway or TP53 alterations, and their correlation to ms3 contribution. Methods: To address this issue, we evaluated 48 OS samples with tumor purity ≥ 20% from 46 patients who had 80x whole genome sequencing (WGS) performed through our pediatric expanded genomics platform. HRD scores were then estimated using scarHRD, a computational tool, which quantifies LOH, LST, and TAI events from WGS data and has shown a good correlation to SNP-array based methods. Results: Of the 48 samples analyzed, 25 samples were from primary sites and 23 from metastatic sites of disease. The median HRD score was 45 with a minimum score of 6 and a maximum score of 79. The 25th, 75th, and 90th percentiles of values were 35.7, 57, and 66.3 respectively. There was a significant difference in HRD score in patients with localized disease (n=22) vs metastatic disease (n=26) at time of diagnosis with median HRD (mHRD) scores of 41 and 51.5 (p = 0.027). The presence of a TP53 structural variant or single nucleotide variant (mHRD=47) was associated with higher HRD scores than TP53 wild-type samples (mHRD=29.5, p=0.04). There was no statistically significant difference in HRD scores in pre vs post-treated samples or primary site vs metastatic site samples. Samples with homologous recombination pathway alterations (n=10) were not associated with increased HRD score. There was a positive correlation (Spearman’s rho=0.425, p=0.001) between the number of mutations attributed to ms3 and HRD score. Conclusions: The range and distribution of HRD scores in our OS cohort closely resemble BRCA1/2 deficient breast cancer. Patients with metastatic disease at diagnosis were found to have higher HRD scores, representing a higher degree of genomic instability and increasing potential to accumulate resistance mutations. Higher HRD scores in TP53 mutated samples may be due to chromosomal instability caused by mechanisms other than HRD. The BRCAness phenotype in OS is now being targeted in a phase II clinical trial, combining the PARP inhibitor olaparib with the ATR inhibitor ceralasertib (NCT0441706), which includes integrated correlative biology to prospectively assess biomarkers of response such as HRD score. Citation Format: Michael David Kinnaman, Julia Pena, Max Levine, Nancy Bouvier, Elli Papaemmanuil, Suzanne Forrest, Katherine Janeway, Paul Meyers, Julia Glade Bender. Analysis of homologous recombination deficiency (HRD) scores in osteosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2590.

Abstract 5003: Gene expression to copy number alterations analysis in colorectal cancers: Organ specificity of transcriptomic impact on copy number alterations

Abstract Background: Colorectal cancers (CRCs) is the third most commonly diagnosed cancer and the second most lethal cancer worldwide. CRCs carry high degrees of genomic instability (GI), which enables cancer evolution and makes prognosis poorer. Thus, GI is an exploitable target for therapy purposes. GI can be caused by gene mutation, alteration in gene expression, epigenetic modification, and other infectious or environmental factors. Several mutated genes involved in Chromosome instability and/or microsatellite instability have been identified. Yet it has not been technically feasible to modify functions of mutated genes (e.g., APC, TP53). From the drug development’ standpoint, inhibition of over-expressed target gene is preferred. However, specific genes involved in GI via expression alterations in CRCs have not been comprehensively identified. The gap in knowledge hinders development of CRC therapies targeting GI in CRC. In a previous study, we developed a data mining strategy (Gene Expression to Copy Number Alterations; “GE-CNA”) and comprehensively identified 1,578 genes that associate with CNA in lung adenocarcinoma, among which 39 were survival-critical (i.e., expression levels correlate with significant differences in patients’ survival). Rationale: Causal genes for GI via expressions have not been comprehensively identified in CRCs. Thus, the mechanistic understanding of GI generation in CRC remains incomplete. Few attempts have been made to understand the organ-specificity of genomic instability in cancer. Main Results: We applied the GE-CNA approach to 592 TCGA CRC datasets, and identified 513 genes whose expression levels associate with CNA. Among these, 27 were survival-critical. Comparison with previous results from lung adenocarcinoma indicated striking differences between lung adenocarcinoma and CRC: (a) overall CNA numbers are higher in lung adenocarcinoma than CRC in all stages, (b) 262 CRC-CNA facilitator genes did not show significant concentration in a specific pathway, and (c) 251 CRC-CNA suppressor genes are concentrated in the following pathways: Interferon Signaling, Antigen Presentation Pathway, Heme Biosynthesis II, Natural Killer Cell Signaling, Retinoic acid Mediated Apoptosis Signaling, JAK/Stat Signaling, Glucocorticoid Receptor Signaling, Heme Biosynthesis from Uroporphyrinogen-III I, and Glutathione Redox Reactions II. Implications: The 27 survival-critical genomic instability genes are potential targets to suppress CRC genomic instability and therefore are targets for CRC drug development. Causal genes for genomic instability via expressions differ among organs. Hence, the “targeting genomic instability and/or aneuploidy” approach will need to be tailored for the specific target organ. Citation Format: Chinthalapally V. Rao, Chao Xu, Yuting Zhang, Adam S. Asch, Hiroshi Y. Yamada. Gene expression to copy number alterations analysis in colorectal cancers: Organ specificity of transcriptomic impact on copy number alterations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5003.

Abstract 3340: Small molecule mediated stabilization of PP2A modulates the homologous recombination pathway and potentiates DNA damage-induced cell death

Abstract High Grade Serous Carcinoma (HGSC) is the most lethal ovarian cancer subtype and accounts for approximately 60% of all ovarian tumors. Despite recent advances in drug development and increased understanding of genetic alterations that drive HGSC progression, mortality has not declined, highlighting the need for novel therapies. PARP inhibitors (PARPi) have become the mainstay of HGSC targeted therapy research given that these tumors are driven by a high degree of genomic instability resulting from the combination of fast DNA replication rates and numerous defects in the DNA-damage response (DDR) pathway. Nonetheless, only ~25% of these patients initially respond to treatment and a significant percentage eventually relapses with resistant disease. Here, we discovered that a Small Molecule Activator of Protein Phosphatase 2A (PP2A) (SMAP-061) induces apoptosis in both established and patient-derived HGSC cell lines as well as in genetically distinct Patient-Derived Xenograft (PDX) mouse models. Interestingly, we also uncovered that several genes that make-up the heterotrimer PP2A tumor suppressor protein are heterozygously lost in more than 95% of HGSC tumors, second only to p53. Mechanistically, we show that stabilization of PP2A protein by SMAP-061 inhibits the Homologous Recombination (HR) pathway via the direct inhibition of RAD51, ultimately leading to chronic accumulation of DNA damage and thus programmed cell death. Furthermore, we found that SMAP-061’s ability to inhibit HR potentiated the effects of PARP inhibition and resulted in synergistic cell death in both HR proficient and deficient models. These studies emphasize the potential of PP2A activators to expand the patient population that can benefit from PARPi therapies and possibly overcome PARPi resistance. In sum, our data highlights a new role of PP2A in regulating the DDR pathway in HGSC and supports the use of SMAPs in both HR proficient and deficient HGSC tumors. Citation Format: Rita A. Avelar, Amy Armstrong, Goutham Narla, Analisa DiFeo. Small molecule mediated stabilization of PP2A modulates the homologous recombination pathway and potentiates DNA damage-induced cell death [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3340.

SATB1, genomic instability and Gleason grading constitute a novel risk score for prostate cancer

Current prostate cancer risk classifications rely on clinicopathological parameters resulting in uncertainties for prognostication. To improve individual risk stratification, we examined the predictive value of selected proteins with respect to tumor heterogeneity and genomic instability. We assessed the degree of genomic instability in 50 radical prostatectomy specimens by DNA-Image-Cytometry and evaluated protein expression in related 199 tissue-microarray (TMA) cores. Immunohistochemical data of SATB1, SPIN1, TPM4, VIME and TBB5 were correlated with the degree of genomic instability, established clinical risk factors and overall survival. Genomic instability was associated with a GS ≥ 7 (p = 0.001) and worse overall survival (p = 0.008). A positive SATB1 expression was associated with a GS ≤ 6 (p = 0.040), genomic stability (p = 0.027), and was a predictor for increased overall survival (p = 0.023). High expression of SPIN1 was also associated with longer overall survival (p = 0.048) and lower preoperative PSA-values (p = 0.047). The combination of SATB1 expression, genomic instability, and GS lead to a novel Prostate Cancer Prediction Score (PCP-Score) which outperforms the current D’Amico et al. stratification for predicting overall survival. Low SATB1 expression, genomic instability and GS ≥ 7 were identified as markers for poor prognosis. Their combination overcomes current clinical risk stratification regimes.

GSA: an independent development algorithm for calling copy number and detecting homologous recombination deficiency (HRD) from target capture sequencing

BackgroundThe gain or loss of large chromosomal regions or even whole chromosomes is termed as genomic scarring and can be observed as copy number variations resulting from the failure of DNA damage repair.ResultsIn this study, a new algorithm called genomic scar analysis (GSA) has developed and validated to calculate homologous recombination deficiency (HRD) score. The two critical submodules were tree recursion (TR) segmentation and filtering, and the estimation and correction of the tumor purity and ploidy. Then, this study evaluated the rationality of segmentation and genotype identification by the GSA algorithm and compared with other two algorithms, PureCN and ASCAT, found that the segmentation result of GSA algorithm was more logical. In addition, the results indicated that the GSA algorithm had an excellent predictive effect on tumor purity and ploidy, if the tumor purity was more than 20%. Furtherly, this study evaluated the HRD scores and BRCA1/2 deficiency status of 195 clinical samples, and the results indicated that the accuracy was 0.98 (comparing with Affymetrix OncoScan™ assay) and the sensitivity was 95.2% (comparing with BRCA1/2 deficiency status), both were well-behaved. Finally, HRD scores and 16 genes mutations (TP53 and 15 HRR pathway genes) were analyzed in 17 cell lines, the results showed that there was higher frequency in HRR pathway genes in high HRD score samples.ConclusionsThis new algorithm, named as GSA, could effectively and accurately calculate the purity and ploidy of tumor samples through NGS data, and then reflect the degree of genomic instability and large-scale copy number variations of tumor samples.

Calculated identification of mutator-derived lncRNA signatures of genomic instability to predict the clinical outcome of muscle-invasive bladder cancer

BackgroundMuscle-invasive bladder cancer (MIBC) is one of the most important type of bladder cancer, with a high morbidity and mortality rate. Studies have found that long non-coding RNA (lncRNA) plays a key role in maintaining genomic instability. However, Identification of lncRNAs related to genomic instability (GIlncRNAs) and their clinical significance in cancers have not been extensively studied yet.MethodsHere, we downloaded the lncRNA expression profiles, somatic mutation profiles and clinical related data in MIBC patients from The Cancer Genome Atlas (TCGA) database. A lncRNA computational framework was used to find differentially expressed GIlncRNAs. Multivariate Cox regression analysis was used to construct a genomic instability-related lncRNA signature (GIlncSig). Univariate and multivariate Cox analyses were used to assess the independent prognostic for the GIlncSig and other key clinical factors.ResultsWe found 43 differentially expressed GIlncRNAs and constructed the GIlncSig with 6 GIlncRNAs in the training cohort. The patients were divided into two risk groups. The overall survival of patients in the high-risk group was lower than that in the low-risk group (P < 0.001), which were further verified in the testing cohort and the entire TCGA cohort. Univariate and multivariate Cox regression showed that the GIlncSig was an independent prognostic factor. In addition, the GIlncSig correlated with the genomic mutation rate of MIBC, indicating its potential as a measure of the degree of genomic instability. The GIlncSig was able to divide FGFR3 wild- and mutant-type patients into two risk groups, and effectively enhanced the prediction effect.ConclusionOur study introduced an important reference for further research on the role of GIlncRNAs, and provided prognostic indicators and potential biological therapy targets for MIBC.

Pan-cancer analyses reveal regulation and clinical outcome association of the shelterin complex in cancer.

Shelterin, a protective complex at telomeres, plays essential roles in cancer. In addition to maintain telomere integrity, shelterin functions in various survival pathways. However, the detailed mechanisms of shelterin regulation in cancer remain elusive. Here, we perform a comprehensive analysis of shelterin in 9125 tumor samples across 33 cancer types using multi-omic data from The Cancer Genome Atlas, and validate some findings in Chinese Glioma Genome Atlas and cancer cell lines from Cancer Cell Line Encyclopedia. In the genomic landscape, we identify the amplification of TRF1 and POT1, co-amplification/deletion of TRF2-RAP1-TPP1 as the dominant alteration events. Clustering analysis based on shelterin expression reveals three cancer clusters with different degree of genome instability. To measure overall shelterin activity in cancer, we derive a shelterin score based on shelterin expression. Pathway analysis shows shelterin is positively correlated with E2F targets, while is negatively correlated with p53 pathway. Importantly, shelterin links to tumor immunity and predicts response to PD-1 blockade immune therapy. In-depth miRNA analysis reveals a miRNA-shelterin interaction network, with p53 regulated miRNAs targeting multiple shelterin components. We also identify a significant amount of lncRNAs regulating shelterin expression. In addition, we find shelterin expression could be used to predict patient survival in 24 cancer types. Finally, by mining the connective map database, we discover a number of potential drugs that might target shelterin. In summary, this study provides broad molecular signatures for further functional and therapeutic studies of shelterin, and also represents a systemic approach to characterize key protein complex in cancer.

Abstract B02: miR-181a initiates and perpetuates oncogenic transformation through the regulation of innate immune signaling

Abstract Genomic instability predisposes cells to malignant transformation; however, the molecular mechanisms that allow for the propagation of cells with a high degree of genomic instability remain unclear. Here we report that miR-181a is able to transform fallopian tube secretory epithelial cells—the precursor cell type for the majority of high-grade serous ovarian cancers—through the inhibition of RB1 and simultaneously drives a cell-protective inhibition of the stimulator-of-interferon-genes (STING) in order to maintain a microenvironment conducive to the propagation of cells with a high-degree of genomic instability. We found that miR-181a inhibition of RB1 leads to profound nuclear defects, genomic instability, and nuclear rupture resulting in a persistence of genomic material in the cytoplasm. While normally, this persistence of genomic material in the cytoplasm induces interferon response through STING to drive cell death, miR-181a directly downregulates STING and prevents apoptosis. The most common mechanism by which oncogenic miRNAs promote tumorigenesis is through the direct inhibition of tumor suppressor genes; however, our studies highlight a new mechanism of oncomiR transformation through the combination of tumor suppressor gene inhibition and abrogation of immune surveillance that initiates and propagates tumor cell survival. Importantly, we found that miR-181a induction in human ovarian tumors is tightly associated with decreased IFNg response and downregulation of lymphocyte infiltration and leukocyte fraction. To date, DNA oncoviruses are the only known inhibitors of STING that allow for cellular transformation; thus, our findings are the first to identify a genetic factor, miR-181a, that can downregulate STING expression and impair signaling in cancer cells, creating a survival advantage. Our studies support the notion that the induction of STING-mediated signaling in cancer cells could lead directly to cancer cell death; however, these effects are abrogated by miR-181a. Given the recent interest in the development of STING agonists as a strategy to harness the immune system to treat cancer, this study introduces a novel patient-selective biomarker as well as potent therapeutic target for development of the most effective combination treatments. Citation Format: Matthew Knarr, Lily J. Kwiatkowski, Michele Dziubinski, Jessica McAnulty, Stephanie Skala, Stefanie Avril, Ronny Drapkin, Analisa DiFeo. miR-181a initiates and perpetuates oncogenic transformation through the regulation of innate immune signaling [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr B02.

Abstract A10: Phenotypic and genomic characterization of intratumoral heterogeneity in high-grade serous ovarian cancer

Abstract High-grade serous ovarian cancer (HGSOC) is the most common subtype of ovarian cancer, accounting for more than 70% of all epithelial ovarian cancers. It is characterized by high degrees of genomic instability and heterogeneity, with the majority of patients eventually acquiring resistance to platinum chemotherapy. The diversity in platinum-resistance mechanisms and limited effective predictive biomarkers mean delivering the best treatment options for patient tumor remains challenging. The purpose of this study is to understand the extent of intratumoral heterogeneity (ITH) in advanced-stage HGSOC and how this changes over time at relapse, to describe the molecular mechanisms behind peritoneal dissemination, and to delineate the link between ITH at the molecular and phenotypic levels. Patients undergoing radical upfront debulking for advanced HGS ovarian cancer underwent tumor mapping of their tumor dissemination patterns (n=50). Biopsies were collected from disseminated tumors (range 4-15, median=9), snap frozen, and placed in short-term cultures. Tumor cultures were treated with cisplatin, apoptosis/viability assayed and IC50 for cisplatin determined. DNA was extracted from frozen tumors (5 tumors per patient plus germline) and Illumina Human OmniExpress genotyping performed. Allele-specific copy number (CN) was quantified using ASCAT. Genomic heterogeneity was quantified as the estimated number of CN aberration events distinct between each pair of deposits. Clonal diversity within a patient’s deposits was calculated using the difference between within-patient and between-patient heterogeneity. When relapsed, patients had paired biopsies collected for genomic and phenotypic analysis. Broad heterogeneity was observed in response to platinum treatment in vitro across cases at the phenotypic level (n=50), with higher variances in apoptosis induction observed in patients with platinum-resistant disease. Genomic analysis of copy number data revealed widespread variations in patterns of evolution for different patients’ tumors, including the relationship between primary deposits and relapsed disease. Variations in CCNE1 CN were observed across multiple tumors in the same patients, and overall higher CCNE1 CN associated with poorer patient outcome (p=0.041). Extensive heterogeneity is observed at the phenotypic and genomic levels in HGSOC patients, which correlates with the subsequent development of platinum-resistant disease. CCNE1 copy number variations across multiple intra-abdominal samples within patients indicate that single-site biopsies do not truly represent overall disseminated HGSOC biology and may have implications for overinterpretation of studies relating to outcome and platinum resistance. Citation Format: Paula Cunnea, Ed Curry, Katherine Nixon, Ratri Wulandari, Kerstin Thol, Chun Hei Kwok, Jennifer Ploski, Iain McNeish, Elizabeth Christie, David Bowtell, Christina Fotopoulou. Phenotypic and genomic characterization of intratumoral heterogeneity in high-grade serous ovarian cancer [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr A10.

Abstract PR09: Frequency and etiology of ctDNA-positive metastatic prostate cancer with BRCA2, ATM, or CDK12 mutations

Abstract Recent sequencing efforts have shown that genomic alterations within DNA damage repair (DDR) pathways are relatively common in metastatic prostate cancer (mPCa). Patients harboring DDR gene aberrations can respond poorly to standard-of-care androgen receptor (AR)-targeted therapy, potentially because defective DNA repair enables rapid tumor evolution. Research has shown that DDR-deficient mPCa is often genetically unstable due to its high mutation frequency; however, the specific differences in mutations and copy number alterations between DDR mutant and nonmutant tumors have not been established. We performed targeted next-generation sequencing on 1,214 cell-free DNA samples from 718 patients with mPCa. In 30 patients with disruption of BRCA2, ATM, or CDK12, we also sequenced archival primary tumor tissue. 118 patients had a deleterious germline or somatic mutation in at least one of the 22 DDR genes on our targeted panel. The most commonly altered were BRCA2 (n=41), ATM (n=20), and CDK12 (n=21), together present in ~14% of patients. Disruption of the intact second allele was detected in 96%, 82%, and 84% of patients with mutations in BRCA2, ATM, and CDK12, respectively. Patients with a mutation in any one of these genes had fewer mutations in TP53 than a control cohort of DDR-intact patients (p&amp;lt;0.05). However, BRCA2-defective patients had frequent compound deletions of tumor-suppressor genes (TP53, PTEN, RB1). In patients with CDK12 mutations, PTEN or RB1 deletion was rare and samples instead exhibited copy number gains of MDM2 and/or CCND1. ATM-deficient cases did not demonstrate the same degree of genomic instability as either BRCA2- or CDK12-defective disease. In summary, patients with BRCA2, ATM, or CDK12 gene disruption possess distinct patterns of genomic alterations. These findings might help clarify why some DDR-deficient mPCa cases represent a more aggressive disease subtype with a poor response to AR-targeted therapy. This abstract is also being presented as Poster A36. Citation Format: Evan Warner, Steven Yip, Matti Annala, Gang Wang, Arkhjamil Angeles, Arshia Beigi, Elena Schönlau, Amanda Wong, Sinja Taavitsainen, Gillian Vandekerkhove, Kevin Beja, Matti Nykter, Daniel Khalaf, Kim Chi, Alexander Wyatt. Frequency and etiology of ctDNA-positive metastatic prostate cancer with BRCA2, ATM, or CDK12 mutations [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr PR09.

Gastric Cancer Registry: A comprehensive patient-reported resource for multidisciplinary and translational genomic approaches to gastric cancer.

432 Background: Gastric cancer (GC) is the fifth leading cancer diagnosis and third frequent cause of cancer death globally. GC results from a cascade of molecular and genetic changes owing to environmental and genetic factors. While there are known risks to GC, more is to be learned about its development so to establish effective screens for early stages of cancer. Methods: The Gastric Cancer Registry (GCR) was built to investigate GC and discover informative genomic biomarkers. The GCR comprises medical, family, and social history and genomics data from patients with GC, a family history of GC, and/or a known mutation in the gene CDH1. Samples of saliva and gastric tumors are collected when available. The GCR allows us to leverage several genome sequencing datasets to construct a complete molecular landscape of GC. Early analysis of GC tissue includes whole exome sequencing (WES) to identify mutations, whole genome sequencing (WGS) for copy number variation, and RNA sequencing (RNAseq) for expression profiling. Results are used to inform of clonal neoantigens, microbiome, and immune cell populations. Saliva will be analyzed with linked reads sequencing to unearth germline mutations not picked up in standard clinical gene panels. Results: Datasets from 455 patients and samples from 159 patients have been collected. In a pilot study, we pinpointed specific mutations using WES and revealed extensive changes in genome copy number involving clinically actionable genes through WGS. Most tumors had a high degree of genomic instability and exhibited candidate markers for treatment decisions and response. Additionally, we found that RNAseq revealed tumor subgroups through gene expression signatures. Conclusions: The GCR is an informative resource enabling the identification of biomarkers for GC. With the GCR we are integrating expertise in translational cancer genomics with molecular biology, statistics, and bioinformatics to build a platform for discovery and the development of tools that will ultimately improve the detection, treatment, and prevention of GC.

Abstract 2562: Functional characterization of Polθ in esophageal squamous cell carcinoma

Abstract Introduction: Wild-type (WT) Polθ, encoded by POLQ gene, is a specialized DNA polymerase which helps to protect cells against genomic instability by allowing DNA double-strand break (DSB) repairs through the alternative end-joining (AltEJ) pathway. In our previous targeted gene sequencing studies, we identified a high frequency of POLQ germline variants associated with esophageal squamous cell carcinoma (ESCC) in an endemic high-risk region of China. Here in this study, we aim to determine the functional impact of POLQ in ESCC, examine the synthetic lethality relationship of POLQ-mediated AltEJ DNA repair pathway and the canonical homologous recombination (HR) pathway, and eventually explore its potential roles as a biomarker or therapeutic target in ESCC. Methods: To determine the functional roles of Polθ, POLQ knock out (KO), FANCD2 KO and POLQ/FANCD2 double KO ESCC cell lines were generated using the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technique. MTT assay was used to assess cell viabilities with or without additional treatments that induced DNA damage or replication stress. DSB repair efficiency was examined by the H2AX foci recovery assay. The single cell gel electrophoresis/Comet assay was used to evaluate the genome stability. The expression level of POLQ was compared between ESCC patients’ samples and paired non-tumoral tissues using Q-PCR. Results: Among all ESCC cell lines we tested, 8 out of 13 (61.5%) exhibited higher levels of POLQ mRNA/protein than the normal immortalized esophageal epithelial cell line NE1. A 2-fold or more elevation of POLQ was found in 17 out of 28 Hong Kong ESCC patients’ tumor samples when compared with their paired non-tumoral tissues. Depletion of POLQ in high-POLQ expressing ESCC cell lines (KYSE180TS and SLMTorT3) only slightly diminished the cell viability. Double KO POLQ and FANCD2, one of the HR genes, significantly sabotaged cell proliferation, as compared with single KO POLQ or FANCD2, especially after treated with Cisplatin or Hydroxyurea. The percentage of H2AX foci positive cells increased considerably upon POLQ KO at 8 hours post 4Gy ionizing radiation, hinting the compromised DSB repair efficiency in POLQ depleted cells. The POLQ-deficient cells also showed a higher degree of genomic instability as suggested by the Comet assay. Conclusions: The results suggested that Polθ is overexpressed in ESCC tumors and cell lines. The Polθ mediated DSB repair pathway may work as a backup plan of HR to prevent the genomic instability in ESCC. Targeting Polθ might be a potential therapeutic approach for the better management of HR-deficient ESCC. Acknowledgments: Research Grants Council Collaborative Research Fund grant number 106150246 and Asian Cancer Research Fund to MLL. We thank Prof. Jean-Sébastien Hoffmann for providing the Polθ antibody. Citation Format: Jian Li, Josephine Ko, Wei Dai, Lvwen Ning, Hoi Yan Ng, Valen Zhuoyou Yu, Maria Lung. Functional characterization of Polθ in esophageal squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2562.

Abstract 2723: Defective homologous recombination DNA repair as therapeutic target in advanced chordoma

Abstract Chordomas are rare tumors of the axial skeleton and skull base with few therapeutic options and no clinically validated molecular drug targets. The value of comprehensive genomic analyses for guiding medical therapy of patients with advanced-stage chordoma is unknown. We performed whole-exome and genome sequencing of tumor and matched germline control samples from 11 patients with locally advanced or metastatic chordoma within the MASTER program, a prospective clinical sequencing program of the German Cancer Consortium. All patients were pretreated and had progressive disease prior to molecular analysis. Genomic profiling showed that advanced chordomas are frequently characterized by genomic patterns indicative of defective homologous recombination (HR) DNA repair. First, DNA copy number profiles showed high numbers of structural variants greater than 10 million base pairs in size in the majority of cases. Second, all patients harbored somatic aberrations of at least 2 genes known to be involved in HR, and 10/11 cases harbored somatic alterations in 3 or more HR pathway genes. For example, 8 patients showed heterozygous BRCA2 deletions, which were associated with heterozygous deletions of ERCC6 in 6 patients and RAD54L in 7 patients, as well as PTEN alterations (heterozygous deletion, heterozygous mutation and deletion of the wildtype allele or loss of heterozygosity). Other recurrently altered HR genes included ATR, CHEK2, FANCC, FANCD2, FANCG, RAD18, RAD51B, and XRCC3. Third, pathogenic germline alterations were detected in 3 patients. A heterozygous BRCA2 frameshift mutation (p.T3085fs*26; ACMG Class 5), a heterozygous NBN frameshift mutation (p.K219Nfs*16; ACMG Class 5), and a heterozygous CHEK2 missense mutation (p.R145W; ACMG Class 4) were accompanied by somatic deletion of the respective wildtype alleles. Fourth, a mutational signature associated with HR deficiency was significantly enriched in 72.7% of samples and coincided with genomic instability. The high prevalence of an HR deficiency “footprint” in chordoma patients prompted us to explore the clinical efficacy of the poly(ADP-ribose) polymerase(PARP) inhibitor olaparib, which is preferentially toxic to HR-incompetent cells. Olaparib treatment of a patient whose tumor showed a prominent exposure to an HR deficiency-associated mutational signature, a high degree of genomic instability, and 13 heterozygous HR gene alterations halted tumor growth for 10 months. Whole-genome analysis at progression revealed a PARP1 p.T910A mutation predicted to disrupt the autoinhibitory PARP1 helical domain, providing novel insight into the mechanisms of PARP inhibitor resistance. In summary, our study has uncovered a key biological feature of advanced chordoma that represents an immediately actionable therapeutic target and provides a rationale for genomics-guided clinical trials of PARP inhibition in this intractable tumor entity. Citation Format: Stefan Gröschel, Daniel Hübschmann, Francesco Raimondi, Peter Horak, Gregor Warsow, Martina Fröhlich, Barbara Klink, Laura Gieldon, Barbara Hutter, Kortine Kleinhenz, David Bonekamp, Oliver Marschal, Priya Chudasama, Jagoda Mika, Marie Groth, Sebastian Uhrig, Stephen Krämer, Christoph Heining, Christoph Heilig, Daniela Richter, Eva Reisinger, Katrin Pfütze, Roland Eils, Stephan Wolf, Christof von Kalle, Christian Brandts, Claudia Scholl, Wilko Weichert, Stephan Richter, Sebastian Bauer, Roland Penzel, Evelin Schröck, Albrecht Stenzinger, Richard Schlenk, Benedikt Brors, Robert Russell, Hanno Glimm, Matthias Schlesner, Stefan Fröhling. Defective homologous recombination DNA repair as therapeutic target in advanced chordoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2723.

Tumor mutation burden and immunogenicity in gastric cancer with HER2 alterations.

4024 Background: Human epidermal growth receptor 2 (HER2) is considered as an oncogenic driver gene in gastric cancer (GC). Immunotherapy has been proven to be effective in GC patients. Previous studies indicated that patients harboring driver mutations were considered as poor candidate for immunotherapy. But the efficacy of immunotherapy for HER2 positive GC has not been defined. We therefore analyzed the immunogenicity of HER2 alterations in GC. Methods: Genomic profiling of DNA from 448 GC was performed using next-generation sequencing on 381 cancer associated genes. The expression of PD-L1 protein was evaluated in 192 GC with the use of an automated immunohistochemical assay (Ventana, SP263). Whole-exome sequencing, copy number variations, RNA-seq and clinical data of 443 GC from The Cancer Genome Altas (TCGA) were also analyzed to further evaluate the immunogenicity of HER2 alterations. TMB was defined as number of somatic non-synonymous mutations in coding regions. HER2 amplification in TCGA was defined as “2” derived from the copy-number analysis algorithms GISTIC. Results: HER2 alterations including amplification, missense and fusion were present in 19.2% (85/443) of TCGA cohort and 11.4% (51/448) of clinical cohort. 14.0% (62/443) of TCGA cohort and 6.0% (27/448) of clinical cohort harbored HER2 amplification. Higher TMB was observed in MSS/MSI-L patients carrying any HER2 alterations in TCGA cohort (P = 0.009). On the contrary, HER2 alterations did not show a higher neoantigen level and HER2 alterations were associated with decreased immunogenicity in terms of immune-related gene mRNA expression and immune infiltrates. In clinical cohort, HER2 alterations was also significantly associated with higher TMB (P = 0.001). Meanwhile, a trend of shrinking proportion of PD-L1 expression was observed in HER2 alteration subgroup (2/23, 8.7%) than HER2 wild-type subgroup (47/169, 27.8%, P = 0.071). Furthermore, HER2 amplification had significant positive associations with HRD score (P = 0.0169) in TCGA cohort which indicated an increased degree of genome instability. Conclusions: Although HER2 alterations in GC were associated with increased TMB, HER2 alterations exhibited poor immunogenicity. These findings indicated that HER2 alterations might confer resistance to immune monotherapy.

TPX2/Aurora kinase A signaling as a potential therapeutic target in genomically unstable cancer cells

Genomic instability is a hallmark feature of cancer cells, and can be caused by defective DNA repair, for instance due to inactivation of BRCA2. Paradoxically, loss of Brca2 in mice results in embryonic lethality, whereas cancer cells can tolerate BRCA2 loss. This holds true for multiple DNA repair genes, and suggests that cancer cells are molecularly “rewired” to cope with defective DNA repair and the resulting high levels of genomic instability. In this study, we aim to identify genes that genomically unstable cancer cells rely on for their survival. Using functional genomic mRNA (FGmRNA) profiling, 16,172 cancer samples were previously ranked based on their degree of genomic instability. We analyzed the top 250 genes that showed a positive correlation between FGmRNA levels and the degree of genomic instability, in a co-functionality network. Within this co-functionality network, a strong cluster of 11 cell cycle-related genes was identified, including TPX2. We then assessed the dependency on these 11 genes in the context of survival of genomically unstable cancer cells, induced by BRCA2 inactivation. Depletion of TPX2 or its associated kinase Aurora-A preferentially reduced cell viability in a panel of BRCA2-deficient cancer cells. In line with these findings, BRCA2-depleted and BRCA2-mutant human cell lines, or tumor cell lines derived from Brca2–/–;p53–/– mice showed increased sensitivity to the Aurora-A kinase inhibitor alisertib, with delayed mitotic progression and frequent mitotic failure. Our findings reveal that BRCA2-deficient cancer cells show enhanced sensitivity to inactivation of TPX2 or its partner Aurora-A, which points at an actionable dependency of genomically unstable cancers.

Association between Genomic Instability and Evolutionary Chromosomal Rearrangements in Neotropical Primates.

During the last decades, the mammalian genome has been proposed to have regions prone to breakage and reorganization concentrated in certain chromosomal bands that seem to correspond to evolutionary breakpoints. These bands are likely to be involved in chromosome fragility or instability. In Primates, some biomarkers of genetic damage may be associated with various degrees of genomic instability. Here, we investigated the usefulness of Sister Chromatid Exchange as a biomarker of potential sites of frequent chromosome breakage and rearrangement in Alouatta caraya, Ateles chamek, Ateles paniscus, and Cebus cay. These Neotropical species have particular genomic and chromosomal features allowing the analysis of genomic instability for comparative purposes. We determined the frequency of spontaneous induction of Sister Chromatid Exchanges and assessed the relationship between these and structural rearrangements implicated in the evolution of the primates of interest. Overall, A. caraya and C. cay presented a low proportion of statistically significant unstable bands, suggesting fairly stable genomes and the existence of some kind of protection against endogenous damage. In contrast, Ateles showed a highly significant proportion of unstable bands; these were mainly found in the rearranged regions, which is consistent with the numerous genomic reorganizations that might have occurred during the evolution of this genus.

Study of Telomere Dysfunction in TP53 Mutant LoVo Cell Lines as a Model for Genomic Instability.

Telomere restriction fragment, 3D quantitative FISH on nuclei, and quantitative FISH on metaphases are complementary approaches that explore telomere dysfunction genomically, cellularly, and chromosomally, respectively. We used these approaches to study association between telomere dysfunction and degree of genomic instability related to TP53 mutations in LoVo isogenic cell lines. We found a strong correlation between degree of genomic instability, telomere dysfunction, and specific mutations of TP53. The use of complementary approaches to study telomere biology is essential to have a comprehensive understanding of telomere involvement in genomic instability.

Abstract 1456: Identifying early genetic steps in malignant transformation of neurofibromatosis type 1- associated plexiform neurofibromas

Abstract BACKGROUND: Neurofibromatosis type 1 (NF1) is a genetic tumor predisposition disorder caused by germline mutations in tumor suppressor NF1. Plexiform neurofibromas (PN) are benign tumors that arise prenatally or early in childhood and affect 30-50% of NF1 population. Somatic inactivation of second copy of NF1 is believed to be primary genetic event leading to PN initiation. NF1 patients have 8-12% lifetime risk of developing malignant peripheral nerve sheath tumor (MPNST), a highly aggressive soft tissue sarcoma, often arising from pre-existing PN and atypical NF (ANF). ANF are pre-malignant tumors that often arise within PN and can transform into MPNST. They are distinct from both PN and MPNST clinically and histologically, thus representing an intermediate step in malignant transformation. Several studies identified deletion of the CDKN2A/2B locus as the most frequent genetic event in ANF, however it is not clear whether other genes or pathways play role in PN transformation into ANF and further into MPNST. In this study, we performed genomic analysis of 16 ANF and 4 MPNST matched with normal DNA obtained from 14 and 4 patients, respectively. METHODS: We performed whole exome sequencing and whole transcriptome RNASeq analyses on Illumina Hi-Seq 2500 platform and copy-number variant (CNV) analysis on Illumina HumanOmniExpressExome-8 SNP-arrays. In addition, we performed deep sequencing of NF1 and validation of select mutations on IonTorrent platform. For select tumors we estimated growth rate and metabolic activity by using volumetric MRI and FDG-PET. RESULTS: We identified inactivation of NF1 in the majority of ANF and all MPNST. We also detected CDKN2A/B locus deletion in the majority of ANF and MPNST (heterozygous in ANF and mostly homozygous in MPNST). We determined that PRC2 genes (EED and SUZ12) were mutated in multiple MPNST but never in ANF. We identified a low number of point mutations and small indels in the genomes of ANF (median 1, range 0-4) and somewhat elevated mutation burden in MPNST (median 23, range 18-31), however none of these mutations were recurrent and none of the mutant genes (other than NF1 and CDKN2A) were present in multiple samples. We found 93 CNV per tumor (median) in ANF that constituted ~2% of their genomes. In comparison, we observed 2,249 CNV (median) in MPNST that comprised ~75% of their genomes. We didn’t detect significant correlation between growth rate or metabolic activity and the degree of genomic instability or mutation burden in the tumors, however the size of the sample set was modest. RNAseq data analysis is pending. CONCLUSIONS: It appears that PN-ANF transition is predominantly if not exclusively driven by heterozygous deletion of the CDKN2A/2B locus. Further progression to MPNST likely involves homozygous loss of CDKN2A/B and complete inactivation of the PRC2 complex. Widespread LOH in MPNST may accelerate inactivation of key gatekeepers. Citation Format: Alexander Pemov, Nancy F. Hansen, Rajesh Patidar, Christine Higham, Eva Dombi, Joseph F. Boland, Settara C. Chandrasekharappa, NIH Intramural Sequencing Center, James C. Mullikin, Margaret Wallace, Javed Khan, Eric Legius, Brigitte Widemann, Douglas R. Stewart. Identifying early genetic steps in malignant transformation of neurofibromatosis type 1- associated plexiform neurofibromas [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 1456. doi:10.1158/1538-7445.AM2017-1456