Genomic Copy Number Changes Research Articles

Single-cell detection of copy number changes reveals dynamic mechanisms of adaptation to antifungals in Candida albicans.

Genomic copy number changes are associated with antifungal drug resistance and virulence across diverse fungal pathogens, but the rate and dynamics of these genomic changes in the presence of antifungal drugs are unknown. Here we optimized a dual-fluorescent reporter system in the diploid pathogen Candida albicans to quantify haplotype-specific copy number variation (CNV) and loss of heterozygosity (LOH) at the single-cell level with flow cytometry. We followed the frequency and dynamics of CNV and LOH at two distinct genomic locations in the presence and absence of antifungal drugs in vitro and in a murine model of candidiasis. Copy number changes were rapid and dynamic during adaptation to fluconazole and frequently involved competing subpopulations with distinct genotypes. This study provides quantitative evidence for the rapid speed at which diverse genotypes arise and undergo dynamic population-level fluctuations during adaptation to antifungal drugs in vitro and in vivo.

HGG-36. ASSESSMENT OF CHROMOSOMAL ABERRATIONS IN DIFFUSE ASTROCYTOMAS OF CHILDHOOD USING THE ARRAY COMPARATIVE GENOMIC HYBRIDIZATION (ACGH+SNP) TECHNIQUE – PRELIMINARY STUDY

Abstract BACKGROUND Comprehensive knowledge of the genomic changes underlying cancer is a key basis for diagnosis, prognosis and targeted therapies. With recent advances, the technique of comparative genomic hybridization (aCGH+SNP) facilitates genome-wide screening for genome copy number changes and rapid examination of numerical and structural chromosomal changes, as well as loss of heterozygosity at high resolution down to several tens of kilobases. METHODS The study included 12 patients aged 6 months to 15 years, operated on at the Department of Pediatric Neurosurgery in Krakow due to paediatric-type diffuse gliomas (according to WHO classification 5th Ed). Tumor tissue samples were collected from all included participants as remnants of routine examination. Pangenomic profiling of diffuse astrocytomas was performed with using SurePrint G3 Cancer CGH+SNP Kit 4x180K (Agilent, Santa Clara USA) at the Molecular Genetics Laboratory of the University Children’s Hospital in Krakow. Genomic DNA was extracted automatically from deep-frozen tumor tissue. Descriptive statistics were used to describe the basic characteristics of the data included in the study due to the small number of participants. RESULTS Of the 12 tumors examined by microarray, 5 patients had abnormalities in the form of diffuse high grade gliomas. In 1 patient, only numerical changes in chromosomes were observed, in 1 case only structural chromosomal abnormalities were detected. Combined numerical and structural aberrations were presented in 3 samples. Additionally, we detected 1 case of triploidy and 1 case of loss of heterozygosity. CONCLUSIONS Significant changes were observed in paediatric-type diffuse high-grade gliomas. Interestingly, we detected new undescribed alterations that were not only of a chromosomal nature, but also concerned individual genes. This may constitute the basis for further research aimed at establishing new diagnostic and prognostic markers, as well as effective therapy. A limitation of this study is the small patient sample, and further research is recommended.

Abstract 4293: Chromosomal instability causes epigenetic aberrations in cancer

Abstract Chromosomal instability and changes in the epigenome are defining features in most metastatic cancers. Our research links chromosomal missegregation, their containment in micronuclei, and subsequent rupture of the micronuclear envelope to histone post-translational modification aberrations. This relationship was across all tested human and murine cancer cell lines, as well as non-transformed cells. Furthermore, we discovered that the observed changes in histone PTMs were caused by either micronuclear rupture or the persistence of histone PTM status during cell division. Moreover, we revealed substantial chromatin accessibility differences in micronuclei. Notably, there is a distinct bias in chromatin accessibility between promoter regions compared to distal and intergenic regions of the genome, which aligned well with observed changes in histone modifications. The induction of chromosomal instability leads to widespread, heterogeneous, and heritable abnormalities in the epigenetic landscape of cancer cells. Therefore, on top of genomic copy number changes, chromosomal instability causes epigenetic reprogramming, which adds another layer to cancer heterogeneity. Citation Format: Albert S. Agustinus, Duaa Al-Rawi, Bhargavi Dameracharla, Ramya Raviram, Bailey S. Jones, Stephanie Stransky, Lorenzo Scipioni, Jens Luebeck, Melody Di Bona, Danguole Norkunaite, Robert M. Myers, Mercedes Duran, Seongmin Choi, Britta Weigelt, Shira Yomtoubian, Andrew McPherson, Eleonore Toufektchan, Kristina Keuper, Paul S. Mischel, Vivek Mittal, Sohrab P. Shah, John Maciejowski, Zuzana Storchova, Enrico Gratton, Peter Ly, Dan Landau, Mathieu F. Bakhoum, Richard P. Koche, Simone Sidoli, Vineet Bafna, Yael David, Samuel F. Bakhoum. Chromosomal instability causes epigenetic aberrations in cancer [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 4293.

Abstract A078: Identifying and targeting the genetic determinants of immune suppression and immunotherapy failure in prostate cancer

Abstract The advent of immunotherapies such as immune checkpoint blockade (ICB) has greatly expanded the therapeutic options available to patients with treatment-refractory solid tumors. However, patients with castration-resistant prostate cancer (CRPC) generally remain unresponsive to ICB therapy due to an immunologically “cold” prostate tumor microenvironment (TME) with limited accessibility to cytotoxic lymphocytes such as Natural Killer (NK) and T cells that can mediate potent anti-tumor immunity. During the course of tumor evolution, CRPCs acquire many genomic alterations and copy number changes that can influence therapy outcomes. Yet the impact of such genetic alterations on the immune TME and response to ICB remains largely unknown, in part due to the lack of models to functionally study them in an immune competent setting. To this end, we previously developed a novel electroporation-based genetically engineered mouse model (EPO-GEMM) to rapidly generate genetically defined prostate tumors de novo within an intact prostate TME (Leibold*, Ruscetti* et al., Cancer Discovery 2020). Here, we applied the EPO-GEMM approach to characterize the immune landscapes of distinct genetic subtypes of CRPC and subsequently identify and target tumor intrinsic mediators of immune suppression for prostate cancer immunotherapy. Using transposon systems to overexpress the MYC oncogene and CRISPR/Cas9 vectors to inactivate tumor suppressor genes (TSGs) commonly altered in CRPC (Pten, P53, Rb1, Apc), we generated a suite of genetically distinct prostate cancer-bearing EPO-GEMMs. Histological analysis revealed genotype-specific immune infiltrates, with MYC-driven tumors generally lacking cytotoxic CD8+ T cells and harboring increased numbers of regulatory T cells (Tregs). A similar correlation was also observed between MYC levels and cytotoxic lymphocyte suppression in CRPC patient samples. In particular, tumors with compound MYC overexpression and p53 loss (MP) displayed drastic NK cell suppression. Cytokine and RNA-seq profiling of primary EPO-GEMM CRPCs, tumor-derived cell lines, and isogenic Myc-CaP cells engineered with TSG losses revealed that MP alterations led to not only inhibition of inflammatory cytokine and interferon signaling and antigen presentation/processing gene expression, but also induction of VEGF signaling and increased angiogenesis in the prostate TME. Treatment of MP tumors with a VEGFR2 antibody to block VEGF signaling significantly reduced tumor growth and increased NK and CD8+T cell infiltration and activation, indicating a phenotypic switch from a traditionally “cold” TME. Our results demonstrate that the genetic configuration of prostate cancer shapes its surrounding TME. Additionally, targeting VEGF signaling may be a mechanism to overcome ICB resistance in prostate cancer. More broadly, we believe that by understanding the tumor intrinsic mechanisms driving immune suppression, we can identify rational immunotherapy combinations for CRPC based on the genetic fingerprint of a patient’s tumor for precision medicine. Citation Format: Katherine C. Murphy, Kelly DeMarco, Yvette Lopez-Diaz, Lin Zhou, Marcus Ruscetti. Identifying and targeting the genetic determinants of immune suppression and immunotherapy failure in prostate cancer [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr A078.

Abstract SY04-01: Understanding and targeting RNA splicing in breast cancer

Abstract Upregulation of SR proteins, a family of 14 essential splicing factors (SFs), often occurs in solid tumors, including in breast, and promotes mammary cell transformation and metastasis in breast cancer models. SR protein alterations often occur without genomic copy number changes, suggesting misregulation at the transcriptional or post-transcriptional levels; however the underlying molecular mechanisms are incompletely understood. Here we investigate the transcriptional and post-transcriptional regulatory mechanisms that control SF-levels in normal mammary cells and their misregulation in cancer cells. We demonstrate the MYC oncogene directly regulates a network of splicing factors that are co-expressed in tumors and act together to regulate their downstream targets and promote cell invasion. In parallel, our work reveals the role of non-coding ‘poison exon’ in regulating SR proteins levels during cell differentiation and in tumors. We uncover an extensive cross-regulatory network used by the SR protein family to control their expression. Finally, we develop splice-switching antisense oligonucleotides to reverse the increased skipping of TRA2β poison exon detected in tumors, leading to selective anti-proliferative effects across a variety of cancer models. In summary, our work provides new insights into the regulatory mechanisms of SR proteins and identifies novel oncogenic spliced isoforms that represent potential biomarkers and targets for therapeutics development. Citation Format: Olga A. Anczukow. Understanding and targeting RNA splicing in breast 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 SY04-01.

A molecular study of pediatric pilomyxoid and pilocytic astrocytomas: Genome-wide copy number screening, retrospective analysis of clinicopathological features and long-term clinical outcome.

Pilocytic Astrocytoma (PA) is the most common pediatric brain tumors. PAs are slow-growing tumors with high survival rates. However, a distinct subgroup of tumors defined as pilomyxoid astrocytoma (PMA) presents unique histological characteristics and have more aggressive clinical course. The studies on genetics of PMA are scarce. In this study, we report one of the largest cohort of pediatric patients with pilomyxoid (PMA) and pilocytic astrocytomas (PA) in Saudi population providing a comprehensive clinical picture, retrospective analysis with long-term follow-up, genome-wide copy number changes, and clinical outcome of these pediatric tumors. We examined and compared genome-wide copy number aberrations (CNAs) and the clinical outcome of the patients with PA and PMA. The median progression free survival for the whole cohort was 156 months and it was 111 months for the PMA, however, not statistically significantly different between the groups (log-rank test, P = 0.726). We have identified 41 CNAs (34 gains and 7 losses) in all tested patients. Our study yielded the previously reported KIAA1549-BRAF Fusion gene in over 88% of the tested patients (89% and 80% in PMA and PA, respectively). Besides the fusion gene, twelve patients had additional genomic CNAs. Furthermore, pathway and gene network analyses of genes in the fusion region revealed alterations in retinoic acid mediated apoptosis and MAPK signaling pathways and key hub genes that may potentially be involved in tumor growth and progression, including BRAF, LUC7L2, MKRN1, RICTOR, TP53, HIPK2, HNF4A, POU5F, and SOX4. Our study is the first report of a large cohort of patients with PMA and PA in the Saudi population that provides detailed clinical features, genomic copy number changes, and outcome of these pediatric tumors and may help better diagnosis and characterization of PMA.

A Methodologically Updated De-Novo Extraction of Copy Number Signatures in Multiple Myeloma: Clinical Significance and Putative Aetiologies

BACKGROUND: The tumor genomes of most Multiple Myeloma (MM) patients are heavily burdened with highly heterogenous copy number (CN) alterations, as detected by multiple molecular methods including whole genome sequencing (WGS), and have been shown to have a strong prognostic and predictive significance for patients' survival. Cutting-edge computational developments have made it possible to analyze complex genomic CN changes by identifying CN alterations' recurrent patterns among large cohorts of patients, named CN signatures (CNS), that are the result of cumulative chromosomic instability (CIN) processes throughout the course of cancer cells evolution. In fact, a robust methodological framework for CNS computation, along with a large compendium of CNS aetiologies have been recently published for many cancer types, notably not including MM (Drews 2022). To our knowledge, the only study that analyzes CNS in MM focused on the role of CNS in predicting chromothripsis events, but remarkably it did not include neither CNS aetiologies nor complete CNS characterization for all the discovered CNS (Maclachlan 2021). AIM: To define a novel methodological framework, based on previous studies, aimed at the identification of CNS in MM, in order to assess both the aetiologies and the biological significance of MM CNS and to evaluate their prognostic impact on MM clinical outcome. The newly extracted CNS defined at diagnosis will be also used as novel disease biomarkers, to develop an improved, aetiology-based MM patients' stratification in different molecular subtypes. METHODS: We calculated the MM genomic distributions of the six essential CN features (segment length, breakpoint per 10 Mb, breakpoint per chromosome arm, segment CN change, CN value, length of oscillating CN states) that have been previously shown to encode patterns of CN alterations, underlying the observed tumor CIN. Starting from 886 WGS generated CN profiles, included in the CoMMpass study, the above mentioned features were computed. Features were then categorized into components, by using mixture models' decomposition and CNS were finally extracted from the components, by using both a Hierarchical Dirichlet Process (HDP) and a Non-Negative Matrix Factorization (NNMF) approach. RESULTS: The main novel characteristics of the developed methodological framework aimed at CNS assessment were 1) the use of a "continuous CN value" feature, which enabled the evaluation of sub-clonal events and 2) the use of a logarithmic scale in "segment length" feature, which favored a higher resolution for categorizing focal and/or gene level CN events, that are very common in MM. Thanks to these implementations, 33 Gaussian mixture components were identified (as compared to 28 detected in Maclachlan 2021). After deriving a Sample x Component - Sum of Posteriors Matrix, the signatures were extracted by applying two parallel state-of-art approaches, namely HDP and NNMF. This allowed the extraction of 9 CNS that were characterized by their component's composition. The signature's exposure levels were correlated to well-known MM biomarkers (e.g. TP53 mut and/or del, 1q CN gain, 13q CN loss, t-IgH, hyperdiploidy), showing that all signatures correlated to at least one of the well known MM biomarkers; in particular, CN.SIG5 exposures was found to correlate to high-risk MM biomarkers (TP53 p<0.001, 1q CN gain p<0.001, t(4;14) p<0.001), thus suggesting its possible involvement in the aetiology of this peculiar genomic configuration. Finally, a survival analysis was performed in patients characterized by high exposure (4th quartile) to the CN.SIG5 (75 patients), as compared to the others (811 patients), showing a significant negative impact of this CNS on both overall (OS p<0.001)) and progression free survivals (PFS p<0.001). Cox-analysis revealed an OS HR= 1.37 p<0.001, PFS HR= 1.16, p<0.001, per 5% increase in exposure. CONCLUSION: By employing a novel bio-informatic approach, based on the use of continuous CN data for CNS extraction, 33 feature's components were identified. We observed that CN.SIG5 significantly affected patients carrying well-known high-risk genomic features, and patients highly exposed to this CNS had decreased PFS and OS. Additional characterizations are needed to unveil the biological meaning of CNS exposure; however, MM CNS, while informing on disease outcome, might be considered as new comprehensive biomarkers in this disease. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

High levels of chromosomal instability facilitate the tumor growth and sphere formation.

Most cancer cells show chromosomal instability (CIN), a condition in which chromosome missegregation occurs at high rates. Growing evidence suggests that CIN is not just a consequence of, but a driving force for, oncogenic transformation, although the relationship between CIN and tumorigenesis has not been fully elucidated. Here we found that conventional two‐dimensional (2D) culture of HeLa cells, a cervical cancer‐derived cell line, was a heterogenous population containing cells with different CIN levels. Although cells with high‐CIN levels (high‐CIN cells) grew more slowly compared with cells with low‐CIN levels (low‐CIN cells) in 2D monolayer culture, they formed tumors in nude mice and larger spheres in three‐dimensional (3D) culture, which was more representative of the in vivo environment. The duration of mitosis was longer in high‐CIN cells, reflecting their higher mitotic defects. Single‐cell genome sequencing revealed that high‐CIN cells exhibited a higher karyotype heterogeneity compared with low‐CIN cells. Intriguingly, the karyotype heterogeneity was reduced in the spheres formed by high‐CIN cells, suggesting that cells with growth advantages were selected, although genomic copy number changes specific for spheres were not identified. When we examined gene expression profiles, genes related to the K‐ras signaling were upregulated, while those related to the unfolded protein response were downregulated in high‐CIN cells in 3D culture compared with 2D culture, suggesting the relevance of these genes for their survival. Our data suggested that, although CIN is disadvantageous in monolayer culture, it promotes the selection of cells with growth advantages under in vivo environments, which may lead to tumorigenesis.

Abstract P3-08-05: Multi-omic profiling of simultaneous ductal carcinoma in situ and invasive breast cancer

Abstract Background: Simultaneous ductal carcinoma in situ (DCIS) and invasive breast cancer (IBC) is very common. However, it is not clear how often these histologies are clonally related or the mechanism by which DCIS evolves into IBC. In this study we evaluated tissue samples of DCIS and IBC by DNA and RNA sequencing from 39 patients with concurrent disease.Methods: Patients were identified from the Swedish Cancer Institute Breast Cancer Database starting sequentially with 2018 backwards to identify candidates with simultaneous DCIS and IBC. Individual pathologic review was performed from the cohort of patients who had had modified radical mastectomy to select 50 cases for analysis of normal breast, DCIS, and invasive disease. Sequencing was performed using the Tempus xT solid tumor assay (DNA-seq of 648 genes at 500x coverage, full transcriptome RNA-seq). The mutations identified by this assay include germline and/or somatic single nucleotide variants, insertions/deletions, and copy number variations. Results: Thirty-nine cases had adequate tissue for both DCIS and IBC for DNA and 31 for RNA analysis. DCIS and IBC were in the same quadrant of the breast for all cases. All IBC cases were ductal carcinoma. 38/39 IBC were estrogen receptor (ER) positive, 33/39 were progesterone receptor (PR) positive. 5 were HER2/neu amplified (three ER+/PR-, 1 ER+/PR+, 1 ER/PR-). 32/39 had Ki67 of 20% or less (IQR 14(9,19.5)). 35/39 were T1 or T2. Three were T3 and one T4. 21/39 were N0, 4/39 N1mic, 9/39 N1a, 3/33 N2a 2/39 N3a.For DCIS 24/39 had both genomic variants and copy number changes (V+C+), 7/39 were V+C-, 5/39 were V-C+ and 3/39 V-C-. For IBC 25/39 were V+C+, 7/39 were V+C-, 6/39 were V-C+, and 1/39 V-C-. In total, there were 36 unique genomic variants seen in DCIS but not the corresponding invasive sample. In contrast, there were 32 total unique variants seen in invasive but not the corresponding DCIS and 38 variants were seen in both samples. The one BRCA variant seen in both samples of a single patient was BRCA1 p.Q284, a known pathogenic variant but gBRCA normal. There were 86 copy number changes seen in DCIS but not IBC, 75 copy number changes seen in IBC but not DCIS and 47 copy number changes seen in both DCIS and IBC. 24/39 patients shared at least one common variant and 22/39 shared at least one copy number change. 4/39 had no copy changes in either sample but all of these had genomic variants. 5/39 had no variants in either sample but all of these had copy number changes. PIK3CA was seen in 2/39 IBC, 2/39 DCIS and 2/39 in both. TMB IQR was 1.6(1.1, 3.4) for IBC and 2.1 (1.1,3.2) for DCIS. All samples tested for MSI were stable. Only one patient had identical genomic variants and copy changes in both DCIS and IBC. The most frequent changes were: RNA sequencing revealed 1178 genes with significant gene expression differences between paired DCIS and IBC (q&amp;lt;0.001). RNA-seq data was quantified using Kallisto and differential gene expression was assessed using DESeq2. Pathway enrichment was performed using rank GSEA against the MSigDB C2 collection (filtered to KEGG, Reactome and Biocarta pathways only). Upregulated pathways in DCIS involved more immunogenic pathways while IBC upregulated pathways involved proliferation and cell cycling. Conclusions: The transition from DCIS to IBC is a dynamic process. How much of these differences are the result of tumor heterogeneity and temporal factors as opposed to truly separate clones will require more study. This could have significant implications for the treatment of DCIS. VariantGain/Loss of FunctionIBC onlyDCIS onlySame in BothTotalTP53Loss53614MAP3K1Loss51511GATA3Gain3249KM2TCLoss0538PTENLoss2237Copy ChangeMCL1 (1q21.2)Gain68822CKS1B (1q21.3)Gain95619ERRB2 (17q12)Gain0459JAK1 (1p31.3)Loss2428IKZF1 (7p12.1)Loss2327 Citation Format: Henry G Kaplan, Anna B Berry, Alexa Dowdell, Brian Piening. Multi-omic profiling of simultaneous ductal carcinoma in situ and invasive breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P3-08-05.

Abstract 587: Genomic copy number profiling of single CTCs reveals clonal evolution in metastatic breast cancer and identifies actionable targets for informing treatment decisions

Abstract Introduction: Genomic profiling of circulating tumor cells (CTCs) is an approach to help to understand tumour heterogeneity and evolution in metastatic breast cancer (MBC) to inform prognosis and guide treatment options. We showed previously that single CTCs provide complementary information on hot spot mutations to those detected in circulating tumor DNA (ctDNA) and the primary tumour tissue, and now extend analyses to include genomic copy number changes. Herein, we report that identification of actionable targets through copy number profiling of single CTCs is achievable at any time point during metastatic progression and could be a valuable tool to guide treatment options. Methods: Fourteen patients (13 12 progressing and 1 2 responding) who were receiving treatment for MBC (12 13 HR pos, 1 HER2 pos, I1 TNBC) were recruited to the study with ethical consent and followed up with a single blood sample. A 7.5ml blood sample was collected into a CellSave Preservative tube and processed and counted within 96 hours of collection using the CELLSEARCH® system and the Circulating Tumor Cell Kit (Menarini Silicon Biosystems). The DEPArray™ System was used to identify and recover individual, intact CTCs of epithelial origin (Cytokeratin 8, 18, 19 +ve, CD45 -ve) and white blood cells (WBCs) as single cells or as a small pool of cells. Whole genome amplification was performed using the Ampli1 WGA kit. Libraries were generated using the Ampli1 LowPass Kit (Menarini Silicon Biosystems) and samples passing QC were analysed f by next generation sequencing on an S5 platform (Thermo fFisher). Results were analysed using the MSBiosuite software and data was compared with in- house informatics workfows. Results: From the 14 patients included in the study, we profiled a total of 97 samples using the Ampli1 LowPass sequencing workflow comprising 79 individual cells (across 14 patients) and 18 pools of approximately 10 cells. Successful genome- wide copy number profiles were generated in 12 of the 14 patients; samples from 2 patients had low QC scores and were inconclusive. In all, 12 patients individual CTCs from 12 patients showed similar copy number profiles, but with some heterogeneity, that revealed gene specific changes not detected by the cell pool. Moreover, 10 of the 12 patients had gene- specific changes (5 with CCND1 amplification, 2 with FGFR1 amplification, 1 with JAK2 amplification and 2 with STK11 deletion) that could have guided treatment decisions, for example offering an FGFR1 or JAK2 inhibitor. Conclusion: This study highlights the genomic heterogeneity in CTCs seen in MBC and the potential of monitoring gene specific changes to identify actionable targets in order to inform treatment decisions. The work requires further evaluation and validation but it may offer a new approach to managing treatment decisions in MBC for those patients with detectable CTCs. Citation Format: Daniel Fernandez Garcia, Georgios Nteliopoulos, Robert Hastings, Amelia Rushton, Karen Page, Luke J. Martinson, Molly Gray, David S. Guttery, Alberto Ferrarini, Nicolò Manaresi, Charles Coombes, Jacqui Shaw. Genomic copy number profiling of single CTCs reveals clonal evolution in metastatic breast cancer and identifies actionable targets for informing treatment decisions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 587.

Title: SNP Microarray Reveals Predicted Outcomes of a Novel High Risk AML Subgroup with ERG Amplification

Increased expression of ERG (ETS-related gene), a proto-oncogene within the ETS (erythroblast transformation specific) family, is associated with an unfavorable clinical outcome in AML patients. A mechanism for ERG over-expression is ERG amplification. ERG amplification is present in 4-6% of adult AML. This study investigates the genetic profile of ERG-amplified AML by assessment of genomic copy number changes and mutational status of AML-related genes and how this correlates to treatment response and overall outcome. Currently copy number analysis is not recommended by ELN or WHO as a diagnostic standard in AML. An institutional retrospective review of 205 adult AML cases from 2015 to 2018 provided two cohorts: a discovery cohort and control cohort. The discovery cohort includes 11 cases (4%) with ERG amplification. All cases in the discovery cohort include either TP53 mutation and/or loss/LOH of 17p. Thus, we developed a control cohort of 39 cases with TP53 abnormalities without ERG amplifications to determine the role TP53 plays in the observed outcomes. The 179 adult AML cases from the TCGA public database comprises the validation cohort, which incorporates both copy number and molecular sequencing data. Approximately 4% of AML cases in the TCGA dataset show ERG amplification. For our discovery and control cohorts, genomic DNA was extracted (Maxwell, Promega, Madison, WI) from fresh bone marrow aspirates. Copy number/LOH analysis was performed using the CytoSNP microarray (Illumina, San Diego, CA). Sequence variants were evaluated in hotspot regions of 49 genes using the ThunderBolts Myeloid Panel library preparation (RainDance Technologies, Billerica, MA) and paired-end sequencing (MiSeq, Illumina, San Diego, CA). Alignment and variant calling was performed with MiSeq Reporter (Illumina) and NextGene (Soft Genetics, State College, PA) and sequences were annotated using VarSeq (Golden Helix, Bozman, MT). SNP microarray analysis of the discovery cohort revealed a distinct pattern of complex cytogenetics including chromothripsis, TP53 loss/LOH and loss of 5q. Molecular sequencing was available for 10 of 11 patients. All specimens analyzed for mutational status demonstrated TP53 loss of function mutations. The majority of cases (9/11) demonstrated complete loss of function of TP53 by mutation in conjunction with a "second hit" in the form of loss due to deletion or LOH of 17p. These nine patients failed to achieve any response with chemotherapy with a median survival time of 3.8 months. The two surviving patients had a single TP53 mutation with an intact second allele. Both of these patients are post-allogeneic stem cell transplant with no evidence of relapse. There is an association of ERG amplification with MDS as 8 of the 11 patients transformed from very-high risk MDS. The interaction between ERG and TP53 allows one to predict treatment response and outcomes based on the presence or absence of an intact TP53 allele. Within the control group, there were no significant differences in survival or response to treatment based on number of intact TP53 alleles. The validation cohort of TCGA patients confirmed the strong positive correlation between ERG amplification and TP53 mutations. There were 8 cases with ERG amplification. Of these, 6 cases had a similar genetic pattern found in the discovery cohort including TP53 mutations, loss of 5q and complex cytogenetics. The median overall survival (OS) of this cohort of ERG amplified + TP53 mutation was 2.5 months. ERG amplification classifies a high-risk population of AML detected by copy number/LOH analysis with a distinct genetic signature including loss of 5q, complex cytogenetics including chromothripsis and loss of function TP53 mutation. The successive loss of the second TP53 allele in the setting of ERG amplification identifies a subset of patients with resistance to cytarabine and a very poor prognosis. These patients are unlikely to benefit from cytotoxic chemotherapy and may have improved outcomes with alternative regimens and/or clinical trial. ERG amplification is associated with AML that transforms from very-high risk MDS. Recognizing these patients before transforming to AML could allow for early transplant. This high-risk subgroup is currently un-recognized by standard cytogenetics and genetic mutation profiling; therefore, we recommend that copy number/LOH analysis become part of the standard diagnostic testing for MDS and AML. Disclosures No relevant conflicts of interest to declare.

Abstract 446: Plasma genome sequencing identifies prostate cancer patients that are sensitive to platinum-based therapy

Abstract Castration-resistant prostate cancer (CRPC) is a lethal disease. A subset of these patients present with atypical clinical characteristics and aggressive disease behavior. These patients, classified as Aggressive Variant Prostate Cancer (AVPC), may derive benefit from platinum-based chemotherapy. However, the identification of AVPC patients is often challenging due to heterogeneity in clinical presentations and potentially in biological drivers, which may not be fully reflected in a biopsy obtained from a single tumor site. To address these challenges, we developed an unbiased genome sequencing method called PEGASUS (Plasma Exome and Genome Analysis by Size-Selection and Unbiased Sequencing) that enables the simultaneous detection of copy number aberrations and exome mutations from circulating-tumor DNA (ctDNA). We applied this method to plasma specimens obtained from 160 CRPC patients with and without AVPC participating in a randomized trial of cabazitaxel alone or in combination with carboplatin. We were able to successfully isolate ctDNA (&amp;gt;2 ng) for genomic profiling using PEGASUS in 91/160 (57%) CRPC patients. Among these 91 CRPC patients, we detected common prostate cancer mutations as well as genomic copy number changes in genes such as RB1, PTEN and TP53 and several other genes associated with DNA repair. One of the most salient features that distinguished the patients’ outcomes was whether they had diploid or aneuploid genomes detected by whole genome profiling of the ctDNA. Patients with aneuploid ctDNA had worse progression-free and overall survival. Overall, our data shows the feasibility of performing whole-genome and exome profiling of ctDNA in CRPC patients to characterize the molecular features associated with distinct clinical presentations. Candidate markers associated to benefit from platinum-based chemotherapy are being evaluated. These results pave the way for future clinical applications in biomarker discovery to assist treatment decisions in prostate cancer patients. Citation Format: Naveen Ramesh, Emi Sei, Pei Ching Tsai, Christopher Logothetis, Paul Corn, Ana Aparicio, Amado J. Zurita, Nicholas E. Navin. Plasma genome sequencing identifies prostate cancer patients that are sensitive to platinum-based therapy [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 446.

Abstract SY31-03: Predictive biomarkers in circulating tumor cells to direct management metastatic castration-resistant prostate cancer (mCRPC)

Abstract Tissue and cfDNA NGS profiling is improving our ability to detect and clinically validate the significance of molecular alterations that drive tumor growth resulting in actionable therapeutic strategies that can improve outcomes for subsets of patients and accelerate the regulatory approval of targeted agents. Confounding our knowledge base are three critical aspects: 1) intra patient and intra-tumor heterogeneity for which the clustering of alterations and signaling pathways cannot be easily disassociated without cellular context, 2) epigenetic and functional cellular biology alterations which perturb genomic pathways presumed to drive tumor growth, and 3) understanding the clinical significance of “variants of uncertain significance” (with respect to changes in protein function) (1) (https://www.ncbi.nlm.nih.gov/pubmed/26582918). Our objective is to improve the outcomes for men with mCRPC starting by identifying unmet needs at decision points in management, credentialing biomarkers to better inform the decision, followed by the analytical and clinical validation of the assay and the biomarker in a sequence of trials in which samples are obtained at the decision point and associated with outcomes, and showing that use of the test result to direct management improves patient outcomes compared to non-use of the test. Our focus is on predictive biomarkers of sensitivity and resistance assessed at the level of the single circulating tumor cell (CTC) through Functional Cell Profiling (FCP) analyzed by 1) protein characterization &amp; localization, 2) cellular and sub-cellular morphology aggregated with machine learned CTC profiles, and 3) low pass whole genome copy number changes. Establishing a phenotype genotype relationship enables a rapid turnaround of test results to inform decision making for an individual patient. Building on the initial results reported by Investigators at JHU (2) showing the association of the presence of the ARV7 (androgen receptor splice variant 7) in CTC using a PCR based assay predicted for resistance to androgen receptor signaling inhibitors (ARSI) but not taxane based chemotherapy, we applied a protein based assay that enabled distinguishing the localization of the splice variant, nuclear vs. cytoplasmic that showed increased specificity, resistance to ARSI, and clinical utility based on a treatment specific interaction that showed in a survival benefit for treatment with a taxane for patients with AR-V7+ CTC (3, 4). The assay, now marketed, has also received a favorable coverage determination from CMS. Later, building on the regulatory precedent for image based phenotyping that enabled rapid cervical cancer screening, we analyzed CTCs based on protein expression and localization and digital pathology features similar to what is applied in facial recognition analyses, to define phenotypically unique cell type (5), studied independently for the relationship to sensitivity to standard of care (SOC) drugs. The aim was to enable rapid test turnaround to enable real time treatment selection decisions. Such profiling has identified unique cell types associated with resistance to ARSi’s and taxanes, and single cell genomic analyses confirmed differences between cell types, providing insight into the underlying mechanisms. More recently we have focused on the development of a phenotypic biomarker of genomic instability, an important driver of tumor progression, that utilizes morphology to identify CTCs with an abnormal number of single cell Large Scale Transitions (LSTs). The resultant biomarker, phenotypic LST (pLST), was then examined in patient CTCs prior to and shortly after treatment with an ARSI or taxane. Those determined to be pLST biomarker positive have worse OS than those who don’t. An important aspect of liquid biopsy analysis is the biologic characterization of the cells that persist, i.e. that are resistant de novo, and which emerging during therapy that have acquired resistance to treatment. In the case of CTCs, we have validated through 5 prospective phase III trials that the elimination of all CTCs on therapy portends a better clinical response than those who have residual CTCs (6). In this context, pLST CTC biomarker positivity ~1 mo after the start of therapy portends a poor outcome whereby fewer than 15% of patients survive beyond 1 yr. Importantly however, is that these pLST+ cells are/were eliminated in patients treated with a PARP inhibitor and had improved PSA response rates7, suggesting that specifically targeting genomic instability is a valid approach where other standard of care drugs fail. Functional Cell Profiling or FCP has also provided an improved understanding of the tumor heterogeneity common in many patients, occurrence of discordance between phenotype and genotype, and the greater biologic learnings of single cell analysis. The framework has allowed us to postulate a novel strategy for analyzing CTCs through FCP for prediction of therapeutic benefit and subclonal disease resistance. Characterizing CTCs in individual patients may provide insight into disease evolution, inform treatment decisions for individual patients and guide new drug development. Longitudinal monitoring of phenotypic CTC subtypes in multiple clinical trials is currently underway.

Unravelling subclonal heterogeneity and aggressive disease states in TNBC through single-cell RNA-seq

Triple-negative breast cancer (TNBC) is an aggressive subtype characterized by extensive intratumoral heterogeneity. To investigate the underlying biology, we conducted single-cell RNA-sequencing (scRNA-seq) of >1500 cells from six primary TNBC. Here, we show that intercellular heterogeneity of gene expression programs within each tumor is variable and largely correlates with clonality of inferred genomic copy number changes, suggesting that genotype drives the gene expression phenotype of individual subpopulations. Clustering of gene expression profiles identified distinct subgroups of malignant cells shared by multiple tumors, including a single subpopulation associated with multiple signatures of treatment resistance and metastasis, and characterized functionally by activation of glycosphingolipid metabolism and associated innate immunity pathways. A novel signature defining this subpopulation predicts long-term outcomes for TNBC patients in a large cohort. Collectively, this analysis reveals the functional heterogeneity and its association with genomic evolution in TNBC, and uncovers unanticipated biological principles dictating poor outcomes in this disease.

2. Distinct genetic signature of a high risk AML subgroup with ERG amplification

Recurrent amplification of 21q22 of the ERG locus has been detected in 6–9% of AML cases and reportedly causes increased gene expression which has been associated with poor overall survival and resistance to cytarabine. The purpose of the study was to determine a genetic profile of ERG-amplified AML by assessment of genomic copy number changes and mutational status of AML related genes.

EP-2295: HNSCC derived cell lines and subclones differ in genomic copy number changes and radiation response

EP-2295: HNSCC derived cell lines and subclones differ in genomic copy number changes and radiation response

The genomic road to invasion\u2014examining the similarities and differences in the genomes of associated oral pre-cancer and cancer samples

BackgroundIt is frequently assumed that pre-invasive lesions are simpler precursors of cancer and will contain a limited subset of the genomic changes seen in their associated invasive disease. Driver mutations are thought to occur early, but it is not known how many of these are present in pre-invasive lesions. These assumptions need to be tested with the increasing focus on both personalised cancer treatments and early detection methodologies.MethodsWe examined genomic copy number changes in 256 pre-invasive and invasive samples from 69 oral cancer patients. Forty-eight samples from 16 patients were further examined using exome sequencing.ResultsEvidence of a shared ancestor of both dysplasia and carcinoma was seen in all but one patient. One-third of dysplasias showed independent copy number events. The remainder had a copy number pattern that was similar to or simpler than that of the carcinoma. All dysplasias examined contained somatic mutations absent in the related carcinoma.Previously observed copy number changes and TP53 mutations were very frequently observed, and almost always shared between dysplasia and carcinoma. Other gene changes were more sporadic. Pathway analysis confirmed that each patient’s disease developed in a different way.Examining the numbers of shared mutations and the rate of accumulation of mutations showed evidence that all samples contain a population of sub-clones, with little evidence of selective advantage of a subset of these.ConclusionsThese findings suggest that most of the genomic changes driving oral cancer occur in the pre-cancerous state by way of gradual random accumulation rather than a dramatic single event.

Abstract MS2-2: MS2-2 The latest breakthroughs on the translational aspects of DNA repair pathways

Abstract Genomic instability exists in the majority of human cancer, and there is an underlying DNA repair defect driving the instability in most cases. There are seven defined DNA repair pathways that, when inactive, can produce genome wide patterns of mutations. Current cancer genomics can provide a significant insight into the underlying DNA repair defect. This information can be used to select targeted therapies or suggest a more likely response to immune checkpoint blockade. To use acquired homologous recombination (HR) deficiency as an example of this paradigm, we have found that large scale genomic rearrangements and segmental copy number changes are found in breast cancers without a known germline mutation and see a strong link to biallelic alterations in homologous recombination genes. We detected a genetic etiology of HR deficiency in 90% of studied HR-deficient cases, whereas HR-proficient tumors only showed biallelic HR gene mutations in &amp;lt;7% of cases tested. The goal now is to develop genomic biomarkers that could be used to identify cancer patients who would benefit from therapy with PARP inhibitors and other novel agents, such as those identified by studies of synthetic lethality. Synthetic lethality in human cancer is a developing area of new therapeutic discovery. HR deficiency is thought to be rescued by at least 3 different DNA repair pathways: base-excision repair, translesion synthesis and alternative end joining – all of which are active in proliferating cancer cells. In addition, even the canonical end-joining mechanism of Ku heterodimer binding to any double-stranded end is another component of handling DNA lesion intermediates. The reason for this variety of synthetic lethality has to be different critical DNA lesion intermediates. Knowledge of the DNA damage processing and the proteins involved in the rescue of lesions that would be repaired by HR can provide the basis for the identification of novel targets. I will review our progress in making RAD52 a potential target along with other initiatives for new drug discovery. Citation Format: Powell SN. MS2-2 The latest breakthroughs on the translational aspects of DNA repair pathways [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr MS2-2.

Genomic instability and proliferation/survival pathways in RB1-deficient malignancies.

Genomic instability (GIN) is a hallmark of most cancer cells. However, compared to most human cancer cell types, the retinoblastoma tumor cells show a relatively stable genome. The fundamental basis of this genomic stability has yet to be elucidated, and the role of certain proteins involved in cell cycle regulation may be the key to the development of these specific genotypes. We examined whether thyroid hormone receptor beta 1 and 2 (TRβ1 and TRβ2), known to regulate tumorigenesis, and PTTG1, a mitotic checkpoint protein, play a role in maintaining genomic stability in retinoblastoma. In order to elucidate the role of these proteins in development of aneuploidy/polyploidy, an indicator of GIN, we first studied comparative GIN in retinoblastomas and multiple RB mutant cancer cell lines using single nucleotide polymorphism (SNP) analysis. We then utilized pLKO lentiviral vectors to selectively modify expression of the targeted cell cycle proteins and interpret their effect on downstream cell cycle proteins and their relative effects on the development of polyploidy in multiple tumor cell lines. The SNP analysis showed that retinoblastomas displayed relatively fewer genomic copy number changes as compared to other RB1-deficient cancer cell lines. Both TRβ1 and TRβ2 knockdown led to accumulation of E2F1 and PTTG1 and increased GIN as demonstrated by an increase in polyploidy. Downregulation of PTTG1 led to a relative decrease in GIN while upregulation of PTTG1 led to a relative increase in GIN. Knockdown of E2F1 led to a downstream decrease in PTTG1 expression. Rb-knockdown also upregulated E2F1 and PTTG1 leading to increased GIN. We showed that Rb is necessary for PTTG1 inhibition and genomic stability. A relatively stable genome in retinoblastoma tumor cells is maintained by TRβ1 and TRβ2-mediated PTTG1 inhibition, counteracting Rb-deficiency-related GIN. TRβ1, TRβ2 and Rb-KD all led to the downstream PTTG1 accumulation, apparently through an activation of E2F1 resulting in extensive genomic instability as seen in other Rb-deficient tumors.

Abstract A34: Targeting genes which are amplified in ovarian cancer

Abstract Over the recent years significant knowledge has been accumulated on which drugs can be therapeutically beneficial for a given patient based on mutational profiling of their tumors using selected gene panels. Much less, however, is known under which conditions targeting of genes which are amplified could be beneficial. Apart from successfully targeting HER2-positive cancers, many of which frequently carry amplified ERBB2 gene, no other targeted therapy based on DNA amplifications or gains of the genes is offered to the patients due to lack of clinical response data. Using mate pair sequencing we have profiled 60 high grade serous ovarian tumors to compare genomic rearrangements and copy number changes. The analyses revealed that these tumors carry highly rearranged genome. Copy number changes amplifications, gains and losses were very common and included genes potentially therapeutically targetable. In contrast, only a few of targetable fusion genes were identified in a set of 60 cases. The tumors were propagated in immunocompromised mice (mouse avatars), sequenced and showed identical to original patient tumor landscape of rearrangements. Using these mouse avatar models we tested whether a therapy inhibiting selected based on genomic amplification targets provided a benefit compared to standard chemotherapy. Mice which harbored tumors showing amplifications of CDKs (CDK2, 4 and 9) and ERBB3 were treated with flavopiridol and pertuzumab respectively, and response was compared to that of standard for ovarian cancer chemotherapy (carboplatin and paclitaxel combination). We observed that application of either agent, significantly inhibited tumor growth in treated mice as compared to untreated. However, neither drug alone caused tumor regression. The treatment regimen then was modified to administer chemotherapy and targeted therapy together. A mouse avatars in which genomic analyses revealed amplification at loci involving AKT3 and RICTOR genes was treated with chemotherapy alone and in combination with MK2206 (AKT inhibitor) or MK8669 (mTOR inhibitor). The high level of both target proteins was confirmed by immunoblotting. Significant regression of tumor was observed after 6 weeks of treatment. Addition of either targeted drug provided an extra benefit in killing tumor cells compared to chemotherapy alone. The mice are followed to study tumor recurrence. These data indicated that genomic information obtained using next generation sequencing can aid therapy decisions by tailoring treatments to individual tumor makeup. Avatar models in conjunction with genomic analyses of the original tumors are invaluable for testing specific targeted therapies selected based on amplification/gains of putative genes-targets and for studying mechanisms of tumor recurrence and therapy resistance. Citation Format: Faye R. Harris, Lin Yang, Xiaonan Hou, Konstantinos Leventakos, Saravut J. Weroha, George Vasmatzis, Irina V. Kovtun. Targeting genes which are amplified in ovarian cancer. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Targeting the Vulnerabilities of Cancer; May 16-19, 2016; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(1_Suppl):Abstract nr A34.