Ongoing Chromosomal Instability Research Articles

Abstract B032: Comprehensive analysis of clonality, segregation errors, chromosomal instability and heterogeneity in cancer cell lines and cancer samples reveals that cancer evolution is often promoted by highly aberrant cellular divisions

Abstract Chromosomal instability (CIN) is the most common form of genomic instability in cancer. CIN leads to structural and numerical chromosomal aberrations and to heterogeneity of such aberrations inside the same tumor. Together with other forms of genomic and epigenomic instability, CIN underlies the most dangerous feature of cancer cells, namely, ability to evolve, allowing cancer cells to evade immunologic surveillance, to metastasize, to become resistant to drug treatments. Studies of chromosomal instability are significantly impeded by the lack of comprehensive approaches for simultaneous detection of different forms of ongoing chromosomal instability and their outcomes. We developed an approach that allows to analyze the clonal composition and chromosomal heterogeneity in cell lines and cancer samples, as well as rates of different forms of ongoing chromosomal instability in cancer cell lines. At the heart our analysis is a multiplex interphase FISH (miFISH) with seven sequential hybridizations of color probe panels resulting in the assessment of a total of 35 probes per single cell. Each probe panel is designed to include the centromeric probe and probes on the p- and q-arm for a given chromosome that allows for the simultaneous assessment of centromeric probes and gene-specific arm probes. This approach was applied to interphase nuclei, metaphase spreads and bi-nucleated/dividing cancer cells in colorectal and cervical cell lines and cancer samples. miFISH allows for automated acquisition of nuclei and metaphase spreads on a BioView spotcounting system. A gallery provides detailed views for semi-automatic as well as manual analysis of the obtained images. A total of 10000-25000 cell images were collected from each slide. The simultaneous evaluation of our strategically chosen centromeric and gene-specific probes within the same cells allows unprecedented insights in the patterns of distribution of genetic material among cancer cells in cancer cell population, and, especially, in dividing cells. We evaluated clonality, clonal and non-clonal chromosomal heterogeneity, ploidy distributions, and occurrence of endoreplication. Assessment of dividing cells based on equal or unequal distribution of miFISH centromeric and gene-specific signals allowed to estimate and compare the rates and outcomes of different forms of ongoing chromosomal instability such as structural rearrangements and amplifications, loss of chromosomes or chromosomal fragments in micronuclei, non-disjunctions, grossly asymmetrical cell divisions (including multipolar mitoses). Conclusions: Gradual changes in chromosomal/genomic content of cancer cells occur due to structural rearrangements, losses of chromosomes and chromosomal fragments in micronuclei, and by relatively rare single chromosome non-disjunctions. Grossly abnormal cell divisions (massive non-disjunctions and structural rearrangements) is the main source for the high level of chromosomal abnormalities and aneuploidy in evolved cancer cell populations. Citation Format: Anna Roschke. Comprehensive analysis of clonality, segregation errors, chromosomal instability and heterogeneity in cancer cell lines and cancer samples reveals that cancer evolution is often promoted by highly aberrant cellular divisions [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Translating Cancer Evolution and Data Science: The Next Frontier; 2023 Dec 3-6; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_2):Abstract nr B032.

Abstract A001: Genomic signatures of past and present chromosomal instability in Barrett’s esophagus and early esophageal adenocarcinoma

Abstract Abstract: The progression of precancerous lesions to malignancy is often accompanied by increasing complexity of chromosomal alterations but how these alterations arise is poorly understood. Here we perform haplotype-specific analysis of chromosomal copy-number evolution in the progression of Barrett’s esophagus (BE) to esophageal adenocarcinoma (EAC) on multiregional whole-genome sequencing data of BE with dysplasia and microscopic EAC foci. We identify distinct patterns of copy-number evolution indicating multigenerational chromosomal instability that is initiated by cell division errors but propagated only after p53 loss. While abnormal mitosis, including whole-genome duplication, underlies chromosomal copy-number changes, segmental alterations display signatures of successive breakage-fusion-bridge cycles and chromothripsis of unstable dicentric chromosomes. Our analysis elucidates how multigenerational chromosomal instability generates copy-number variation in BE cells, precipitates complex alterations including DNA amplifications, and promotes their independent clonal expansion and transformation. In particular, we suggest sloping copy-number variation as a signature of ongoing chromosomal instability that precedes copy-number complexity. These findings suggest copy-number heterogeneity in advanced cancers originates from chromosomal instability in precancerous cells and such instability may be identified from the presence of sloping copy-number variation in bulk sequencing data. Citation Format: Chunyang Bao. Genomic signatures of past and present chromosomal instability in Barrett’s esophagus and early esophageal adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Translating Cancer Evolution and Data Science: The Next Frontier; 2023 Dec 3-6; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_2):Abstract nr A001.

Genomic signatures of past and present chromosomal instability in Barrett’s esophagus and early esophageal adenocarcinoma

The progression of precancerous lesions to malignancy is often accompanied by increasing complexity of chromosomal alterations but how these alterations arise is poorly understood. Here we perform haplotype-specific analysis of chromosomal copy-number evolution in the progression of Barrett’s esophagus (BE) to esophageal adenocarcinoma (EAC) on multiregional whole-genome sequencing data of BE with dysplasia and microscopic EAC foci. We identify distinct patterns of copy-number evolution indicating multigenerational chromosomal instability that is initiated by cell division errors but propagated only after p53 loss. While abnormal mitosis, including whole-genome duplication, underlies chromosomal copy-number changes, segmental alterations display signatures of successive breakage-fusion-bridge cycles and chromothripsis of unstable dicentric chromosomes. Our analysis elucidates how multigenerational chromosomal instability generates copy-number variation in BE cells, precipitates complex alterations including DNA amplifications, and promotes their independent clonal expansion and transformation. In particular, we suggest sloping copy-number variation as a signature of ongoing chromosomal instability that precedes copy-number complexity. These findings suggest copy-number heterogeneity in advanced cancers originates from chromosomal instability in precancerous cells and such instability may be identified from the presence of sloping copy-number variation in bulk sequencing data.

Exploiting a living biobank to delineate mechanisms underlying disease-specific chromosome instability.

Chromosome instability (CIN) is a cancer hallmark that drives tumour heterogeneity, phenotypic adaptation, drug resistance and poor prognosis. High-grade serous ovarian cancer (HGSOC), one of the most chromosomally unstable tumour types, has a 5-year survival rate of only ~30% - largely due to late diagnosis and rapid development of drug resistance, e.g., via CIN-driven ABCB1 translocations. However, CIN is also a cell cycle vulnerability that can be exploited to specifically target tumour cells, illustrated by the success of PARP inhibitors to target homologous recombination deficiency (HRD). However, a lack of appropriate models with ongoing CIN has been a barrier to fully exploiting disease-specific CIN mechanisms. This barrier is now being overcome with the development of patient-derived cell cultures and organoids. In this review, we describe our progress building a Living Biobank of over 120 patient-derived ovarian cancer models (OCMs), predominantly from HGSOC. OCMs are highly purified tumour fractions with extensive proliferative potential that can be analysed at early passage. OCMs have diverse karyotypes, display intra- and inter-patient heterogeneity and mitotic abnormality rates far higher than established cell lines. OCMs encompass a broad-spectrum of HGSOC hallmarks, including a range of p53 alterations and BRCA1/2 mutations, and display drug resistance mechanisms seen in the clinic, e.g., ABCB1 translocations and BRCA2 reversion. OCMs are amenable to functional analysis, drug-sensitivity profiling, and multi-omics, including single-cell next-generation sequencing, and thus represent a platform for delineating HGSOC-specific CIN mechanisms. In turn, our vision is that this understanding will inform the design of new therapeutic strategies.

Tracking the evolution of esophageal squamous cell carcinoma under dynamic immune selection by multi-omics sequencing

Intratumoral heterogeneity (ITH) has been linked to decreased efficacy of clinical treatments. However, although genomic ITH has been characterized in genetic, transcriptomic and epigenetic alterations are hallmarks of esophageal squamous cell carcinoma (ESCC), the extent to which these are heterogeneous in ESCC has not been explored in a unified framework. Further, the extent to which tumor-infiltrated T lymphocytes are directed against cancer cells, but how the immune infiltration acts as a selective force to shape the clonal evolution of ESCC is unclear. In this study, we perform multi-omic sequencing on 186 samples from 36 primary ESCC patients. Through multi-omics analyses, it is discovered that genomic, epigenomic, and transcriptomic ITH are underpinned by ongoing chromosomal instability. Based on the RNA-seq data, we observe diverse levels of immune infiltrate across different tumor sites from the same tumor. We reveal genetic mechanisms of neoantigen evasion under distinct selection pressure from the diverse immune microenvironment. Overall, our work offers an avenue of dissecting the complex contribution of the multi-omics level to the ITH in ESCC and thereby enhances the development of clinical therapy.

Conventional and Spectral Karyotyping of Murine Cerebellar Granule Neuron Progenitors.

Karyotyping remains an invaluable tool to researchers exploring the cause and consequence of genomic instability in biologic systems. It allows investigators to survey the entire chromosome complement in individual cells and in a single experiment, visualize, and measure different forms or features of instability such as aneuploidy, ongoing chromosomal instability, DNA damage/mis-repair, telomere erosion, chromosome mis-segregation, or defects in cell cycle progression. This chapter describes the combined use of conventional (DAPI-banding) and spectral karyotyping (SKY) to characterize genomic instability in murine cerebellar granule neuron progenitors (CGNPs), using CGNPs with conditional deletion of Atr as a positive control for chromosomal rearrangements. Protocols for preparing slides (metaphase spreads) from fixed cell suspension, DAPI-banding, and spectral karyotyping (SKY) are included. Pertinent aspects of image acquisition and analysis are detailed. These protocols can likely be adapted to other tissue types (murine or human).

Chromosomal Instability Characterizes Pediatric Medulloblastoma but Is Not Tolerated in the Developing Cerebellum.

Medulloblastoma is a pediatric brain malignancy that consists of four transcriptional subgroups. Structural and numerical aneuploidy are common in all subgroups, although they are particularly profound in Group 3 and Group 4 medulloblastoma and in a subtype of SHH medulloblastoma termed SHHα. This suggests that chromosomal instability (CIN), the process leading to aneuploidy, is an important player in medulloblastoma pathophysiology. However, it is not known if there is ongoing CIN in medulloblastoma or if CIN affects the developing cerebellum and promotes tumor formation. To investigate this, we performed karyotyping of single medulloblastoma cells and demonstrated the presence of distinct tumor cell clones harboring unique copy number alterations, which is suggestive of ongoing CIN. We also found enrichment for processes related to DNA replication, repair, and mitosis in both SHH medulloblastoma and in the highly proliferative compartment of the presumed tumor cell lineage-of-origin, the latter also being sensitive to genotoxic stress. However, when challenging these tumor cells-of-origin with genetic lesions inducing CIN using transgenic mouse modeling, we found no evidence for large chromosomal aberrations in the cerebellum or for medulloblastoma formation. We therefore conclude that without a background of specific genetic mutations, CIN is not tolerated in the developing cerebellum in vivo and, thus, by itself is not sufficient to initiate medulloblastoma.

Abstract 1606: Chromosomal instability drives spatial and temporal phenotypic diversity in Schwann cancer cells

Abstract Background Malignant peripheral nerve sheath tumors (MPNSTs) are rare, highly aggressive soft tissue sarcomas derived from Schwann cells with poor prognosis. Previous studies, although limited in scale and depth, have shown that these tumors are typically genomically complex, with a moderate burden of single nucleotide variants but extensive copy number aberrations (CNAs). Methods We applied a multi-omics approach to scrutinize the evolution and heterogeneity of a primary MPNST and five recurrence regions from one patient with an NF1 phenotype. Techniques utilized included bulk whole genome sequencing (WGS), single-cell DNA sequencing (scDNA-seq), single-cell RNA sequencing (scRNA-seq), simultaneous genome and transcriptome sequencing (G&T-seq), spatial transcriptomics, and laser capture microdissection (LCM)-based spatial genomics. Results We find significant heterogeneity in copy number profiles, suggestive of ongoing chromosomal instability and evolution. We perform in-depth tumor phylogenetic reconstruction from bulk WGS data. scDNA-seq revealed further CNA heterogeneity across and within regions, allowing us to refine the tree down to single-cell resolution. De novo somatic variant calling in scDNA-seq data confirms the reconstructed tree and identifies additional subclonal mutations. We also profile different populations of tumor and non-tumor cells and confirmed this with a genotyping approach. CNA profiles inferred from scRNA-seq reflect the within-region heterogeneity seen in scDNA-seq. We find tumor cells with similar CNA profiles typically cluster together by their gene expression profiles as well, implying that the majority of gene expression heterogeneity in this tumor is underpinned by copy number changes. By using CNA profiles as a native barcoding system, we link genetic subclones to clusters of cells and their transcriptomes to characterize the effects of gene dosage. Finally, we explore the spatial relationships between tumor subclones using LCM. We detect heterogeneity within tissue sections evidencing local clonal expansions and CNA events that follow spatial distributions. Using spatial transcriptomics, we show tumor cells are homogeneously admixed with tumor microenvironment populations. Conclusions Our work demonstrates the power of combining spatial multi-omics at the single-cell and bulk levels to study cancer evolution. This enables a spatio-temporal representation of a tumor’s development with annotation of genetic and transcriptomic events. Citation Format: Haixi Yan, Jonas Demeulemeester, Annelien Verfaillie, Cristina Cotobal Martin, Tom Kaufmann, Roland Schwarz, Thierry Voet, Simone Zaccaria, Adrienne Flanagan, Maxime Tarabichi, Peter Van Loo. Chromosomal instability drives spatial and temporal phenotypic diversity in Schwann cancer cells [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 1606.

Abstract IA12: Cancer evolution: Chromosomal instability and immune evasion

Abstract Evidence supports complex subclonal relationships in solid tumors, manifested as intratumor heterogeneity. Parallel evolution of subclones, with distinct somatic events occurring in the same gene, signal transduction pathway or protein complex, suggests constraints to tumor evolution that might be therapeutically exploitable. Data from TRACERx, a longitudinal lung cancer evolution study will be presented. Drivers of tumor heterogeneity change during the disease course and contribute to the temporally distinct origins of lung cancer driver events. APOBEC driven mutagenesis appears to be enriched in subclones in multiple tumor types. Oncogene, tumor suppressor gene and drug-induced DNA replication stress are found to drive APOBEC mutagenesis. On-going chromosomal instability, manifested as Mirrored Subclonal Allelic Imbalance (MSAI) is found to be a major driver of intratumor heterogeneity across cancer types, contributing to parallel evolution and selection. Subclonal driver events, evidence of ongoing selection within subclones, combined with genome instability driving cell-to-cell variation is likely to limit the efficacy of targeted monotherapies, suggesting a need for new approaches to drug development and integration of cancer immunotherapeutic approaches. Multiple adaptive mechanisms to neo-antigen evolution have been found in TRACERx highlighting cancer chromosomal instability driving immune evasion and HLA loss and loss of clonal neo-antigens as well as epigenetic repression of neo-antigens. The clonal neo-antigenic architecture may act as a tumor vulnerability to mitigate resistance and treatment failure. Citation Format: Charles Swanton. Cancer evolution: Chromosomal instability and immune evasion [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2021 Oct 5-6. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(1 Suppl):Abstract nr IA12.

Spatial and temporal intratumour heterogeneity has potential consequences for single biopsy-based neuroblastoma treatment decisions

Intratumour heterogeneity is a major cause of treatment failure in cancer. We present in-depth analyses combining transcriptomic and genomic profiling with ultra-deep targeted sequencing of multiregional biopsies in 10 patients with neuroblastoma, a devastating childhood tumour. We observe high spatial and temporal heterogeneity in somatic mutations and somatic copy-number alterations which are reflected on the transcriptomic level. Mutations in some druggable target genes including ALK and FGFR1 are heterogeneous at diagnosis and/or relapse, raising the issue whether current target prioritization and molecular risk stratification procedures in single biopsies are sufficiently reliable for therapy decisions. The genetic heterogeneity in gene mutations and chromosome aberrations observed in deep analyses from patient courses suggest clonal evolution before treatment and under treatment pressure, and support early emergence of metastatic clones and ongoing chromosomal instability during disease evolution. We report continuous clonal evolution on mutational and copy number levels in neuroblastoma, and detail its implications for therapy selection, risk stratification and therapy resistance.

Chromosomal copy number heterogeneity predicts survival rates across cancers

Survival rates of cancer patients vary widely within and between malignancies. While genetic aberrations are at the root of all cancers, individual genomic features cannot explain these distinct disease outcomes. In contrast, intra-tumour heterogeneity (ITH) has the potential to elucidate pan-cancer survival rates and the biology that drives cancer prognosis. Unfortunately, a comprehensive and effective framework to measure ITH across cancers is missing. Here, we introduce a scalable measure of chromosomal copy number heterogeneity (CNH) that predicts patient survival across cancers. We show that the level of ITH can be derived from a single-sample copy number profile. Using gene-expression data and live cell imaging we demonstrate that ongoing chromosomal instability underlies the observed heterogeneity. Analysing 11,534 primary cancer samples from 37 different malignancies, we find that copy number heterogeneity can be accurately deduced and predicts cancer survival across tissues of origin and stages of disease. Our results provide a unifying molecular explanation for the different survival rates observed between cancer types.

P53 induces senescence in the unstable progeny of aneuploid cells

ABSTRACT Aneuploidy is the condition of having an imbalanced karyotype, which is associated with tumor initiation, evolution, and acquisition of drug-resistant features, possibly by generating heterogeneous populations of cells with distinct genotypes and phenotypes. Multicellular eukaryotes have therefore evolved a range of extrinsic and cell-autonomous mechanisms for restraining proliferation of aneuploid cells, including activation of the tumor suppressor protein p53. However, accumulating evidence indicates that a subset of aneuploid cells can escape p53-mediated growth restriction and continue proliferating in vitro. Here we show that such aneuploid cell lines display a robust modal karyotype and low frequency of chromosomal aberrations despite ongoing chromosome instability. Indeed, while these aneuploid cells are able to survive for extended periods in vitro, their chromosomally unstable progeny remain subject to p53-induced senescence and growth restriction, leading to subsequent elimination from the aneuploid pool. This mechanism helps maintain low levels of heterogeneity in aneuploid populations and may prevent detrimental evolutionary processes such as cancer progression and development of drug resistance.

Abstract IA16: Cancer evolution, immune evasion and metastasis

Abstract Increasing evidence supports complex subclonal relationships in solid tumours, manifested as intratumour heterogeneity. Parallel evolution of subclones, with distinct somatic events occurring in the same gene, signal transduction pathway or protein complex, suggests constraints to tumour evolution that might be therapeutically exploitable. Data from TRACERx, a longitudinal lung cancer evolution study will be presented. Drivers of tumour heterogeneity change during the disease course and contribute to the temporally distinct origins of lung cancer driver events. APOBEC driven mutagenesis appears to be enriched in subclones in multiple tumour types. Oncogene, tumour suppressor gene and drug induced DNA replication stress are found to drive APOBEC mutagenesis. Phylogenetic tracking detects minimal residual disease and clonal evolution of disease from primary to metastatic sites, presenting opportunities for drug development. On-going chromosomal instability, manifested as Mirrored Subclonal Allelic Imbalance (MSAI) is found to be a major driver of intratumour heterogeneity across cancer types, contributing to parallel evolution and selection. The finding of subclonal driver events, evidence of ongoing selection within subclones, combined with genome instability driving cell-to-cell variation is likely to limit the efficacy of targeted monotherapies, suggesting the need for new approaches to drug development and clinical trial design and integration of cancer immunotherapeutic approaches. Multiple adaptive mechanisms to neo-antigen evolution have been found in TRACERx that emphasise the role of cancer chromosomal instability driving immune evasion and HLA/MHC loss and loss of clonal neo-antigens as well as epigenetic repression of neo-antigens. The clonal neo-antigenic architecture may act as a tumour vulnerability, targeting multiple clonal neo-antigens present in each tumour to mitigate resistance and treatment failure. Citation Format: Charles Swanton. Cancer evolution, immune evasion and metastasis [abstract]. In: Proceedings of the AACR Virtual Special Conference on Tumor Heterogeneity: From Single Cells to Clinical Impact; 2020 Sep 17-18. Philadelphia (PA): AACR; Cancer Res 2020;80(21 Suppl):Abstract nr IA16.

Exploiting aneuploidy-imposed stresses and coping mechanisms to battle cancer.

Aneuploidy, an irregular number of chromosomes in cells, is a hallmark feature of cancer. Aneuploidy results from chromosomal instability (CIN) and occurs in almost 90% of all tumours. While many cancers display an ongoing CIN phenotype, cells can also be aneuploid without displaying CIN. CIN drives tumour evolution as ongoing chromosomal missegregation will yield a progeny of cells with variable aneuploid karyotypes. The resulting aneuploidy is initially toxic to cells because it leads to proteotoxic and metabolic stress, cell cycle arrest, cell death, immune cell activation and further genomic instability. In order to overcome these aneuploidy-imposed stresses and adopt a malignant fate, aneuploid cancer cells must develop aneuploidy-tolerating mechanisms to cope with CIN. Aneuploidy-coping mechanisms can thus be considered as promising therapeutic targets. However, before such therapies can make it into the clinic, we first need to better understand the molecular mechanisms that are activated upon aneuploidization and the coping mechanisms that are selected for in aneuploid cancer cells. In this review, we discuss the key biological responses to aneuploidization, some of the recently uncovered aneuploidy-coping mechanisms and some strategies to exploit these in cancer therapy.

Karyotypic Flexibility of the Complex Cancer Genome and the Role of Polyploidization in Maintenance of Structural Integrity of Cancer Chromosomes.

Ongoing chromosomal instability in neoplasia (CIN) generates intratumor genomic heterogeneity and limits the efficiency of oncotherapeutics. Neoplastic human cells utilizing the alternative lengthening of telomeres (ALT)-pathway, display extensive structural and numerical CIN. To unravel patterns of genome evolution driven by oncogene-replication stress, telomere dysfunction, or genotoxic therapeutic interventions, we examined by comparative genomic hybridization five karyotypically-diverse outcomes of the ALT osteosarcoma cell line U2-OS. These results demonstrate a high tendency of the complex cancer genome to perpetuate specific genomic imbalances despite the karyotypic evolution, indicating an ongoing process of genome dosage maintenance. Molecular karyotyping in four ALT human cell lines showed that mitotic cells with low levels of random structural CIN display frequent evidence of whole genome doubling (WGD), suggesting that WGD may protect clonal chromosome aberrations from hypermutation. We tested this longstanding hypothesis in ALT cells exposed to gamma irradiation or to inducible DNA replication stress under overexpression of p21. Single-cell cytogenomic analyses revealed that although polyploidization promotes genomic heterogeneity, it also protects the complex cancer genome and hence confers genotoxic therapy resistance by generating identical extra copies of driver chromosomal aberrations, which can be spared in the process of tumor evolution if they undergo unstable or unfit rearrangements.

Altering microtubule dynamics is synergistically toxic with spindle assembly checkpoint inhibition.

Chromosomal instability (CIN) and aneuploidy are hallmarks of cancer. As most cancers are aneuploid, targeting aneuploidy or CIN may be an effective way to target a broad spectrum of cancers. Here, we perform two small molecule compound screens to identify drugs that selectively target cells that are aneuploid or exhibit a CIN phenotype. We find that aneuploid cells are much more sensitive to the energy metabolism regulating drug ZLN005 than their euploid counterparts. Furthermore, cells with an ongoing CIN phenotype, induced by spindle assembly checkpoint (SAC) alleviation, are significantly more sensitive to the Src kinase inhibitor SKI606. We show that inhibiting Src kinase increases microtubule polymerization rates and, more generally, that deregulating microtubule polymerization rates is particularly toxic to cells with a defective SAC. Our findings, therefore, suggest that tumors with a dysfunctional SAC are particularly sensitive to microtubule poisons and, vice versa, that compounds alleviating the SAC provide a powerful means to treat tumors with deregulated microtubule dynamics.

Ongoing chromosomal instability and karyotype evolution in human colorectal cancer organoids.

Chromosome segregation errors cause aneuploidy and genomic heterogeneity, which are hallmarks of cancer in humans. A persistent high frequency of these errors (chromosomal instability (CIN)) is predicted to profoundly impact tumor evolution and therapy response. It is unknown, however, how prevalent CIN is in human tumors. Using three-dimensional live-cell imaging of patient-derived tumor organoids (tumor PDOs), we show that CIN is widespread in colorectal carcinomas regardless of background genetic alterations, including microsatellite instability. Cell-fate tracking showed that, although mitotic errors are frequently followed by cell death, some tumor PDOs are largely insensitive to mitotic errors. Single-cell karyotype sequencing confirmed heterogeneity of copy number alterations in tumor PDOs and showed that monoclonal lines evolved novel karyotypes over time in vitro. We conclude that ongoing CIN is common in colorectal cancer organoids, and propose that CIN levels and the tolerance for mitotic errors shape aneuploidy landscapes and karyotype heterogeneity.

Spatial and temporal clonal evolution of intrahepatic cholangiocarcinoma

Intrahepatic cholangiocarcinoma (ICC) is the second-most lethal primary liver cancer. Little is known about intratumoral heterogeneity (ITH) and its impact on ICC progression. We aimed to investigate the ITH of ICC in the hope of helping to develop new therapeutic strategies. We obtained 69 spatially distinct regions from six operable ICCs. Patient-derived primary cancer cells (PDPCs) were established for each region, followed by whole-exome sequencing (WES) and multi-level validation. We observed widespread ITH for both somatic mutations and clonal architecture, shaped by multiple mechanisms, like clonal "illusion", parallel evolution and chromosome instability. A median of 60.3% of mutations were heterogeneous, among which 85% of the driver mutations were located on the branches of tumor phylogenetic trees. Many truncal and clonal driver mutations occurred in tumor suppressor genes, such as TP53, SMARCB1 and PBRM1 that are involved in DNA repair and chromatin-remodeling. Genome doubling occurred in most cases (5/6) after the accumulation of truncal mutations and was shared by all intratumoral sub-regions. In all cases, ongoing chromosomal instability is evident throughout the evolutionary trajectory of ICC. The recurrence of ICC1239 provided evidence to support the polyclonal metastatic seeding in ICC. The change of mutation landscape and internal diversity among subclones during metastasis, such as the loss of chemoresistance mediator, can be used for new treatment strategies. Targeted therapy against truncal alterations, such as IDH1, JAK1, and KRAS mutations and EGFR amplification, was developed in 5/6 patients. Integrated investigations of spatial ITH and clonal evolution may provide an important molecular foundation for enhanced understanding of tumorigenesis and progression in ICC. We applied multiregional whole-exome sequencing to investigate the evolution of intrahepatic cholangiocarcinoma (ICC). The results revealed that many factors, such as parallel evolution and chromosome instability, may participate and promote the branch diversity of ICC. Interestingly, in one patient with primary and recurrent metastatic tumors, we found evidence of polyclonal metastatic seeding, indicating that symbiotic communities of multiple clones existed and were maintained during metastasis. More realistically, some truncal alterations, such as IDH1, JAK1, and KRAS mutations and EGFR amplification, could be promising treatment targets in patients with ICC.

Abstract A182: Apobec-mutagenesis drives mutational heterogeneity while copy number alterations drive transcriptomic and proteogenomic heterogeneity in metastatic lung adenocarcinoma and thymic carcinoma

Abstract Background: Molecular characterization of metastatic tumors to investigate intra- and intertumor heterogeneity has proven more difficult than early stage disease as opportunities for surgical removal are limited. Rapid/warm autopsy programs that are conducted soon after death minimize tissue ischemia, thereby enabling procurement of high-quality viable tissue. To our knowledge, integrated proteo-genomics studies to characterize tumor heterogeneity of metastatic lung adenocarcinoma and thymic carcinoma are lacking. Methods: We sought to characterize and decipher mechanisms of intra- and intertumor heterogeneity of Stage IV lung adenocarcinoma and thymic carcinoma through whole exome sequencing, RNA sequencing, copy number estimation, and mass spectrometry-based proteomics of 38 tumors from five rapid/warm autopsy patients. The autopsy procedure was initiated between 2-4 hours of death. Results: One lung adenocarcinoma (RA003) and one thymic carcinoma (RA006) patient had high mutation burden and extreme mutational (SNV) heterogeneity. This was likely driven by APOBEC-mutagenesis. We constructed phylogenetic trees showing truncal, shared, and private SNVs and copy number alterations (CNAs). Interestingly, we discovered a pattern of recurrent, parallel but opposite arm level CNAs in different metastatic branches within a subset of patients (RA000, RA004, and RA006). In contrast, RA003 had only truncal CNAs and the least heterogeneity at the CNA level. Assessment of intra- and intertumor heterogeneity at the transcriptomic and proteomic levels revealed high concordance in gene expression and protein abundance within tumors from patient RA003 compared to patient RA004, potentially reflecting the downstream effect of CNA vs. APOBEC-induced mutations. Further analysis of cis vs. trans effects of CNA on gene expression and protein abundance in all the metastatic tumors revealed many trans-acting hot spots and limited cis effects, which is contrary to copy number effects on gene expression in primary lung adenocarcinoma tumors. Trans-acting hot spots differed between two major metastatic lineage of patient RA004, suggesting copy number changes may be responsible for high intermetastatic proteomic heterogeneity in this patient. Conclusions: Our results demonstrate ongoing mutational and chromosomal instability throughout the metastatic process of lung adenocarcinoma and thymic carcinoma. APOBEC mutagenesis was likely the primary driver of SNV heterogeneity. However, intertumor heterogeneity of metastatic disease at the level of the transcriptome and proteome occurred in association with CNA rather than mutational heterogeneity. APOBEC mutagenesis and CNA are complementary mechanisms of tumor evolution and heterogeneity affecting tumor metastasis. Citation Format: Nitin Roper, James Gao, Rouf Banday, Tapan Maity, Xu Zhang, Abhilash Venugopalan, Rajesh Patidar, Sivasish Sindiri, Constance Cultraro, Alexandr Goncearenco, Anna Panchenko, Romi Biswas, Jing Wang, Stephen Hewitt, David Kleiner, Bing Zhang, Javed Khan, Ludmila Prokunina-Olsson, Udayan Guha. Apobec-mutagenesis drives mutational heterogeneity while copy number alterations drive transcriptomic and proteogenomic heterogeneity in metastatic lung adenocarcinoma and thymic carcinoma [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A182.

Abstract 4387: The clonal composition of colorectal cancer cell lines is defined by the maintenance of specific genomic imbalances, not by ongoing chromosomal instability

Abstract Tumor type specific genomic imbalances are the defining feature of carcinomas. It remains elusive to which extent intratumor heterogeneity and the clonal composition of cancer cell populations influences the genetic makeup and the phenotype of tumors. Many studies aimed at understanding the idiosyncrasies of cancer rely on the analysis of cell lines derived from primary tumors. In an attempt to decipher patterns of chromosomal heterogeneity we analyzed the widely used colorectal cancer cell lines DLD-1, HCT116, H508, SW620, HT-29 and SW480 using FISH and gene expression profiling. First, we applied a four color probe set that targets the oncogenes EGFR, CCND1, CDX2 and TERC and enumerated 200 metaphase spread, which included chromosome counts, and 5,000 interphase cells. Not surprisingly, the mismatch repair deficient cell lines DLD-1 and HCT116 were the most stable, whereas the aneuploid cell lines displayed a higher degree of instability. These results prompted us to apply our recently developed multiplex interphase FISH approach that allows copy number analysis of 12 gene loci in individual cells to 11 single cell derived clones from both HT-29 and SW480. SW480 was considerably more instable than HT-29. The parental lines consisted of major clones, which were propagated in the single cell derived lines. These analyses were complemented by gene expression profiling of the single cell derived clones and the parental cell lines using the Nanostring technology which measures expression levels of 770 cancer-associated genes. The SW480 clones separated into two distinct clusters that reflect discrete ploidy levels that were also present in the parental line. We then explored whether mutations of the BRAF (HT-29) and KRAS (SW480) genes observed in the parental lines were maintained in the derived clones; this was the case in all instances. Finally, we reconstructed phylogenetic trees of tumor evolution using a specifically developed algorithm, termed FISHTrees. Our meticulous analysis of the clonal composition of these colorectal cancer models shows that, despite a certain degree of chromosomal instability, specific genomic imbalances are maintained. The results challenge the concept of ongoing chromosomal instability in cancer cell populations. This suggests a karyotype evolution that is driven by the necessity to arrive at a different plateau of chromosomal copy number as the driving force of carcinogenesis. Citation Format: Darawalee Wangsa, Madison Schiefer, Daniel Bronder, Hesed Padilla-Nash, Irianna Torres, Lidia Warner, Yue Hu, E Michael Gertz, Russell Schwartz, Alejandro A. Schäffer, Daniela Hirsch, Timo Gaiser, Rüdiger Meyer, Jordi Camps, Kerstin Heselmeyer-Haddad, Thomas Ried. The clonal composition of colorectal cancer cell lines is defined by the maintenance of specific genomic imbalances, not by ongoing chromosomal instability [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 4387. doi:10.1158/1538-7445.AM2017-4387