Whole-genome Doubling Research Articles

Integrative Profiling of T790M-Negative EGFR-Mutated NSCLC Reveals Pervasive Lineage Transition and Therapeutic Opportunities.

Despite the established role of EGFR tyrosine kinase inhibitors (TKIs) in EGFR-mutated NSCLC, drug resistance inevitably ensues, with a paucity of treatment options especially in EGFR T790M-negative resistance. We performed whole-exome and transcriptome analysis of 59 patients with first- and second-generation EGFR TKI-resistant metastatic EGFR-mutated NSCLC to characterize and compare molecular alterations mediating resistance in T790M-positive (T790M+) and -negative (T790M-) disease. Transcriptomic analysis revealed ubiquitous loss of adenocarcinoma lineage gene expression in T790M- tumors, orthogonally validated using multiplex IHC. There was enrichment of genomic features such as TP53 alterations, 3q chromosomal amplifications, whole-genome doubling and nonaging mutational signatures in T790M- tumors. Almost half of resistant tumors were further classified as immunehot, with clinical outcomes conditional on immune cell-infiltration state and T790M status. Finally, using a Bayesian statistical approach, we explored how T790M- and T790M+ disease might be predicted using comprehensive genomic and transcriptomic profiles of treatment-naïve patients. Our results illustrate the interplay between genetic alterations, cell lineage plasticity, and immune microenvironment in shaping divergent TKI resistance and outcome trajectories in EGFR-mutated NSCLC. Genomic and transcriptomic profiling may facilitate the design of bespoke therapeutic approaches tailored to a tumor's adaptive potential.

ECOA-5. Integrative 3D spatial characterization of genomic and epigenomic intratumoral heterogeneity in glioblastoma

Treatment failure in glioblastoma is often attributed to intratumoral heterogeneity (ITH), which fosters tumor evolution and selection of therapy-resistant clones. While genomic alterations are known contributors to ITH, emerging studies highlight functional roles for epigenomic ITH which integrates differentiation status, stochastic events, and microenvironmental inputs. Here, we have established a novel platform for integrative characterization of genomic and epigenomic ITH of glioblastoma in three-dimensional (3-D) space. In collaboration with neurosurgeons and biomedical imaging experts, we utilize 3-D surgical neuro-navigation to safely acquire ~10 tumor samples per patient representing maximal anatomical diversity. We conduct whole-exome sequencing, RNA sequencing, and assay for transposase-accessible chromatin using sequencing (ATAC-Seq) on each sample. The spatial location of each sample is mapped by its 3-D coordinates, allowing 360-degree visualization of genomic and epigenomic ITH for each patient. We demonstrate this approach on 8 patients with primary IDH-WT glioblastoma (83 spatially mapped samples), providing unprecedented insight into their spatial organization at the genomic and epigenomic levels. We link genetically defined tumor subclones to patterns of open chromatin and gene regulation, revealing underlying transcription factor binding at active promoters and enhancers. We also identify ITH in whole-genome doubling and focal oncogene amplification events in multiple patients, which we then link with epigenomic ITH. Further, to study microenvironmental inputs and their contribution to epigenomic ITH, we conduct deconvolution of RNA sequencing and ATAC-Seq data by analyzing feature co-variation. We resolve the 3-D spatial organization of immune, neural, and other nontumor cell types present in glioblastoma, characterizing their functional states and interactions with tumor cells. This work provides the most comprehensive spatial characterization of genomic and epigenomic ITH to date in glioblastoma. As a resource for further investigation, we have developed an interactive data sharing platform – The 3D Glioma Atlas – that enables 360-degree visualization of both genomic and epigenomic ITH.

Integrated synteny- and similarity-based inference on the polyploidization-fractionation cycle.

Whole-genome doubling, tripling or replicating to a greater degree, due to fixation of polyploidization events, is attested in almost all lineages of the flowering plants, recurring in the ancestry of some plants two, three or more times in retracing their history to the earliest angiosperm. This major mechanism in plant genome evolution, which generally appears as instantaneous on the evolutionary time scale, sets in operation a compensatory process called fractionation, the loss of duplicate genes, initially rapid, but continuing at a diminishing rate over millions and tens of millions of years. We study this process by statistically comparing the distribution of duplicate gene pairs as a function of their time of creation through polyploidization, as measured by sequence similarity. The stochastic model that accounts for this distribution, though exceedingly simple, still has too many parameters to be estimated based only on the similarity distribution, while the computational procedures for compiling the distribution from annotated genomic data is heavily biased against earlier polyploidization events—syntenic ‘crumble’. Other parameters, such as the size of the initial gene complement and the ploidy of the various events giving rise to duplicate gene pairs, are even more inaccessible to estimation. Here, we show how the frequency of unpaired genes, identified via their embedding in stretches of duplicate pairs, together with previously established constraints among some parameters, adds enormously to the range of successive polyploidization events that can be analysed. This also allows us to estimate the initial gene complement and to correct for the bias due to crumble. We explore the applicability of our methodology to four flowering plant genomes covering a range of different polyploidization histories.

Genomic analysis of primary malignant melanoma of the esophagus.

e21515 Background: Primary malignant melanoma of the esophagus (PMME) is an exceedingly rare disease but behave more aggressively and have a poorer prognosis. No specific histopathological classification and therapy strategies have been established for this type of disease due to lack of both tumor biological and genetics perception to PMME. Methods: We performed whole exome sequencing on 29 dissections including 23 primary tumors and 6 metastatic lymph nodes from 18 PMME patients. The genetic characteristics including somatic mutations and copy number variation (CNV) of PMME were compared to 398 skin cutaneous melanoma (SKCM), 67 mucosal melanoma (MM) and 79 uveal melanoma (UVM). Using multi-region sequencing, we reconstructed the phylogenetic structure and inferred the evolutionary mode. Results: We found that PMME/MM have similar genomic patterns to SKCM but distinct from UVM. Frequently mutated driver genes such as MUC16, BRAF, NRAS, NF1, KMT2C, POLE, PTPRT, RANBP2 appeared in both SKCM and PMME/MM cohorts. Of note, pathway enrichment analysis using identified driver genes revealed that melanoma pathway and MAPK signaling pathway were among the top affected pathways in MM/PMME as well as in SKCM. UVR-associated single base substitution signature 7 (SBS7) was not a dominant signature (median: 0.17, range:0̃0.79) in PMME/MM but 99 percent (89/90) of them do have it. An overall concordant trend in genome instability was found between PMME and SKCM. Significant CNV events at arm level showed the similar changes in PMME and SKCM, including amplification regions of 1q, 6p, 7p, 8q, 15q, 20p and deletion of 10p, 10q, 11p. CDKN2A loss appeared as a shared focal event by PMME and SKCM. To elucidate the evolution trajectory of spatially separated samples, we also performed multi-region sequencing, and the phylogenetic analysis suggested that whole genome doubling (WGD) was an early and clonal event, which preceded the majority of somatic mutations and CNV. Most driver genes such as NF1, RANBP2 and MUC16 mutated after WGD except TP53. Above findings suggested that WGD might be capable of promoting ongoing genome instability and shaping the evolution in PMME. Moreover, parallel evolution of RANBP2 was recurrently observed indicating the existence of constraints and selection. Conclusions: Our data showed that PMME/MM exhibited genetic features very similar to SKCM so it might not be excluded from treatments currently used for common SKCM.

Whole-Genome Doubling Affects Pre-miRNA Expression in Plants.

Whole-genome doubling (polyploidy) is common in angiosperms. Several studies have indicated that it is often associated with molecular, physiological, and phenotypic changes. Mounting evidence has pointed out that micro-RNAs (miRNAs) may have an important role in whole-genome doubling. However, an integrative approach that compares miRNA expression in polyploids is still lacking. Here, a re-analysis of already published RNAseq datasets was performed to identify microRNAs’ precursors (pre-miRNAs) in diploids (2x) and tetraploids (4x) of five species (Arabidopsis thaliana L., Morus alba L., Brassica rapa L., Isatis indigotica Fort., and Solanum commersonii Dun). We found 3568 pre-miRNAs, three of which (pre-miR414, pre-miR5538, and pre-miR5141) were abundant in all 2x, and were absent/low in their 4x counterparts. They are predicted to target more than one mRNA transcript, many belonging to transcription factors (TFs), DNA repair mechanisms, and related to stress. Sixteen pre-miRNAs were found in common in all 2x and 4x. Among them, pre-miRNA482, pre-miRNA2916, and pre-miRNA167 changed their expression after polyploidization, being induced or repressed in 4x plants. Based on our results, a common ploidy-dependent response was triggered in all species under investigation, which involves DNA repair, ATP-synthesis, terpenoid biosynthesis, and several stress-responsive transcripts. In addition, an ad hoc pre-miRNA expression analysis carried out solely on 2x vs. 4x samples of S. commersonii indicated that ploidy-dependent pre-miRNAs seem to actively regulate the nucleotide metabolism, probably to cope with the increased requirement for DNA building blocks caused by the augmented DNA content. Overall, the results outline the critical role of microRNA-mediated responses following autopolyploidization in plants.

Next-Generation Sequencing of 487 Esophageal Adenocarcinomas Reveals Independently Prognostic Genomic Driver Alterations and Pathways

To delineate recurrent oncogenic driver alterations and dysregulated pathways in esophageal adenocarcinoma and to assess their prognostic value. We analyzed a large cohort of patients with lower esophageal and junctional adenocarcinoma, prospectively sequenced by MSK-IMPACT with high-quality clinical annotation. Patients were subdivided according to treatment intent, curative versus palliative, which closely mirrored clinical staging. Genomic features, alterations, and pathways were examined for association with overall survival using Cox proportional hazard models, adjusted for relevant clinicopathologic factors knowable at the time of diagnosis. Analysis of 487 patients revealed 16 oncogenic driver alterations, mostly amplifications, present in ≥5% of patients. Patients in the palliative-intent cohort, compared with those in the curative-intent cohort, were more likely to have metastatic disease, ERBB2 amplifications, Cell-cycle and RTK-RAS pathway alterations, as well as a higher fraction of genome altered and rate of whole-genome doubling. In multivariable analyses, CDKN2A alterations, SMAD4 alterations, KRAS amplifications, Cell-cycle and TGFβ pathways, and overall number of oncogenic drivers were independently associated with worse overall survival. ERBB2 amplification was associated with improved survival, presumably due to trastuzumab therapy. Our study suggests that higher levels of genomic instability are associated with more advanced disease in esophageal adenocarcinoma. Furthermore, CDKN2A, KRAS, and SMAD4 represent prognostic biomarkers, given their strong association with poor survival.

A survey of cancer genome signatures identifies genes connected to distinct chromosomal instability phenotypes.

Certain breast and ovarian cancers are characterised by high levels of chromosomal instability. We established a suite of eleven SNP array-based signatures of various forms of chromosomal instability. To understand what biological mechanisms might underpin these signatures, we generated and assembled genetic-feature data including allele-specific expression, fusion genes, gene expression, methylation, somatic coding mutations and protein expression. For each signature, we extracted a compendium of significantly associated genetic features using machine learning. We established an association between subchromosomal allelic imbalance-based measures and DNA repair genes. Numerical chromosomal instability and chromothripsis were found to have distinct genetic associations but shared a relationship to mitotic processes, while whole-genome doubling was characterised by TP53 mutation, and high AURKA and GINS1 expression. Furthermore, we identified two genetically distinct forms of tandem duplicator phenotypes. These findings identify potentially novel genomic targets for validation and drug development for specific subsets of breast and ovarian cancer.

High prevalence of TP53 loss and whole-genome doubling in early-onset colorectal cancer

The global incidence of early-onset colorectal cancer (EO-CRC) is rapidly rising. However, the reason for this rise in incidence as well as the genomic characteristics of EO-CRC remain largely unknown. We performed whole-exome sequencing in 47 cases of EO-CRC and targeted deep sequencing in 833 cases of CRC. Mutational profiles of EO-CRC were compared with previously published large-scale studies. EO-CRC and The Cancer Genome Atlas (TCGA) data were further investigated according to copy number profiles and mutation timing. We classified colorectal cancer into three subgroups: the hypermutated group consisted of mutations in POLE and mismatch repair genes; the whole-genome doubling group had early functional loss of TP53 that led to whole-genome doubling and focal oncogene amplification; the genome-stable group had mutations in APC and KRAS, similar to conventional colon cancer. Among non-hypermutated samples, whole-genome doubling was more prevalent in early-onset than in late-onset disease (54% vs 38%, Fisher’s exact P = 0.04). More than half of non-hypermutated EO-CRC cases involved early TP53 mutation and whole-genome doubling, which led to notable differences in mutation frequencies between age groups. Alternative carcinogenesis involving genomic instability via loss of TP53 may be related to the rise in EO-CRC.

Deep sequencing reveals the genomic characteristics of lung adenocarcinoma presenting as ground-glass nodules (GGNs).

BackgroundThe concept of multi-step progression from atypical adenomatous hyperplasia (AAH) to invasive adenocarcinoma (ADC) has been proposed, and ground-glass nodules (GGNs) may play a critical role during the early lung tumorigenesis. We present the first comprehensive description of the genomic architecture of GGNs to unravel the genetic basis of GGN.MethodsWe investigated 30 GGN-like lungs ADC by performing >1,000× whole-exome sequencing (WES) and characterized the genomic variations and evaluate the relationship between the clinicopathologic and molecular characteristics in this disease.ResultsDespite the low somatic mutation burden, GGNs exhibited high intratumor heterogeneity (ITH) characterized by the proportion of subclonal mutations. Different mutagenesis shaped the genomes of GGN during cancer evolution and were mostly featured by molecular clock-like signatures that occur in clonal mutations and defective DNA mismatch signatures that occur in subclonal mutations. Moreover, 10.7–67.1% clonal mutations occurred after whole-genome doubling (WGD), indicating that WGD could be a frequent truncal event in GGNs. Samples with WGD showed higher genomic instability but lower ITH. These GGNs were characterized by recurrent focal copy-number changes that are highly associated with tumorigenesis, with only two genes (EGFR and RBM10) that were recurrently mutated. Additionally, GGNs with different pathological subtypes or computed tomography (CT) features exhibited distinct genetic characteristics. Lepidic predominant or pure GGNs in CT images carried a lower mutation burden and had a relatively stable genome than nonlepidic or mixed GGNs. GGNs with RBM10 mutations tended to accompany a pathologically lepidic pattern, indicating RBM10 may drive the distinct subtype of lung cancer with better prognosis.ConclusionsThese findings facilitated interpreting the genomic characteristics of GGNs, provided insight into the early stages of lung cancer evolution, and possessed potential clinical significance.

Genetic and molecular subtype heterogeneity in newly diagnosed early- and advanced-stage endometrial cancer

ObjectiveTo characterize and compare the molecular subtypes and profiles of prospectively-accrued newly-diagnosed early- and advanced-stage endometrial cancers (ECs). MethodsEC patients consented to an IRB-approved protocol of massively parallel sequencing of 410–468 cancer-related genes; 175 ECs of 7 histologic types (n = 135 FIGO stages I/II, n = 40 FIGO stages III/IV) were included. Previously reported sequencing data from 99 additional advanced-stage ECs were retrieved for comparisons. ResultsIrrespective of histologic type, all 175 ECs could be stratified into the molecular subtypes, with 75 (43%) being of p53 wild-type, 49 (28%) MMR-deficient, 39 (22%) p53 abnormal and 12 (7%) of POLE molecular subtypes. Subtype distribution, mutational and copy number profiles varied according to histologic type. In endometrioid ECs, genetic alterations varied according to histologic grade. Potential therapeutic targets, including high tumor mutational burden, ERBB2 amplification and PIK3CA hotspot mutations, were found across histologic types in 63% (n = 110) of all ECs. Compared to their early-stage counterparts, advanced-stage endometrioid ECs had a significantly higher fraction of genome altered (median 0.1% vs 12%, p < 0.001) and ARID1B mutations (0% vs 11%, p = 0.01), and advanced-stage serous ECs harbored more frequent ERBB2 amplification (18% vs 8%, p > 0.05) and PIK3CA mutations (46% vs 27%, p > 0.05). Whole-genome doubling was found in advanced- but not early-stage carcinosarcomas and clear cell carcinomas. ConclusionsOur findings demonstrate the molecular heterogeneity within and across histologic types of EC and the increased genomic complexity of advanced-stage ECs. Molecular subtypes are present across EC histologic types and may help stratify EC patients for prognostic and therapeutic purposes.

Whole-genome doubling confers unique genetic vulnerabilities on tumour cells.

SummaryWhole genome doubling (WGD) occurs early in tumorigenesis and generates genetically unstable tetraploid cells that fuel tumor development1,2. Cells that undergo WGD (WGD+) must adapt to accommodate their abnormal tetraploid state; however, the nature of these adaptations, and whether they confer vulnerabilities that can subsequently be exploited therapeutically, is unclear. Using sequencing data from ~10,000 primary human cancer samples and essentiality data from ~600 cancer cell lines, we show that WGD gives rise to common genetic traits that are accompanied by unique vulnerabilities. We reveal that WGD+ cells are more dependent on spindle assembly checkpoint signaling, DNA replication factors, and proteasome function than WGD– cells. We also identify KIF18A, which encodes for a mitotic kinesin, as being specifically required for the viability of WGD+ cells. While loss of KIF18A is largely dispensable for accurate chromosome segregation during mitosis in WGD– cells, its loss induces dramatic mitotic errors in WGD+ cells, ultimately impairing cell viability. Collectively, our results reveal new strategies to specifically target WGD+ cancer cells while sparing the normal, non-transformed WGD– cells that comprise human tissue.

Reviving the Autopsy for Modern Cancer Evolution Research.

Simple SummaryMedical autopsies have a long history, but are currently experiencing a revival in order to progress modern cancer evolution research. Research autopsies represent an unparalleled opportunity to collect large volumes of cancer tissue accessible from multiple sites in a metastatic cancer patient’s body—not usually feasible during the standard clinical course. These collections enable researchers to unravel tumour evolution and heterogeneity questions. Many institutions around the world have recognised the value of these tissues, and have established rapid cancer autopsy programmes. Our article discusses a comprehensive collection of 24 rapid cancer autopsy programmes from across the globe.Outstanding questions plaguing oncologists, centred around tumour evolution and heterogeneity, include the development of treatment resistance, immune evasion, and optimal drug targeting strategies. Such questions are difficult to study in limited cancer tissues collected during a patient’s routine clinical care, and may be better investigated in the breadth of cancer tissues that may be permissible to collect during autopsies. We are starting to better understand key tumour evolution challenges based on advances facilitated by autopsy studies completed to date. This review article explores the great progress in understanding that cancer tissues collected at autopsy have already enabled, including the shared origin of metastatic cells, the importance of early whole-genome doubling events for amplifying genes needed for tumour survival, and the creation of a wealth of tissue resources powered to answer future questions, including patient-derived xenografts, cell lines, and a wide range of banked tissues. We also highlight the future role of these programmes in advancing our understanding of cancer evolution. The research autopsy provides a special opportunity for cancer patients to give the ultimate gift—to selflessly donate their tissues towards better cancer care.

Pervasive chromosomal instability and karyotype order in tumour evolution.

Chromosomal instability in cancer consists of dynamic changes to the number and structure of chromosomes1,2. The resulting diversity in somatic copy number alterations (SCNAs) may provide the variation necessary for tumour evolution1,3,4. Here we use multi-sample phasing and SCNA analysis of 1,421 samples from 394 tumours across 22tumour types to show that continuous chromosomal instability results in pervasive SCNA heterogeneity. Parallel evolutionary events, which cause disruption in the same genes (such as BCL9,MCL1, ARNT (also known as HIF1B), TERT and MYC) within separate subclones, were present in 37% of tumours. Most recurrent lossesprobably occurred before whole-genome doubling, that was foundas a clonal event in 49% of tumours. However, loss of heterozygosity at the human leukocyte antigen (HLA) locus and loss of chromosome 8p to a single haploid copy recurred at substantial subclonal frequencies, even in tumours with whole-genome doubling, indicating ongoing karyotype remodelling. Focal amplifications that affected chromosomes 1q21 (which encompasses BCL9,MCL1 and ARNT), 5p15.33 (TERT), 11q13.3 (CCND1), 19q12 (CCNE1) and 8q24.1 (MYC) were frequently subclonal yet appeared to be clonal within single samples. Analysis of an independent series of 1,024 metastatic samples revealed that 13 focal SCNAs were enriched in metastatic samples, including gains in chromosome 8q24.1 (encompassing MYC) in clear cell renal cellcarcinoma and chromosome 11q13.3 (encompassing CCND1) in HER2+ breast cancer. Chromosomal instability may enable the continuous selection of SCNAs, which are established as ordered events that often occur in parallel, throughout tumour evolution.

Abstract 5459: Combining targeted sequencing and ultra-low pass WGS for accurate SCNA detection on tumor sample of biased aneuploidy

Abstract Background: Somatic copy number alteration (SCNA) as a common genomic abnormality in tumor tissue has been routinely screened in cancer research due to its role in tumorigenesis. One popular approach of genome scale SCNA detection is ultra-low pass WGS. However, when dealing with tumor sample of highly biased aneuploidy, this method makes biased normalization of sequencing throughput and consequently yields inaccurate SCNA result. Recent pan-cancer study revealed that whole genome doubling (WGD), a typical example of severe aneuploidy, occurs in 56% of tumor samples, much higher than previous estimation. This observation highlights the imminent need for developing more accurate SCNA detection method especially when dealing with tumor with high aneuploidy. Here, we present a targeted sequencing guided SCNA detection method that allows accurate SCNA detection through improved normalization method. Method: Both tumor and normal tissue samples were subjected to targeted sequencing (genomic coverage: 300kb) and 2X ultra-low pass WGS. Genomic regions were binned and corresponding depth was calculated. GC bias was corrected using LOWSS regression. Single nucleotide polymorphism (SNP) was detected from targeted sequencing. We then selected all genomics regions that have stable sequencing depth as well as stable VAF of heterozygous SNP. Among them, regions of lowest depth were defined as diploid and used for sequencing throughput normalization. To compare SCNA results from different methods, we defined CN-VAF conflict index to represent the deviation of VAF of heterozygous SNP from diploid region. Results and conclusion: We applied our method to a total number of 29 NSCLC samples. In comparison with CNVkit and ControlFreeC results, 18 tumor samples shown consistent SCAN results. For the rest of 11 samples, CNVkit and ControlFreeC resulted in overall higher SCNA across genome comparing to our method. Correlation analysis shown that the inconsistency of SCNA status between methods positively correlates with the difference of baseline estimation (Pearson coef: CNVkit: 0.93, ControlFreeC: 0.91). For samples with inconsistent SCNA results, CN-VAF conflict index indicated that our method made more accurate SCNA estimation through improved baseline estimation. In addition, experimental validation using ddPCR shown consistent SCNA results with our method. It is worth mentioning that the key step of our method is the estimation of baseline depth which depends on the length of genomic region, the amount of SNP within the genomic region and the fluctuation of both depth and SNP VAF in the region. If sufficient tumor samples with experimentally validated aneuploidy are available, machine learning approach may make further improved estimation. Lastly, it is worth mentioning that this article is not intended to propose using both targeted sequencing and ultra-low WGS in routine NGS experiment, but rather as a complementary approach to evaluate the heterogeneity of aneuploidy tumor. Citation Format: Cheng Yan, Weihua Guo, Yufei Yang. Combining targeted sequencing and ultra-low pass WGS for accurate SCNA detection on tumor sample of biased aneuploidy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5459.

The Underlying Tumor Genomics of Predominant Histologic Subtypes in Lung Adenocarcinoma.

The purpose of the study is to genomically characterize the biology and related therapeutic opportunities of prognostically important predominant histologic subtypes in lung adenocarcinoma (LUAD). We identified 604 patients with stage I to III LUAD who underwent complete resection and targeted next-generation sequencing using the Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets platform. Tumors were classified according to predominant histologic subtype and grouped by architectural grade (lepidic [LEP], acinar or papillary [ACI/PAP], and micropapillary or solid [MIP/SOL]). Associations among clinicopathologic factors, genomic features, mutational signatures, and recurrence were evaluated within subtypes and, when appropriate, quantified using competing-risks regression, with adjustment for pathologic stage and extent of resection. MIP/SOL tumors had higher tumor mutational burden (p < 0.001), fraction of genome altered (p= 0.001), copy number amplifications (p= 0.021), rate of whole-genome doubling (p= 0.008), and number of oncogenic pathways altered ( p < 0.001) as compared with LEP and ACI/PAP tumors. Across all tumors, mutational signatures attributed to APOBEC activity were associated with the highest risk of postresection recurrence: SBS2 (p= 0.021) and SBS13 (p= 0.005). Three oncogenic pathways (p53, Wnt, Myc) were altered with statistical significance in MIP/SOL tumors. Compared with LEP and ACI/PAP tumors, MIP/SOL tumors had a higher frequency of targetable BRAF-V600E mutations (p= 0.046). Among ACI/PAP tumors, alterations in the cell cycle (p < 0.001) and PI3K (p= 0.002) pathways were associated with recurrence; among MIP/SOL tumors, only PI3K alterations were associated with recurrence (p= 0.049). These results provide the first in-depth assessment of tumor genomic profiling of predominant LUAD histologic subtypes, their associations with recurrence, and their correlation with targetable driver alterations in patients with surgically resected LUAD.

Thermodynamic energetics underlying genomic instability and whole-genome doubling in cancer

Genomic instability contributes to tumorigenesis through the amplification and deletion of cancer driver genes. DNA copy number (CN) profiling of ensembles of tumors allows a thermodynamic analysis of the profile for each tumor. The free energy of the distribution of CNs is found to be a monotonically increasing function of the average chromosomal ploidy. The dependence is universal across several cancer types. Surprisal analysis distinguishes two main known subgroups: tumors with cells that have or have not undergone whole-genome duplication (WGD). The analysis uncovers that CN states having a narrower distribution are energetically more favorable toward the WGD transition. Surprisal analysis also determines the deviations from a fully stable-state distribution. These deviations reflect constraints imposed by tumor fitness selection pressures. The results point to CN changes that are more common in high-ploidy tumors and thus support altered selection pressures upon WGD.

Origin, conservation, and loss of alternative splicing events that diversify the proteome in Saccharomycotina budding yeasts.

Many eukaryotes use RNA processing, including alternative splicing, to express multiple gene products from the same gene. The budding yeast Saccharomyces cerevisiae has been successfully used to study the mechanism of splicing and the splicing machinery, but alternative splicing in yeast is relatively rare and has not been extensively studied. Alternative splicing of SKI7/HBS1 is widely conserved, but yeast and a few other eukaryotes have replaced this one alternatively spliced gene with a pair of duplicated, unspliced genes as part of a whole genome doubling (WGD). We show that other examples of alternative splicing known to have functional consequences are widely conserved within Saccharomycotina. A common mechanism by which alternative splicing has disappeared is by replacement of an alternatively spliced gene with duplicate unspliced genes. This loss of alternative splicing does not always take place soon after duplication, but can take place after sufficient time has elapsed for speciation. Saccharomycetaceae that diverged before WGD use alternative splicing more frequently than S. cerevisiae, suggesting that WGD is a major reason for infrequent alternative splicing in yeast. We anticipate that WGDs in other lineages may have had the same effect. Having observed that two functionally distinct splice-isoforms are often replaced by duplicated genes allowed us to reverse the reasoning. We thereby identify several splice isoforms that are likely to produce two functionally distinct proteins because we find them replaced by duplicated genes in related species. We also identify some alternative splicing events that are not conserved in closely related species and unlikely to produce functionally distinct proteins.

Distinct genomic instability landscape of lung adenocarcinoma associated with treatment and metastasis.

9534 Background: Lung adenocarcinoma (LUAD) is the most common subtype of non-small cell lung cancer (NSCLC). Genomic instability, defined as genome-wide copy number alterations, is a key pathogenic signature which occurs at the early stage of most cancers and is associated with an increased risk of recurrence or death. We examined the pattern of genomic instability in primary and metastatic LUAD. Methods: We performed deep targeted sequencing (425 genes) of 3395 tissue samples and whole exome sequencing (WES) of 60 tissue samples from LUAD patients. Whole-genome doubling (WGD) and arm level aneuploidy were analyzed to uncover correlation with clinical phenotypes and other genomic alterations including driver mutations, tumor mutation burden (TMB), and microsatellite instability (MSI). Results: Overall, targeted sequencing revealed that WGD occurred in 64.33% LUAD samples, which was comparable with WES results. Compared to primary site, metastasis exhibited higher proportion of WGD (1.14 fold). Specifically, liver metastasis has the highest WGD percentage among metastasis sites (~87.5%; 1.40 fold increase compared to primary). Interestingly, patients who received tyrosine kinase inhibitors (TKI) had higher frequency of WGD than patients without TKI treatment. In addition, TMB was higher in WGD+ patients but MSI status was not significantly different between groups. Arm-level aneuploidy was prevalent in this cohort. The most common amplification events were 7p gain (62%), 5p gain (54%), and 8q gain (53%); top deletion events were 19p loss (47%), 15q loss (42%), and 10 q loss (41%). Patients with EGFR or TP53 mutation were more likely to have aneuploidy compared to wildtypes. Subgroup analysis showed distinct patterns of aneuploidy among metastasis sites, suggesting organ-specific alterations. Evolution analysis showed 7p gain was an early event common in primary tumor whereas metastatic tumor had multiple distinct evolutionary trajectories following 7p gain. Several copy number signatures were associated with specific TKI and chemotherapies. For example, TKI-naïve tumors lacked 7p gain but had 19p loss as the most common alteration. Conclusions: The genomic landscape of LUAD was characterized by widespread large-scale copy number alterations including WGD and chromosomal aneuploidy. Metastasis had elevated level of aneuploidy compared to primary tumors which were specific to metastatic site. Copy number signature associated with different treatments may contribute to distinct long-term survival and side effects among patients.

Tetraploidization of Immortalized Myoblasts Induced by Cell Fusion Drives Myogenic Sarcoma Development with DMD Deletion.

Whole-genome doubling is the second most frequent genomic event, after TP53 alterations, in advanced solid tumors and is associated with poor prognosis. Tetraploidization step will lead to aneuploidy and chromosomic rearrangements. The mechanism leading to tetraploid cells is important since endoreplication, abortive cytokinesis and cell fusion could have distinct consequences. Unlike processes based on duplication, cell fusion involves the merging of two different genomes, epigenomes and cellular states. Since it is involved in muscle differentiation, we hypothesized that it could play a role in the oncogenesis of myogenic cancers. Spontaneous hybrids, but not their non-fused immortalized myoblast counterparts they are generated from, induced tumors in mice. Unstable upon fusion, the hybrid genome evolved from initial mitosis to tumors with a highly rearranged genome. This genome remodeling finally produced targeted DMD deletions associated with replicative stress, isoform relocalization and metastatic spreading, exactly as observed in human myogenic sarcomas. In conclusion, these results draw a model of myogenic oncogenesis in which cell fusion and oncogene activation combine to produce pleomorphic aggressive sarcomas.

PD18-09 GENOMIC BIOMARKERS FOR IMMUNE-CHECKPOINT BLOCKADE RESPONSE IN METASTATIC UPPER TRACT UROTHELIAL CARCINOMA

You have accessJournal of UrologyBladder Cancer: Upper Tract Transitional Cell Carcinoma I (PD18)1 Apr 2020PD18-09 GENOMIC BIOMARKERS FOR IMMUNE-CHECKPOINT BLOCKADE RESPONSE IN METASTATIC UPPER TRACT UROTHELIAL CARCINOMA Andrew W Silagy*, Renzo G DiNatale, Diego Chowell, Vlad Makarov, Eugene Pietzak, David Solit, Michael Berger, A Ari Hakimi, Eduard Reznik, Dean Bajorin, Timothy A Chan, and Jonathan A Coleman Andrew W Silagy*Andrew W Silagy* More articles by this author , Renzo G DiNataleRenzo G DiNatale More articles by this author , Diego ChowellDiego Chowell More articles by this author , Vlad MakarovVlad Makarov More articles by this author , Eugene PietzakEugene Pietzak More articles by this author , David SolitDavid Solit More articles by this author , Michael BergerMichael Berger More articles by this author , A Ari HakimiA Ari Hakimi More articles by this author , Eduard ReznikEduard Reznik More articles by this author , Dean BajorinDean Bajorin More articles by this author , Timothy A ChanTimothy A Chan More articles by this author , and Jonathan A ColemanJonathan A Coleman More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000000861.09AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Patients with metastatic upper tract urothelial carcinoma (UTUC) have limited treatment options: cisplatin-based adjuvant chemotherapy has been shown to provide a survival benefit, however renal insufficiency is a contraindication. More recently, some patients receiving second line immune-checkpoint blockade (ICB) for urothelial carcinoma appeared to respond in trials, however immunogenic biomarkers to predict this population have not been described. Therefore, we aimed to identify genomic biomarkers associated with overall survival (OS) in patients treated with ICB. METHODS: Next-generation targeted DNA sequencing (MSK-IMPACTÒ) was performed on 131 pretreatment tumor samples from 121 patients. Using our validated pipeline, tumor mutation burden (TMB), copy-number alterations (CNAs), microsatellite instability (MSI) scores and HLA class I diversity (germline and somatic) were recorded. The primary endpoint was OS, calculated as the time from the date of metastasis to death or last follow-up. Survival estimates were computed using the Kaplan-Meier method and associations with OS were tested using Cox regression models. RESULTS: The study cohort consisted of 121 patients, with 62 receiving ICB therapy (Table 1). A higher fraction of the genome bearing CNAs (FCNAg) was associated with adverse OS in univariate analysis (HR: 1.01 [1.00, 1.02], p=0.03). This finding was specific to patients treated with ICB (interaction, p=0.001) and was independent of other prognostic variables including whole-genome doubling (Figure 1). Biomarkers of response to ICB reported for other tumor types, such as TMB (HR: 0.99 [0.96, 1.01], p=0.2) and HLA homozygosity (HR: 1.2 [0.57, 2.52], p=0.6), were not associated with OS. Resection of the primary tumor was also associated with improved OS. CONCLUSIONS: A reduced fraction of CNAs in the genome was significantly associated with favorable outcomes in patients receiving ICB therapy for metastatic UTUC. Source of Funding: None © 2020 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 203Issue Supplement 4April 2020Page: e379-e379 Advertisement Copyright & Permissions© 2020 by American Urological Association Education and Research, Inc.MetricsAuthor Information Andrew W Silagy* More articles by this author Renzo G DiNatale More articles by this author Diego Chowell More articles by this author Vlad Makarov More articles by this author Eugene Pietzak More articles by this author David Solit More articles by this author Michael Berger More articles by this author A Ari Hakimi More articles by this author Eduard Reznik More articles by this author Dean Bajorin More articles by this author Timothy A Chan More articles by this author Jonathan A Coleman More articles by this author Expand All Advertisement PDF downloadLoading ...