Tumor Evolution Research Articles

Abstract LB286: Extrachromosomal DNA in lung cancer from never smokers

Abstract Extrachromosomal DNA (ecDNA), circular DNA that resides outside of the linear chromosome, is a potent form of oncogene amplification in cancer. The structure and properties of ecDNA are markedly different from those of linear DNA, characterized by features like non-Mendelian inheritance and an altered epigenetic landscape. A prior pan-cancer analysis ​​indicated patients with cancers harboring ecDNA have statistically worse overall survival when compared to cancer patients with other types of focal amplification. Moreover, its unique characteristics also make it a promising target for therapy. Recently, efforts have intensified to profile ecDNA across many cancer types, yet comprehensive studies of ecDNA in lung cancer in never smokers (LCINS) is lacking. Given that LCINS is partially driven by somatic copy number alterations, we hypothesized that ecDNA-driven focal amplifications significantly influence LCINS tumor evolution. As part of the Sherlock-Lung project (current data freeze, 1217 samples), we comprehensively profiled 871 treatment-naive samples using a robust computational pipeline that refined copy number segmentation by accounting for tumor purity and ploidy. ecDNA was present in 151 (17.3%) of tumor samples, with a third of ecDNA-positive samples harboring more than one distinct ecDNA amplicon. Consistent with prior reports, no ecDNA was found in bulk sequenced blood or matched normal lung tissues. These ecDNA often included genes (84%), frequently overlapping with known oncogenes like MDM2, TERT, and MYC, which showed higher expression levels compared to non-amplified or other amplified genes. Notably, most MDM2 amplifications (36/45) were on ecDNA. ecDNA was significantly associated with whole genome doubling, tumor stage, and several endogenous mutational signatures, including APOBEC (SBS2 and SBS13), and clock-like signature (SBS1). Intriguingly, ecDNA-positive tumors were associated with significantly worse survival compared to tumors without any focal amplifications, though tumors with other amplification types (e.g. breakage-fusion-bridge, linear amplification, or complex events) had similar survival impacts. The unprecedented size of our cohort and the integration of clinical, epidemiologic, and multi-omics data allowed us to perform the first in-depth characterization of ecDNA in LCINS. We reconstructed the genomic regions involved in ecDNA and revealed the full spectrum of simple and complex ecDNAs across cancer histologies as well as the clinical and functional impact of ecDNA on LCINS. Citation Format: Azhar Khandekar, Tongwu Zhang, Marcos Diaz-Gay, Jens Luebeck, Ludmil Alexandrov, Maria Landi. Extrachromosomal DNA in lung cancer from never smokers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB286.

Abstract NG02: Distinct genomic and immunologic tumor evolution in germline TP53-driven breast cancers

Abstract NG02: Distinct genomic and immunologic tumor evolution in germline TP53-driven breast cancers

Genetic intratumour heterogeneity and clonal evolution in extramammary Paget's disease.

All tumour cells in a patient have shared and non-shared genetic alterations, and the diversity of mutations is described as intratumoural heterogeneity (ITH). Multiregion sequencing is a genome sequencing analytical technique used for multiple, spatially-separated biopsy tissues that may further our understanding of ITH and tumour evolution. Although genetic mutations in extramammary Paget's disease (EMPD) have recently been detected by next-generation sequencing analysis, there have been no reports of ITH based on multiregion sequencing in EMPD. Thus, we clarified the landscape of ITH and tumour evolution in EMPD. We performed whole-exome sequencing on 35 tissues (30 tumour tissues and five normal skin samples as a paired control), collected from five patients with EMPD. The rate of private mutations was significantly higher than that of ubiquitous and shared mutations. Ubiquitous mutations were not present in driver genes, and most driver genes exhibited private and shared mutations. The most frequent base substitution was C>T in almost all lesions, and most mutational signatures corresponded to signature 1, 2, 3, and 8. The types of proposed aetiology in most lesions were based on age and AID/APOBEC family and BRCA1/BRCA2 mutations. Evolutionary trees were characterized by short trunks and long branches due to the extremely high ratio of private mutations. In contrast, pathogenic factors, such as base substitutions, mutational signatures, and proposed aetiology, were shared. Tumour evolution in EMPD appears to be characterized by a high level of genetic ITH with shared background factors.

Targeting APOBECs in cancer: It’s about timing

APOBEC3 cytidine deaminases have emerged as key drivers of mutagenesis in a wide spectrum of tumor types and are now appreciated to play a causal role in driving tumor evolution and drug resistance. As efforts to develop APOBEC3 inhibitors progress, understanding the timing and consequences of APOBEC3-mediated mutagenesis in distinct clinical contexts will be critical for guiding the development of anti-cancer therapeutic strategies.

Characterization of tumor evolution by functional clonality and phylogenetics in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is a molecularly heterogeneous solid malignancy, and its fitness may be shaped by how its tumor cells evolve. However, ability to monitor tumor cell evolution is hampered by the presence of numerous passenger mutations that do not provide any biological consequences. Here we develop a strategy to determine the tumor clonality of three independent HCC cohorts of 524 patients with diverse etiologies and race/ethnicity by utilizing somatic mutations in cancer driver genes. We identify two main types of tumor evolution, i.e., linear, and non-linear models where non-linear type could be further divided into classes, which we call shallow branching and deep branching. We find that linear evolving HCC is less aggressive than other types. GTF2IRD2B mutations are enriched in HCC with linear evolution, while TP53 mutations are the most frequent genetic alterations in HCC with non-linear models. Furthermore, we observe significant B cell enrichment in linear trees compared to non-linear trees suggesting the need for further research to uncover potential variations in immune cell types within genomically determined phylogeny types. These results hint at the possibility that tumor cells and their microenvironment may collectively influence the tumor evolution process.

A multiparameter liquid biopsy approach allows to track melanoma dynamics and identify early treatment resistance

Melanoma heterogeneity is a hurdle in metastatic disease management. Although the advent of targeted therapy has significantly improved patient outcomes, the occurrence of resistance makes monitoring of the tumor genetic landscape mandatory. Liquid biopsy could represent an important biomarker for the real-time tracing of disease evolution. Thus, we aimed to correlate liquid biopsy dynamics with treatment response and progression by devising a multiplatform approach applied to longitudinal melanoma patient monitoring. We conceived an approach that exploits Next Generation Sequencing (NGS) and droplet digital PCR, as well as the FDA-cleared platform CellSearch, to analyze circulating tumor DNA (ctDNA) trend and circulating melanoma cell (CMC) count, together with their customized genetic and copy number variation analysis. The approach was applied to 17 stage IV melanoma patients treated with BRAF/MEK inhibitors, followed for up to 28 months. BRAF mutations were detected in the plasma of 82% of patients. Single nucleotide variants known or suspected to confer resistance were identified in 70% of patients. Moreover, the amount of ctDNA, both at baseline and during response, correlated with the type and duration of the response itself, and the CMC count was confirmed to be a prognostic biomarker. This work provides proof of principle of the power of this approach and paves the way for a validation study aimed at evaluating early ctDNA-guided treatment decisions in stage IV melanoma. The NGS-based molecular profile complemented the analysis of ctDNA trend and, together with CMC analysis, revealed to be useful in capturing tumor evolution.

Evolution of Testicular Germ Cell Tumors in the Molecular Era With Histogenetic Implications.

The current WHO classification of testicular germ cell tumors is based on the pathogenesis of the tumors driven by different genomic events. The germ cell neoplasia in situ is the precursor lesion for all malignant germ cell tumors. The current understanding of pathogenesis is that the developmental and environmental factors with the erasure of parental genomic imprinting lead to the development of abnormal gonocytes that settle in the "spermatogonial Niche" in seminiferous tubules. The abnormal primordial germ cells in the seminiferous tubules give rise to pre-GCNIS cells under the influence of TPSY and OCT4 genes. The whole genome duplication events give rise to germ cell neoplasia in situ, which further acquires alterations in 12p along with NRAS and KRAS mutations to produce seminoma. A subset of seminomas acquires KIT mutation and does not differentiate further. The remaining KIT-stable seminomas differentiate to nonseminomatous GCTs after obtaining recurrent chromosomal losses, epigenetic modification, and posttranscriptional regulation by multiple genes. Nonseminomatous germ cell tumors also develop directly from differentiated germ cell neoplasia in situ. TP53 pathway with downstream drivers may give rise to somatic-type malignancies of GCT. The GCTs are remarkably sensitive to cisplatin-based combination chemotherapy; however, resistance to cisplatin develops in up to 8% of tumors and appears to be driven by TP53/MDM2 gene mutations. Serum and Plasma miRNAs show promise in diagnosing, managing, and following up on these tumors. The mechanisms underlying the development of most tumors have been elucidated; however, additional studies are required to pinpoint the events directing specific characteristics. Advances in identifying specific molecular markers have been seen recently and may be adopted as gold standards in the future.

Multi-omic profiling of simultaneous ductal carcinoma in situ and invasive breast cancer

PurposeThe progression of ductal carcinoma in situ (DCIS) to invasive breast carcinoma (IBC) in humans is highly variable. To better understand the relationship between them, we performed a multi-omic characterization of co-occurring DCIS and IBC lesions in a cohort of individuals.MethodsFormalin-fixed paraffin-embedded tissue samples from 50 patients with co-occurring DCIS and IBC lesions were subjected to DNA-seq and whole transcriptome RNA-seq. Paired DCIS and IBC multi-omics profiles were then interrogated for DNA mutations, gene expression profiles and pathway analysis.ResultsMost small variants and copy number variations were shared between co-occurring DCIS and IBC lesions, with IBC exhibiting on average a higher degree of additional mutations. However, 36% of co-occurring lesions shared no common mutations and 49% shared no common copy number variations. The most frequent genomic variants in both DCIS and IBC were PIK3CA, TP53, KMT2C, MAP3K1, GATA3 and SF3B1, with KMT2C being more frequent in DCIS and TP53 and MAP3K1 more frequent in IBC, though the numbers are too small for definitive conclusions. The most frequent copy number variations were seen in MCL1, CKSB1 and ERBB2. ERBB2 changes were not seen in IBC unless present in the corresponding DCIS. Transcriptional profiles were highly distinct between DCIS and IBC, with DCIS exhibiting upregulation of immune-related signatures, while IBC showed significant overexpression in genes and pathways associated with cell division and proliferation. Interestingly, DCIS and IBC exhibited significant differential expression of different components of extracellular matrix (ECM) formation and regulation, with DCIS showing overexpression of ECM-membrane interaction components while IBC showed upregulation of genes associated with fibronectin and invadopodia.ConclusionWhile most co-occurring DCIS and IBC were mutationally similar and suggestive of a common clonal progenitor, transcriptionally the lesions are highly distinct, with IBC expressing key pathways that facilitate invasion and proliferation. These results are suggestive of additional levels of regulation, epigenetic or other, that facilitate the acquisition of invasive properties during tumor evolution.

Abstract 7353: Exploring intrapatient heterogeneity in metastatic breast cancer: Insights into tumor evolution from a warm procurement trial

Abstract Metastatic breast cancers show variable clinical responses due to inherent heterogeneity, both within tumors and among patients. Studies mainly examine molecular diversity across different patients' tumors and genetic variance within a single tumor. Yet, the variability among multiple tumors or metastases in a single patient remains underexplored. Our study investigates this intrapatient heterogeneity, examining cell and tumor evolution from a single genetic source in metastatic breast cancer. In a warm procurement trial involving 6 patients with multi-site tumors, samples were collected for detailed molecular analysis. Whole exome sequencing (WES) and single-cell RNA sequencing (scRNA-seq) were used to analyze genetic similarities and disparities. WES data revealed mutational patterns and copy number alterations, showing homogeneity and heterogeneity in tumors within patients. Further, phylogenetic analysis with scRNA-seq variant calls paired with transcriptional phenotypes uncovered a MYC-enriched subclonal population in a metastatic site. Integrating WES and scRNA-seq data provided a clearer understanding of the genomic and phenotypic features in and between patients. Our WES study revealed significant interpatient variations in genetic drivers and CNA profiles. Among five patients, two showed APOBEC and HR mutations, linked to increased tumor mutations. Despite general genetic homogeneity, there were exceptions. Tumors in Patient 3's upper lymph nodes had distinct genetics compared to abdominal tumors. Unique genetic patterns were also noted in the pancreas and mesentery of Patients 3 and 7, respectively. Genetic findings were supported by transcriptional analysis, showing distinct expression patterns in specific sites. Phylogenetic analysis highlighted intratumor heterogeneity, as shown by variations in phylogenetic composition and gene expression across samples. Notably, in patients with the most samples (Patients 3 and 7), a conserved cell clade, evolved early and primarily located in a single site, displayed a high proliferation gene expression signature closely associated with Myc activity. This underscores the importance of early clonal expansions in tumor evolution and suggests potential therapeutic targets in these early, proliferative cells. Our study highlights the vital role of intrapatient heterogeneity in understanding cancer complexity and treatment resistance. By analyzing genetic and phenotypic variability in multi-site tumors within patients, and Myc's key role in tumor growth and evolution, our research offers new insights into tumor evolution. This underscores the need for personalized oncology treatments based on each patient's tumor genetics. Citation Format: Isaac Bishara, Xuan Liu, Jason Griffiths, Jiayi Liu, Patrick Cosgrove, Jasmine R. McQuerry, Feng Chi, Pierre Wallet, Vince K. Grolmusz, Benjamin Copeland, Lance Pflieger, Jinfeng Chen, Sumana Majumdar, Grace Ronquillo, Terron Crowder, Rena Emond, Rachel Factor, Eliza Barragan, David Bowtell, Adam Cohen, Daniel D. Schmolze, Peter Lee, Kena Ihle, Lusine Tumyan, Aritro Nath, James Waisman, Andrea Bild, Jeff Chang. Exploring intrapatient heterogeneity in metastatic breast cancer: Insights into tumor evolution from a warm procurement trial [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 7353.

Abstract 3382: Unveiling gastric premalignancy: A comprehensive study of tumor initiation and evolution using organoid models

Abstract Tumor initiation and evolution is still a poorly characterized process. Organoid models recapitulate three-dimensional tissue structure, differentiation, and disease pathogenesis. These models can help to better characterize pre-neoplastic processes and tumor evolution, with implications for early cancer detection and personalized therapeutic strategies for patients with gastric cancer (GC). In our research, we performed whole exome sequencing (WES) on 24 GC patient tumors, uncovering distinct mutation frequencies in well-known tumor suppressor genes -APC, TP53, and ARID1A (18.75%, 37.5%, 43.75%, respectively) -compared to The Cancer Genome Atlas (TCGA) PanCancer Altas Cohort (n=440) (13.48%, 50.56%, 22.47% respectively). To gain a deeper understanding of how these mutated genes promote gastric premalignancy, we generated multiple isogenic gastric organoid lines with mutant APC (AT-dmut) or ARID1A (ArT-dmut) on a TP53 mutant background as well as TP53 mutant-only lines (T-mut) using our established CRISPR/Cas9 genome-editing platform and two HLA patient-derived normal gastric organoid lines (HL-PDOs). After confirmation of functional gene knockouts, we focused on elucidating carcinogenic changes via a two-pronged approach: (1) a comparative analysis of growth and morphology between these mutant organoid models and the parental normal gastric organoids and (2) an in-depth examination of mutational and transcriptional changes in our edited AT-mut, ArT-mut and T-mut organoid models. We cultured each edited organoid line for up to 350 days and collected histological and molecular data at multiple time points. Preliminary analysis shows an increased growth rate of all edited cell lines compared to parental normal lines. The ArT-mut and AT-mut cell lines exhibit distinct morphological and 3D structural alterations, while T-mut cell lines do not. We expect to find additional mutation gains in these cells’ lines compared to the parental normal gastric organoids that promote gastric phenotype with the greatest gain of alternations at the 350-day timepoint. These additional mutations and transcriptional changes will help us gain insights into the pathways altered in gastric cancer initiation and early progression that may be specific to HLAs. Our overall goal is to enhance our understanding of the molecular mechanisms underlying gastric premalignancy among HLAs, ultimately contributing to advancements in early detection and tailored therapeutic interventions for HLA patients with GC and advancing cancer health equity. Citation Format: Alexa Morales Arana, Nicole B. Halmai, Hongyong Zhang, Paul Lott, Ana Estrada, University of California Minority Patient-DerivedXenograft Development and Trial (UCaMP) Center, Luis Carvajal-Carmona. Unveiling gastric premalignancy: A comprehensive study of tumor initiation and evolution using organoid models [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 3382.

Abstract 1617: Tumor evolution during therapy in ovarian cancer

Abstract The evolution of tumors from diagnosis to relapse in ovarian high-grade serous carcinoma (HGSC) patients is poorly understood. Consequently, it is unknown whether diagnostic samples are representative enough to guide treatment directions at relapse, or whether acquired changes during therapy provide targets in patients with relapses. To answer these questions and characterize tumor evolution during treatment, we used data from >700 tissue and plasma samples from 200 HGSC patients belonging to the observational, longitudinal DECIDER trial (NCT04846933). Mutations from whole-genome sequencing data from tissue samples (2-12 per patient) were used to construct phylogenetic trees. The trees were corrected by the copy-number alterations and curated to integrate circulating tumor DNA (ctDNA) exome- and targeted-sequencing data from plasma samples. Evolutionary states presenting within and between sample heterogeneity were evaluated through clonal complexity and divergence. Additionally, genetic differences between persistent and non-persistent subclones were studied to identify features affecting treatment resistance. Our results indicate that the majority of subclones survive chemotherapy treatment and major subclonal replacement takes place in only a few patients. Within-sample heterogeneity was higher in plasma samples than in ascites, highlighting the importance of ctDNA samples to estimate persistent subclones. Additionally, our recently reported evolutionary states (Lahtinen et al. Cancer Cell 2023) were often altered by the treatment induced selection pressure. At late relapses, subclone compositions typical of maintaining evolutionary state tumors were less common, and a higher number of acquired subclones were detected, suggesting further evolution of the persisting clones. In summary, chemotherapy does not suppress heterogeneity in HGSC tumors. Interestingly, patients with the worst prognosis have a stable composition of multiple clones in their tumors, and tumors with low heterogeneity responded better to primary treatment. Relapse samples show lower within-sample heterogeneity and further evolution. Furthermore, we detected several evolutionary trajectories from stable, persistent cancer subclone populations to highly dynamic subclone structures. This renders diagnostic samples suitable to guide relapse treatment decisions in a subset of patients with HGSC but not for all. Our characterization of the dynamics of tumor clonal heterogeneity during treatment allows to define better therapeutic interventions for HGSC patients with relapsed disease. Citation Format: Sampsa Kalervo Hautaniemi, Jaana Oikkonen, Alexandra Lahtinen, Mai T.N. Nguyen, Giovanni Marchi, Kari Lavikka, Anna Rajavuori, Sakari Hietanen, Anni Virtanen, Johanna Hynninen. Tumor evolution during therapy in ovarian 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 1617.

Abstract 419: Evolutionary trajectories of complex genome rearrangements in osteosarcoma

Abstract Whole-genome sequencing (WGS) studies have revealed that osteosarcoma genomes are riddled by remarkably intricate forms of structural variants (SV), collectively known as complex genomic rearrangements (CGR). Yet, most CGR detected in osteosarcoma remain unexplained, hinting at the possibility of undiscovered mechanisms that might lead to novel therapeutic strategies. To elucidate the mechanisms underpinning cancer genome complexity in osteosarcomas and the downstream consequences of CGR during tumor evolution, we uniformly processed exiting osteosarcoma WGS data sets and performed high-depth (>120x) multi-regional short- and long-read WGS for hundreds of osteosarcomas. We found that whole genome doubling and diverse types of CGR are frequent clonal events in most osteosarcomas. Through the integration of multi-regional WGS data, we discovered that in tumors with whole-genome doubling, cancer genome evolution and oncogene amplification are the consequence of multi-generational CGR triggered by whole-genome doubling and involving multiple mechanisms, especially chromothripsis and breakage-fusion-bridge cycles. By contrast, in tumors without whole-genome doubling, biallelic inactivation of TP53 and oncogene amplification frequently occur as part of a cascade of genomic instability initiated by a single double-strand break upstream of the TP53 locus. This process results in clonal diversification and rapid tumor growth suggestive of punctuated evolution. In addition, we found that subclonal CGRs are also frequent events. Indeed, clonal expansions triggered by the acquisition of subclonal CGR often colonize distant tumor regions, suggesting that CGR act as subclonal driver events. Finally, we show that some recurrent genomic alterations are prognostic of poor survival, which we validate in independent cohorts. In sum, using WGS data, we have elucidated the mechanisms underpinning the complexity and evolution of osteosarcoma genomes. Our results indicate that diverse types of CGR, such as chromothripsis, are triggered by whole genome doubling and occur throughout tumor evolution, which primes the cancer genome for ongoing genomic instability, rapid karyotype evolution and clonal diversification. These results have implications for our understanding of the molecular basis of intra-tumor heterogeneity and drug resistance in osteosarcomas and other cancers driven by genomic instability. Citation Format: Isidro Cortes Ciriano, Jose Espejo Valle-Inclán, Solange de Noon, Katherine Trevers, Hillary Elrick, Adrienne M. Flanagan. Evolutionary trajectories of complex genome rearrangements in osteosarcoma [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 419.

Abstract 1201: Pervasive HLA disruption fuels cancer evolution

Abstract Disruption of the human leukocyte antigen (HLA) molecules has important implications for immune evasion and tumor evolution. However, although genomic loss of HLA is frequent in non-small cell lung cancer (NSCLC) and in some types of breast cancer, the extent and importance of transcriptomic disruption to HLA presentation, including transcript repression and alternative splicing (AS), remains unclear. To address this, we developed MHC Hammer, a tool to call HLA allele-specific mutations, loss of heterozygosity (LOH), transcriptional repression, and alternative splicing. We first assessed HLA allelic expression and the prevalence of AS in normal tissue. Applying MHC Hammer to 510 normal lung samples and 407 normal breast samples from the Genotype-Tissue Expression cohort, we identified extensive heterogeneity in HLA allelic expression depending on both the allele and tissue type. In addition, HLA allelic AS, occurring in at least 20% of the allele transcripts, was observed in 19% of normal lung and 13% of normal breast samples. Next, we applied MHC Hammer to 413 TRACERx NSCLCs and 743 TCGA breast tumours. While mutations were uncommon, HLA LOH was a frequent event, occurring in 32% of lung adenocarcinomas (LUAD) and 57% of lung squamous cell carcinomas (LUSC). We observed a similar rate of HLA LOH in estrogen receptor positive (ER+) and negative (ER-) tumors (ER+: 14%, ER-: 15%), while in triple negative breast tumours we observed a higher rate of LOH (31%). Due to the heterogeneity in HLA expression observed in the normal samples, to measure tumor specific HLA repression and alternative splicing, we restricted our analysis to tumours with WES, tumor and tumor-adjacent normal RNAseq data. This resulted in a cohort of 88 NSCLCs from the TRACERx study as well as 44 ER+ breast tumours from the TCGA cohort. 61% of LUAD, 75% of LUSC and 35% of ER+ tumours harbored class I HLA transcriptional repression that could not be explained by underlying genomic loss. 27% of LUAD, 7% of LUSC and 55% of ER+ breast tumor patients exhibited no HLA disruption. Tumor specific AS affecting HLA exons 2, 3 or 4, potentially disrupting HLA antigen presentation, was identified in 8% of LUADs, 11% of LUSC and 7% of ER+ breast tumours. Tumor specific AS of exon 5, yielding soluble HLA molecules without a transmembrane domain was identified in 20% of LUAD, 14% of LUSCs and 18% of ER+ breast tumours. LUAD and LUSC tumor regions without HLA LOH or repression were enriched for somatic HLA AS events and alleles with somatic HLA AS had a higher neoantigen burden than those without. We found no relationship between survival and HLA expression or AS in the normal tissue. However, consistent with the importance of HLA dysfunction in tumor evolution, LUADs with low HLA expression in at least one tumor region were associated with shorter disease-free survival, HLA LOH was more common in LUAD’s that metastasized and HLA repression was enriched in LUSC primary regions seeding metastases. Citation Format: Clare Puttick, Thomas P. Jones, Michelle Leung, Oriol Pich, Felipe Galvez Cancino, Jiali Liu, Manuel Varas-Godoy, Andrew Rowan, Carlos Martinez-Ruiz, Robert Bentham, Krijn K. Dijkstra, Rachel Rosenthal, Nnennaya Kanu, Kevin Litchfield, Roberto Salgado, David A. Moore, Peter Van Loo, Mariam Jamal-Hanjani, Sergio A. Quezada, Nicholas McGranahan, Charles Swanton. Pervasive HLA disruption fuels cancer evolution [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 1201.

Abstract 6225: Tumor evolution through selection by ECM stiffness

Abstract In this study, we used experimental evolution to investigate the extent to which the mechanical stiffness of the tumor extracellular matrix (ECM) exerts selection pressure on a genetically diverse population of breast cancer cells. Polyacrylamide hydrogels were fabricated and conjugated with collagen type I to create the ECM with different stiffness. MDA-MB-231 cells were continuously cultured on the hydrogels of the same corresponding stiffness. Growth rate was determined at certain time points by harvesting and counting the cells. Abundance of clonal MDA-MB-231 cells labeled with heritable DNA barcodes underwent the same selection was identified with sequencing of DNA barcode tags. Cellular phenotypes were characterized through immunofluorescence staining and confocal microscopy. 3D migration of the cancer cells was examined using Boyden chamber assay. Cellular contractility was measured through traction force microscopy. Whole genome transcriptome sequencing (RNA-seq), assay for transposase-accessible chromatin with sequencing, and reduced representation bisulfite sequencing were performed for transcriptomic and epigenetic analysis. Sustained culture of MDA-MB-231 breast cancer cells overall three months on soft ECM resulted in an increased proliferation rate and beta1 integrin-dependent spreading area of the cells. Such changes did not occur in clonal cells with low genetic variation and did not occur in ancestral cells on stiff substrates. Barcode analysis revealed two distinct surviving populations post-genetic selection, with one demonstrating robust proliferation on both soft and stiff ECM, and the other exhibiting preferential adaptation to the soft matrix. The soft-selected populations exhibited nuclear localization of the transcriptional regulator yes-associated protein and less wrinkled nuclei compared with ancestral populations when both populations were grown on soft ECM. Aggressive migration was observed in the soft-selected populations, indicating a more malignant selected phenotype. The soft-selected cells showed increased RhoA activity and contractile tension on a soft substrate compared to the ancestral cells. RNA-seq analysis revealed systematic differences between ancestral, stiff-selected, and soft-selected populations corresponding to enriched biological processes related to cell adhesion and ECM organization. Significant differences in DNA methylation and chromatin accessibility were observed in soft selected populations which were correlated with changes in RNA expression. ECM stiffness exerts selection pressure on genetically variable cancer cell populations, selecting specific genetic variants in a cell population optimally adapted for that stiffness. Selected variants on soft ECM tend to be highly migratory, proliferative and generate high traction even on soft ECM. Our results highlight the importance of ECM stiffness-driven evolution in cancer development. Citation Format: Ting-Ching Wang, Suchitaa Sawhney, Daylin Morgan, Richa Rashmi, Marcos R. Estecio, Amy Brock, Irtisha Singh, Richard L. Bennett, Charles F. Baer, Jonathan D. Licht, Tanmay P. Lele. Tumor evolution through selection by ECM stiffness [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 6225.

Abstract 3289: APOBEC3A drives tumor evolution through activation of ERVs in non small cell lung cancer

Abstract Acquired drug resistance to even the most effective anti-cancer targeted therapies remains an unsolved clinical problem. Although many drivers of acquired drug resistance have been identified, the underlying molecular mechanisms shaping tumor evolution during treatment are incompletely understood. We recently demonstrated that lung cancer targeted therapies commonly used in the clinic induce the expression of cytidine deaminase APOBEC3A (A3A), leading to sustained mutagenesis in drug-tolerant cancer cells persisting during therapy. Preventing therapy-induced A3A mutagenesis by gene deletion decreased the chromosomal aberration events and delayed the emergence of drug resistance. Thus, inhibition of A3A may represent a potential therapeutic strategy to prevent acquired resistance. Understanding the regulatory mechanisms of A3A may provide alternative approaches to inhibit A3A mutagenesis. Using a panel drug screen, we found that DNA methyltransferase (DNMT) inhibitor remarkably induces A3A in non-small cell lung cancer cells. RNA-seq profiling revealed that targeted therapy as well as DNMT inhibitor treatment activate expression of distinct classes non-coding repetitive RNA elements, including endogenous retrovirus (ERVs), in drug-tolerant persister cells. Activation of intracellular viral sensing pathways by introduction of exogenous nucleic acids mimicking viral elements induced A3A expression. While the transcription factor NFkB was acutely activated by targeted therapy, TBK1-IRF3 signaling was activated at later time points. These findings suggest that reactivation of ERVs may underlie A3A mutagenic activity and tumor evolution during targeted therapy. Citation Format: Hideko Isozaki, Ramin Sakhtemani, Naveed Nikpour, Susanna Monroe, Michael Lawrence, Aaron Hata. APOBEC3A drives tumor evolution through activation of ERVs in non small cell lung 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 3289.

Abstract 869: Single-cell DNA replication dynamics in genomically unstable cancers

Abstract Background: DNA replication and cell cycle regulation are frequently disrupted as part of a cancer’s progression toward uncontrolled proliferation, generating somatic copy number alterations (CNAs) and producing intratumoral heterogeneity that drives subsequent evolution. Structural variation and CNAs have been shown to impact epigenetic and chromatin states, but our ability to assess their impact on DNA replication timing (RT) and cell proliferation rates remains limited. Single-cell whole genome sequencing (scWGS) is a powerful method for studying clonal heterogeneity and CNAs, and has the potential to provide greater insight into DNA replication dynamics in aneuploid populations. However, computational identification of S-phase cells and distinguishing inherited somatic CNAs from transient DNA replication changes remain challenging. Methods: We present a new method, PERT, which uses a Bayesian framework to model read depth as a combination of somatic copy number, replication, and sequencing bias, enabling estimation of DNA replication profiles and cell cycle phase from scWGS data. Unlike previous approaches, PERT provides unbiased estimates of RT and cell cycle phase which allows for analysis of previously uncharacterized cell types using any scWGS platform. These unique properties enable PERT to perform novel analysis such as estimating clone-specific proliferation rates and studying the interplay between RT and somatic CNAs during tumor evolution. We applied PERT to a cohort of >50,000 scWGS cells obtained from a collection of genomically unstable breast and ovarian cell lines, xenografts and primary cancer tissues. Results: Clone RT profiles correlated with future copy number changes in serially passaged cell lines. Cell type was the strongest determinant of RT heterogeneity, while whole genome doubling (WGD) and mutational signature had weaker RT associations but were associated with accumulation of late S-phase cells. Recurrent CNAs affecting chromosome X had striking impact on RT, with loss of the inactive X allele shifting replication earlier, and loss of inactive Xq resulting in reactivation of Xp. Analysis of time series xenografts illustrated that cell cycle distributions approximate clone proliferation. This relationship enabled us to observe that highly proliferative clones were the most chemosensitive in cisplatin-treated xenografts and, separately, present novel evidence that WGD leads to slower proliferation. Conclusions: Our analysis implicates cell type as the strongest determinant of RT with chrX being the locus of highest RT variation due to X-inactivation. Separately, quantification of S-phase cells enables interrogation of the on- and off-treatment fitness of genetically distinct subclones. This work leads to better understanding of how DNA replication dynamics drive and are further modulated by genomic instability. Citation Format: Adam C. Weiner, Marc Williams, Hongyu Shi, Ignacio Vazquez-Garcia, Sohrab Salehi, Nicole Rusk, Sohrab P. Shah, Andrew McPherson. Single-cell DNA replication dynamics in genomically unstable cancers [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 869.

Abstract 1205: Inferring allele-specific copy number aberrations and tumor phylogeography from spatially resolved transcriptomics

Abstract Tumors evolve through the acquisition of somatic mutations over time and across space. While the temporal aspects of tumor evolution can be inferred by applying phylogenetic techniques to bulk or single-cell DNA sequencing data, the spatial aspects of tumor evolution remain understudied. Spatially resolved transcriptomics (SRT) measures gene expression at thousands of spatial locations in a tumor, but does not directly measure genetic aberrations. Thus, SRT data has not yet been fully utilized for tumor evolution studies. We introduce CalicoST, an algorithm that simultaneously infers allele-specific copy number aberrations (CNAs) and derives a phylogeographic model of tumor evolution from SRT data. By modeling CNA-induced perturbations in both gene expression and allele frequencies, CalicoST identifies important types of CNAs - including copy-neutral loss of heterozygosity (CNLOH) and mirrored subclonal CNAs - that are invisible to total copy number analysis. On SRT data from nine patients from the Human Tumor Atlas Network (HTAN), CalicoST achieves an average accuracy of 86%, approximately 21% higher than existing methods. On two of these patients with multiple adjacent slices, CalicoST reconstructs a tumor phylogeography that describes the spread of cancerous clones in three-dimensional space. CalicoST analysis of multiple SRT slices from a cancerous prostate organ reveals five spatially coherent clones, with mirrored subclonal CNAs distinguishing clones on the two sides of the prostate, and a bifurcating phylogeography in both genetic and physical space that suggests a polyclonal cancer. Citation Format: Cong Ma, Metin Balaban, Jingxian Liu, Siqi Chen, Li Ding, Benjamin J. Raphael. Inferring allele-specific copy number aberrations and tumor phylogeography from spatially resolved transcriptomics [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 1205.

Abstract 5219: Loss of chromosome Y is unrelated to the composition of the tumor microenvironment and patient prognosis in muscle-invasive urothelial bladder cancers

Abstract Background: Loss of chromosome Y (LOY) occurs commonly in cancers of male patients. It was recently proposed to be associated with cancer aggressiveness and altered T-cell function in bladder cancer and it was found to be linked to poor prognosis in a TCGA bladder cancer patient cohort of 300 patients based on a combined analysis of RNAs from Y-chromosome genes. In this study, we aimed to clarify the clinical relevance of LOY in a large cohort of urothelial bladder cancer patients by using fluorescence in-situ hybridization (FISH). Design: X and Y chromosomes were analyzed by FISH on a tissue microarray (TMA) containing 1,819 urothelial carcinomas of the urinary bladder from male patients including 487 patients who had undergone cystectomy for muscle-invasive disease and for which follow-up data were available. Data of tumor microenvironment parameters were obtained in a previous study. Results: LOY was found in 26.0% of 1,479 analyzable cancers. In the subset of non-invasive cancers, the frequency of LOY increased from pTaG2 (14.5%) to pTG3 (28.6%) but these differences were not significant (p=0.1015). In muscle-invasive cancers, the frequency of LOY increased slightly from pT2 (25.4%) to pT4 cancers (32.8%) but this association was again not significant (p=0.1868). Within pT2-4 cancers, LOY was associated with venous invasion (p=0.0010) but unrelated to pT, pN, and L-status, as well as to overall, recurrence-free, and cancer-specific survival. Muscle-invasive urothelial carcinomas with and without LOY did not show significant differences in the number of CD8 positive lymphocytes, the fraction of CD8 positive intraepithelial lymphocytes, the number of macrophages and dendritic cells, as well as the fraction of t helper and t regulatory cells. Conclusion: FISH is the gold standard for determining LOY. That LOY was neither associated with histopathological parameters of cancer aggressiveness and patient prognosis nor with any parameters describing the tumor microenvironment strongly argues against a driving role of LOY for bladder cancer progression and cancer associated immune reaction. The slight increase of LOY from pTaG2 to pT4 cancers is consistent with a continuous (but clinically irrelevant) loss of the dispensable Y chromosomes during tumor evolution over time. Citation Format: Martina Kluth, Henning Plage, Sebastian Hofbauer, Kira Furlano, Sarah Weinberg, Antonia Franz, Annika Fendler, Florian Roßner, Simon Schallenberg, Sefer Elezkurtaj, Niclas C. Blessin, Maximilian Lennartz, Andreas H. Marx, Henrik Samtleben, Margit Fisch, Michael Rink, Marcin Slojewski, Krystian Kaczmarek, Thorsten Ecke, Stefan Koch, Nico Adamini, Ronald Simon, Guido Sauter, Joachim Weischenfeldt, Tobias Klatte, Sarah Minner, David Horst, Thorsten Schlomm, Dr. med Henrik Zecha Zecha. Loss of chromosome Y is unrelated to the composition of the tumor microenvironment and patient prognosis in muscle-invasive urothelial bladder cancers [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 5219.

Abstract 2541: The landscape of circular extrachromosomal amplification in pediatric cancers

Abstract Circular extrachromosomal DNA (ecDNA, also known as double minutes, dm) has emerged as a driver of oncogenic copy number amplification, intratumoral heterogeneity, tumor evolution and therapy resistance. We recently screened the whole genomes of 481 medulloblatomas for ecDNA and concluded that medulloblastoma tumors harboring ecDNA are more than twice as likely to progress within 5 years of diagnosis. To evaluate the frequency and clincical impact of ecDNA across the spectrum of childhood cancers, we performed a retrospective analysis of 1671 tumors from 1482 patients across 47 pediatric tumor types from two large cloud pediatric cancer genomics resources, the Children’s Brain Tumor Network and St. Jude Cloud. ecDNA was detected in 15 pediatric tumor types, including ETMR (100%, n=4), osteosarcoma (47%, n=57), rhabdomyosarcoma (40%, n=35), neuroblastoma (30%, n=106), high-grade glioma (20%, n=157), and retinoblastoma (19%, n=32). Patients with tumors containing ecDNA had significantly poorer 5-year overall survival. In sequential samples from the same tumor at diagnosis and relapse, we observe evidence of ecDNA evolution including sequence instability and generation of new high-copy ecDNA amplification not detected at initial diagnosis. These results comprise the most detailed survey to date of the landscape of extrachromosomal amplifications across childhood cancers. Citation Format: Sunita Sridhar, Owen S. Chapman, Shanqing Wang, Aditi Dutta, Jill P. Mesirov, Lukas Chavez. The landscape of circular extrachromosomal amplification in pediatric cancers [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 2541.

Abstract 6243: Rapid autopsy reveals the interplay of tumor heterogeneity at genomic, transcriptomic, and spatial dimensions

Abstract Incomplete understanding of tumor heterogeneity across primary and metastatic sites often lead to ineffective cancer treatments. To comprehensively interrogate tumor heterogeneity at genomic, transcriptomic, and spatial levels, we established a novel rapid autopsy protocol to obtain multiregional tri-omic data consisted of whole exome sequencing, mRNA-sequencing, and multiplexed immunohistochemistry on primary and metastatic tumors and associated microenvironments (TMEs). We applied this protocol to six patients with various primary tumor types: non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), urothelial cancer, and one mixed case with both NSCLC and SCLC. A total of 50 tumor samples with paired tri-omic data were obtained across 16 different primary and metastatic organs to interrogate heterogeneities underlying tumor evolution and treatment resistance mechanisms. We constructed mutation-based phylogeny trees to reveal structural differences in tumor evolution trajectories across patients and infer heterogeneous tumor antigen landscape. We uncovered highly entropic phylogenetic structures having unique variants gained at each metastasis as well as low entropic structures with high sharing across sites. The mixed case had a clonal sweep structure with two metastases developed prior to the rest of the metastasis. In this case, older clones were shown to have strong association with APOBEC mutation signatures, enhanced subclonal mutation burden and loss of human leukocyte antigen heterozygosity as mechanisms of immune evasion. Integrating transcriptome and spatial profiles, we revealed that tumor intrinsic expressions and extrinsic immune infiltration levels covary with tissue types rather than with mutation-based phylogenetic clusters. Across patients, samples that had high similarities in mutational landscape varied in their pro-tumor expression programs and the cellular composition of TMEs. In particular, lung samples across patients had the highest effector T cell population and major histocompatibility complex (MHC) class I, II presentations compared to other tissue types. Contrasting small cell carcinoma (SCC) and adenocarcinoma (Adeno) samples from lung tissues further revealed that SCC had more immunosuppressive TMEs with downregulation of MHC class II. Liver metastasis samples were further characterized to have the most aggressive phenotypes enriched in genomic instability measured by aneuploidy compared to other metastatic sites. In sum, we present a novel technology and computational framework that enables a three-dimensional profiling of tumor heterogeneity. This work suggests that single-site biopsies are insufficient to capture tumor heterogeneity and calls for multi-modal integrations to design treatments that can overcome tumor-specific resistance mechanisms. Citation Format: Ning Yao, Rami Vanguri, Juber Patel, Jayon Lihm, Andrew Fisher, Irene Jarchum, Shigeaki Umeda, Hao Li, Scott Ely, Travis Hollmann, Christine Iacobuzio-Donahue, Benjamin Greenbaum. Rapid autopsy reveals the interplay of tumor heterogeneity at genomic, transcriptomic, and spatial dimensions [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 6243.