Tumor Genome Research Articles

Single-mitosis dissection of acute and chronic DNA mutagenesis and repair.

How chronic mutational processes and punctuated bursts of DNA damage drive evolution of the cancer genome is poorly understood. Here, we demonstrate a strategy to disentangle and quantify distinct mechanisms underlying genome evolution in single cells, during single mitoses and at single-strand resolution. To distinguish between chronic (reactive oxygen species (ROS)) and acute (ultraviolet light (UV)) mutagenesis, we microfluidically separate pairs of sister cells from the first mitosis following burst UV damage. Strikingly, UV mutations manifest as sister-specific events, revealing mirror-image mutation phasing genome-wide. In contrast, ROS mutagenesis in transcribed regions is reduced strand agnostically. Successive rounds of genome replication over persisting UV damage drives multiallelic variation at CC dinucleotides. Finally, we show that mutation phasing can be resolved to single strands across the entire genome of liver tumors from F1 mice. This strategy can be broadly used to distinguish the contributions of overlapping cancer relevant mutational processes.

Exploring the tumor genomic landscape of aggressive prostate cancer by whole-genome sequencing of tissue or liquid biopsies.

Treatment resistance remains a major issue in aggressive prostate cancer (PC), and novel genomic biomarkers may guide better treatment selection. Circulating tumor DNA (ctDNA) can provide minimally invasive information about tumor genomes, but the genomic landscape of aggressive PC based on whole-genome sequencing (WGS) of ctDNA remains incompletely characterized. Thus, we here performed WGS of tumor tissue (n = 31) or plasma ctDNA (n = 10) from a total of 41 aggressive PC patients, including 11 hormone-naïve, 15 hormone-sensitive, and 15 castration-resistant patients. Across all variant types, we found progressively more altered tumor genomic profiles in later stages of aggressive PC. The potential driver genes most frequently affected by single-nucleotide variants or insertions/deletions included the known PC-related genes TP53, CDK12, and PTEN and the novel genes COL13A1, KCNH3, and SENP3. Etiologically, aggressive PC was associated with age-related and DNA repair-related mutational signatures. Copy number variants most frequently affected 14q11.2 and 8p21.2, where no well-recognized PC-related genes are located, and also frequently affected regions near the known PC-related genes MYC, AR, TP53, PTEN, and BRCA1. Structural variants most frequently involved not only the known PC-related genes TMPRSS2 and ERG but also the less extensively studied gene in this context, PTPRD. Finally, clinically actionable variants were detected throughout all stages of aggressive PC and in both plasma and tissue samples, emphasizing the potential clinical applicability of WGS of minimally invasive plasma samples. Overall, our study highlights the feasibility of using liquid biopsies for comprehensive genomic characterization as an alternative to tissue biopsies in advanced/aggressive PC.

Habitat escalated adaptive therapy (HEAT): a phase 2 trial utilizing radiomic habitat-directed and genomic-adjusted radiation dose (GARD) optimization for high-grade soft tissue sarcoma

BackgroundSoft tissue sarcomas (STS), have significant inter- and intra-tumoral heterogeneity, with poor response to standard neoadjuvant radiotherapy (RT). Achieving a favorable pathologic response (FPR ≥ 95%) from RT is associated with improved patient outcome. Genomic adjusted radiation dose (GARD), a radiation-specific metric that quantifies the expected RT treatment effect as a function of tumor dose and genomics, proposed that STS is significantly underdosed. STS have significant radiomic heterogeneity, where radiomic habitats can delineate regions of intra-tumoral hypoxia and radioresistance. We designed a novel clinical trial, Habitat Escalated Adaptive Therapy (HEAT), utilizing radiomic habitats to identify areas of radioresistance within the tumor and targeting them with GARD-optimized doses, to improve FPR in high-grade STS.MethodsPhase 2 non-randomized single-arm clinical trial includes non-metastatic, resectable high-grade STS patients. Pre-treatment multiparametric MRIs (mpMRI) delineate three distinct intra-tumoral habitats based on apparent diffusion coefficient (ADC) and dynamic contrast enhanced (DCE) sequences. GARD estimates that simultaneous integrated boost (SIB) doses of 70 and 60 Gy in 25 fractions to the highest and intermediate radioresistant habitats, while the remaining volume receives standard 50 Gy, would lead to a > 3 fold FPR increase to 24%. Pre-treatment CT guided biopsies of each habitat along with clip placement will be performed for pathologic evaluation, future genomic studies, and response assessment. An mpMRI taken between weeks two and three of treatment will be used for biological plan adaptation to account for tumor response, in addition to an mpMRI after the completion of radiotherapy in addition to pathologic response, toxicity, radiomic response, disease control, and survival will be evaluated as secondary endpoints. Furthermore, liquid biopsy will be performed with mpMRI for future ancillary studies.DiscussionThis is the first clinical trial to test a novel genomic-based RT dose optimization (GARD) and to utilize radiomic habitats to identify and target radioresistance regions, as a strategy to improve the outcome of RT-treated STS patients. Its success could usher in a new phase in radiation oncology, integrating genomic and radiomic insights into clinical practice and trial designs, and may reveal new radiomic and genomic biomarkers, refining personalized treatment strategies for STS.Trial registrationNCT05301283.Trial statusThe trial started recruitment on March 17, 2022.

Phosphatidylserine externalization as immune checkpoint in cancer.

Cancer is the second leading cause of mortality worldwide. Despite recent advances in cancer treatment including immunotherapy with immune checkpoint inhibitors, new unconventional biomarkers and targets for the detection, prognosis, and treatment of cancer are still in high demand. Tumor cells are characterized by mutations that allow their unlimited growth, program their local microenvironment to support tumor growth, and spread towards distant sites. While a major focus has been on altered tumor genomes and proteomes, crucial signaling molecules such as lipids have been underappreciated. One of these molecules is the membrane phospholipid phosphatidylserine (PS) that is usually found at cytosolic surfaces of cellular membranes but can be rapidly and massively shuttled to the extracellular leaflet of the plasma membrane during apoptosis to serve as a limiting factor for immune responses. These immunosuppressive interactions are exploited by tumor cells to evade the immune system. In this review, we describe mechanisms of immune regulation in tumors, discuss if PS may constitute an inhibitory immune checkpoint, and describe current and future strategies for targeting PS to reactivate the tumor-associated immune system.

Ginsenoside Rh4 alleviates gastrointestinal mucositis and enhances chemotherapy efficacy through modulating gut microbiota

BackgroundGastrointestinal mucositis stands as one of the most severe side effects of irinotecan (CPT-11). however, only palliative treatment is available at present. Therefore, there is an urgent need for adjunctive medications to alleviate the side effects of CPT-11. PurposeIn this study, our objective was to explore whether ginsenoside Rh4 could serve as a modulator of the gut microbiota and an adjunctive agent for chemotherapy, thereby alleviating the side effects of CPT-11 and augmenting its anti-tumor efficacy. Study designA CPT-11-induced gastrointestinal mucositis model was used to investigate whether ginsenoside Rh4 alleviated CPT-11-induced gastrointestinal mucositis and enhanced the anti-tumor activity of CPT-11. MethodsIn this study, we utilized CT26 cells to establish a xenograft tumor model, employing transcriptomics, genomics, and metabolomics techniques to investigate the impact of ginsenoside Rh4 on CPT-11-induced gastrointestinal mucositis and the effect on the anti-tumor activity of CPT-11. Furthermore, we explored the pivotal role of gut microbiota and their metabolites through fecal microbiota transplantation (FMT) experiments and supplementation of the key differential metabolite, hyodeoxycholic acid (HDCA). ResultsThe results showed that ginsenoside Rh4 repaired the impairment of intestinal barrier function and restored intestinal mucosal homeostasis in a gut microbiota-dependent manner. Ginsenoside Rh4 treatment modulated gut microbiota diversity and upregulated the abundance of beneficial bacteria, especially Lactobacillus_reuteri and Akkermansia_muciniphila, which further regulated bile acid biosynthesis, significantly promoted the production of the beneficial secondary bile acid hyodeoxycholic acid (HDCA), thereby alleviating CPT-11-induced gut microbiota dysbiosis. Subsequently, ginsenoside Rh4 further alleviated gastrointestinal mucositis through the TGR5-TLR4-NF-κB signaling pathway. On the other hand, ginsenoside Rh4 combination therapy could further reduce the weight and volume of colon tumors, promote tumor cell apoptosis, and enhance the anti-tumor activity of CPT-11 by inhibiting the PI3K-Akt signaling pathway, thus exerting a synergistic anti-tumor effect. ConclusionIn summary, our findings confirm that ginsenoside Rh4 can alleviate CPT-11-induced gastrointestinal mucositis and enhance the anti-tumor activity of CPT-11 by modulating gut microbiota and its related metabolites. Our study validates the potential of ginsenoside Rh4 as a modulator of the gut microbiota and an adjunctive agent for chemotherapy, offering new therapeutic strategies for addressing chemotherapy side effects and improving chemotherapy efficacy.

Measurement of ctDNA Tumor Fraction Identifies Informative Negative Liquid Biopsy Results and Informs Value of Tissue Confirmation.

Liquid biopsy (LBx) for tumor profiling is increasingly used, but concerns remain regarding negative results. A lack of results may truly reflect tumor genomics, or it may be a false negative that would be clarified by tissue testing. A method of distinguishing between these scenarios could help clarify when follow-on tissue testing is valuable. Here we evaluate circulating tumor DNA (ctDNA) tumor fraction (TF), a quantification of ctDNA in LBx samples, for utility in identifying true negative results. We assessed concordance between LBx and tissue-based results, stratified by ctDNA TF, in a real-world genomic dataset of paired samples across multiple disease types. We also evaluated the frequency of tissue results identifying driver alterations in patients with lung cancer after negative LBx in a real-world clinicogenomic database. The positive percent agreement and negative predictive value between liquid and tissue samples for driver alterations increased from 63% and 66% for all samples to 98% and 97% in samples with ctDNA TF ≥1%. Among 505 patients with lung cancer with no targetable driver alterations found by LBx who had subsequent tissue-based profiling, 37% had a driver, all of which had ctDNA TF <1%. Patients with lung cancer with negative LBx and ctDNA TF ≥1% are unlikely to have a driver detected on confirmatory tissue testing; such informative negative results may benefit instead from prompt treatment initiation. Conversely, negative LBx with ctDNA TF <1% will commonly have a driver identified by follow-up tissue testing and should be prioritized for reflex testing.

Abstract 4347: Cell-type specific transcriptional regulation of APOBEC3A in cancers

Abstract APOBEC3s (A-H) are a family of seven cytosine deaminases that inactivate viruses as part of the innate immune response. However, APOBEC3A and APOBEC3B have been found to mediate mutagenesis in tumor genomes by deaminating ssDNA that transiently arise during transcription and DNA replication. The mechanisms that trigger these enzymes to generate high mutation burdens in some cancers and affect disease progression and clinical outcomes are largely unknown. Here, we explored cell-type-specific transcriptional regulation of APOBEC3s using single-nucleus/cell RNA-seq (sn/scRNA-seq) data (&amp;gt;100 samples and &amp;gt;300,000 cells) from four tumor types - bladder, lung, breast, and kidney - that display different levels of APOBEC-induced mutation burden. Our analyses revealed that among seven members of the APOBEC3 family, APOBEC3A is expressed in a cell-type-specific manner. The expression was predominant in KRT13 and CDH12 epithelial cell-types and inflamed macrophages. The APOBEC3A+ epithelial cell populations were abundant in the bladder, moderate in the lung and breast, and nearly absent in the kidney tumors. APOBEC3A expression correlated with interferon and pro-inflammatory signaling in KRT13+ cells. Transcription factors ELF3 and ATF3 were significantly upregulated in APOBEC3A+ KRT13 cells. Pseudotime analysis of sn/scRNA-seq data revealed that APOBEC3A cells are in epithelial-mesenchymal transition. Finally, several gene markers defining APOBEC3A+ KRT13 cell types were identified. Ongoing analyses include investigations into clinical correlates, cell-type-specific mutation burdens, and validation through in vitro functional approaches. Our results propose that APOBEC3A is turned on in specific epithelial cell types by multiple signaling pathways during tumorigenesis, thereby driving tumor heterogeneity, particularly in high mutation-burden cancers. Citation Format: Dhanusha Yesudhas, Bilal Lone, Kelly Butler, Arup Chakraborty, Abdul Rouf Banday. Cell-type specific transcriptional regulation of APOBEC3A in 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 4347.

Abstract 3892: Systematic generation of a clinicogenomic harmonized oncologic real-world dataset (MSK-CHORD)

Abstract Clinical data storage in unstructured notes and siloed datasets present a major challenge for large-scale cancer informatics. Whether natural language processing (NLP) combined with multimodal integration across datasets can produce a mineable resource and improve discovery of relationships between tumor genomics and clinical phenotypes is unknown. We hypothesized that NLP could automatically annotate a pan-cancer corpus of 82,464 patients with tumor genomic sequencing. To develop algorithms to annotate free-text reports, we leveraged the AACR Project GENIE Biopharma Collaborative (BPC), a structured curation of EMR from five cancer types (non-small cell lung (NSCLC), breast, colorectal, prostate, and pancreatic cancer), to train and validate several Transformer and rule based-based NLP models. After automating the generation of NLP annotations alongside medication, demographic, tumor registry, survival, and tumor genomic sequencing data, we tested whether clinicogenomic relationships not apparent in the smaller BPC cohort might be discoverable in the larger cohort. In 5-fold cross-validation, NLP Transformers accurately annotated the presence of cancer (AUC=0.99), cancer progression (AUC=0.97), and sites of disease (AUC=0.99) from radiology reports, and presence of prior outside treatment (AUC=0.98) and hormone receptor (HR) and HER2 receptor status (AUC=0.98, 0.98) from clinician notes. In addition, rule-based models, trained on non-BPC data and validated on the whole BPC cohort, annotated smoking status from clinician notes (ACC=0.95), and Gleason score (ACC=1.0), PD-L1 status (ACC=0.98), and mismatch repair deficiency (ACC=0.98) from histopathology reports. NLP annotations were merged with genomic and other structured clinical data to create a Clinicogenomic, Harmonized Oncologic Real-world Dataset (MSK-CHORD). Finally, we tested if associations not apparent in the BPC might be discoverable in MSK-CHORD. We found positive associations between Gleason score and gene-level alterations in prostate cancer including TP53, PTEN and BRCA2 (q&amp;lt;0.1), none of which were adequately powered for detection in the BPC. We found PD-L1 status was associated with better survival following immunotherapy treatment in NSCLC, but only in the larger MSK-CHORD was this association statistically significant. In breast cancer, NF1 mutations were associated with prior therapy in both cohorts, but this association was only significant in MSK-CHORD. The infrastructure generating MSK-CHORD uses a combination of on-premise and cloud computing resources and open-source development operation applications to automate processes. Once annotations are created, data is imported into a local instance of cBioPortal, where researchers can visualize data and perform analyses. The system generating MSK-CHORD demonstrates how large-scale data delivery and integration can fuel cancer research. Citation Format: Christopher J. Fong, Karl Pichotta, Thinh Tran, Michele Waters, Tom Fu, Mono Pirun, Mirella Altoe, Brooke Mastrogiacomo, Anisha Luthra, Mehnaj Ahmed, Arfath Pasha, Armaan Kohli, Raymond Lim, Tom Pollard, Darin Moore, Benjamin Gross, Avery Wang, Calla Chennault, Ritika Kundra, Ramyasree Madupuri, Ino de Bruijn, Aaron Lisman, Walid K. Chatila, Subhiksha Nandakumar, Anika Begum, Doori Rose, Kenneth L. Kehl, Deborah Schrag, Michael Berger, Jian Carrot-Zhang, Pedram Razavi, Bob Li, Peter Stetson, Nikolaus Schultz, Justin Jee. Systematic generation of a clinicogenomic harmonized oncologic real-world dataset (MSK-CHORD) [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 3892.

Abstract 4341: Non canonical mitochondrial hotspot mutations in cancer

Abstract The vast majority of recurrent somatic mutations arising in tumors affect protein-coding genes in the nuclear genome. Here, through population-scale analysis of 15,619 whole tumor genomes, we report the discovery of highly recurrent mutations affecting both the small (12S, MT - RNR1) and large (16S, MT - RNR2) RNA subunits of the mitoribosome. In total, we identified 70 recurrent, hotspot mutations affecting either subunit of the mitoribosome. Compared to non-hotspot positions, rRNA hotspots preferentially affect positions participating in Watson-Crick base pairing, and tend to arise at positions under strong purifying selection in the germline. Consistent with their putative selection, hotspot rRNA alleles arose at higher heteroplasmy than silent mutations under neutral selection. Transcriptional and metabolomic profiling of the m.1227G&amp;gt;A MT - RNR1 hotspot revealed a heteroplasmic dosage-dependent shift in energy generation towards glycolysis. Mutations disrupting the mitoribosome therefore represent a novel class of functional mutations under positive selection in cancer genomes. Citation Format: Sonia Boscenco, Jaqueline Tait-Mulder, Minsoo Kim, Cerise Tang, Mark Zucker, Tricia Park, Wei Wei, Patrick F. Chinnery, Payam A. Gammage, Ed Reznik. Non canonical mitochondrial hotspot mutations in cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4341.

Abstract 1771: NYU Langone Genome PACT - Genome Profiling of Actionable Cancer Targets (LG-PACT) for clinical patient molecular diagnostics and treatment

Abstract Next-generation sequencing (NGS) for the detection of somatic variants has become the tool of choice in a variety of molecular oncology fields and in the clinic. Its use ranges from sequencing entire tumor genomes and transcriptomes to targeted clinical diagnostic gene panels. The NYU Langone Genome PACT assay is a qualitative in vitro diagnostic test that uses targeted next generation sequencing of formalin-fixed paraffin-embedded (FFPE) tumor tissue matched with normal specimens from patients to detect gene alterations in a 607-gene panel. Indications for testing are cancer (solid tumors or hematological malignancies) where a mutational profile from multiple genes would be informative for disease stratification, prognosis, or treatment options including targeted therapies and eligibility for clinical trials. The test is intended to provide information on somatic mutations including point mutations, copy number aberrations, and small insertions/deletions (indels) for diagnostic and treatment decisions. LG-PACT is a United States Food and Drug Administration (FDA) approved diagnostic test. The clinical interpretation of sequencing data of molecular tumor markers from NGS encompasses automated variant calling tools with human interpretation. The final mostly manual review of data is intensive, involving highly trained scientists, encompassing literature review, interpretation and tier classification by pathologists, who then provide a complete molecular diagnostic report to the treating oncologists. Since January 2022 to present (October 2023) we have provided clinical genomic reports for 1029 oncological cases from 124 different cancers and their subtypes, including Brain (489 cases incl. meningioma, glioma and glioblastoma), Gastrointestinal (123), Lung (114), among others. We first present the technical challenges of validating an NGS oncological diagnostic targeted assay for appropriate clinical grade accuracy and sensitivity acceptable for patient care. We show how copy number alterations provide a more comprehensive description of the tumors genomic profile. We then outline the actionability of targeted panel sequencing for our current patient cohort. Where analysis of variant detection has led to 47% (490) of our clinical tumor samples containing known tier 1 therapeutic variants. We conclude with presenting case studies that identify both the clinical utility and informatic challenges of variant calling specific to gene panel sequencing e.g., i) the capture of potential targetable rare and novel indels and multivariant mutations in exons 19 and 20 of EGFR, and ii) a unique KIT tandem duplication event in a gastrointestinal stromal tumor (GIST). We demonstrate the value in precision oncology for multiple cancer types and how the capture of unique variants can provide better targeted treatment options to cancer patients. Citation Format: Varshini Vasudevaraja, Yiying Yang, Jonathan Serrano, Kazimierz Wrzeszczynski, Matija Snuderl. NYU Langone Genome PACT - Genome Profiling of Actionable Cancer Targets (LG-PACT) for clinical patient molecular diagnostics and treatment [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 1771.

Abstract 2422: Liquid biopsy approaches for monitoring metastatic pancreatic cancer in immunotherapy treated patients

Abstract Introduction: Pancreatic cancer has a poor prognosis especially when identified at advanced stages. Globally in 2020, &amp;gt;450,000 people died from the disease. For those with locally advanced or metastatic cancer, the standard of care treatment is chemotherapy. Preliminary studies show that some patients with advanced-stage disease respond to immune checkpoint blockade treatment. Determining response to therapy using imaging techniques can be challenging. There is a clinical unmet need for noninvasive approaches that can provide a real-time assessment of response to therapy in these patients. We evaluated two methods for assessing response to therapy using circulating cell-free DNA (cfDNA) in patients with metastatic pancreatic cancer treated with immune checkpoint inhibition and radiation as part of the CheckPAC study (NCT02866383). Methods: For all patients with available samples (n=40), we evaluated one pre-treatment blood draw and one on-treatment blood draw (26-65 days after treatment start). We extracted cfDNA from each sample and performed low-coverage 1-2x whole genome sequencing (WGS). We evaluated tumor and matched buffy coat for the 34 patients who had &amp;gt; 10% tumor purity using WGS with at least 60x and 30x coverage, respectively. We compiled the mutated bases in the tumor genome and examined these positions in the cfDNA. We counted the number of mutant observations from cfDNA WGS and divided them by the total number of observations at these bases to calculate whole-genome mutant allele frequency (MAF). We categorized responders based on &amp;gt;30% drop in MAF from baseline. In a tissue independent approach, we detected changes in genome-wide cfDNA fragmentation patterns using the DNA evaluation of fragments for early interception (DELFI) approach. We evaluated survival using DELFI scores of on-treatment samples. Samples were designated DELFI high (above median) or DELFI low (below median.) Log-Rank tests were used to compare survival curves. Results: For the mutation based approach, molecular responders (n=7) had a median progression free survival (PFS) of 485 days compared to 57 days for non-responders (n=9) (p=0.02). Molecular responders had a median overall survival (OS) of 694 days compared to 200 days for non-responders (p=0.062). Patients with DELFI low scores after therapy (n=14) had a longer median PFS than those with DELFI high scores (n=14) (307 days versus 70 days, respectively) (p=0.013). DELFI low patients also had a longer median OS than DELFI high patients (627 vs 158 days, respectively) (p&amp;lt;0.0001). Conclusions: These analyses suggest that WGS mutation-based and fragmentation cfDNA approaches can identify individuals with metastatic pancreatic cancer who respond to immune checkpoint inhibition. Incorporation of these molecular methods for evaluation of tumor burden may provide information for patient-physician decision making to improve patient quality of life. Citation Format: Carolyn Anna Hruban, Inna Chen, Daniel C. Bruhm, Shashikant Koul, Jennie Yao, Susann Theile, Kavya Boyapati, Nicholas A. Vulpescu, Akshaya Annapragada, Leonardo Ferreira, Stephen Cristiano, Zachariah H. Foda, Vilmos Adleff, Julia Johansen, Jillian Phallen, Robert B. Scharpf, Victor E. Velculescu. Liquid biopsy approaches for monitoring metastatic pancreatic cancer in immunotherapy treated patients [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 2422.

Abstract 3986: Exploring nonsense-mediated decay inhibition as a novel approach to improve immunogenicity in small cell lung cancer

Abstract Cancer immunotherapy has revolutionized the field of oncology and is one of the most promising treatments for lung cancer. Small cell lung cancer (SCLC) is highly aggressive and patients often relapse due to chemotherapy resistance. In the recent years, chemotherapy has been combined with immune checkpoint inhibition (ICI) for SCLC treatment, leading to improved responses in some patients, but also leaving a high number of non-responders to address. Paradoxically, SCLC has one of the highest tumor mutational burden, including remarkably abundant frameshifts (FS) which could create highly immunogenic neoantigens (NeoAg). However, FS-harbouring transcripts are often degraded via Nonsense-Mediated Decay (NMD). We therefore aimed to inhibit the NMD pathway to increase the immunogenicity of SCLC. Our lab has so far profiled the mutational landscape of more than 400 SCLC patients and generated over 100 patient-derived xenotransplant models. We analyzed genome and transcriptome sequencing data of multiple tumors to interrogate the NMD target repertoire in SCLC. We determined tumor-specific mutations and predicted NeoAg based on the HLA-I-binding ability of mutant peptides. Upon NMD inhibiton, we validated the expression and presentation of NeoAg via transcriptomics, proteomics and HLA-I-immunopeptidomics. NMD-sensitive mutated transcripts included a wide range of somatic alterations beyond FS-mutations, suggesting an overall increase of potential tumor-specific NeoAg. We furthermore evaluated immunogenicity after NMD perturbation employing in vitro co-cultures as well as immunocompetent in vivo mouse models. Inhibition of NMD in tumor cells resulted in an HLA-I-dependent enhancement of T cell-mediated tumor killing in vitro, and in vivo experiments showed reduced tumor growth accompanied by increased immune infiltration upon controlled induction of NMD inhibition. Altogether, NMD inhibition in SCLC led to an upregulation of NeoAg expression in tumor cells accompanied by enhanced T cell responses in vitro and in vivo. Our strategy provides a novel approach for the treatment of low immunogenic tumors which could be combined with ICI to enhance the efficacy of immunotherapy. Citation Format: Lucia A. Torres Fernández, Volker Böhm, Lukas Maas, Anna Schöllhorn, Joel Kaufmann, Joshua D'Rozario, Laura Kaiser, Niels Gehring, Martin Peifer, Cécile Gouttefangeas, Roman K. Thomas, Julie George. Exploring nonsense-mediated decay inhibition as a novel approach to improve immunogenicity in 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 3986.

Abstract 1627: Persistence and dynamics of mutation, selection, and epistasis during the somatic evolution of low-risk, high-risk, and metastatic prostate cancer

Abstract Driver genes and mutations responsible for prostate cancer tumorigenesis have been identified, but research has not previously quantified the effects of prostate cancer driver mutations, or how the magnitude of those effects vary with cancer stage and epistatic interactions among genes. Using 2699 prostate cancer tumor exomes, genomes, and panel sequences (1648 primary tumors and 1051 metastatic samples), we calculated the cancer effect size (CES)—the each mutation confers to lower-risk (Grade Group 1/2) and higher-risk (Grade Group 3/4/5) tumorigenesis and metastasis. The lower-risk, higher-risk, and metastatic tumors exhibited similar neutral rates at which mutations were introduced into DNA, with these underlying gene mutation rates generally increasing from lower-risk to higher risk to metastatic tumors. However, lower risk, higher risk, and metastatic tumors noticeably differed in the genes and specific somatic variants that were most highly selected for within each group. Therefore, to assess whether differences in natural selection that we observed among low-risk, high-risk, and metastatic tumor types were at least partly due to epistatic interactions, we applied a model of pairwise epistasis that quantified the effect of the presence or absence of a mutation in one gene on the selection for mutations in another gene. We found significant evidence of epistatic effects among driver genes. Our results indicate a very early role for SPOP in the development of prostate cancer: strength of selection for SPOP mutations is tightly associated with the degree to which the mutation ablates contact with the BRD3, and these disruptive mutations in turn increase the selection for mutations in several other tumor suppressors and oncogenes such as RHOA. Previously identified gene-gene interactions such as that between SPOP and PIK3CA were supported by this analysis, and we also identified novel gene-gene interactions such as those between AR and MUC16 occurring during metastasis. In conclusion, we have demonstrated that rates of mutation and strengths of selection on these driver mutations vary dynamically along the trajectories to lower-risk, higher-risk, and metastatic prostate cancer, and that powerful selective epistasis among driver genes provides an explanation for these differences. Because precision therapeutic targeting of somatic variant genes is fundamentally targeting the selective benefit obtained from that variant at the time of treatment, understanding these changes in stage-specific and somatic genotype-specific selective strengths will enable better therapeutic targeting in translational and clinical science. Citation Format: Moein Rajaei, Alexander Yang, Christopher N. Cross, J Nick Fisk, Elizabeth B. Perry, Jeffrey D. Mandell, Stephen G. Gaffney, Takafumi N. Yamaguchi, Julie Livingstone, Vincent L. Cannataro, Peter Humphrey, Jose Costa, Paul C. Boutros, Jeffrey P. Townsend. Persistence and dynamics of mutation, selection, and epistasis during the somatic evolution of low-risk, high-risk, and metastatic prostate 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 1627.

Abstract 5377: Investigating human leukocyte antigen (HLA) class 1 leukemogenic pathways in acute myelogenous leukemia

Abstract Background: Aging, smoking and germline predisposition are known to accelerate leukemogenesis. However, the the ability for myeloid clonal pathogenic variants (MCPV) to elicit an antigen-driven host antileukemia immune response is not well characterized. A better understanding on how cellular immunity selectively discriminates leukemia-related “neopeptides” could uncover mechanisms supporting tumor genome evolution. Methods: 122 patients with acute myelogenous leukemia (AML) were included and 111 had available protein coding sequences. A neopeptide library was created using protein FASTA coding extracted for individual myeloid clonal mutations via National Center for Biotechnology Information (NCBI). Patient class 1 HLA typing was obtained through Tempus, and Genomic Testing Cooperative laboratories. Prediction of number and strength of AML neopeptide bindings to MHC class I molecules were obtained via NetMHCPan-1. Analysis was performed with K-means algorithm assigning age at diagnosis, detected MCPV, number of neopeptides binding (as continuous variable), overall survival (OS) and event-free survival (EFS) as variables. Unsupervised data input allowed 3 cluster partitions for differential survivals allowing HLA class 1 peptide binding aggregation with potential roles in leukemogenesis and patient outcome. Descriptive statistics were performed with SAS. Results: Sixty-six different MCPV were detected. Of 111 patients, 70 (63%) with available MCPV produced at least 1 MHC I neopeptide binding. Frequently observed variants were P53 (13%), FLT3 (9.2%), WT1 (7%), DNMT3A (6.5%), RAS (5%), ASXL1 (4.3%), RUNX1 (4.2%), NPM1 (4.2%) and IDH1/2 (2.6%). Cluster 2 demonstrated a lack of favorable and higher proportion of adverse ELN 2022 subgroups. Cluster 3 exhibited a higher of P53 [p=0.03] and MDS-related mutations. OS was 79, 239, and 1551 days for cluster 2,1 and 3, respectively [p=0.0001]. EFS was 134, 363 and 451 days for cluster 2,1 and 3, respectively [p=0.0001]. Interestingly, Cluster 2 and 3 assembled the highest probability for peptide binding [1 v 2 v 3 = 1.76% v 36.6% v 62%, p=0.01]. Lastly, albeit in low frequencies, higher diversity of DNA repairing enzymes was seen in cluster 2. Conclusions: Different MCPV arising in AML genome are variably immunogenic and leukemia-related neopeptide binding’s ability is differentially expressed in our 3 clusters. Frequent neopeptide bindings in clusters 2 and 3 may reflect higher AML immunogenicity resulting in genomic instability. It is possible that less “edited” genomes in cluster 1 preserve higher chemosensitivity resulting in better OS, while highly edited genomes in cluster 2 and 3 are less chemo-sensitive and circumvent host immunosurveillance. Further expansion of cluster 2 is necessary to better understand the role of DNA repairing enzymes in younger patients’ outcomes, especially when genomic instability is observed. Citation Format: Paul Sackstein, Sarah Mudra, Lacey Williams, Garrett Diltz, Rachel Zemel, Sravanti Teegavarapu, Martha Mims, Catherine Lai, Kimberley Doucette, Anne Renteria, Olga Timofeeva, Gustavo A. Rivero. Investigating human leukocyte antigen (HLA) class 1 leukemogenic pathways in acute myelogenous leukemia [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 5377.

Abstract 5610: Investigating the role of BRCA2 in the development of pediatric rhabdomyosarcoma

Abstract Introduction: In a large-scale exome sequencing study of pediatric rhabdomyosarcoma (RMS) patients, we identified a statistical enrichment of patients with germline variants in BRCA2 (Li, et al. JNCI, 2020), which prompted this study to assess the role of heterozygous BRCA2 variants in the development of pediatric RMS in both precursor cell and mouse models. Methods: We engineered BRCA2 loss of function (LoF) variants in lines of immortalized mesenchymal stem cells, a potential precursor cell to RMS, using CRISPR and Prime Editing (PE). CRISPR was used to induce frameshift alleles in Exons 10 and 11 and with PE, we created two RMS patient specific LOF variants in Exons 5 and 17. We generated a genetically engineered mouse model (GEMM) with heterozygous excision of Exon 11 of Brca2. This allele is now being crossed into two different previously established GEMMs of RMS: a myoblast-specific (MyoD1-Cre) conditional loss of p53, and a gain-of-function mutant p53, LSL-p53R172H (Nakahata, et al. Dis. Model. Mech., 2022), to assess changes in the pace of tumorigenesis and metastatic potential. Through an X01 agreement with the Gabriella Miller Kids First Program, we have submitted paired tumor-normal samples for additional whole-genome sequencing and RNA sequencing (tumor only) on &amp;gt;500 patients with RMS, including those with germline variants in BRCA2. Results: Using CRISPR, we generated cell lines heterozygous for the frameshift variants c.1345dup, c.5680del, and c.5680dup. With Prime Editing, we created heterozygous lines mimicking two specific variants detected in the RMS cohort, c.462_463del and c.7857G&amp;gt;A. Generation of a C57BL6 GEMM with a heterozygous removal of Exon 11 in Brca2 has been successful. No homozygous mice were obtained following 7 heterozygous crosses (50 pups), a departure from Mendelian ratios (p=0.0001) consistent with embryonic lethality. Heterozygous Brca2 mice derived from crosses into two RMS models are being monitored for tumor development. We have completed tumor-normal whole-genome and tumor RNA sequencing for 609 RMS patients, including 228 subjects not previously analyzed. We will directly assess whether there is evidence for mutation or loss of expression of the remaining BRCA2 allele in tumors and whether these tumor genomes exhibit signatures of DNA repair deficiency. Conclusions: The goal of this project is to determine the molecular basis for the enrichment in germline BRCA2 LOF variants in pediatric malignancies, including RMS. Ongoing experiments using the engineered heterozygous BRCA2 in vitro cell line models will assess genomic stability, homologous recombination proficiency, and DNA replication fork stability. Assessment of the effect of heterozygous Brca2 variants in GEMMs of RMS, and subsequent analysis of additional sequencing data, will further elucidate what role this gene may play in increasing risk or progression of pediatric cancers. Citation Format: P. Adam Weinstein, Ryan Zabriskie, Cem Ozdemir, Kyle M. Miller, Olga Medina-Martinez, Jason D. Heaney, Aniko Sabo, Philip J. Lupo, Sharon E. Plon. Investigating the role of BRCA2 in the development of pediatric rhabdomyosarcoma [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 5610.

Abstract 6924: Structural variant dynamics in melanoma: Unraveling tumor heterogeneity and evolution

Abstract Melanoma is characterized by significant intratumoral heterogeneity and complex evolutionary dynamics. This diversity in genomic alterations leads to the emergence of various subclonal populations within a single tumor. In our research, we established a new model system composed of 24 single-cell-derived clonal sublines (C1-C24), originating from the M4 melanoma model. This model was developed using a genetically engineered hepatocyte growth factor (HGF)-transgenic mouse. We employed Trisicell (Triple-toolkit for single-cell intratumor heterogeneity inference), a cutting-edge computational tool for scalable analysis of intratumor heterogeneity and evaluation based on single-cell RNA mutations. This enabled us to construct a phylogeny tree, revealing melanoma’s intricate branching evolutionary patterns. These patterns show ancestral clones evolving into genetically distinct subclones, which demonstrate varied phenotypic traits such as drug sensitivity or resistance, cellular plasticity, and immunogenicity. In our study, we conducted long-read sequencing on these clonal sublines in the phylogeny and identified structural variants (SVs) using Severus, a tool optimized for phasing in long-read sequencing. The types of SVs we discovered include deletions, insertions, amplifications, translocations, and inversions. We explored their roles in subclonal evolution, particularly focusing on how they disrupt genes and accumulate during melanoma progression. Our initial data from eleven sublines indicated a higher prevalence of ancestral SVs, shared by all sublines, compared to subline-specific SVs, representing later events. Notably, the individual sublines showed a higher rate of gene disruption by SVs, hinting at potential functional selection. Our analysis further revealed that SVs common to all sublines are linked with genes in key cell growth pathways, such as Rap1, Hippo, and calcium signaling pathways. In contrast, subline-specific SVs primarily affect genes involved in neurophysiological pathways, such as glutamatergic synapse and morphine addiction pathways. These findings suggest that different genes, associated with various pathways, are disrupted at distinct stages of melanoma progression, providing insights into the genetic factors that may predispose individual melanocytes to melanomagenesis. This methodology presents a comprehensive tool for characterizing tumor genomes and understanding their relationship with disease progression and therapy resistance. We are continuing our analysis to map SVs across the entire mutation-based phylogeny of all sublines, which will further our understanding of melanoma’s genomic landscape. Citation Format: Xiwen Cui, Ayse G. Keskus, Farid R. Mehrabadi, Salem Malikic, Mikhail Kolmogorov, Chi-Ping Day, Glenn Merlino, S. Cenk Sahinalp. Structural variant dynamics in melanoma: Unraveling tumor heterogeneity and 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 6924.

A novel tumor mutation-related long non-coding RNA signature for predicting overall survival and immunotherapy response in lung adenocarcinoma

BackgroundImmunotherapy has changed the treatment landscape for lung cancer. This study aims to construct a tumor mutation-related model that combines long non-coding RNA (lncRNA) expression levels and tumor mutation levels in tumor genomes to detect the possibilities of the lncRNA signature as an indicator for predicting the prognosis and response to immunotherapy in lung adenocarcinoma (LUAD). MethodsWe downloaded the tumor mutation profiles and RNA-seq expression database of LUAD from The Cancer Genome Atlas (TCGA). Differentially expressed lncRNAs were extracted based on the cumulative number of mutations. Cox regression analyses were used to identify the prognostic lncRNA signature, and the prognostic value of the five selected lncRNAs was validated by using survival analysis and the receiver operating characteristic (ROC) curve. We used qPCR to validate the expression of five selected lncRNAs between human lung epithelial and human lung adenocarcinoma cell lines. The ImmuCellAI, immunophenoscore (IPS) scores and Tumor Immune Dysfunction and Exclusion (TIDE) analyses were used to predict the response to immunotherapy for this mutation related lncRNA signature. ResultsA total of 162 lncRNAs were detected among the differentially expressed lncRNAs between the Tumor mutational burden (TMB)-high group and the TMB-low group. Then, five lncRNAs (PLAC4, LINC01116, LINC02163, MIR223HG, FAM83A-AS1) were identified as tumor mutation-related candidates for constructing the prognostic prediction model. Kaplan‒Meier curves showed that the overall survival of the low-risk group was significantly better than that of the high-risk group, and the results of the GSE50081 set were consistent. The expression levels of PD1, PD-L1 and CTLA4 in the low-risk group were higher than those in the high-risk group. The IPS scores and TIDE scores of patients in the low-risk group were significantly higher than those in the high-risk group. ConclusionOur findings demonstrated that the five lncRNAs (PLAC4, LINC01116, LINC02163, MIR223HG, FAM83A-AS1) were identified as candidates for constructing the tumor mutation-related model which may serve as an indicator of tumor mutation levels and have important implications for predicting the response to immunotherapy in LUAD.

Abstract 136: Determining the genomic basis of PAX3-FOXO1 mediated transformation in rhabdomyosarcoma

Abstract Rhabdomyosarcoma (RMS) is the most common pediatric soft tissue sarcoma. Despite rigorous scientific advances and clinical trials, the survival rate for high-risk RMS has not increased above 20% in the last three decades. A deeper understanding of the basic biology driving RMS tumorigenesis is needed to discover novel therapeutic targets. RMS can be divided into two major histologic subtypes: embryonal RMS (ERMS) and alveolar RMS (ARMS). ARMS accounts for 20% of pediatric cases and has poorer survival. The majority of ARMS tumors harbor t(2;13)(q35;q14) or t(1;13)(p36;q14) chromosomal translocations resulting in the PAX3-FOXO1 (P3F) and PAX7-FOXO1 (P7F) fusion oncoproteins, respectively. ARMS tumors that lack oncofusions more closely resemble ERMS. Classifying on fusion status more accurately describes the disease and highlights the significance of P3/7F. ARMS tumor genomes only contain an average of 6.4 somatic mutations suggesting the oncofusion protein is the key oncogenic driver and a potential candidate for therapeutic intervention. RMS is commonly thought to arise from skeletal muscle progenitor cells that fail to terminally differentiate. However, RMS occurs throughout the body in sites devoid of skeletal muscle, underscoring the potential for alternative cells of origin. Our lab established that endothelial progenitors are indeed a cell of origin for ARMS by generating a genetically engineered mouse model with endothelial cell specific P3F expression coupled with Cdkn2a loss. We further generated a human model system derived from TP53-null human induced pluripotent stem cells (iPSCs), in which we direct differentiation to endothelial cell fate and then force P3F expression. P3F expression during endothelial cell directed differentiation blocked endothelial maturation and instead reprogrammed cells shifting cell fate to skeletal muscle-like cells that form ARMS tumors in mice. Generation of iPSC-derived ARMS (iARMS) occurs over 15 days and provides a unique model to determine the kinetics and specific genomic contributions facilitated by P3F during RMS transformation. To investigate the genomic changes occurring during iARMS transformation I utilized CUT&amp;RUN for P3F and H3K27ac to assess P3F occupancy and enhancer landscape, ATAC-seq to assess chromatin accessibility, and RNA-seq to assess gene expression changes at timepoints throughout the transformation event. These results define the fundamental genomic changes needed for transformation into FP-RMS by P3F and uncovers the key determinants of ARMS cell identity. The iARMS system provides a unique platform to uncover how P3F establishes and maintains RMS cell state and to dissect the specific dependencies required to maintain the ARMS cell state. Understanding the fundamental mechanism of P3F-driven tumorigenesis could provide greater resolution into the key disease determinants to focus pre-clinical efforts on pivotal targets. Citation Format: Bradley T. Stevens, Yang Zhang, Randolph K. Larsen, Grace E. Adkins, Jack D. Hopkins, Darden W. Kimbrough, Matthew R. Garcia, Brian J. Abraham, Mark E. Hatley. Determining the genomic basis of PAX3-FOXO1 mediated transformation in rhabdomyosarcoma [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 136.

Abstract 3664: Evaluation of on-treatment tumor molecular dynamics using plasma whole genome sequencing (pWGS) and whole exome sequencing (pWES) of circulating tumor DNA (ctDNA) in patients (pts) with metastatic non-small cell lung cancer (NSCLC): An exploratory analysis of KEYNOTE-598

Abstract Background: ctDNA-based pWES is a promising tool to examine tumor genomics using a non-invasive liquid biopsy to serially monitor changes during tumor evolution and treatment. Here, we evaluate on-treatment tumor molecular dynamics using ctDNA-based pWGS and pWES in pts with metastatic NSCLC that responded to pembrolizumab (pembro) monotherapy and later experienced PD in KEYNOTE-598 (NCT03302234). Methods: In KEYNOTE-598, pts had metastatic NSCLC (PD-L1 tumor proportion score ≥50%) and received first-line pembro ± ipilimumab for ≤35 cycles. Cell-free DNA (cfDNA) was isolated and sequenced from plasma samples at baseline (BL), during on-treatment response (cycle 5 [C5]), and at PD (discontinuation [D]) from pts with objective response to pembro monotherapy and subsequent PD. ctDNA burden (tumor fraction [TF]) was estimated using low-pass pWGS by quantifying tumor content in cfDNA as a function of aneuploidy. All samples with TF &amp;gt;1.5% underwent high-depth pWES. ctDNA burden was estimated from pWES data using maximum somatic allele frequency (MSAF). Correlation between TF and MSAF was evaluated using the Spearman coefficient (r). Variant allele frequency (VAF) and normalized VAF (nVAF) were used to determine variant clonality. Results: In total, 23 pts with initial response to pembro and subsequent PD had plasma samples available from BL, C5, and D. Samples from 22 pts had TF &amp;gt;1.5% by pWGS and underwent pWES. MSAF by pWES was correlated with TF by pWGS at BL (r = 0.68), C5 (0.80), and D (0.59). ctDNA TFs at BL and D (mean = 8.1% and 4.9%, respectively) were higher than C5 (2.0%). In a pooled variant-level analysis (nVAF &amp;gt;0.25) of samples from pts with high TF (≥2.5%) at both BL and D (n = 10), 786 (46%) of variants were present at BL and D, 391 (23%) were present at BL only, and 540 (32%) were present at D only. Variants shared between BL and D had higher clonality (mean nVAF = 0.46) than BL-only (0.38) or D-only variants (0.4). In general, shared variants had similar nVAFs at BL and D but in some pts nVAFs increased at D, suggesting evolution of tumor subclones during treatment. TP53 was mutated in 9 of 10 pts with high TF and was shared at BL and D in 6 pts (mean nVAF = 0.49), present at BL only in 1 pt (0.11) and at D only in 2 pts (0.43). KRAS G12V and Q61H hotspot mutations were observed in 2 pts at D-only and may be related to immune editing. Conclusions: Comprehensive pWES is feasible with sufficient ctDNA burden (TF ≥2.5%). ctDNA dynamics demonstrated molecular profiles characteristic of recurrence with high TF at BL and D and were consistent with radiographic determination of response/progression. Tumor variants at BL and D generally had a common clonal trunk with clones disappearing or appearing de novo. Citation Format: Minita Shah, EJ Dettman, Andrew Albright, Cai Chen, Julie Kobie, Vinay Varadan, Ayman Samkari, Carol Peña, Razvan Cristescu, Z Alexander Cao. Evaluation of on-treatment tumor molecular dynamics using plasma whole genome sequencing (pWGS) and whole exome sequencing (pWES) of circulating tumor DNA (ctDNA) in patients (pts) with metastatic non-small cell lung cancer (NSCLC): An exploratory analysis of KEYNOTE-598 [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 3664.

Abstract 3951: Timing the development of chemoresistance in relapsed pediatric cancer

Abstract Survivors of pediatric cancer face lifelong battles with severe morbidities, which includes a significant risk of recurrence. Pre-existing genetic variation within primary tumors may offer certain cell populations an evolutionary advantage, increasing the likelihood that some will be resistant to treatment. But the inherent complexity of the tumor means finding these markers of resistance has yet to be fully explored across childhood cancer. To address this, we developed a pipeline that combines clonal evolution reconstruction with mutational signature extraction at the subclonal level to elucidate changes in mutational processes. Using this pipeline to analyze 1,743 pediatric tumor genomes we found a common pattern of parallel evolution in most pediatric cancers, irrespective of their tissue of origin. Further, we detected therapy-associated mutational signatures linked to specific treatments such as temozolomide, platinum, fluorouracil, and thiopurine in post-treatment tumor subpopulations. Of 235 samples with known treatment exposure, 37.9% displayed one or more treatment-associated signatures. In the context of the phylogenetic tree, we identified specific subpopulations with therapy signatures. In multi-sample cases, resistant subpopulations had ancestors present in primary tumors at the time of diagnosis, suggesting certain subpopulations possessed the ability to withstand chemotherapy-induced pressures from an early stage and then expanded following treatment. Finally, we observed differences in the timing of mutational signatures between antimetabolite and alkylating agent treatments, reflecting the mechanisms of these drugs. Antimetabolite agents showed subclonal mutational signatures occurring in later branches, likely due to dependence on cell cycling. In contrast, alkylating agents displayed signatures in both early and late subpopulations, as they are unaffected by cell cycle state. Thus, investigating mutational signatures at the subclonal level unveils new insights into the clonal dynamics of pediatric cancers and the development of chemoresistance. This research offers valuable insights for identifying pediatric cancer patients at greater risk of recurrence, enabling the development of more effective treatment plans to enhance their quality of life and overall survival rates. Citation Format: Sasha Blay, Mehdi Layeghifard, Scott Davidson, David Chen, Astra Schwertschkow, Vijay Ramaswamy, Michael Taylor, Elli Papaemmanuil, Anita Villani, David Malkin, Ludmil Alexandrov, Mark Cowley, Adam Shlien. Timing the development of chemoresistance in relapsed pediatric 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 3951.