Copy Number Loss Research Articles

Abstract 2446: Genomic landscape of longitudinally collected preinvasive airway lesions

Abstract Lung carcinoma in situ (CIS) lesions, as precursors to squamous cell carcinoma, exhibit microscopic uniformity while displaying diverse clinical trajectories. Half of these lesions progress to invasive cancer, while the remaining half either regress or remain indolent. Previous genomic profiling efforts have elucidated progression-specific genomic changes against a backdrop of inherent heterogeneity and notable chromosomal instability signatures. Here, we present a novel approach by analyzing longitudinally collected samples of preinvasive airway lesions, encompassing both progressive and indolent trajectories. Our findings reveal that genomic integrity and cell cycle dynamics constitute key distinctions between progressive and indolent lesions. Notably, a decrease in clonal diversity and an inverse correlation between TCRA-Tcell fraction and genomic instability are observed as lesions approach invasion. Somatic mutations in cancer driver genes BRAC2, CSMD3, and MUC16 are found significantly associated with lesion progression. Furthermore, progressive lesions exhibit a higher frequency of copy number changes compared to indolent lesions. Copy number loss in 1p, 3p, 9p, and 17p are found to be significantly associated with lesion progression. Phylogenetic analysis further confirms positive selection for copy number loss in these specific genomic regions, providing insights into the evolutionary trajectory of alterations in progressive lesions. The comprehensive understanding derived from these genomic analyses is anticipated to advance early detection strategies, mitigate overtreatment, and facilitate the development of preventative therapies targeting early clonal events in lung cancer. Citation Format: Ahmed Alhendi, Lukas Kalinke, Adam Pennycuick, Kate Davies, Pascal Durrenberger, Kate Gowers, Kristiana Grigoriadis, Nicholas McGranahan, Sam Janes. Genomic landscape of longitudinally collected preinvasive airway lesions [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 2446.

Abstract 3908: DHX9 inhibition as a novel therapeutic for cancer with loss-of-function mutations in DNA damage repair genes BRCA1 and BRCA2

Abstract DHX9 is a DEAH-box RNA helicase which can unwind regions of double-stranded DNA and RNA but has a greater propensity for secondary structures such as DNA/RNA hybrids (R-loops), circular RNA and DNA/RNA G-quadruplexes. Given the delicate balance of R-loop formation and resolution in maintaining efficient transcription and replication, the ability of DHX9 to unwind R-loops is important in helping to maintain genomic stability. In addition, DHX9 can interact and regulate a large variety of proteins, including key proteins in DNA damage repair pathways such as BRCA1, ATR, Ku86, and WRN. Previously we demonstrated that DHX9 inhibition was efficacious in microsatellite high (MSI-H) CRC xenograft tumor models. Efficacy attributed to DHX9 loss in MSI-H correlates with defective DNA repair pathways such as mismatch repair (MMR). Here we report results of preclinical studies in ovarian and breast cancer models that indicate patients with Loss-of-Function (LOF) mutations in the DNA damage repair genes BRCA1 and/or BRCA2, may also benefit from DHX9 inhibitor treatment. DHX9 small molecule inhibitor ATX968 was tested for anti-proliferative activity in a large cell panel of 300 cell lines, and bioinformatic analyses was performed to identify molecular variants that co-associate with sensitivity or resistance cell proliferation outcomes. Notably, selective dependency on DHX9 was observed in both ovarian and breast cancer cell lines that exhibit BRCA LOF, as defined by somatic mutations including single-nucleotide variants and/or copy number loss in BRCA1 and/or BRCA2. DHX9 inhibition leads to increased RNA/DNA secondary structures such as R-loops and G-quadruplexes, resulting in subsequent DNA damage and increased replication stress. Cell lines that exhibit BRCA LOF appear unable to resolve this replication stress and show S-G2 phase cell cycle arrest prior to onset of apoptosis. Furthermore, a potent and selective DHX9 inhibitor tool compound was dosed orally in vivo to assess DHX9 dependency within multiple human xenografts representing triple negative breast cancer and high-grade serous ovarian cancer with BRCA LOF. In all models, the tool DHX9 inhibitor was dosed orally at 100 mg/kg BID and it was well tolerated for a period of up to 28 days. Robust and significant tumor growth inhibition and regression was observed in multiple BRCA LOF models with minimal tumor growth inhibition observed in BRCA1 and BRCA2 wild type xenograft models. These results extend the opportunity for DHX9 inhibition to provide therapeutic benefit for patients with solid tumors beyond what was previously reported for MSI-H CRC. Together, this preclinical data package validates DHX9 as a tractable new target with potential utility as a novel treatment for patients with defective DNA repair, such as MMR and BRCA1 and/or BRCA2 LOF, across multiple tumor types including colorectal, breast and ovarian cancer. Citation Format: Jennifer B. Castro, Matthew H. Daniels, Sunaina Nayak, Monique Laidlaw, David Brennan, Brian T. Johnston, Jie Wu, Anugraha Raman, Chuang Lu, Stephen J. Blakemore, Serena J. Silver, P. Ann Boriack-Sjodin, Kenneth W. Duncan, Jason A. Sager, Robert A. Copeland. DHX9 inhibition as a novel therapeutic for cancer with loss-of-function mutations in DNA damage repair genes BRCA1 and BRCA2 [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 3908.

Abstract 148: Multiomic sequencing reveals diversity in clonal landscape and genomic alterations in a lung metastatic PDX osteosarcoma model

Abstract Introduction: Osteosarcoma is a primary malignant bone tumor characterized by the production of spindle cells resulting in immature bone formation. The lung is the most frequent site of metastatic disease and relapse occurs in more than 30 % of patients. Recent results show that relapse is due to a subset of cells with different phenotypic and genetic signatures conferring advantage to drive progression and drug resistance within the intra-tumorally heterogeneous population. Thus, developing a defined in vivo model that can identify the rare genetic subpopulation and recapitulate clonal evolution is crucial. In this study, we used whole exome, barcode and RNA sequencing to characterize the landscape of genomic alterations and also track and identify the clonal subpopulations, clonal and genetic drivers of lungs metastasis. Methods: We injected barcoded PDX (OS17) cells intravenously in ten SCID mice to track the clonal subpopulation of cells that metastasize to the lungs. Metastasized lung nodules were collected following death or euthanasia. All samples collected were snap frozen in liquid nitrogen and stored at -80°C. DNA and RNA was extracted and analyzed by PCR, NGS, WES and RNA sequencing to map clonality, mutational and evolutionary profiles. Results: Four (40%) of the mice developed lung metastases with three mice having multiple metastatic lung nodules. Surgery to remove the metastatic nodules was performed 216 ± 55.5 days following tumor cell injection. Nine metastatic lung nodules from three mice were used for sequencing and analysis. A Shannon-Weaver and Jaccard similarity index show a diversity in clonal architecture between the lung nodules. A total of 15,394 somatic variants was identified including 12,547 single nucleotide variants (SNV), 4054 synonymous, 2662 missense, 28 nonsense mutations and 1639 somatic indels. The SNVs accounted for 81% of all variants followed by CNV 11 % and indels 8% respectively. Synonymous and missense mutations were the most frequently observed in all samples. Copy number loss was two-fold compared to copy number gain across all tumors. Furthermore, the deletions span longer genomic distances compared to amplifications. The mutational spectrum showed that the highest substitutions were C>T/G>A followed by T>C/A>G; the least substitutions were T>A/A>T. Also, we identified a large set of genes with genomic aberrations. These include amplifications in VEGF, RB1, RUNX, PARP4, ICAM3, EGFR, BRCA2, COLA6A1, COLA6A2, CCND3, CDKN2D etc. Conclusions: In the current model, we identified multiple tumorigenic seeding clones and the diverse set of genes mutated in osteosarcoma. Further analysis of the mutational signature resulted in the identification of potential genes that act as drivers in the lung metastatic process. Acknowledgments: Swim Across America, the Foster Foundation and the Barbara Epstein Foundation. Citation Format: Sylvester Jusu, Wendong Zhang, Zhongting Zhang, Xu Zhaohui, Sankaranarayanan Kannan, Yifei Wang, Zhou Xin, Yi Yanhua, Michael Roth, Jonathan Gill, Richard Gorlick. Multiomic sequencing reveals diversity in clonal landscape and genomic alterations in a lung metastatic PDX osteosarcoma model [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 148.

Abstract 5845: Cytarabine resistance leads to a unique mutational signature

Abstract The prognosis of recurrent T-cell acute lymphoblastic leukemia (T-ALL) is poor, mainly due to chemotherapy resistance. The mechanism(s) leading to chemoresistance at relapse are incompletely understood. We previously characterized a mouse strain that is hypomorphic for the DNA replication factor Mcm2. Mcm2hypo mice develop T-ALL due to acquired DNA copy number variations (CNV) involving tumor suppressor or oncogenes. We hypothesize that treatment of Mcm2hypo cells with specific chemotherapy agents may reveal CNV that lead to chemoresistance. We treated two Mcm2hypo T-ALL cell lines (designated 2883 and 2869) and an Mcm2 WT T-ALL cell line (designated 7298) with cytarabine (ARAC), an active agent often incorporated in T-ALL treatment regimens. After one year of treatment with gradually increasing ARAC concentrations, we obtained ARAC resistant cell lines (designated with the suffix CR) that tolerate drug concentrations up to 10,000X that of parental cell lines. These samples were characterized by sparse WGS (for CNV assessment), RNA-Seq and WES. Both the 2883CR and 2869CR cell lines had bi-allelic mutations involving Dck, which is the rate-limiting enzyme in the cytidine salvage pathway, and is thought to be required for metabolism of ARAC. In both cases, one allele was deleted while the second allele had a splice site/region mutation leading to partial intron retention and resultant frameshift. The 7298CR cell line had homozygous deletion of Dck. Western blotting showed absence of WT Dck protein in all three cell lines. Further analysis of WES data revealed thousands of acquired single nucleotide variants (SNV) in both Dck mutant Mcm2hypo cell lines. These mutations occurred in a specific trinucleotide context (C>T in a GCG context, T>C in a GTC context, T>G mutations in GTC and GTT context, and C>G in a GCC context) generating a mutational signature that is not present in the COSMIC database. In addition, the parental 2883 and 2869 cell lines showed fewer mutations (ie, hundreds vs thousands) in a similar context (C>T in a GCG context, T>C in a GTC context, T>G mutations in GTC and GTT context, but not the C>G in GCC context). A GEMINI (Genotoxic Mutational Signature Identified After Clonal Expansion In Vitro) assay of untreated 2883CR and 2869CR cells demonstrated that ongoing ARAC exposure was not required for the mutational signature. We hypothesize that the basic signature (C>T in a GCG context, T>C in a GTC context, T>G mutations in GTC and GTT context) can be produced by Mcm2 deficiency, and this signature is amplified and modified (to include a C>G in GCC context) by Dck inactivation. In summary, we identified three mechanisms responsible for Dck inactivation associated with ARAC resistance: copy number loss, splice site mutations, and splice region mutations. Moreover, Dck inactivation leads to a specific mutation signature. These findings add to emerging data that exposure to specific chemotherapy agents can lead to specific mutational signatures. Citation Format: Dengchao Cao, Mianmian Yin, Zhenhua Zhang, Taimour Baslan, Jack Zhu, Ryan Bertoli, Paul Meltzer, Peter Aplan. Cytarabine resistance leads to a unique mutational signature [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 5845.

Abstract 874: Expanded detection of BAP1 alterations in cancer and comparison of tumor type-specific gene expression scores

Abstract BAP1 is a tumor suppressor gene that was originally studied in uveal melanoma (UVM), kidney renal cell clear cell carcinoma (KIRC), and malignant mesothelioma (MESO). Early analyses focused on single-nucleotide variants, but other alteration types such as larger indels and gene-level copy number (CN) loss can also lead to loss of BAP1 expression. By incorporating these, we can improve our assessment of cancer type-specific consequences of BAP1 loss and broaden our findings beyond the initial small subset of cancer types which were studied in the context of mutations alone. We performed integrated multi-omic analyses using data from The Cancer Genome Atlas (TCGA), for 33 cancer types and more than 10,000 individuals. By combining and manually reviewing existing variant calls and new calls derived from a de novo local realignment pipeline across multiple independent variant callers including indel callers, we increased detection of high-quality somatic variant calls by 30% from 85 to 111, including 14 indels ≥5bp. Including CN loss alterations, we found a total of 1561 samples from 32 cancer types to be BAP1-altered, with alterations being predominantly CN-driven. We confirmed that expression of BAP1 was lower in altered versus unaltered tumors (p<0.001). Alteration frequencies across cancer types were highly variable, ranging from 0% in thyroid cancer to >30% in KIRC, cholangiocarcinoma (CHOL), UVM, MESO, lung squamous carcinoma (LUSC), and head-neck squamous cell carcinoma (HNSC). Tumor types such as CHOL, UVM, MESO, and liver hepatocellular carcinoma (LIHC) were relatively more mutation-driven whereas other tumor types like KIRC, LUSC, and HNSC were almost entirely CN-driven. To better understand the tissue and context-specific consequences of BAP1 alteration, we performed differential expression analyses across BAP1-altered and -unaltered samples. For each cancer type, we separately computed per-sample BAP1 alteration signature z-scores summarizing expression of the set of differentially expressed genes. In UVM, we observed distinct patterns of RNA expression, consistent with established literature on BAP1 loss and serving as a baseline for comparison for the biological effects of BAP1 loss in other less-studied cancer types. The BAP1 altered scores for 66% of cancer types were positively correlated with the UVM BAP1 altered signature, suggesting shared biological responses and affected pathways including immune responses, epithelial-mesenchymal transition, oxidative phosphorylation, and proliferation – all known roles of BAP1. In LIHC, we noted enrichment of bile duct gene expression in BAP1-altered tumors, suggesting a possible role for BAP1 in maintaining cell identity distinguishing hepatocytes and cholangiocytes. Our findings broadly emphasize the improvements that are gained by using new computational approaches in large cancer-genome studies such as TCGA. Citation Format: Ian R. Sturgill, Jesse R. Raab, Katherine A. Hoadley. Expanded detection of BAP1 alterations in cancer and comparison of tumor type-specific gene expression scores [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 874.

Abstract 4345: Evaluation of the prevalence and impact of molecular variation in the three-prime repair exonuclease 1 TREX1 across mammalian species and in human malignancies

Abstract The three-prime repair exonuclease 1, TREX1, degrades cytosolic DNA to prevent aberrant immune activation. Inactivation of TREX1 results in DNA accumulation in the cytosol and subsequent induction of the cGAS-STING DNA sensing pathway, downstream interferon-mediated innate immune signaling, and inflammation. Correspondingly, germline pathogenic mutational defects in the TREX1 gene lead to hereditary autoimmune and autoinflammatory disorders. While TREX1 mutations are also reported in some tumors, their consequences in cancer remain poorly understood. To assess the functional importance of amino acid residues in TREX1, we analyzed protein sequences of the functional TREX1 isoform, TREX1b, from 168 mammalian species. We also conducted a survey of genomic variant databases COSMIC, ClinVar, TCGA, LOVD, Decipher, and gnomAD for disease-associated TREX1 mutations. Finally, we modeled TREX1 mutations occurring in human tumors to evaluate predicted impact on protein stability and function using structure-based approaches FoldX and INPS-3D, and compared results to available predictions from AlphaMissense and COSMIC-3D. In the Cancer Genome Atlas (TCGA), 16 out of 32 cancer histologies have tumor samples bearing protein-altering TREX1 variants, with bladder cancer and melanoma having the highest frequency of TREX1 variants at 2.7% of tumor samples. Moreover, all 32 cancer types have some samples with loss of one or both copies of TREX1; frequencies of heterozygous loss range from 0.6% (thymoma) to 86% (kidney clear cell and lung squamous cell carcinomas). We also observed TREX1 copy gain with lower frequency than instances of copy loss, but also occurring in 31 out of 32 studies. Multiple tumors have a combination of a TREX1 protein sequence variant and copy number gain or loss, suggesting that their potential interplay may increase functional impact of TREX1 variants. Out of 15 cancer-associated TREX1 variants, 5 (33%) were predicted to be pathogenic by AlphaMissense. For FoldX and INPS-3D mutagenesis modeling, we used the published experimental 3D crystal structure of the wild type (WT) human TREX1 (PDB: 7TQQ). The changes in Gibbs free energy of folding ΔΔG between WT and mutant predicted by FoldX and INPS-3D both correlated strongly with the AlphaMissense pathogenic probability scores (Pearson r=0.69 and 0.73, respectively). We will discuss implications of TREX1 variation and conservation across mammalian species and potential effects of germline TREX1 variants in hereditary autoimmune and autoinflammatory disorders and on somatic and germline TREX1 variants in human cancer cells and tumor samples. Citation Format: Mariam M. Konate, Marwa Shekfeh, Julia Krushkal. Evaluation of the prevalence and impact of molecular variation in the three-prime repair exonuclease 1 TREX1 across mammalian species and in human malignancies [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 4345.

Abstract 6916: InnoPanelTMand InnoPediaTM: resources of engineered cell lines and xenografts for drug discovery

Abstract The Development of precision medicine relies on well-characterized experimental models that carry naive, induced, or engineered genomic and epigenetic features targetable by certain treatments. Patient-derived xenografts (PDX) and cell lines can serve as such in vivo and in vitro models by harboring abnormalities such as somatic and driver mutations, dysregulated gene expression and silencing, microsatellite instability, gene fusions, copy number amplification and loss of heterozygosity. In addition, cell lines can also be engineered to introduce sometimes rare-occurring abnormalities that the engineered cell line shares an identical genetic background with its parental cell line, thus providing a clearly contrasting pair to evaluate treatment specificity. With the advancement of disease research and drug discovery, existing cell lines can no longer meet the needs of various types of drug discovery. To evaluate drug efficacy more quickly and accurately, Kyinno has constructed and identified more than 4,000 engineered cell lines, including overexpressed cell lines constructed through transposon systems or lentivirus infection, gene-edited cell lines constructed through CRISPR-Cas9 technology, and reporter cell lines targeting multiple signaling pathways and membrane receptors. Besides, InnoPanelTM, a panel of over 1000 engineered cell lines was established and characterized to cover a wide range of heavily targeted genes such as CLDN18.1, CLDN18.2, LAG3, OX40, PD-L1, PD-L2, and TIM3. Cell line families were also developed for genes with multiple known driver mutations. All cell lines are annotated with background information, engineering details, functional characterization, and efficacy studies. Furthermore, we developed xenografts for several engineered cell lines and showed that the combination of cell lines and xenografts can accelerate drug development. Additionally, all of this information can be accessed in our free database, InnoPediaTM. The database also offers browsing, searching, and analytical capabilities for over 600 commonly used tumor cell lines and thousands of engineered cell lines with essential genomic and pharmacological data, cell line efficacy data for nearly 2000 common anti-tumor drugs, and comprehensive details for over 100 standard antibodies. Our engineered cell lines are now registered in the online cell line database Cellosaurus, which is available on the ExPASy server. Citation Format: Siyu Li, Chang Liu, Na Wang, Yuqing Hu, Jinying Ning, Feng Hao. InnoPanelTMand InnoPediaTM: resources of engineered cell lines and xenografts for drug discovery [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 6916.

Abstract 4338: A pan-cancer single-cell analysis of intratumoral copy number diversity and evolution

Abstract Aneuploidy is a hallmark of human cancers and studies have shown that cancer patients harbor many differences in their copy number aberrations (CNAs) reflecting interpatient heterogeneity (IPH). While IPH has been studied extensively, the intratumoral heterogeneity (ITH) of CNAs within a tumor remains understudied. Here, we conducted a pan-cancer analysis of 94 human tumor samples at single cell genomic resolution, representing seven major cancer types: bladder, breast, colon, glioblastoma, kidney, lung, and ovary. In total, 62,646 aneuploid cells were analyzed by single cell copy number profiling and bulk exome sequencing was performed. In most cancer types, increased subclonal diversity was associated with higher CNA burden, whole genome doubling (WGD) events, and TP53 mutations. In all tumors, our data show that cancer cells share a clonal set of truncal CNAs or mutations, suggesting these tumors evolved from a single ancestral cell. In 18/94 cases we identified subclonal WGD events, which resulted in a higher number of copy number losses after tetraploidization. Macro-spatial sampling showed that increased clonal diversity also correlated with increased spatial geographical diversity. We found that many cancers evolved CNAs in short punctuated bursts of evolution, and that this was not restricted to a specific cancer type. Collectively, these data show that copy number ITH is universally associated with molecular features across these human cancer types. Citation Format: Hanghui Ye, Thomas McDonald, Emi Sei, Darlan Conterno Minussi, Min Hu, Chenling Tang, Junke Wang, Kaile Wang, Anna Casasent, Hui Chen, Franziska Michor, Nicholas Navin. A pan-cancer single-cell analysis of intratumoral copy number diversity 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 4338.

Abstract 5036: Analytical and clinical validity of Northstar Select, a quantitatively enhanced liquid biopsy assay for comprehensive genomic profiling of solid tumors

Abstract Background: Liquid biopsy assays have emerged as vital tools in precision oncology. Among these assays, Comprehensive Genomic Profiling (CGP) tests in particular have improved treatment selection for late-stage solid tumor cancer patients by allowing for the detection via circulating tumor DNA (ctDNA) of somatic mutations in the tumor, many of which have corresponding targeted therapies. However, technical restrictions in the performance of such tests have limited their utility when ctDNA signal is low. BillionToOne’s Northstar Select assay is a new CGP test that achieves increased performance by addressing these limitations. Innovation: By using Quantitative Counting Template (QCT) technology, Northstar Select is able to track the yield of multiple processing steps, contributing to the higher sensitivity of the assay by maximizing the number of decoded molecules after sequencing. In addition, it employs chemistry and panel design optimizations, as well as novel algorithms for SNV, indel, CNV, fusion, and MSI calling to increase sensitivity and specificity. Analytical Performance: The performance of Northstar Select surpasses that of other commercially available tests for multiple mutation types, detecting more pathogenic and actionable mutations at low tumor DNA fraction while maintaining similarly high specificity. In analytical validation (N=103), limit of detection was determined to be 0.13-0.16% variant allele fraction (VAF) for SNVs and indels, 2.11 copies for copy number amplifications, 1.8 copies for copy number losses, 0.35% VAF for fusions, and 0.07% tumor fraction for MSI. Clinical Performance: Northstar Select was determined to have high clinical utility as well, with a study of clinical samples (N=481) from a variety of tumor types yielding <2% of reports with 0 mutations detected. A head-to-head comparison with other commercial liquid biopsy assays on a subset of the clinical samples (N=192) showed that the assay detected 115 additional pathogenic mutations in total, leading to a higher proportion of patients with at least one pathogenic mutation detected (90%) compared to comparators (79%). Conclusion: These results demonstrate that the Northstar Select assay is an innovative and high-performing diagnostic for treatment selection in oncology, leveraging quantitative approaches in process design, chemistry, and bioinformatics to offer superior analytical and clinical performance compared to the current landscape of liquid biopsy CGP tests. Citation Format: Xavier Bower, Jan Wignall, Joyce Zhu, Naomi Searle, Matthew Varga, Emilio Rosas-Linhard, Jason Luong, Esther Lin, Marie Simon, John ten Bosch, Wen Zhou. Analytical and clinical validity of Northstar Select, a quantitatively enhanced liquid biopsy assay for comprehensive genomic profiling of solid tumors [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 5036.

Abstract 3505: Classification of cancer subtypes by cfDNA fragmentomics analysis

Abstract Introduction Fragmentation patterns of cell-free DNA (cfDNA) from whole genome sequencing offer insights into nucleosome occupancy, providing a tool to infer epigenomic and transcriptomic information. The nucleosome complex, the primary protector of cfDNA, is reflected in the size distribution of cfDNA fragments, with a mode fragment size of 167 bp corresponding to the wrapping of DNA around a single nucleosome. Nucleosome occupancy patterns at transcription factor binding sites (TFBS) exhibit differences between circulating tumor DNA in cancer patients and normal plasma controls, as well as among different tumor subtypes. Therefore, cfDNA fragmentomics can be employed for cancer subtyping analysis. Methods We utilized the Griffin framework to classify tumor subtypes based on nucleosome profiling of cancer-specific TFBS and tumor subtype-specific chromatin accessibility regions from low-pass whole genome sequencing data of cfDNA. For prostate cancer subtyping, AR and ASCL1 binding sites were used to distinguish between androgen receptor-dependent prostate cancer (ARPC) and neuroendocrine prostate cancer (NEPC). ER and ERBB2 were employed for breast cancer subtyping to differentiate between ER-positive and ER-negative tumor subtypes. Results Nucleosome profiling patterns at AR binding sites (ARBS) were compared among over 500 prostate cancer plasma samples and 42 normal plasma backgrounds. The ARBS nucleosome profiling abnormality score (ARBS score) was quantified by comparing its GC-corrected fragment central coverage with the normal background using a standard Z-score.Majority of prostate cancer samples with a high ARBS score could be classified as ARPC. For 12 samples with high tumor fraction but low ARBS score and no AR copy number amplification, we found that 8 had a high ASCL1 nucleosome profiling abnormality score. All these samples also exhibited RB1 copy number loss, suggesting that these 8 samples likely belong to NEPC. Nucleosome profiling of 171 breast cancer plasma samples at breast cancer-specific ER binding sites revealed that majority of samples had a high ERBS abnormality score, indicating likely ER-positive breast cancer. Conclusion Our results demonstrate that nucleosome profiling of tumor cfDNA exhibits distinct patterns across different tumor types. Integrating nucleosome profiling at cancer-specific transcription factor binding sites can facilitate cancer subtyping, highlighting its potential clinical utility for patient stratification. Citation Format: Lisha Zhu, Chao Dai, Shuang Gan, Shidong Jia, Pan Du. Classification of cancer subtypes by cfDNA fragmentomics analysis [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 3505.

Abstract 861: Integrative multi-omics analysis enables a comprehensive characterization of prostate cancer and unveils metastasis-associated candidate biomarkers

Abstract Prostate cancer (PCa), a common malignancy in men, presents a complex and variable disease landscape, requiring precise evaluation for personalized treatment decisions. Urologists employ a range of assessments, including clinical staging, PSA levels, imaging, Gleason scores, and emerging biomarkers. Nevertheless, the need for additional biomarkers to enhance precision medicine continues. This study utilizes extensive omics datasets to thoroughly profile PCa. We conducted an in-depth analysis of primary PCa data from the multi-omics dataset from The Cancer Genome Atlas studies to identify molecular subtypes showing significant prognostic differences. We conducted integrative clustering analysis on a dataset comprising 341 primary prostate adenocarcinomas with comprehensive multi-omics data, including substitutions, small insertions/deletions, copy number alterations, DNA methylation, gene expression profiles, micro-RNA expression, reverse-phase protein arrays, and microbiome data. The analysis revealed seven distinct clusters (C1 to C7) based on multi-omics features. At the genomic level, recurrent copy number losses and DNA methylation/demethylation were the most prominent alterations across clusters. When looking at survival curves, the 7 clusters could be clearly partitioned into two groups. Therefore, to further investigate multi-omics features specifically linked to prognosis, we grouped clusters with similar prognoses into two macro-clusters. Clusters C1 to C4 formed MC1 (favorable prognosis), while C5 to C7 constituted MC2 (poor prognosis). A comprehensive comparison between MC1 and MC2 revealed molecular differences associated with survival. We performed computational validation of candidate prognostic biomarkers using two external datasets. By utilizing these selected features, we could stratify patients in both the validation datasets into groups with significant differences in survival. The analysis identified six features consistently associated with prognosis across both datasets, including CCNB1, PCNA, RAD51, FOXM1, miR-503-5p (positively associated with risk), and miR-7704 (negatively associated with risk). The practical applicability of these biomarkers was explored through immunohistochemical analysis on biopsy samples obtained at diagnosis and from metastatic sites. Based on their potential relevance according to the literature, we investigated three candidate biomarkers derived from the multi-omics analysis: FOXM1, CCNB1 and RAD51. The expression of these proteins was found to correlate with PCa progression and mark the metastatic stage. In summary, this study highlights the potential of translating comprehensive multi-omics bioinformatic analyses into practical approaches for understanding the molecular mechanisms of tumors and identifying prognostic markers. Citation Format: Matteo Villa, Giorgio Cazzaniga, Maddalena Bolognesi, Valentina Crippa, Federica Malighetti, Andrea Aroldi, Giorgio Bozzini, Fabio Pagni, Rocco Piazza, Luca Mologni, Daniele Ramazzotti. Integrative multi-omics analysis enables a comprehensive characterization of prostate cancer and unveils metastasis-associated candidate biomarkers [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 861.

Abstract 1256: RPL22 is a tumor suppressor in MSI-high cancers and a key splicing regulator of MDM4

Abstract Microsatellite instability high (MSI-H) tumors are marked by 30% or more mutations in microsatellite regions and are generated by somatic or germline alterations in DNA mismatch repair machinery. Despite a high tumor mutational burden, MSI-H tumors do not frequently harbor mutations in TP53. Notably, a frameshift mutation (p.K15fs) in the gene RPL22 is among the most recurrent mutations in MSI-H tumors. RPL22 and its paralog, RPL22L1, form part of the large subunit of the 60S ribosome and are known to affect protein synthesis as well as splicing of genes and transcription factors that affect development and tumorigenesis. Here we explore the result of RPL22 loss on the regulation of MDM4, an upstream target of p53. MDM4 protein expression is mediated through alternative mRNA splicing of its sixth exon, which leads to two isoforms, the exon 6-inclusive MDM4-FL and the exon 6-exclusive MDM4-S, which is prone to degradation. To date, no mutational events have been associated with this alternative splicing event. We show the prevalence of RPL22fs mutations is more than 70% in certain MSI-H cell lines (e.g. colon, endometrial) and tumors (e.g. stomach adenocarcinoma) in the CCLE and TCGA. RPL22 copy number loss and RPL22 frameshift mutations are strongly correlated with MDM4 exon 6 inclusion in TCGA samples, and RPL22 knockout increases MDM4 exon 6 inclusion and MDM4 protein in multiple cell lines. RPL22 loss increases cell proliferation and augments resistance to the MDM inhibitor Nutlin-3a. Downstream targets of p53 (p21, BBC3) are downregulated due to RPL22 loss and Nutlin-3a inhibition, suggesting mutation of RPL22 and subsequent overexpression of MDM4 promotes resistance to Nutlin-3a. CLIP-seq revealed that RPL22 binds the MDM4 3’-UTR. We find that RPL22 loss is associated with an alternative 3’-splicing event in RPL22L1 adjacent to exon 3 and results in the expression of the full-length transcript of RPL22L1. In summary, we identify RPL22 as a key modulator of MDM4 splicing through an alternative splicing switch in exon 6. Furthermore, RPL22 represses the expression of its paralog, RPL22L1, by mediating the splicing of a cryptic exon corresponding to a truncated functional transcript. Therefore, we propose that damaging mutations in RPL22 drive oncogenic MDM4 induction and reveal a common splicing circuit in MSI-H tumors that may inform therapeutic targeting of the MDM4-p53 axis and oncogenic RPL22L1 induction. Our results also suggest that the presence of RPL22 may act as a rheostat to maintain MDM4 exon 6 skipping under normal cellular contexts. Citation Format: Hannah N. Weinstein, Kevin Hu, Lisa Fish, Yih-An Chen, Paul Allegakoen, Keliana S. Hui, Julia H. Pham, Maria B. Baco, Hanbing Song, Andrew O. Giacomelli, Francisca Vazquez, Mahmoud Ghandi3, Hani Goodarzi, Franklin W. Huang. RPL22 is a tumor suppressor in MSI-high cancers and a key splicing regulator of MDM4 [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 1256.

Abstract 969: Tumor-agnostic ctDNA monitoring in patients with metastatic HR+/HER2- breast cancer receiving first-line CDK4/6 inhibitor and endocrine therapy

Abstract Introduction: CDK4/6-inhibitors (-i) in combination with endocrine therapy are first-line treatment for metastatic HR+/HER2- breast cancer (mBC). Baseline circulating tumor DNA (ctDNA) burden and changes on therapy are prognostic in mBC. GuardantINFINITY is a tumor-agnostic platform which measures aggregated epigenomic methylation signals that can distinguish ctDNA from non-tumor derived signals and permits the detection of somatic alterations in a 753 NGS panel. Longitudinal measurement of ctDNA quantity and characterization of emerging mutations may permit early identification of resistance and allow for intervention prior to clinical/radiographic progression. Methods: Patients with HR+/HER2- mBC receiving CDK4/6i were enrolled in a prospective cohort study from 2018 onwards. Plasma samples were collected at baseline (prior to initiation of CDK4/6i) and regularly in clinical follow up. Samples were analyzed using the GuardantINFINITY platform. Clinical information, pathologic characteristics, and dates of clinical progression were collected. Results: 57 patients with 350 clinical timepoints (median of 5 per patient, range 2-15) were evaluated. Median follow up was 28.6m (range: 0.9-64.3m). Median age at diagnosis of mBC was 59 (range: 38-88). CDK4/6i received included 42/57 (73%) palbociclib, 14/57 (25%) ribociclib and 1/57 (2%) abemaciclib; 40/57 (70%) were in combination with an aromatase inhibitor and 17/57 (30%) with fulvestrant. 5/350 (1.4%) of samples failed analysis related to low cfDNA input. ctDNA was detected (by methylation) in 49/57 (86%) at baseline (1 fail, 7 not detected), and 208/345 (60%) of all samples. Median methylation-based tumour fraction (TF) at baseline was 0.0132 (range: 0.000875-0.433); a higher baseline methylation TF (≥1%) was associated with worse CDK4/6i-PFS (14.7m vs. 26.7m; HR: 2.27, 95%CI: 1.12-4.62, p=0.023). 19/49 patients had ctDNA clearance, which was associated with improved CDK4/6i-PFS (34.4m vs. 13.9m; p<0.0001). In longitudinal sampling, methylation TF rose prior to clinical progression and genomic alterations associated with CDK4/6i-resistance were detected, including copy-number loss in RB1. Conclusion: Baseline methylation TF and ctDNA clearance as measured by GuardantINFINITY were prognostic in this cohort. Longitudinal sampling permitted genomic characterization and detection of alterations conferring acquired-resistance prior to clinical progression. Clinical correlates of ctDNA detection, clearance, and further serial genomic analyses will be presented. Citation Format: Mitchell J. Elliott, Jesús Fuentes-Antrás, Aaron Dou, Nancy Gregorio, Elizabeth Shah, Emily Van de Laar, Geethika Yalamanchili, Leylah M. Drusbosky, Eitan Amir, Michelle B. Nadler, Celeste Yu, Hal K. Berman, Lillian L. Siu, Philippe L. Bedard, David W. Cescon. Tumor-agnostic ctDNA monitoring in patients with metastatic HR+/HER2- breast cancer receiving first-line CDK4/6 inhibitor and endocrine therapy [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 969.

Abstract 5890: ZNF451 and doxorubicin resistance

Abstract Precision medicine most often utilizes mutations in patient selection, however targetable mutations are exceedingly rare in high-grade serous ovarian cancer (OV). Aneuploidy and chromosome instability are major contributors to such genomic changes in OV. Genes are pervasively disrupted by copy-number alterations: 39% of all genes in the median solid tumor of all cancer types, and 67% in OV. A common second-line OV chemotherapeutic is peggylated-liposomal doxorubicin, a topoisomerase-II poison. To address which genes in OV contribute to doxorubicin sensitivity and resistance, we performed a CRISPR-Cas9 screen on 19,114 human genes in the presence or absence of doxorubicin. Positive control genes involved in drug-efflux and DNA repair were significant hits. The gene with both the highest significance and greatest magnitude of doxorubicin sensitization upon knockout was the E3 SUMO2/3 ligase ZNF451. ZNF451 exhibits a copy-number loss in 14% of patients and corresponds to ZNF451 protein reduction. ZNF451 is an attractive target for future combination therapies with doxorubicin to induce conditional lethality. ZNF451 may be valuable to study as a prognostic biomarker for topoisomerase-II inhibitor chemotherapy. Citation Format: Amy Rees, Alexandra Blackman, Della Evans, Joe Delaney. ZNF451 and doxorubicin resistance [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 5890.

Optical Genome Mapping Reveals Genomic Alterations upon Gene Editing in hiPSCs: Implications for Neural Tissue Differentiation and Brain Organoid Research.

Genome editing, notably CRISPR (cluster regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated protein 9), has revolutionized genetic engineering allowing for precise targeted modifications. This technique's combination with human induced pluripotent stem cells (hiPSCs) is a particularly valuable tool in cerebral organoid (CO) research. In this study, CRISPR/Cas9-generated fluorescently labeled hiPSCs exhibited no significant morphological or growth rate differences compared with unedited controls. However, genomic aberrations during gene editing necessitate efficient genome integrity assessment methods. Optical genome mapping, a high-resolution genome-wide technique, revealed genomic alterations, including chromosomal copy number gain and losses affecting numerous genes. Despite these genomic alterations, hiPSCs retain their pluripotency and capacity to generate COs without major phenotypic changes but one edited cell line showed potential neuroectodermal differentiation impairment. Thus, this study highlights optical genome mapping in assessing genome integrity in CRISPR/Cas9-edited hiPSCs emphasizing the need for comprehensive integration of genomic and morphological analysis to ensure the robustness of hiPSC-based models in cerebral organoid research.

Abstract A007: DNA methylation analysis comparing high-grade serous ovarian cancer to the normal ovary with adjustment for cell type variation identifies tumor-specific methylation alterations

Abstract A007: DNA methylation analysis comparing high-grade serous ovarian cancer to the normal ovary with adjustment for cell type variation identifies tumor-specific methylation alterations

Abstract IA016: Molecular diversity of uterine serous carcinoma

Abstract Uterine serous carcinoma (USC) is a biologically aggressive subtype of endometrial cancer with a high recurrence rate and poor prognosis disproportionately affecting black women. Whole-exome-sequencing analysis revealed that up to 10% of USC harbor features of ultra/hypermutation due to a deficiency of polymerase e (POLE) and/or mismatched-repair genes (MMRd). These tumors are notable for having a high number of single nucleotide variants (SNV), no LOH segments or copy-number variants (CNV) and a peculiar genetic signature characterized by an increased rate of C > A transversions. In contrast, the overwhelming majority of USC (~ 90%) are POLE/MMR proficient, have a low number of SNV and demonstrate alteration in genes including TP53, PIK3CA mutation/amplification, ERBB2 amplification, CCNE1 amplification, FBXW7 mutation/deletion, PPP2R1A mutation, and somatic mutations involving homologous recombination genes. Frequent mutations in CHD4/Mi2b, a member of the NuRD-chromatin-remodeling-complex, and TAF1, an element of the core TFIID transcriptional machinery are also detected in a significant number of USC. Somatic CNVs, a sign of genomic instability, are identified in the majority of USC with multiple copy number gains and copy number losses, with the most frequent events taking place in pathways similar to those found from analysis of SNVs. In addition to loss of TP53, frequent deletion of a small segment of chromosome 19 containing MBD3, a member of the NuRD-chromatin-modification-complex, and frequent amplification of chromosome segments containing PIK3CA, ERBB2 (encoding for the HER2/neu receptor) and CCNE1 (a target of FBXW7-mediated ubiquitination) are commonly detected. Overall, these findings suggest that both somatic point mutations (SNV) and somatic CNV play a major role in the pathogenesis of USC. Importantly, the frequent mutation of DNA damage, chromatin remodeling, cell cycle and cell proliferation pathways in USC including the overexpression/amplification of HER2/neu, CCNE1 and the tumor-associated calcium signal transducer 2 (TACSTD2/TROP2) genes, suggest novel targets for therapy against this lethal variant of endometrial cancer. Citation Format: Alessandro D. Santin. Molecular diversity of uterine serous carcinoma [abstract]. In: Proceedings of the AACR Special Conference on Endometrial Cancer: Transforming Care through Science; 2023 Nov 16-18; Boston, Massachusetts. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(5_Suppl):Abstract nr IA016.

Abstract B012: Application of shallow whole genome sequencing to identify therapeutic opportunities in p53abn endometrial cancers

Abstract Background: Shallow whole genome sequencing (sWGS) can be used to detect copy number (CN) aberrations. In high-grade serous ovarian (HGSOC) it has been used to identify CN signatures to direct therapy, recognizing two signatures associated with homologous recombination deficiency (HRD). p53abn endometrial carcinomas (EC) represent the most lethal subtype of EC with molecular similarities to HGSOC. Recent data have shown improved outcomes in patients with p53abn EC when adjuvant chemotherapy is used in addition to radiation, however, overall prognosis is still poor and therapeutic advances are urgently needed. Our aim was to apply sWGS with targeted sequencing to assess CN signatures in a cohort of p53abn ECs to identify additional prognostic or predictive stratification. Methods: sWGS was performed on formalin fixed paraffin embedded (FFPE) tissue from 215 p53abn ECs and 188 samples had targeted panel sequencing for 275 cancer related genes. CN variations, common mutations and CN signatures were derived, and associations to clinicopathologic and outcomes data were assessed. Results: 187 p53abn ECs had sufficient quality DNA to generate CN signatures (including 96 serous, 39 endometrioid, 33 carcinosarcoma, 12 clear cell and 7 other), with 5 distinct CN signatures identified. Signature 5 was associated with HRD due to CN loss of BRCA1 and 2 and was found in 35 (19%) of patients. Signatures 3 and 4 were associated with whole genome duplications, with mutations in PIK3CA defining Signature 3. 28% of cases had CCNE1 amplification, and this was enriched with carcinosarcoma histotype (p= 0.0019). 34% of patients had either HER2 gene amplification or HER2 overexpression by immunohistochemistry (IHC), which was seen across all histotypes, and was associated with worse outcomes (p=0.017). The most common mutations beyond TP53 were PIK3CA (32%), PPP2R1A (29%), PIK3R1 (18%), FBXW7 (16%) and PTEN (16%). There was no significant difference in outcomes observed between the CN signatures (35% of patients had recurred and 22% had died from disease at 3 years in total cohort), nor in the distribution of histotypes across signatures. After adjusting for stage, there was no difference in outcomes between histotypes. sWGS in combination with selective sequencing and IHC identified targetable pathways in 84% of patients with p53abn EC (HRD-PARP inhibitors, HER2-anti HER2 therapy, CCNE1-Wee1 inhibitor, PPP2R1A/FBXW7-PKMYT1 inhibitor). Conclusions: sWGS is a relatively inexpensive tool that can be performed on standard FFPE material and identified therapeutic opportunities in 84% of p53abn EC patients. Outcomes were uniformly poor in this cohort of p53abn ECs highlighting the need for improved treatment options beyond surgery and conventional chemoradiation. Further research is needed to determine the efficacy of treatments targeting these identified pathways within p53abn ECs. Citation Format: Amy Jamieson, Juliana Sobral de Barros, Dawn R. Cochrane, Maxwell Douglas, Sameer Shankar, Samuel Leung, Branden Lynch, Janine Senz, Blake Gilks, David G. Huntsman, Jessica N. McAlpine. Application of shallow whole genome sequencing to identify therapeutic opportunities in p53abn endometrial cancers [abstract]. In: Proceedings of the AACR Special Conference on Endometrial Cancer: Transforming Care through Science; 2023 Nov 16-18; Boston, Massachusetts. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(5_Suppl):Abstract nr B012.

Genomic analysis of primary epithelial neoplasms of the seminal vesicle identifies a subset of mucinous cystic tumours driven by KRAS mutations.

Carcinomas of the seminal vesicle are exceedingly rare, with a limited number of cases described in the literature. Reported cases span a relatively wide morphological spectrum, and their genomic features remain unexplored. In this study, we interrogated five primary epithelial neoplasms of the seminal vesicle using a targeted DNA sequencing platform (OncoPanel, 447 genes). The tumours included one adenocarcinoma with intestinal phenotype presenting after external beam radiation (for prostatic adenocarcinoma), one carcinoma with Müllerian-type clear cell phenotype, two mucinous tumours resembling low-grade mucinous neoplasms of the appendix (LAMN) and one mucinous cystadenoma. The post-radiation mucinous adenocarcinoma had genomic findings consistent with bi-allelic inactivation of TP53, as well as multiple copy-number changes with regional and chromosomal arm-level copy-number losses. The Müllerian-type clear cell carcinoma exhibited a complex copy-number profile with numerous regional and arm-level copy-number changes, as well as focal amplification events, including copy-number gain of 8q and amplification of a region within 20q13. Both low-grade mucinous tumours resembling LAMN harboured hot-spot gain-of-function KRAS variants (p.G12V and p.G13D) as the only genomic alteration. No genomic alterations were detected inthe lesion diagnosed as mucinous cystadenoma. Our results suggest that primary low-grade mucinous neoplasms of the seminal vesicle may represent a distinct entity equivalent to appendiceal counterparts, driven by gain-of-function variants of RAS GTPases. The remaining tumours showed genomic features that closely resembled those of neoplasms with comparablephenotypes and/or biological characteristics arising in other sites, suggesting that they could be managed similarly, with special considerations related to their anatomical location.

Copy number variations within fibroblast growth factor 13 gene influence growth traits and alternative splicing in cattle

Previous researches revealed a copy number variation (CNV) region in the bovine fibroblast growth factor 13 (FGF13) gene. However, its effects remain unknown. This study detected the various copy number types in seven Chinese cattle breeds and analysed their population genetic characteristics and effects on growth traits and transcription levels. Copy number Loss was more frequent in Caoyuan Red cattle and Xianan cattle than in the other breeds. Association analysis between CNV and growth traits of Qinchuan indicated that the CNV was significantly related to chest depth, hip width and hucklebone width (P < 0.05). Additionally, the growth traits of individuals with copy number Loss were significantly inferior to those with copy number Gain or Median (P < 0.05). Besides, we found two splicing isoforms, AS1 and AS2, in FGF13 gene, which resulted from alternative 5′ splicing sites of intron 1. These isoforms showed varied expression levels in various tissues. Moreover, CNV was significantly and negatively associated with the mRNA expression of AS1 (r = −0.525, P < 0.05). The CNVs in bovine FGF13 gene negatively regulated growth traits and gene transcription. These observations provide new insights into bovine FGF13 gene, delivering potentially useful information for future Chinese cattle breeding programs.