Analysis Of Somatic Mutations Research Articles

Analysis of somatic mutations and key driving factors of cervical cancer progression.

We investigated the somatic mutations and key driving factors of cervical cancer by whole exome sequencing . We found 22,183 somatic single nucleotide variations (SNVs) in 52 paired samples. Somatic SNVs in cervical cancer were significantly higher than those in high-grade intraepithelial lesion and low-grade squamous intraepithelial lesion groups (P < 0.05). C → T/G accounted for 44.12% of base substitution. Copy number variation (false discovery rate < 0.05) was found in 57 chromosome regions. The three regions with significant differences between cervical cancer and non-cervical cancer groups were 1q21.1, 3q26.33, and 13q33.1, covering genes related to tumor proliferation, differentiation, and apoptosis. The frequency of human papillomavirus (HPV) insertion/integration and the number of "tCw" mutations in the cervical cancer group were significantly higher than those in the non-cervical cancer group (P < 0.05). The total number of mutations was positively correlated with the number of "tCw" mutations (R 2 = 0.7967). HPV insertion/integration (OR = 2.302, CI = 1.523-3.589, P = 0.0005), APOBEC enrichment (OR = 17.875, CI = 2.117-150.937, P = 0.001), and HLA-B*39 in HLA-I (OR = 6.435, CI = 0.823-48.919, P = 0.0042) were risk factors for cervical cancer, while HLA-DQB1*05 in HLA-II was a protective factor (OR = 0.426, CI = 0.197-0.910, P = 0.032). Conclusively, HPV insertion/integration, APOBEC mutagenesis, and HLA polymorphisms are high-risk factors for cervical cancer and may be causes of genome instability and somatic mutations. This study provides experimental data for revealing the molecular mechanism of cervical cancer.

Mitigating age-related somatic mutation burden.

Genomes are inherently unstable and require constant DNA repair to maintain their genetic information. However, selective pressure has optimized repair mechanisms in somatic cells only to allow transmitting genetic information to the next generation, not to maximize sequence integrity long beyond the reproductive age. Recent studies have confirmed that somatic mutations, due to errors during genome repair and replication, accumulate in tissues and organs of humans and model organisms. Here, we describe recent advances in the quantitative analysis of somatic mutations in vivo. We also review evidence for or against a possible causal role of somatic mutations in aging. Finally, we discuss options to prevent, delay or eliminate de novo, random somatic mutations as a cause of aging.

Analysis of Somatic Mutations in Senescent Cells Using Single-Cell Whole-Genome Sequencing

Analysis of Somatic Mutations in Senescent Cells Using Single-Cell Whole-Genome Sequencing

Analysis of Somatic Mutations in Senescent Cells Using Single-Cell Whole-Genome Sequencing.

Somatic mutations accumulate in multiple organs and tissues during aging and are a known cause of cancer. Cellular senescence is a possible cause of functional decline in aging, yet also acts as an anticancer mechanism in vivo. Here, we compared somatic mutation burden between early passage and deeply senescent human fibroblasts using single-cell whole-genome sequencing. The results show that single-nucleotide variants (SNVs) and small insertions and deletions (INDELs) are increased in senescent cells by about twofold but have the same mutational signature as early passage cells. The increase in SNVs and INDELs can be explained by increased replication errors due to the increased number of cell divisions senescent cells are likely to have undergone. By contrast, a stark increase of aneuploidies was observed in deeply senescent cells, with about half of all senescent cells affected but none of the early passage cells analyzed. These results indicate that large chromosomal events rather than small base substitutions or insertions and deletions could be mechanistically linked to cellular senescence.

Dysfunction of ubiquitin protein ligase MYCBP2 leads to cell resilience in human breast cancers.

Breast cancer is the most common type of cancer among women worldwide, and it is estimated that 294000 new diagnoses and 37000 deaths will occur each year in the United States alone by 2030. Large-scale genomic studies have identified a number of genetic loci with alterations in breast cancer. However, identification of the genes that are critical for tumorgenicity still remains a challenge. Here, we perform a comprehensive functional multi-omics analysis of somatic mutations in breast cancer and identify previously unknown key regulators of breast cancer tumorgenicity. We identify dysregulation of MYCBP2, an E3 ubiquitin ligase and an upstream regulator of mTOR signaling, is accompanied with decreased disease-free survival. We validate MYCBP2 as a key target through depletion siRNA using in vitro apoptosis assays in MCF10A, MCF7 and T47D cells. We demonstrate that MYCBP2 loss is associated with resistance to apoptosis from cisplatin-induced DNA damage and cell cycle changes, and that CHEK1 inhibition can modulate MYCBP2 activity and caspase cleavage. Furthermore, we show that MYCBP2 knockdown is associated with transcriptomic responses in TSC2 and in apoptosis genes and interleukins. Therefore, we show that MYCBP2 is an important genetic target that represents a key node regulating multiple molecular pathways in breast cancer corresponding with apparent drug resistance in our study.

Identification of a novel NPM1 mutation in acute myeloid leukemia.

e19019 Background: Nucleophosmin (NPM1) is predominant nucleolar localization. NPM1-mutant acute myeloid leukemia (AML) account for 25-35% of adult AML patients, has been defined as a distinct AML entity in the 2022 WHO classification. The majority of NPM1 mutations reported affect exon 12 (classical type A mutation), account for 75-80% of adult NPM1-mutated AML cases, which generate an extra nuclear export signal (NES) in the C-terminal leading to aberrant cytoplasmic localization. Similar aberrant cytoplasmic translocation has also been reported in NPM1 mutants that identified in exons 9 and 11. Identification of novel non-type A mutants is paramount for diagnosis, risk stratification, and prognosis for targeted population. Methods: Somatic mutation analysis is performed on 566 AML patients using next-generation sequencing (NGS). Immunofluorescence and immunohistochemistry were used to identify the cytological localization of mutant NPM1. HEK-293T cells transiently transfected with plasmids for ectopic expression of the GFP-mutated NPM1 fusion proteins were treated with the specific Crm1/XPO1 inhibitor leptomycin B and to evaluate the NES dependence of their subcellular localization. Results: We identified one AML patient with NPM1 mutant located on exon 5. This patient was a 59-year-old female with de novo AML, with an 18-nucletide in-frame insertion at position 405 (c.405_406insGCCCTGGAACTGGGGAAC, named MutSong) in the middle of exon 5 [variant allele fraction (VAF), 11.9%]. It generated a new NPM1 mutant protein (p.135insALELGN), and containing a leucine-rich NES. Notably, different with all NPM1 mutant in other exons reported so far, our exon 5 mutant retained the functional c-terminal but insert one leucine-rich NES, similar finding was recently reported in NPM1 mutants that identified in exon 5 but on different position. This NPM1 mutant in exon 5 resulted in cytoplasmic localization both by confocal and immunohistochemistry in AML patient sample. HEK-293T overexpressing the new GFP-NPM1 exon 5 fusion protein indicated the aberrant localization in the cytoplasm and partially in nucleoli. Moreover, NES-dependent cytoplasmic localization was inhibited by exportin-1 inhibitor leptomycin B, indicated that the novel NES generated by exon 5 mutant is responsible for cytoplasmic localization. This patient carried WT1, IKZF1, JAK2, and NUP98 mutations, and was intravenously treated with daunorubicin plus cytarabine for induction followed by 3 cycles of intermediate-dose cytarabine as consolidation. Then the patient received allogeneic hematopoietic stem cell transplantation (allo-HSCT). Unfortunately, the patient died of severe pulmonary infection and viral encephalitis 14 months after the initial remission, 7 months after allo-HSCT. Conclusions: Our finding of the novel NPM1 mutation in exon 5 supported that beside exon 12, exon 5 mutant is another NPM1 “born to be exported” mutant critical for leukemogenesis.

Identification of Driver Epistatic Gene Pairs Combining Germline and Somatic Mutations in Cancer.

Cancer arises from the complex interplay of various factors. Traditionally, the identification of driver genes focuses primarily on the analysis of somatic mutations. We describe a new method for the detection of driver gene pairs based on an epistasis analysis that considers both germline and somatic variations. Specifically, the identification of significantly mutated gene pairs entails the calculation of a contingency table, wherein one of the co-mutated genes can exhibit a germline variant. By adopting this approach, it is possible to select gene pairs in which the individual genes do not exhibit significant associations with cancer. Finally, a survival analysis is used to select clinically relevant gene pairs. To test the efficacy of the new algorithm, we analyzed the colon adenocarcinoma (COAD) and lung adenocarcinoma (LUAD) samples available at The Cancer Genome Atlas (TCGA). In the analysis of the COAD and LUAD samples, we identify epistatic gene pairs significantly mutated in tumor tissue with respect to normal tissue. We believe that further analysis of the gene pairs detected by our method will unveil new biological insights, enhancing a better description of the cancer mechanism.

Prevalence and clinical expression of germ line predisposition to myeloid neoplasms in adults with marrow hypocellularity

AbstractSystematic studies of germ line genetic predisposition to myeloid neoplasms in adult patients are still limited. In this work, we performed germ line and somatic targeted sequencing in a cohort of adult patients with hypoplastic bone marrow (BM) to study germ line predisposition variants and their clinical correlates. The study population included 402 consecutive adult patients investigated for unexplained cytopenia and reduced age-adjusted BM cellularity. Germ line mutation analysis was performed using a panel of 60 genes, and variants were interpreted per the American College of Medical Genetics and Genomics/Association for Molecular Pathology guidelines; somatic mutation analysis was performed using a panel of 54 genes. Of the 402 patients, 27 (6.7%) carried germ line variants that caused a predisposition syndrome/disorder. The most frequent disorders were DDX41-associated predisposition, Fanconi anemia, GATA2-deficiency syndrome, severe congenital neutropenia, RASopathy, and Diamond-Blackfan anemia. Eighteen of 27 patients (67%) with causative germ line genotype were diagnosed with myeloid neoplasm, and the remaining with cytopenia of undetermined significance. Patients with a predisposition syndrome/disorder were younger than the remaining patients and had a higher risk of severe or multiple cytopenias and advanced myeloid malignancy. In patients with myeloid neoplasm, causative germ line mutations were associated with increased risk of progression into acute myeloid leukemia. Family or personal history of cancer did not show significant association with a predisposition syndrome/disorder. The findings of this study unveil the spectrum, clinical expressivity, and prevalence of germ line predisposition mutations in an unselected cohort of adult patients with cytopenia and hypoplastic BM.

RaggedExperiment: the missing link between genomic ranges and matrices in Bioconductor.

The RaggedExperiment R / Bioconductor package provides lossless representation of disparate genomic ranges across multiple specimens or cells, in conjunction with efficient and flexible calculations of rectangular-shaped summaries for downstream analysis. Applications include statistical analysis of somatic mutations, copy number, methylation, and open chromatin data. RaggedExperiment is compatible with multimodal data analysis as a component of MultiAssayExperiment data objects, and simplifies data representation and transformation for software developers and analysts. Measurement of copy number, mutation, single nucleotide polymorphism, and other genomic attributes that may be stored as VCF files produce "ragged" genomic ranges data: i.e. across different genomic coordinates in each sample. Ragged data are not rectangular or matrix-like, presenting informatics challenges for downstream statistical analyses. We present the RaggedExperiment R/Bioconductor data structure for lossless representation of ragged genomic data, with associated reshaping tools for flexible and efficient calculation of tabular representations to support a wide range of downstream statistical analyses. We demonstrate its applicability to copy number and somatic mutation data across 33 TCGA cancer datasets.

Molecular genomic landscape of pediatric solid tumors in Chinese patients: implications for clinical significance.

Pediatric solid tumors are significantly different from adult tumors. Studies have revealed genomic aberrations in pediatric solid tumors, but these analyses were based on Western populations. Currently, it is not known to what extent the existing genomic findings represent differences in ethnic backgrounds. DESIGN: We retrospectively analyzed the basic clinical characteristics of the patients, including age, cancer type, and sex distribution, and further analyzed the somatic and germline mutations of cancer-related genes in a Chinese pediatric cohort. In addition, we investigated the clinical significance of genomic mutations on therapeutic, prognostic, diagnostic, and preventive actions. Our study enrolled 318 pediatric patients, including 234 patients with CNS tumors and 84 patients with non-CNS tumors. Somatic mutation analysis showed that there were significant differences in mutation types between CNS tumors and non-CNS tumors. P/LP germline variants were identified in 8.49% of patients. In total, 42.8% patients prompted diagnostic, 37.7% patients prompted prognostic, 58.2% patients prompted therapeutic, and 8.5% patients prompted tumor-predisposing and preventive, and we found that genomic findings might improve clinical management. Our study is the first large-scale study to analyze the landscape of genetic mutations in pediatric patients with solid tumors in China. Genomic findings in CNS and non-CNS solid pediatric tumors provide evidence for the clinical classification and individualized treatment of pediatric tumors, and they will facilitate improvement of clinical management. Data presented in this study should serve as a reference to guide the future design of clinical trials.

Longitudinal somatic mutation analysis of individual human CD8 +T cells by a UMI-based single cell mutation detection (USCMD) method

Abstract Somatic mutation is a contributing factor for cancer and aging. DNA-based single-cell mutation detection methods have been developed, but are hindered by the limited number of cells examined as well as by errors from PCR amplification and sequencing methods. Here, we developed a UMI-based scRNAseq mutation detection (USCMD) method that allows for the examination of up to 10,000 cells per sample with reduced PCR and sequencing errors, and which also filters potential errors of RNA polymerase II. We applied USCMD to analyze the scRNAseq data from human CD8 +T cells collected from 8 donors with 2 visits over a period of 9 years (aged from 30 to 69 years at first visit). We found that some novel missense mutations were associated with changes (increase or decrease) in the number of cells carrying them over time, and were able to identify clonally related cells based on shared somatic mutations. Together, this USCMD method improves the accuracy of mutational analysis of scRNAseq data and allows parallel transcriptional assessment of mutated and unmutated cells to understand their potential functional consequences.

Basement membrane-related regulators for prediction of prognoses and responses to diverse therapies in hepatocellular carcinoma

BackgroundHepatocellular carcinoma (HCC) remains a global health threat. Finding a novel biomarker for assessing the prognosis and new therapeutic targets is vital to treating this patient population. Our study aimed to explore the contribution of basement membrane-related regulators (BMR) to prognostic assessment and therapeutic response prediction in HCC.Material and methodsThe RNA sequencing and clinical information of HCC were downloaded from TCGA-LIHC, ICGC-JP, GSE14520, GSE104580, and CCLE datasets. The BMR signature was created by the Least Absolute Shrinkage and Selection Operator (LASSO) algorithm and used to separate HCC patients into low- and high-risk groups. We conducted analyses using various R 4.1.3 software packages to compare prognoses and responses to immunotherapy, transcatheter arterial chemoembolization (TACE), and chemotherapeutic drugs between the groups. Additionally, stemness indices, molecular functions, and somatic mutation analyses were further explored in these subgroups.ResultsThe BMR signature included 3 basement membrane-related genes (CTSA, P3H1, and ADAM9). We revealed that BMR signature was an independent risk contributor to poor prognosis in HCC, and high-risk group patients presented shorter overall survival. We discovered that patients in the high-risk group might be responsive to immunotherapy, while patients in the low-risk group may be susceptible to TACE therapy. Over 300 agents were screened to identify effective drugs for the two subgroups.ConclusionOverall, basement membrane-related regulators represent novel biomarkers in HCC for assessing prognosis, response to immunotherapy, the effectiveness of TACE therapy, and drug susceptibility.

Classification related to immunogenic cell death predicts prognosis, immune microenvironment characteristics, and response to immunotherapy in lower-grade gliomas.

Immunogenic cell death (ICD) is a form of cell death that elicits immune responses against the antigens found in dead or dying tumor cells. Growing evidence implies that ICD plays a significant role in triggering antitumor immunity. The prognosis for glioma remains poor despite many biomarkers being reported, and identifying ICD-related biomarkers is imminent for better-personalized management in patients with lower-grade glioma (LGG). We identified ICD-related differentially expressed genes (DEGs) by comparing gene expression profiles obtained across Genotype-Tissue Expression (GTEx) and The Cancer Genome Atlas (TCGA) cohorts. On the foundation of ICD-related DEGs, two ICD-related clusters were identified through consensus clustering. Then, survival analysis, functional enrichment analysis, somatic mutation analysis, and immune characteristics analysis were performed in the two ICD-related subtypes. Additionally, we developed and validated a risk assessment signature for LGG patients. Finally, we selected one gene (EIF2AK3) from the above risk model for experimental validation. 32 ICD-related DEGs were screened, dividing the LGG samples from the TCGA database into two distinct subtypes. The ICD-high subgroup showed worse overall survival (OS), greater immune infiltration, more active immune response process, and higher expression levels of HLA genes than the ICD-low subgroup. Additionally, nine ICD-related DEGs were identified to build the prognostic signature, which was highly correlated with the tumor-immune microenvironment and could unambiguously be taken as an independent prognostic factor and further verified in an external dataset. The experimental results indicated that EIF2AK3 expression was higher in tumors than paracancerous tissues, and high-expression EIF2AK3 was enriched in WHO III and IV gliomas by qPCR and IHC, and Knockdown of EIF2AK3 suppressed cell viability and mobility in glioma cells. We established novel ICD-related subtypes and risk signature for LGG, which may be beneficial to improving clinical outcome prediction and guiding individualized immunotherapy.

Identification of a m6A-related ferroptosis signature as a potential predictive biomarker for lung adenocarcinoma

BackgroundBoth N6-methyladenosine (m6A) and ferroptosis-related genes are associated with the prognosis of lung adenocarcinoma. However, the predictive value of m6A-related ferroptosis genes remains unclear. Here, we aimed to identify the prognostic value of m6A-related ferroptosis genes in lung adenocarcinoma.MethodsLung adenocarcinoma sample data were downloaded from the University of California Santa Cruz Xena and Gene Expression Omnibus databases. Spearman’s correlation analysis was used to screen for m6A-related ferroptosis genes. Univariate Cox regression, Kaplan–Meier, and Lasso analyses were conducted to identify prognostic m6A-related ferroptosis genes, and stepwise regression was used to construct a prognostic gene signature. The predictive value of the gene signature was assessed using a multivariate Cox analysis. In the validation cohort, survival analysis was performed to verify gene signature stability. The training cohort was divided into high- and low-risk groups according to the median risk score to assess differences between the two groups in terms of gene set variation analysis, somatic mutations, and tumor immune infiltration cells.ResultsSix m6A-related ferroptosis genes were used to construct a gene signature in the training cohort and a multivariate Cox analysis was conducted to determine the independent prognostic value of these genes in lung adenocarcinoma. In the validation cohort, Kaplan–Meier and receiver operating characteristic analyses confirmed the strong predictive power of this signature for the prognosis of lung adenocarcinoma. Gene set variation analysis showed that the low-risk group was mainly related to immunity, and the high-risk group was mainly related to DNA replication. Somatic mutation analysis revealed that the TP53 gene had the highest mutation rate in the high-risk group. Tumor immune infiltration cell analysis showed that the low-risk group had higher levels of resting CD4 memory T cells and lower levels of M0 macrophages.ConclusionOur study identified a novel m6A-related ferroptosis-associated six-gene signature (comprising SLC2A1, HERPUD1, EIF2S1, ACSL3, NCOA4, and CISD1) for predicting lung adenocarcinoma prognosis, yielding a useful prognostic biomarker and potential therapeutic target.

Genetic and Epigenetic Study of Monozygotic Twins Affected by Parkinson’s Disease

Background: Genetic and epigenetic modifiers of age at onset of Parkinson’s disease (PD) are largely unknown. It remains unclear whether DNA methylation (DNAm) age acceleration is linked to age at onset in PD patients of different ethnicities with a similar genetic background. We aim to characterize the clinical, genomic and epigenomic features of three pairs of Chinese monozygotic twins discordant for PD onset by up to 10 years. Methods: We conducted whole genome sequencing, multiplex ligation-dependent probe amplification and genome-wide DNAm array to evaluate the three pairs of Chinese monozygotic twins discordant for age at onset of PD (families A–C). Results: We identified two heterozygous PRKN mutations (exon 2–4 deletion and p.Met1Thr) in PD affected members of one family. Somatic mutation analyses of investigated families did not reveal any variants that could explain the phenotypic discordance in the twin pairs. Of note, our epigenetic study revealed that the twins with earlier-onset had a trend of faster DNAm age acceleration than the later-onset/asymptomatic twins, but without statistical significance. Conclusion: The link between DNAm age acceleration and PD onset in Chinese patients should be interpreted with cautious, and need to be further verified in an extended PD cohort with similar genetic background.

Spectra and characteristics of somatic mutations induced by ionizing radiation in hematopoietic stem cells

Spectra and frequencies of spontaneous and X-ray-induced somatic mutations were revealed with mouse long-term hematopoietic stem cells (LT-HSCs) by whole-genome sequencing of clonal cell populations propagated invitro from single isolated LT-HSCs. SNVs and small indels were the most common types of somatic mutations, and increased up to twofold to threefold by whole-body X-irradiation. Base substitution patterns in the SNVs suggested a role of reactive oxygen species in radiation mutagenesis, and signature analysis of single base substitutions (SBS) revealed a dose-dependent increase of SBS40. Most of spontaneous small deletions were shrinkage of tandem repeats, and X-irradiation specifically induced small deletions out of tandem repeats (non-repeat deletions). Presence of microhomology sequences in non-repeat deletions suggested involvement of microhomology mediated end-joining repair mechanisms as well as nonhomologous end-joining in radiation-induced DNA damages. We also identified multisite mutations and structural variants (SV), i.e., large indels, inversions, reciprocal translocations, and complex variants. The radiation-specificity of each mutation type was evaluated from the spontaneous mutation rate and the per-Gy mutation rate estimated by linear regression, and was highest with non-repeat deletions without microhomology, followed by those with microhomology, SV except retroelement insertions, and multisite mutations; these types were thus revealed as mutational signatures of ionizing radiation. Further analysis of somatic mutations in multiple LT-HSCs indicated that large fractions of postirradiation LT-HSCs originated from single LT-HSCs that survived the irradiation and then expanded invivo to confer marked clonality to the entire hematopoietic system, with varying clonal expansion and dynamics depending on radiation dose and fractionation.

Abstract 2033: Assessment of human reference genomes on cancer somatic mutation detection in tumor-normal paired reference samples using whole genome short-read sequencing data

Abstract A typical cancer somatic mutation identification workflow involves using a standard human reference genome for aligning the sequencing reads and annotating the uncovered somatic variants. The completeness and the correctness of the reference genome, and the haplotype representations may all impact the sequencing read alignments and subsequent somatic variant identifications. However, the exact impacts of the underlying references being used on somatic mutation analysis has not been investigated. In this study, we selected 3 human reference genomes, including GRCh38, T2TCHM13, and a personalized genome (PG) that was derived from a normal cell line, and 12 tumor normal paired replicates of Illumina short read sequencing data for the HCC1395 breast cancer cell line and a matched normal cell line, and aim to illustrate and quantify the effects of the reference genomes on the accuracy of somatic SNV/SV mutation detection using various somatic variant callers. Our analysis shows that the use of a personalized haplotype-specific genome assembly, rather than an unrelated genome such as GRCh38 or T2TCHM13 as a reference, not only improves read alignments for short-read sequencing data but also ameliorates the detection accuracy of somatic SNVs and SVs. While most somatic mutation calls are identical or equivalent, we identify GRCh38 and T2TCHM13 specific somatic mutations that may need to be avoided for further pursuit of incorrect treatment options. We uncover novel somatic mutations only when personalized genome assembly is used as a reference, some of which overlap with genes involving with pathways related to cancer invasion and metastasis (e.g., CDH23, ST14 etc.). The predicted somatic SVs are found more precise. Such results may provide additional interventional target choices for patients, researchers or clinicians, and physicians to look into further for personalized patient care. Citation Format: Chunlin Xiao, Valerie Schneider. Assessment of human reference genomes on cancer somatic mutation detection in tumor-normal paired reference samples using whole genome short-read sequencing data [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2033.

Abstract 3330: Development of a genome-wide multiomic atlas of early-stage lung cancer enables identification of novel methylation biomarkers for disease detection beyond TCGA

Abstract Lung cancer is the leading cause of cancer mortality, mainly due to diagnosis at advanced stages. Early detection reduces mortality and screening by CT is recommended for a high-risk population, however, uptake is low. Biomarkers for early-stage detection may enhance screening. Liquid biopsy and blood circulating tumor DNA (ctDNA) are established plasma surrogates of tumor tissue but their use in early detection remains challenging due to the high noise level of current techniques. We constructed a novel multiomic atlas through whole genome sequencing (WGS) to map all genetic and epigenetic changes associated with early-stage lung cancer as a tool to identify markers. Biospecimens from 48 early-stage lung cancer cases (58% stage I) and 29 cancer-free controls, all high-risk by USPSTF, were acquired from academic (UBC, Vanderbilt, Cleveland Clinic) and commercial biobanks. Cases had tumor and normal lung tissue, whole blood (WB) and plasma samples. Controls had WB and plasma samples. EpiCheck sequencing (ECS) which combines methylation-sensitive restriction endonuclease digestion to detect differential methylation with WGS for multiomic analyses was performed on each sample. Extracted DNA was digested, underwent standard library preparation and sequenced at an average depth of 600x for cfDNA and 80x for tissue and WB. A subset also underwent WG bisulfite sequencing (BS) and standard WGS for comparison. Data analysis was performed using customized software. ECS outperformed BS. Mapping rate was 99.6%, 99.7% and 85.7% and unique mapping rate was 94.1%, 94.3% and 81.4% for WGS, ECS, and BS samples, respectively. Copy number integrity showed Pearson correlations of 0.9 for ECS and 0.67 for BS. Somatic mutation analysis in tissue identified a subset of cases with relatively high ctDNA shedding in plasma that were associated with larger tumors, older age and squamous cell carcinoma histology. This subset was further used to identify tumor derived plasma-based markers and assess fragmentation with high confidence. Shorter ctDNA fragments were observed in some, but not all cases. Additional analysis of WB and plasma identified host derived blood-based markers using methylation and copy number. This orthogonal approach enabled detection of small, low shedding tumors. A discovery panel of 87 markers showed 100% sensitivity and 97% specificity to discriminate cases vs controls. Only 11% of these markers are represented in the TCGA data. ECS generates high integrity sequencing data superior to BS, enabling genome-wide multiomic analyses (methylation, mutation, copy number, fragmentomics), with methylation signatures beyond the scope of TCGA. Results, which require validation, underscore the potential of the EpiCheck lung cancer atlas as a development platform of novel blood biomarkers for early-stage lung cancer detection. Citation Format: Revital Knirsh, Stephen Lam, Anna McGuire, Peter J. Mazzone, Stephen Deppen, Eric Grogan, Fabien Maldonado, Orna Savin, Shacade Danan, Sarah Zaouch, Nimrod Axelrad, Dvir Netanely, Aharona Shuali, Catherine A. Schnabel, Adam Wasserstrom, Danny Frumkin. Development of a genome-wide multiomic atlas of early-stage lung cancer enables identification of novel methylation biomarkers for disease detection beyond TCGA [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3330.

Abstract 131: Clonal expansion in bile duct associated with chronic inflammation

Abstract Introduction: Chronic inflammation has been implicated in cancer development in many tissues and has recently been associated with promoted clonal expansion in non-cancer tissues. Primary sclerosing cholangitis (PSC) is a chronic progressive disease of unknown etiology affecting the bile ducts and has been associated with a substantially increased risk of cholangiocarcinoma. Clonal dynamics in the normal bile duct and PSC epithelium remains poorly understood because of the limited accessibility to normal/inflamed bile ducts. In this study, we established a method to detect somatic mutations accumulated in the bile duct epithelium and investigated a mutation rate in normal bile duct cells and clonal expansion in PSC epithelium. Methods: We established single cell-derived organoids from bile, which were subjected to whole genome sequencing to measure the age-related mutation rate in normal epithelial cell in the bile ducts. We next collected multiple samples from perihilar bile ducts in patients with PSC who underwent total hepatectomy for liver transplantation, for which we performed whole exome sequencing to investigate clonal expansion in PSC-involved bile ducts. Results: In total, 14 single cell-derived organoid lines were established from 2 patients with PSC and 3 control individuals. Based on the whole genome sequencing of those organoids, the number of mutations found in non-PSC bile duct epithelium increases with age at an annual mutation rate of 38.2 mutations per genome per year, which did not substantially differ from the rate in the PSC epithelium, suggesting that the inflammatory process in PSC hardly affected the mutations in PSC epithelium. To further investigate clonal expansion in the PSC epithelium, a total of 126 bulk organoid samples from perihilar bile ducts in 3 PSC patients were analyzed. Somatic mutations were detected in all samples (21 [4-101]/exome/sample on average), some of which showed high variant allele frequencies. In 2 PSC cases, a small number of somatic mutations (n=12[case 1], 11 and 8[case 2]) were shared between samples that were 13 mm distant apart from each other, suggesting a clonal expansion in PSC epithelium. Expanded clones harbored mutations in known cancer-driver genes, such as ARID2 and PIK3CA, suggesting their role in the expansion in this case. Conclusions: Here, we newly established the method to detect somatic mutations accumulated in the normal/inflamed bile duct epithelium using an organoid-based analysis of clonal somatic mutations. Our results revealed clonal expansion existing in PSC epithelium and open up further potential for approaching carcinogenesis in the bile duct. Citation Format: Hirona Maeda, Nobuyuki Kakiuchi, Takashi Ito, Eri Ogawa, Masahiro Shiokawa, Norimitsu Uza, Hiroko Tanaka, Yasuhito Nannya, Hideki Makishima, Hiroaki Yasuda, Yuzo Kodama, Satoru Miyano, Seishi Ogawa. Clonal expansion in bile duct associated with chronic inflammation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 131.

Abstract 775: Integrative genomic and transcriptomic profiling of myeloma precursors in a prospective longitudinal observational study (ORIGIN)

Abstract Background: Monoclonal gammopathy of undetermined significance (MGUS) and smoldering myeloma (SMM) are myeloma precursors (MPD). A better understanding of the oncogenic/immune programs underlying progression may reveal exploitable targets for patients (pts) at high risk of progression and aid new intervention strategies. We designed a longitudinal Observational prospective Research study In monoclonal Gammopathies leadINg to myeloma (ORIGIN) to collect well-annotated, longitudinal samples (sps) for integrative genomic/transcriptomic analysis. Methods: Pts were accrued meeting MGUS/SMM IMWG definition. BM sps were partitioned into CD138-positive and -negative compartments and DNAs/RNAs extracted for bulk RNA and whole-exome sequencing (WES). Matched germline controls were included for somatic mutation and DNA copy number analysis. A number of tools and algorithms were applied, and the data were thoroughly analyzed and integrated. Results: From 12/2015-03/2022, 249 pts were accrued and 205 eligible (99 MGUS/106 SMM). Median follow up time is 33 months (6-49 months). As of 11/2022, 21 pts progressed (prog) on ORIGIN study (14% 15/106 SMM, 3% 3/99 prog to SMM from MGUS and 1% 3/205 to AL amyloidosis). To increase analytic power, genomic data of 166 SMM baseline sps from SMM pts on treatment studies were added. Altogether, genomic data of 496 sps were analyzed. Briefly, WES was performed on 170 sps from 150 pts and RNA-Seq was performed on enriched tumor/TME cells in 326 sps from 187 pts. Relative to MGUS, we observed higher mutation load in SMM and more frequent somatic mutations in known drivers of myeloma: KRAS (14% vs. 2%), NRAS (7% vs. 2%) leading to a shorter time to myeloma as well as genes recurrently mutated in other cancers: FOXO3 (7% vs 0%). We observed increased aneuploidy levels in SMM vs MGUS and in prog vs. non-prog including both gains and losses of multiple chromosomes. Transcriptome analysis of CD138+ cells revealed clearly distinct expression profiles between SMM and MGUS, and between prog and non-prog. The pathway activity of oxidative phosphorylation and MYC was significantly increased in SMM vs. MGUS and in prog vs. non-prog, suggesting their potential roles in driving progression. We noted a greater degree of heterogeneity in TME cell compositions in SMM vs MGUS sps and in prog vs. non-prog: ie, SMM and prog sps formed multiple distinct clusters exhibiting differential abundance of cytotoxic T cells, neutrophils, fibroblasts and myeloid cells. Conclusions: Together, our integrative analysis of genomic and transcriptome data in ORIGIN demonstrates extensive changes in both CD138+ cells and the immune microenvironment and co-evolution of the ecosystems during disease progression. This study is an invaluable resource and lays the molecular foundation for the community for future biomarker and target discovery. Citation Format: Minghao Dang, Chutima Kunacheewa, Hans C. Lee, Krina K. Patel, Sheeba K. Thomas, Melody Becnel, Donna M. Weber, Pei Lin, Zuzana Berkova, Mei Huang, David A. Berrios, Hima Bansal, Andrew Futreal, Cristhiam Rojas Hernandez, Vahid Afshar-Khargan, Michael Kroll, Peter Kuhn, Robert Z. Orlowski, Linghua Wang, Elisabet E. Manasanch. Integrative genomic and transcriptomic profiling of myeloma precursors in a prospective longitudinal observational study (ORIGIN) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 775.