Somatic Single Nucleotide Variants Research Articles

Mugen-UMAP: UMAP visualization and clustering of mutated genes in single-cell DNA sequencing data

BackgroundThe application of Uniform Manifold Approximation and Projection (UMAP) for dimensionality reduction and visualization has revolutionized the analysis of single-cell RNA expression and population genetics. However, its potential in single-cell DNA sequencing data analysis, particularly for visualizing gene mutation information, has not been fully explored.ResultsWe introduce Mugen-UMAP, a novel Python-based program that extends UMAP’s utility to single-cell DNA sequencing data. This innovative tool provides a comprehensive pipeline for processing gene annotation files of single-cell somatic single-nucleotide variants and metadata to the visualization of UMAP projections for identifying clusters, along with various statistical analyses. Employing Mugen-UMAP, we analyzed whole-exome sequencing data from 365 single-cell samples across 12 non-small cell lung cancer (NSCLC) patients, revealing distinct clusters associated with histological subtypes of NSCLC. Moreover, to demonstrate the general utility of Mugen-UMAP, we applied the program to 9 additional single-cell WES datasets from various cancer types, uncovering interesting patterns of cell clusters that warrant further investigation. In summary, Mugen-UMAP provides a quick and effective visualization method to uncover cell cluster patterns based on the gene mutation information from single-cell DNA sequencing data.ConclusionsThe application of Mugen-UMAP demonstrates its capacity to provide valuable insights into the visualization and interpretation of single-cell DNA sequencing data. Mugen-UMAP can be found at https://github.com/tengchn/Mugen-UMAP

Temporal evolution and inter-patient heterogeneity in primary and recurrent head and neck squamous cell carcinoma

BackgroundHead and neck squamous cell carcinomas (HNSCCs) are heterogeneous in terms of origin and aetiology. In addition, there is uncertainty about the genetic evolution from initial diagnosis to recurrence after primary treatments and further disease progression following systemic treatment. Changes in the genetic profile have implications on the selection of appropriate treatments for patients, especially in the era of targeted therapies and immunotherapies.MethodsWe analysed a cohort of nine HNSCC patients with metachronous recurrence. All patients had paired primary and recurrent samples suitable for whole-exome sequencing, while transcriptomic data from seven patients could be analysed (multiple recurrent samples collected at different time points were available for three patients).ResultsAt the genomic level, the recurrences shared a fraction of the somatic single nucleotide variants (SNVs) with the index primary tumours, but they also acquired many additional mutations, while losing only a few others. A similar behaviour was also observed when examining the changes of mutational signatures between primary and recurrent samples. Overall, recurrences appeared thus more genetically diverse than the respective primary tumours. The transcriptomic analysis showed that recurrent samples had lower immune cell presence, which was also confirmed by the multiplex immunofluorescence (IF) histology assays performed on the PhenoCycler platform. Several genes related to immune response were significantly downregulated compared to the primary samples.ConclusionsOur results underline the importance of analysing multiple samples per patient to obtain a more complete picture of the patient’s tumour and advocate a re-biopsy in the event of recurrence and treatment failure, in order to select the most appropriate therapeutic strategy.

Identifying cancer cells from calling single-nucleotide variants in scRNA-seq data.

Single-cell RNA sequencing (scRNA-seq) data are widely used to study cancer cell states and their heterogeneity. However, the tumour microenvironment is usually a mixture of healthy and cancerous cells and it can be difficult to fully separate these two populations based on transcriptomics alone. If available, somatic single-nucleotide variants (SNVs) observed in the scRNA-seq data could be used to identify the cancer population and match that information with the single cells' expression profile. However, calling somatic SNVs in scRNA-seq data is a challenging task, as most variants seen in the short-read data are not somatic, but can instead be germline variants, RNA edits or transcription, sequencing, or processing errors. In addition, only variants present in actively transcribed regions for each individual cell will be seen in the data. To address these challenges, we develop CCLONE (Cancer Cell Labelling On Noisy Expression), an interpretable tool adapted to handle the uncertainty and sparsity of SNVs called from scRNA-seq data. CCLONE jointly identifies cancer clonal populations, and their associated variants. We apply CCLONE on two acute myeloid leukaemia datasets and one lung adenocarcinoma dataset and show that CCLONE captures both genetic clones and somatic events for multiple patients. These results show how CCLONE can be used to gather insight into the course of the disease and the origin of cancer cells in scRNA-seq data. Source code is available at github.com/HaghverdiLab/CCLONE.

Abstract Mo075: Cigarette smoking causes somatic mutations in cardiac endothelial cells

Cigarette smoking (CS) is a major contributor to cardiovascular diseases (CVDs), responsible for one in every four CVD-related deaths. Despite this, the precise mechanisms through which smoking induces CVDs remain elusive. The chemicals in cigarette smoke, particularly oxidants, are known to instigate oxidative stress and activate the vessel wall. Alongside the dysregulation of cellular functions, oxidative stress can lead to DNA damage. Accumulation of DNA damage and somatic mutations is a recognized hallmark of aging, a significant risk factor for various age-related diseases, including CVDs, neurodegenerative diseases, and cancer. Recent studies have indicated a substantial increase in somatic mutations, with mutational signatures reflective of oxidative stress, in bronchial epithelial cells from smokers. Given their proximity in the blood circulation, these pathophysiological findings could naturally extend to the coronary system. Therefore, we hypothesized that CS induces somatic mutations and injuries in cardiac endothelial cells (ECs), laying the mechanisms for cigarette smoking-induced CVDs. To test this hypothesis, we conducted single-cell whole-genome sequencing (scWGS) on 105 cardiac endothelial cells isolated from postmortem hearts of 6 normal individuals, 3 smokers, and 4 smokers with atherosclerotic cardiovascular disease (ASCVD). Our results revealed a significant increase in somatic single nucleotide variants (SNVs) and insertions and deletions (indels) in cardiac ECs from smokers and those with ASCVD. These excess somatic mutations exhibited Catalogue Of Somatic Mutations In Cancer (COSMIC) mutational signatures, including SBS4, SBS29, SBS40, SBS92, and ID3, indicative of DNA damage and mutagenesis associated with cigarette smoking, oxidative stress, and formaldehyde exposure. Further analysis of single-nucleus RNA sequencing (snRNA-seq) data from postmortem hearts of 3 normal individuals, 4 smokers, and 1 smoker with ASCVD demonstrated upregulated pathways involved in apoptosis, EC migration, and vascular development. In conclusion, our study demonstrates that chemicals in cigarette smoke exert direct genotoxic effects on cardiac ECs, mirroring their impact on bronchial epithelial cells. Cigarette smoking significantly accelerates the accumulation of somatic mutations in cardiac ECs, resulting in apoptosis and vascular damage. Consequently, this damage leads to a secondary activation of angiogenesis for vascular repair.

Low-level brain somatic mutations in exonic regions are collectively implicated in autism with germline mutations in autism risk genes

Low-level somatic mutations in the human brain are implicated in various neurological disorders. The contribution of low-level brain somatic mutations to autism spectrum disorder (ASD), however, remains poorly understood. Here, we performed high-depth exome sequencing with an average read depth of 559.3x in 181 cortical, cerebellar, and peripheral tissue samples to identify brain somatic single nucleotide variants (SNVs) in 24 ASD subjects and 31 controls. We detected ~2.4 brain somatic SNVs per exome per single brain region, with a variant allele frequency (VAF) as low as 0.3%. The mutational profiles, including the number, signature, and type, were not significantly different between the ASD patients and controls. Intriguingly, when considering genes with low-level brain somatic SNVs and ASD risk genes with damaging germline SNVs together, the merged set of genes carrying either somatic or germline SNVs in ASD patients was significantly involved in ASD-associated pathophysiology, including dendrite spine morphogenesis (p = 0.025), mental retardation (p = 0.012), and intrauterine growth retardation (p = 0.012). Additionally, the merged gene set showed ASD-associated spatiotemporal expression in the early and mid-fetal cortex, striatum, and thalamus (all p < 0.05). Patients with damaging mutations in the merged gene set had a greater ASD risk than did controls (odds ratio = 3.92, p = 0.025, 95% confidence interval = 1.12–14.79). The findings of this study suggest that brain somatic SNVs and germline SNVs may collectively contribute to ASD-associated pathophysiology.

Comprehensive molecular characterization of TFE3-rearranged renal cell carcinoma

TFE3-rearranged renal cell cancer (tRCC) is a rare form of RCC that involves chromosomal translocation of the Xp11.2 TFE3 gene. Despite its early onset and poor prognosis, the molecular mechanisms of the pathogenesis of tRCC remain elusive. This study aimed to identify novel therapeutic targets for patients with primary and recurrent tRCC. We collected 19 TFE3-positive RCC tissues that were diagnosed by immunohistochemistry and subjected them to genetic characterization to examine their genomic and transcriptomic features. Tumor-specific signatures were extracted using whole exome sequencing (WES) and RNA sequencing (RNA-seq) data, and the functional consequences were analyzed in a cell line with TFE3 translocation. Both a low burden of somatic single nucleotide variants (SNVs) and a positive correlation between the number of somatic variants and age of onset were observed. Transcriptome analysis revealed that four samples (21.1%) lacked the expected fusion event and clustered with the genomic profiles of clear cell RCC (ccRCC) tissues. The fusion event also demonstrated an enrichment of upregulated genes associated with mitochondrial respiration compared with ccRCC expression profiles. Comparison of the RNA expression profile with the TFE3 ChIP-seq pattern data indicated that PPARGC1A is a metabolic regulator of the oncogenic process. Cell proliferation was reduced when PPARGC1A and its related metabolic pathways were repressed by its inhibitor SR-18292. In conclusion, we demonstrate that PPARGC1A-mediated mitochondrial respiration can be considered a potential therapeutic target in tRCC. This study identifies an uncharacterized genetic profile of an RCC subtype with unique clinical features and provides therapeutic options specific to tRCC.

Comprehensive genomic analysis reveals clonal origin and subtype-specific evolution in a case of sporadic multiple meningiomas.

Meningioma is the most common primary intracranial tumor in adults, with up to 10% manifesting as multiple tumors. Data on the genomic and molecular changes in sporadic multiple meningiomas are scarce, leading to ongoing debates regarding their evolutionary processes. A comprehensive genetic analysis of a large number of lesions, including precursor lesions, is necessary to explore these two possible origins: clonal and independent. In the present study, we performed whole-exome sequencing and analyzed somatic single-nucleotide variants (SNVs), insertions/deletions (INDELs), and copy number alterations (CNAs) in a patient with sporadic multiple meningiomas. These meningiomas included two mass-forming lesions of different histological subtypes (transitional and chordoid) and two small meningothelial nests. Genetic analysis revealed CNAs on chromosomes 22q and Y as common abnormalities in the two largest tumors. Furthermore, we identified SNV/INDELs unique to each focus, with NF2 mutation prevalent in the transitional meningioma and CREBBP mutation in the chordoid meningioma. Loss of chromosome 22 was detected in two small meningothelial nests. Overall, we elucidated the clonal origin and subtype-specific evolution of multiple meningiomas in this case. CNAs may serve as the initial driving event in meningioma development.

Paired comparison of the analytical performance between the Oncomine™ Comprehensive Assay v3 and whole-exome sequencing of ovarian cancer tissue

BackgroundNext-generation sequencing (NGS) has been implemented in clinical oncology as a personalized medicine tool to identify targetable genetic alterations and to guide treatment decisions. However, the optimal NGS test strategy and target genes for clinical use are still being discussed. The aim was to compare the performance of the Oncomine™ Comprehensive Assay v3 (OCAv3) (targeted gene panel) and whole-exome sequencing (WES) to investigate somatic single and multiple nucleotide variants and small indels in ovarian cancer patients.Methods and resultsGenomic DNA was isolated from fresh frozen samples of five high-grade serous (HGSC) and three clear cell ovarian (oCCC) cancer patients. Exome sequencing libraries were prepared by using the Ion AmpliSeq Exome RDY kit, whereas libraries for OCAv3 were prepared using by Ion AmpliSeq™ Library Kit Plus. Sequencing was performed using the Ion S5XL System (Thermo Fisher Scientific). When including only variants classified as pathogenic, likely pathogenic or unknown significance based on ClinVar database verdicts and comparing overlapping regions covered both by the OCAv3 assay and WES, 23 variants were detected by both assays. However, OCAv3 detected additionally two variants: ARID1A: p.Gln563Ter and TP53: p.Ser261ValfsTer84 that have not passed WES filtering criteria due to low coverage.ConclusionsWith the present treatment possibilities, OCAv3 panel testing provided higher diagnostic yield due to better coverage. Our study emphasizes that WES, although offering the potential to identify novel findings in genes not covered by OCAv3, might overlook variants in genes relevant for OC.

Integrated analysis of -omic landscapes in breast cancer subtypes

The subtypes of breast cancer exhibit diverse histology, molecular features, therapeutic response, aggressiveness, and patient outcomes. Multi-omics high-throughput technologies, which are widely used in cancer research, generated waste amounts of multimodal omic datasets calling for new approaches of integrated analyses to uncover patterns of transcriptomic, genomic, and epigenetic changes in breast cancer subtypes and connect them to disease clinical characteristics. Here, we applied multi-layer self-organizing map (ml-SOM) algorithms to PAM50-classified TCGA breast cancer samples to disentangle the diversity of the effects of gene expression, methylation, copy number, and somatic single nucleotide variation in the disease subtypes. Furthermore, we studied the association of perturbed gene modules with survival, prognosis, and other clinical characteristics. Our findings highlight the power of multi-omic analyses to offer a better understanding of the molecular diversity of breast cancer subtypes compared to single-omic analyses. Moreover, they highlight the complex subtype-characteristic associations between gene expression and epigenetic/genomic factors and their implications for survival and clinical outcomes.

Genetic landscape of colorectal cancer (CRC) across genetic ancestries: Implications for early cancer detection (ECD).

e15527 Background: Blood-based ECD has the potential to reduce mortality rates by improving strategies for adoption of and adherence to screening. As health disparities in the incidence of CRC across minority populations are known to exist, we evaluated the genomic landscape in patients with CRC across genetic ancestries. Methods: De-identified patients (N = 16,337) with stage I-IV CRC had personalized, tumor-informed commercial ctDNA testing (SignateraTM) based on whole exome sequencing. Genetic ancestry determined via supervised local ancestry inference (pmid36042219) included African (AFR, n = 1697), Ashkenazi Jewish (ASJ, n = 1192), East Asian (EAS, n = 2247), European (EUR, n = 9726), Latino (LAT, n = 1291), and South Asian (SAS, n = 184) ancestries. Somatic driver mutations and pathogenic germline variants (PVs) in CRC-associated genes were evaluated. Somatic single nucleotide variants were used for mutational signature inference. Results: Across ancestries, 47-67% of the patients were male and most were aged &gt; 50 years. Among 35 to 50-year-olds, microsatellite instability (MSI) rates were higher in LAT (12.9%, p = 0.04) and SAS (28.3%, p &lt; 0.001) vs EUR (8.2%). Among &gt; 50-year-olds, MSI rates were lower in AFR (10.7%, p &lt; 0.001), EAS (7.9%, p &lt; 0.001), and LAT (10.4%, p &lt; 0.001) vs EUR (16.0%). The POLE signature was more common in AFR (2%, p = 0.001) vs EUR (1.1%). There were significant differences in the frequency of driver mutations and PV rates between non-EUR vs EUR ancestry groups (Table, *p &lt; 0.05, **p &lt; 0.001). Gene-level enrichment was observed in MLH1 for SAS (8.7%, p &lt; 0.001) and LAT (1.8%, p = 0.03), CHEK2 for AFR (0.4%, p = 0.03) and EAS (0.1%, p &lt; 0.001), and PMS2 (1.4%, p = 0.006) for AFR. Among patients aged ≤50 years, similar findings were observed, with differences driven by MSI rates and germline findings in Lynch syndrome (LS) genes. Conclusions: This is the first study to investigate genomic differences between multiple ancestry groups in the largest cohort to date, including EAS, LAT, and SAS populations. The differences observed between ancestries could provide insight into differences in both hereditary risk and subsequent tumor initiation and growth. Findings from this study may provide information for developing risk stratification and prevention strategies for the early detection of cancer and provide the rationale for precision treatment based on ancestry. [Table: see text]

Application of ctDNA sequencing in patients with metastasized lung cancer.

e20033 Background: Circulating tumor DNA (ctDNA) is a form of cell-free DNA (cfDNA) found in blood that originates from tumor cells, which has emerged as a promising non-invasive biomarker in monitoring cancer prognosis and precision medicine with the advancement of deep sequencing. Here we compared the mutational landscape between solid tumor and cfDNA in metastasis-free and metastasized lung cancer patients using targeted NGS to investigate the feasibility of ctDNA sequencing in monitoring cancer prognosis. Methods: Blood-derived cfDNA samples and formalin-fixed paraffin embedded tissue were collected from 217 Chinese patients with metastatic lung cancer (n = 39) or metastasis-free lung cancer (n = 178). Blood samples were collected after surgery. Panel sequencing targeting 680 cancer-related genes was performed on both samples. Somatic single nucleotide variations and indels were analyzed. Results: Among the 178 metastasis-free patients, 89.3% of the patients have at least one mutation both detected in tumor and blood samples. On average, 50.4% of tumor mutations were detected in blood for each patients. EGFR, TP53, CSMD3, LRP1B and SYNE1 were the top five most frequently mutated genes, with the proportion of tumor mutation found in blood of 89.1%, 80.2%, 70.0%, 61.3% and 64.3% respectively. In the metastasized cohort, all 39 individuals have at least one tumor mutations found in blood, achieving a concordance of 100%. On average, 59.9% of tumor mutations were detected in blood for each patients. Notably, the proportion of tumor mutations detected in blood for each patients is significantly higher than the metastasis-free cohort (unpaired T-test, p = 0.0167). EGFR, TP53, RBM10, PIK3CA and CSMD3 were the top five most frequently mutated genes, with the proportion of tumor mutation found in blood of 93.5%, 80.8%, 57.1%, 83.3% and 42.9% respectively. Moreover, the proportion of patients having EGFR p.L858R mutation in metastasized cohort was higher than the metastasis-free cohort (54.3% vs 32.8%). While within the metastasized cohort, blood samples detected a higher proportion of EGFR p.T790M (12.9% vs 7.69%) but a lower proportion of EGFR exon19 deletion (p.E746_A750del, 19.4% vs 27.7%) comparing to tumor sample, which can indicate drug resistance. Conclusions: We demonstrated a high mutation detection concordance between tumor and blood using targeted NGS, with an exceptional performance especially in driver oncogenes like EGFR and TP53. Our findings showed that metastasized lung cancer patients exhibit a higher positive concordance rate compared to metastasis-free cohort using ctDNA. We also showed a varied mutational landscape between ctDNA and tumor sequencing in metastasized lung cancer patients. Overall, this study not only reaffirms the viability of using blood as an alternative of tissue biopsy, but also provides new insights on application of ctDNA monitoring lung cancer metastasis and its clinically targetable mutations.

Prognostic and predictive value of ctDNA-based MRD and actionable biomarkers in patients with resectable colorectal cancer: CIRCULATE-Japan GALAXY.

3609 Background: The role of biomarker-guided therapy for resectable colorectal cancer (CRC) remains unestablished. Circulating tumor DNA (ctDNA) has emerged as a minimally invasive biomarker for detecting molecular residual disease (MRD) and predicting recurrence. Tumor-informed ctDNA testing offers the advantage of tumor sequencing and MRD detection in a single workflow, suggesting its potential for facilitating integration of biomarker-guided therapy into perioperative treatment. Methods: GALAXY, a large-scale prospective observational study, monitored post-surgical ctDNA MRD status in patients with clinical stage II to IV or relapsed CRC (UMIN000039205). Tumor tissue and matched normal DNA were processed for whole-exome sequencing (WES) to identify and track patient-specific somatic single nucleotide variants (SNVs), for ctDNA detection in the associated patients’ plasma samples. Actionable biomarkers having OncoKB therapeutic levels 1-3 were explored and identified separately from the Signatera test (through WES data). Results: Of the 4,590 patients enrolled between May 2020, and October 2022, 2,695 who were not enrolled in randomized trials and had available WES data were included in this analysis. Identified actionable biomarkers included RAS/ BRAF wild-type in 1,369 (50.8%) patients, tumor mutational burden (TMB) high in 256 (10.3%), MSI high in 236 (9.5%), BRAF V600E in 176 (6.5%), KRAS G12C in 61 (2.3%), ERBB2 amplification in 47 (1.7%), TP53 Y220C in 33 (1.2%), NTRK1/ 2/ 3 fusion in 1 (0.04%), and RET fusion in 1 (0.04%). ctDNA positivity during the MRD window (2 to 10 weeks post-surgery) varied from 5.1% in those with TMB high and MSI high to 29.8% in those with ERBB2 amplification. ERBB2 amplification was the only actionable biomarker significantly associated with ctDNA positivity during the MRD window (odds ratio 2.20). Positive ctDNA MRD status correlated with significantly worse DFS across all actionable biomarkers, with hazard ratios ranging from 8.46 for TP53 Y220C to 5.53e+8 for BRAF V600E. In MSI high disease, recurrence was observed in only 3 of 225 (1.3%) patients with negative ctDNA compared to 5 of 12 (41.7%) with positive ctDNA during the MRD window. Adjustment by the propensity score revealed that adjuvant chemotherapy provided survival benefits in ctDNA-positive patients with pathological stage II and III disease across various biomarkers. In patients with one or more actionable mutations selected among the16-plex of the ctDNA MRD assay, those variants were detected at time of disease recurrence. Conclusions: Our study highlights the importance of ctDNA-based MRD as both a prognostic and predictive tool for CRC across different actionable biomarkers. These findings suggest the potential of MRD-driven targeted therapeutic strategies for CRC with specific actionable biomarkers. Clinical trial information: UMIN000039205.

Single-nucleus multi-omic profiling of polyploid heart nuclei identifies fusion-derived cardiomyocytes in the human heart.

Understanding the mechanisms of polyploidization in cardiomyocytes is crucial for advancing strategies to stimulate myocardial regeneration. Although endoreplication has long been considered the primary source of polyploid human cardiomyocytes, recent animal work suggests the potential for cardiomyocyte fusion. Moreover, the effects of polyploidization on the genomic-transcriptomic repertoire of human cardiomyocytes have not been studied previously. We applied single-nuclei whole genome sequencing, single nuclei RNA sequencing, and multiome ATAC + gene expression (from the same nuclei) techniques to nuclei isolated from 11 healthy hearts. Utilizing post-zygotic non-inherited somatic mutations occurring during development as "endogenous barcodes," to reconstruct lineage relationships of polyploid cardiomyocytes. Of 482 cardiomyocytes from multiple healthy donor hearts 75.7% can be sorted into several developmental clades marked by one or more somatic single-nucleotide variants (SNVs). At least ~10% of tetraploid cardiomyocytes contain cells from distinct clades, indicating fusion of lineally distinct cells, whereas 60% of higher-ploidy cardiomyocytes contain fused cells from distinct clades. Combined snRNA-seq and snATAC-seq revealed transcriptome and chromatin landscapes of polyploid cardiomyocytes distinct from diploid cardiomyocytes, and show some higher-ploidy cardiomyocytes with transcriptional signatures suggesting fusion between cardiomyocytes and endothelial and fibroblast cells. These observations provide the first evidence for cell and nuclear fusion of human cardiomyocytes, raising the possibility that cell fusion may contribute to developing or maintaining polyploid cardiomyocytes in the human heart.

Molecular Characterization and Therapeutic Opportunities in KRAS Wildtype Pancreatic Ductal Adenocarcinoma.

To investigate the molecular characteristics of and potential for precision medicine in KRAS wildtype pancreatic ductal adenocarcinoma (PDAC). We investigated 27 patients with KRASWT PDAC at our institution. Clinical data were obtained via chart review. Tumor specimens for each subject were interrogated for somatic single nucleotide variants, insertion and deletions, and copy number variants by DNA sequencing. Gene fusions were detected from RNA-seq. A patient-derived organoid (PDO) was developed from a patient with a MET translocation and expanded ex vivo to predict therapeutic sensitivity prior to enrollment in a phase 2 clinical trial. Transcriptomic analysis showed our cohort may be stratified by the relative gene expression of the KRAS signaling cascade. The PDO derived from our patient harboring a TFG-MET rearrangement was found to have in vitro sensitivity to the multi-tyrosine kinase inhibitor crizotinib. The patient was enrolled in the phase 2 SPARTA clinical trial and received monotherapy with vebrelitinib, a c-MET inhibitor, and achieved a partial and durable response. KRASWT PDAC is molecularly distinct from KRASMUT and enriched with potentially actionable genetic variants. In our study, transcriptomic profiling revealed that the KRAS signaling cascade may play a key role in KRASWT PDAC. Our report of a KRASWT PDAC patient with TFG-MET rearrangement who responded to a cMET inhibitor further supports the pursuit of precision oncology in this sub-population. Identification of targetable mutations, perhaps through approaches like RNA-seq, can help enable precision-driven approaches to select optimal treatment based on tumor characteristics.

Abstract PO3-06-05: Association of tumor-derived extracellular vesicles with circulating tumor DNA alterations in metastatic breast cancer patients: exploring differences in inflammatory breast cancer

Abstract Background: Liquid biopsy provides a real-time assessment of metastatic breast cancer (MBC). Recently, the complementary prognostic value of tumor-derived extracellular vesicles (tdEVs) and circulating tumor cells (CTCs) has been reported. We have previously confirmed the strong prognostic significance of CTCs and tdEVs in inflammatory breast cancer (IBC). While previous studies have reported the association of CTCs with circulating tumor DNA (ctDNA) alterations in MBC, no evidence is available for tdEVs. This study aimed to analyze the association of tdEVs with ctDNA alterations, to investigate the molecular pathways underlying their presence. Moreover, we explored potential differences of this association in patients (pts) with IBC to provide a comprehensive liquid biopsy-based portrait of this aggressive subtype of BC. Methods: Blood samples were collected from 355 pts with MBC before starting a new line of therapy at Northwestern University (Chicago, IL) between 2016 and 2021 (NU16B06 trial). For CTCs and tdEVs analysis, 7.5 mL of blood was processed with the CellSearch® system. The ACCEPT software was applied to CellSearch® images to automatically enumerate CTCs and tdEVs. Positivity cutoff was ≥5 for CTCs, while tdEV cutoff points were &amp;lt; 20 (low), 20-79 (intermediate), and ≥80 (high). For ctDNA analysis, matched plasma samples (± 1 month) were analyzed with the Guardant360™ NGS platform for the detection of somatic single nucleotide variants (SNVs) and copy number variations (CNVs), which were classified into oncogenic pathways based on defined profiles generated on the Cancer Genome Atlas database (p53, PI3K, ER, RTK, RAS, RAF, WNT, MYC, cell cycle, notch). Associations between ctDNA pathways alterations and tdEVs were tested in the overall population by multinomial logistic regression and corrected for significant clinical characteristics. Differences in liquid biopsy features between IBC and non-IBC subgroups were analyzed through Fisher's exact test. Results: Of the 355 MBC pts, 210 (62%) had HR+ BC, 61 (17%) had HER2+ BC, and 68 (20%) had triple-negative BC. Eighty-three (23%) had a diagnosis of IBC and had a numerically lower tdEV count. Also, lower tdEVs were detected in HER2+ BC as compared with HR+. Significantly higher tdEVs were observed among pts with lobular histology, liver, and bone metastases. CTC count was significantly associated with tdEV number. A matched plasma sample for ctDNA analysis was available for 175 pts (62 IBC and 113 non-IBC). In the overall population, SNVs in ER pathway were associated with intermediated/high levels of tdEVs (p=0.004 and 0.008). A similar association was observed for CNVs in the cell cycle pathway (p=0.008 and 0.007). Moreover, associations with ≥80 tdEVs were observed for PI3K SNVs and CNVs (p=0.039 and 0.004, respectively), RTK CNVs (p=0.023), and MYC CNVs (p=0.001). In multivariable analysis, clinical characteristics associated with higher tdEVs were lobular histology (p=0.014 for 20-79 and &amp;lt; 0.001 for ≥80) and bone metastases (p=0.019 for 20-79 and 0.002 for ≥80). When considering clinical variables of interest, only ER SNVs and MYC CNVs were significantly associated with intermediate (p=0.031) and high (p=0.022) tdEV counts, respectively. While the association with ER SNVs and cell cycle CNVs was significant both in the IBC and non-IBC subgroups, others were specific for a certain subgroup: higher tdEVs were significantly associated with PI3K SNVs only in non-IBC pts whereas the significant association with CNVs in PI3K and MYC pathways was observed only in pts with IBC. Conclusion: Detection of tdEVs was associated with particular genomic profiles. These alterations seem to be different in IBC further underlying a different biology of this BC subtype. Additional studies are needed to explore how to integrate different liquid-biopsy based biomarkers in the management of pts with MBC. Citation Format: Eleonora Nicolò, Lorenzo Gerratana, Lorenzo Foffano, Laura Munoz Arcos, Mara Serena Serafini, Maroua Manai, Letizia Pontolillo, Nadia Bayou, Elisabetta Molteni, Amanda Kaylan Strickland, Youbin Zhang, Paolo D’Amico, Andrew Davis, Jeannine Donahue, Huiping liu, William Gradishar, Ami Shah, Giuseppe Curigliano, Carolina Reduzzi, Massimo Cristofanilli. Association of tumor-derived extracellular vesicles with circulating tumor DNA alterations in metastatic breast cancer patients: exploring differences in inflammatory breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-06-05.

Abstract PS12-02: Exploration of ctDNA Dynamics in the PACE Trial: A Randomized Phase II Study of Fulvestrant, Palbociclib, and Avelumab for HR+/HER2- Metastatic Breast Cancer

Abstract Background The PACE trial for HR+/HER2- metastatic breast cancer (MBC) prospectively evaluated whether continuation of the CKD4/6 inhibitor (CDK4/6i) palbociclib (P) beyond progression on prior CDK4/6i and aromatase inhibitor (AI), with a change in endocrine therapy (ET) to fulvestrant (F), improved outcomes beyond change to F alone, as well as explored the activity of a palbociclib, fulvestrant, and avelumab (P+F+A) triplet. The primary analysis did not show improvement in progression-free survival (PFS) with F+P versus F alone. In this analysis, an evaluation of serial translational PACE samples was performed to gain insights into the mutational landscape of HR+/HER2- MBC after prolonged exposure to CDK4/6i, evaluate early mutational dynamics with ongoing therapy, and identify potential genetic markers predictive of treatment response. Additionally, the identification of acquired mutations at the time of tumor progression could provide valuable information about mechanisms of treatment resistance. Methods PACE is a multicenter randomized open-label investigator-initiated phase II trial. Eligible patients (pts) had HR+/HER2- evaluable MBC with prior progression on AI and any CDK4/6i after &amp;gt; 6 months (mo) of therapy in the MBC setting, or during/within 12 mo in the adjuvant setting, with no more than 1 prior line of chemotherapy for MBC. Pts were randomized 1:2:1 to F alone, F+P, or F+P+A, with tumor assessments every 8 weeks. Correlative samples including circulating tumor DNA (ctDNA) were collected at baseline (BL), times of tumor assessments (C3D1), and end of study/progression (EOT). Targeted NGS (Guardant360) to evaluate mutations (somatic single nucleotide variants and indels) was performed. Kaplan-Meier survival curves were used for assessing PFS and the log-rank test for comparisons. McNemar’s test with continuity correction was used to test the changes in the frequency of mutations. Results Of the 220 randomized pts, 211 contributed ctDNA samples (200 at BL, 129 at C3D1, 146 at EOT). Characteristics of the correlative science population were similar to the intention to treat population. At BL, the most common mutations observed were ESR1 (57%), TP53 (38%), PIK3CA (36%), GATA3 (20%) ATM (12%) and Rb1 (12%). The presence of a PIK3CA mutation vs WT-PIK3CA at BL was associated with shorter PFS in the F alone arm (p=0.001) but not in the F+P or F+P+A arms. ESR1 mutations present at baseline were D538G (34%), Y537S (21%), Y537N (15%), E380Q (10%) and L536H (4%). At C3D1, the most common mutations observed were ESR1 (47%), TP53 (33%), PIK3CA (26.1), GATA3 (18.1), ATM (13%) and PTEN (8.6%). Including only BL and C3D1 matched samples (N=124), the prevalence of ESR1 mutations decreased after 2 cycles of treatment (57% at BL vs 44% at C3D1), predominantly observed in the F+P+A arm. For those in any arm with at least 6 months of disease stability on study (N=53), the presence of an ESR1 D538G mutation decreased during therapy, with 38% having a mutation at BL, 24% at C3D1, and no pts gaining ESR1 D538G at C3D1. The prevalence of PIK3CA mutations also decreased on treatment (32% at BL to 24% at C3D1), with the greatest decrease in the F+P arm. A predictive model correlating early genomic dynamics and associations with outcome will be presented. Conclusions The PACE trial offers one of the largest clinical trial cohorts describing the genomic landscape of HR+/HER2- MBC post-CDK4/6 exposure. Results from this correlative study provide a comprehensive mutational landscape post-CDK4/6i, demonstrate associations between mutations and clinical outcomes, and suggest the potential value of early ctDNA dynamics in this setting. Citation Format: Rinath Jeselsohn, Jingxin Fu, Yue Ren, Reshma Mahtani, Cynthia Ma, Angela DeMichele, Massimo Cristofanilli, Jane Meisel, Kathy Miller, Yara Abdou, Elizabeth Riley, Rubina Qamar, Priyanka Sharma, Sonya Reid, Naomi Ko, Yuan Liu, Harold Burstein, Michelle DeMeo, Sara Tolaney, Erica Mayer. Exploration of ctDNA Dynamics in the PACE Trial: A Randomized Phase II Study of Fulvestrant, Palbociclib, and Avelumab for HR+/HER2- Metastatic Breast Cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PS12-02.

Abstract PO4-01-02: High throughput analysis in HER2 positive locally advanced breast cancer: pCR and mutational status

Abstract Background Pathologic complete response (pCR) after neoadjuvant treatment (NAD) is a strong prognostic biomarker associated with improved survival in breast cancer patients, especially with HER2-positive and Triple negative subtypes. PIK3CA is mutated in up to 20% of HER2 positive breast cancers, contributes to anti-HER-2 resistance and may be predictive of the lack of response to anti HER2 NAD. PIK3CA mutations in breast cancer occur primarily at hotspots E545K at exon 9 and H1047R at exon 20. Next generation sequencing has improved our knowledge regarding the biology of the mechanisms behind treatment resistance. The aim of this study is to evaluate the genomic landscape of tissue samples obtained from locally advanced breast cancer patients treated in an neoadjuvant setting, in order to correlate the genomic analysis to response and clinical outcome. Methods DNA extracted from the archival 84 formalin-fixed paraffin-embedded (FFPE) samples from core needle biopsies was subjected to deep sequencing using the TruSight Oncology (TSO) 500 panel (Illumina, San Diego, USA; 523 genes, size: 1.94 Mb), following the manufacturer’s protocol, which assesses microsatellite instability (MSI) status, tumor mutation burden (TMB), recurrent somatic copy number variations (sCNV), somatic single nucleotide variants (SNVs). Libraries were sequenced on a NovaSeq 6000 instrument (Illumina) to reach a minimum of 500 × read depth. All samples were evaluated for RNA single cell transcriptional profile. 73 of 84 patients were treated with traditional neoadjuvant chemotherapy plus trastuzumab meanwhile 13 of 84 received neoadjuvant chemotherapy plus trastuzumab and pertuzumab. Statistical analysis was performed to detect the genes whose mutation involved in the progression and/or non-response of the disease using the Statistical Package for Social Science (SPSS), release 23.0. Continuous variables were expressed as mean ± SD or median [range], categorical variables were displayed as frequencies and the appropriate parametric (Student‘s t-test) non-parametric test (X2-test or Mann-Whitney test) was used to assess significance of the differences between subgroups (patients with or without pCR). Results In total we analyzed samples of 84 patients. The median age was 48 years. 64 of 84 (76.2%) were triple positive, 66 (78.6%) with G3 and 29 (34.5%) with stage III. The most common frequently mutated genes were PIK3CA (16/84, 19%) and BRCA1 (7/84, 8.3%). The pathogenic variant of BRCA1 (sBRCA1) had a significant association with resistance to treatment: we found a pCR in only 14.3% of sBRCA1 versus 62.3% in sBRCA1 wild type (wt) patients (p=0.014). We also identified 11 somatic mutations in hereditary genes (ATM, BRCA1, BRCA2, PALB2, CHEK2, RAD51C, RAD51D). From a germline standpoint we detected 7 pathogenic variants (two affecting BRCA1, two affecting CHEK2, one affecting PALB2, 7/84, 8.3%), which will be clinically confirmed. Among patients with a mutated PI3KCA, 43.8% obtained a pCR versus 61.8% of patients with PI3KCA wt (p=0.19). This data probably presents an imbalance due to the sample size A PTEN mutation was identified in 2% of pts (2/84) which obtained no benefit in terms of pCR after NAD versus 59.8% in wt patients (p=0.09). Conclusions In this preliminary report, genetic drivers such as BRCA1 status may have a clinical implication for prognosis and treatment response after NAD in locally advanced HER2 positive breast cancer. More samples are needed to evaluate the significance of PI3K and PTEN mutations in this patient setting. We are waiting for the results of the RNA single cell transcriptional profile of these samples. Event free survival data is not yet mature at this moment. We would like to acknowledge the contribution of Multi-specialistic Biobank Research Core Facility G-STeP, Fondazione Policlinico Universitario “A. Gemelli” IRCCS (Biobank-FPG) who provided the bio-resources. Citation Format: Ida Paris, Angelo Minucci, Daniele Generali, simona Nanni, Tatiana D'angelo, Francesco Pavese, Alessandra Fabi, Maria De Bonis, Katia Cannita, Alessia Piermattei, Giordana Tiberi, Paola Fuso, Luisa Carbognin, Silvia Rotondaro, Camilla Nero, Flavia Giacomini, Tina Pasciuto, Maria Marrazzo, Luciano Giaco', Antonino Mulè, Riccardo Masetti, Gianluca Franceschini, Giorgia Garganese, Diana Giannarelli, Giovanni Scambia. High throughput analysis in HER2 positive locally advanced breast cancer: pCR and mutational status [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-01-02.

Comprehensive characterization of somatic mutations associated with chimeric RNAs in human cancers.

Chimeric RNAs, which can arise from gene recombination at the DNA level or non-canonical splicing events at the RNA level, have been identified as important roles in human tumors. Dysregulated gene expression caused by somatic mutations and altered splicing patterns of oncogenes or tumor suppressor genes can contribute to the development of tumors. Therefore, investigating the formation mechanism of chimeric RNAs via somatic mutations is critical for understanding tumor pathogenesis. This project is the first to propose studying the association between somatic single nucleotide variants and chimeric RNAs, identifying around 2900 somatic SNVs affecting chimeric RNAs in pan-cancer level. The somatic SNVs on chimeric RNAs were commonly observed in various types of tumor tissues, providing a valuable resource for future study. Additionally, these SNVs show distinct tumor specificity, and those with high frequency had a significant impact on the survival time of patients with tumors. Further research revealed that somatic SNVs associated with chimeric RNA (chiR-SNVs) were typically found within 10 nt of the junction site of chimeric RNAs and had a particularly significant effect on chimeric RNAs from different chromosomes. The enrichment analysis revealed that chiR-SNVs were significantly overrepresented in oncogenes and genes related to RNA binding proteins involved in RNA splicing, which could imply that chiR-SNVs may disrupt the process of RNA splicing and induce the occurrence of chimeric RNAs. This study sheds light on the potential molecular interaction mechanism between somatic SNVs and chimeric RNAs, which opens up a new avenue for researching disease pathway and tumorigenesis development.

Abstract 2850: Noncoding regulatory mutations as driving event for the oncogenic core regulatory circuitries of neuroblastoma

Abstract Background and Aim: Neuroblastoma (NB) is the most aggressive solid tumor of infancy and arises from neural-crest-derived progenitor cells. It is composed by cell types reflecting developmental stages of the adrenergic lineage: mesenchymal (MES) and adrenergic (ADRN) cell types. These cell identities result from the activity of NB specific Core Regulatory Circuitries (CRCs), transcription factor (TF) sets that co-occupy regulatory regions together impacting the expression of their own genes and those of lineage-specific ones governing cell state transitions and cellular identities. Our hypothesis is that noncoding somatic single nucleotide variants (SNVs) located in NB CRCs TF binding sites (TFBSs) underlie NB CRCs activity, affecting cellular differentiation and promoting tumor onset. We aim to investigate such pattern of regulatory elements and identify putative driver SNVs, exploring their role and characterizing their action mechanism. Methods: Transcriptionally active TFBSs (aTFBSs) bound by ADRN CRC TFs (GATA3, HAND2, ISL1, TBX2, MYCN, PHOX2B, ASCL1 and LMO1) were identified integrating 27 ChIP-seq and 11 ATAC-seq experiments in 6 NB ADRN cell lines (SKNBE2C, Kelly, NGP, COGN415, LAN5 and NB1643). In particular, aTFBSs were defined as ChIP-seq peaks overlapping ATAC-seq signals in the same cell line. SNVs from 317 NB whole genomes were mapped on aTFBSs and the enrichment of mutations in core regions respect to flanking ones was tested using Fisher test. Observed mutation rate in aTFBSs was compared with background mutation rate due to local sequence context. SNVs in mutated aTFBSs were selected and their impact on TF binding affinity was evaluated with FABIAN-variant and motifbreakR tools. aTFBSs target-genes were identified through promoter capture HiC (CHiC) in ADRN NB cells (SKNBE2C and SHSY5Y). Results: We found a significant (FDR &amp;lt; 0.05) mutation enrichment in aTFBSs of ISL1, PHOX2B, TBX2, GATA3 and MYCN, suggesting a common somatic mutation burden for a subset of ADRN CRC TFBSs in ADRN NB cells. Starting from 839 SNVs falling in significantly mutated aTFBSs, we selected 269 mutations impacting the binding of ADRN CRC TFs. This mutations subset mapped in aTFBSs interacting with genes that significantly enriched pathways involved in neuronal development and migration (FDR &amp;lt; 0.05), thus suggesting their possible role in affecting cell identity definition. As an example, chr6:137753475:G&amp;gt;T mapped in aTFBSs interacting with the promoter of OLIG3, which takes part in neuronal differentiation. Interestingly, chr6:137753475:G&amp;gt;T was predicted to break the binding to ISL1 (FABIAN score: -0.24), a key regulator of ADRN CRC. Conclusions: These results demonstrate that somatic noncoding SNVs can act synergistically to promote NB onset, affecting CRCs activity and, in turn, the normal cell identity definition. Citation Format: Mario Capasso, Vincenzo Aievola, Vito Alessandro Lasorsa, Annalaura Montella, Ferdinando Bonfiglio, Marianna Avitabile, Teresa Maiorino, Matilde Tirelli, Giuseppe D'Alterio, Matthias Fischer, Frank Westermann, Achille Iolascon. Noncoding regulatory mutations as driving event for the oncogenic core regulatory circuitries of neuroblastoma [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 2850.

Abstract 4336: Homologous recombination repair deficiency, and not recurrence, determines mutational burden and clonal dynamics in high grade serous ovarian cancer

Abstract High grade serous ovarian cancer (HGSOC) is the most lethal gynecologic malignancy, killing more than 9,000 women each year in the United States. Nearly 80% of patients with HGSOC will experience recurrence within 5 years, but little is known about the mechanisms that drive this process. Up to 60% of the genome in an HGSOC tumor is impacted by structural variant mutations and pronounced intratumoral heterogeneity. Intratumor heterogeneity is believed to be a key feature of recurrence and therapy resistance with some evidence indicating diverse and complex mechanisms of the seeding of metastatic sites and recurrent tumors. We included 35 HGSOC patients for whom paired chemo-naive and chemoresistant tumors as well as germline DNA was available. Whole genome sequencing was used to identify somatic single nucleotide variants (SNV) insertion/deletion variants (indels), and structural variants (SVs) in each tumor. The number of somatic SVs and indels was higher in tumors with homologous recombination repair deficiency (HRD; P=0.000252 SVs, P=0.00385 Indel) but was not affected by recurrent status (P=0.826 SV, P=0.145 SNV/indel). Clonal composition and dynamics were measured using SNVs/indels as tumors progressed from chemo-naïve primary samples to recurrent chemo-resistant tumors. Surprisingly, few changes were observed in clonal abundance and complexity through progression. When this analysis was repeated with SVs homologous recombination repair proficient tumors tend to be polyclonal while HRD tumors tend to be monoclonal. Five denovo structural variant signatures were identified in our cohort and led to three distinct structural variant classes tumors defined by deletion, tumors defined by duplication, and tumors defined by copy number neutral changes (inversions, translocations and balanced complex events). Patients with tumors defined by deletions and copy number neutral changes carry pathogenic SVs correlating with reduced survival (P=0.00023), while DNA gains are less subject to pathogenic alterations. Each class displayed distinct regions of the chromosome that was frequently affected by large scale SV events (&amp;gt;5Mb), which was also observed in the HRP/HRD status. Although no regions were notably altered across recurrence, GO analysis revealed that recurrent tumors have a significantly reduced immune response, which was not seen in the primary tumors. Within a subset of tumors, we are plan to utilize Oxford Nanopore Technologies ultra long read sequencing which will allow for confirmation of SVs observed in the WGS as well as identification of more complex events that cannot be deciphered using WGS techniques. Citation Format: Michael A. Diaz, Nicole Gull, Pei-Chen Peng, Kate Lawrenson, Bobbie J. Rimel, Jenny Lester, Beth Karlan, Simon A. Gayther, Michelle R. Jones. Homologous recombination repair deficiency, and not recurrence, determines mutational burden and clonal dynamics in high grade serous ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4336.