Allele Frequencies Research Articles

Linking CDH1 SNPs to gastric cancer risk: a comprehensive analysis of rs16260, rs13689, and rs9929218.

Single nucleotide polymorphisms (SNPs) are linked to carcinogenesis. Pathogenic variants in the CDH1 gene are associated with gastric cancer. This study examines the genotype and allele frequencies of three SNPs (rs16260, rs13689, and rs9929218) in the CDH1 gene and their relationship with gastric cancer risk. The study involved 105 gastric cancer patients with pathology results and 105 healthy controls. Clinical, histopathological, and demographic data were collected and compared between the two groups. No significant differences were found for rs16260 (-160 C > A) and rs9929218 (G > A) between patients and controls (p > 0.05). For rs13689 (T > C), the T allele frequency was 90% in patients versus 69% in controls, while the C allele frequency was 10% in patients versus 31% in controls. A significant difference was observed for this SNP, with a higher T allele frequency in patients (OR = 4.03 CI95% 2.4-6.7, p < 0.0001) compared with controls, suggesting a fourfold increased risk of gastric cancer. Genotype frequencies were 80% wild-type (TT) and 20% heterozygous-type (TC) in patients, and 58% TT, 22% TC, and 20% mutant-type (CC) in controls (p < 0.0001). The frequencies of non-C allele carriers (TT) were present in 80% of patients versus 58.1% of controls (OR = 2.88 CI95% 1.56-5.34, p = 0.0006). This study is the first to link the rs13689 SNP's T allele and TT genotype with increased gastric cancer risk. Our results suggest that thers13689 T allele may contribute significantly to disease susceptibility, while thers16260 CC genotype and rs9929218 GG genotype may influence risk in smokers.

Association of BRAF V600E allele frequency with clinicopathologic outcomes in papillary thyroid cancer.

BRAF V600E mutation is the most common genetic driver of papillary thyroid cancer (PTC), where it is found with various allele frequency (AF), reflecting the proportion of cells carrying the mutant and wild-type gene alleles. To determine whether BRAF V600E AF can improve prognostication and inform initial surgical management of PTC. Retrospective cohort study (2016-2019). UCLA health. Consecutive patients with Bethesda V/VI nodules and isolated BRAF V600E mutation who underwent surgery with histopathology showing PTC. Blinded ThyroSeq v3 molecular analysis after completion of initial management and follow-up. Aggressive histopathology and cancer persistence/recurrence. Of 73 patients, the median BRAF V600E AF was 25.5% (IQR, 16.7-34.3%). Higher median AF was seen in patients classified as American Thyroid Association (ATA) high-risk (37%) vs. intermediate-risk (25.3%, p<0.01) and low-risk (24.7%, p<0.01), largely attributed to higher AF in patients with gross extrathyroidal extension (ETE) (40.1% vs. 25.2% without gross ETE, p=0.02). No differences in AF were observed on the basis of lymph node positivity or presence of aggressive variants of PTC. A higher BRAF V600E AF was also found in patients with tumors ≥2cm vs. <2cm (median 32.0% vs. 24.4%, p<0.01). Over 4.1 years of follow-up, disease persistence/recurrence was found in 7 patients (9.4%) and was associated with higher median AF than those without recurrence (35.3% vs. 25.2%, p=0.02). Higher AF was associated with poorer recurrence-free survival (AF≥35%, HR 7.40, CI 1.4-38.1). Higher AF was associated with gross ETE and increased recurrence risk. This may inform initial management in patients with PTC harboring an isolated BRAF V600E mutation.

Ferroptosis regulates hemolysis in stored murine and human red blood cells.

Red blood cell (RBC) metabolism regulates hemolysis during aging in vivo and in the blood bank. However, the genetic underpinnings of RBC metabolic heterogeneity and extravascular hemolysis at population scale are incompletely understood. Based on the breeding of 8 founder strains with extreme genetic diversity, the Jackson laboratory diversity outbred population can capture the impact of genetic heterogeneity in like fashion to population-based studies. RBCs from 350 outbred mice, either fresh or stored for 7 days, were tested for post-transfusion recovery, as well as metabolomics and lipidomics analyses. Metabolite and lipid Quantitative Trait Loci (QTL) mapped >400 gene-metabolite associations, which we collated into an online interactive portal. Relevant to RBC storage, we identified a QTL hotspot on chromosome 1, mapping on the region coding for the ferrireductase Steap3, a transcriptional target to p53. Steap3 regulated post-transfusion recovery, contributing to a ferroptosis-like process of lipid peroxidation, as validated via genetic manipulation in mice. Translational validation of murine findings in humans, STEAP3 polymorphisms were associated with RBC iron content, lipid peroxidation and in vitro hemolysis in 13,091 blood donors from the Recipient Epidemiology and Donor Evaluation Study. QTL analyses in humans identified a network of gene products (FADS1/2, EPHX2, LPCAT3, SLC22A16, G6PD, ELOVL, PLA2G6) associated with lower levels of oxylipins. These polymorphisms were prevalent in donors of African descent and were linked to allele frequency of hemolysis-linked polymorphisms for Steap3 or p53. These genetic variants were also associated with lower hemoglobin increments in thousands of single-unit transfusion recipients from the vein-to-vein database.

Longitudinal ultra-sensitive mutation burden sequencing for precise minimal residual disease assessment in AML.

Relapse is one of the major challenges in clinical treatment of acute myeloid leukemia (AML). Though minimal residual disease (MRD) monitoring plays a crucial role in quantitative assessment of the disease, molecular MRD analysis has been mainly limited to patients diagnosed with gene fusions and NPM1 mutations. Here, we report a longitudinal ultra-sensitive mutation burden (UMB) monitoring strategy for accurate MRD analysis in AML patients regardless of genetic abnormality types. Using a Quantitative Blocker Displacement Amplification (QBDA) sequencing panel with limit of detection below 0.01% variant allele frequency (VAF), a hazard ratio of 14.8 (p < 0.001) is observed in cumulative incidence of relapse analysis of 20 patients with ≥ 2 samples during complete remission (CR). The ROC area under curve (AUC) is 0.98 when predicting relapse within 30 weeks of CR timepoint 2 (N = 20). Furthermore, we demonstrate quantitating VAF below 0.01% is essential for accurate relapse prediction.

Abstract A037: Improved liquid biopsy assay performance using novel highly accurate sequencing-by-binding (SBB) technology

Abstract Liquid biopsy is revolutionizing the field of early cancer detection research through non-invasive detection of tumor DNA in the blood. However, existing liquid biopsy assays are limited in their sensitivity for ctDNA detection at low variant allele frequencies (VAFs), with most relying on extreme sequencing depth and computational error correction to separate the true ctDNA signal from background errors. This limitation is particularly problematic in the area of early cancer detection and minimal residual disease monitoring, in which expected ctDNA allele frequencies are extremely low. Novel strategies are therefore needed to help improve liquid biopsy assay sensitivity and reduce per-sample sequencing requirements. Here we describe PacBio’s application of the Onso short-read sequencing system to enable detection of ctDNA at low VAFs using the SeraCare Complete ctDNA Mutation Mix reference standard. The Onso system makes use of a novel sequencing by binding (SBB) method to achieve 10- to 100-fold greater quality scores, with ≥90% of reads at Q40 or above. We performed targeted capture and sequencing of libraries prepared from the SeraCare reference mix diluted into WT human DNA at the following VAFs: 0.00% (WT), 0.05%, 0.10%, and 0.25%, and compared the sensitivity at each VAF for SBB compared to a competitor method using sequencing by synthesis (SBS) at varying sequencing depths. At equivalent sequencing depth (12,000X) we observed superior recall for low frequency variants (VAF = 0.05%, 0.1%) in the reference sample for Onso compared to SBS. Furthermore, SBB was able to achieve comparable sensitivity results to SBS using four- fold less sequencing (6,000X vs. 24,000X), and without the use of computational error correction. Combining SBB with error correction methods boosted sensitivity even further, suggesting an additive value for these technologies. Comparison of false positive variant calls in the WT samples showed a dramatic decrease in false positives for Onso (n=2) compared to SBS (n=49) across 10 subsampling replicates, and observed allele frequencies for Onso were closer to the expected values. In conclusion, Onso achieved superior sensitivity for ctDNA detection at compared to SBS at equivalent sequencing depths, and achieved similar performance with four- fold less sequencing and without the use of error correction. This improvement was due in part to the reduction in false positive variant calling in the WT sample, which lowers the limit of detection to maximize variant detection while maintaining high specificity. Taken together, our results demonstrate the potential of SBB to improve upon existing methods of liquid biopsy and better enable research on early cancer detection and minimal residual disease. Citation Format: Alexandra Sockell, Dan Nasko, Young Kim, Kristi Kim, Jonas Korlach. Improved liquid biopsy assay performance using novel highly accurate sequencing-by-binding (SBB) technology [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr A037.

Droplet Digital PCR: A New Molecular Method to Detect G1105S/V Mutations in Plasmopara viticola CesA3 Gene

Plasmopara viticola is the causal agent of Grapevine Downy Mildew (GDM), which is a devastating disease of grapevines in humid temperate regions. The most employed method for protecting grapevines against GDM is the application of chemical fungicides. In Spain, Carboxylic Acid Amides (CAAs) are a fungicide group currently utilized in GDM control. In P. viticola, resistance to CAAs is conferred by G1105S and G1105V mutations in the CesA3 gene. Droplet digital polymerase chain reaction (ddPCR) is an innovative technique that combines PCR and droplet microfluidics to disperse the sample into thousands of water-in-oil droplets in which an amplification reaction is individually performed. In this study, we set up a ddPCR protocol to quantify S1105 and V1105 mutations conferring resistance to CAAs in P. viticola. The optimal PCR conditions were established, and the sensitivity and precision of the protocol were assessed. Four P. viticola populations coming from commercial vineyards in northern Spain were analyzed, and different allele frequencies were found in the analyzed samples corresponding to the different fungicide management strategies, ranging from 7.72% to 100%. Knowing the level of mutated alleles allows for designing resistance management strategies suited for each location. This suggests that similar ddPCR assays could be developed for studying mutations implicated in fungicide resistance in other fungicide groups and plant pathogens.

Two fitness inference schemes compared using allele frequencies from 1,068,391 sequences sampled in the UK during the COVID-19 pandemic.

Throughout the course of the SARS-CoV-2 pandemic, genetic variation has contributed to the spread and persistence of the virus. For example, various mutations have allowed SARS-CoV-2 to escape antibody neutralization or to bind more strongly to the receptors that it uses to enter human cells. Here, we compared two methods that estimate the fitness effects of viral mutations using the abundant sequence data gathered over the course of the pandemic. Both approaches are grounded in population genetics theory but with different assumptions. One approach, tQLE, features an epistatic fitness landscape and assumes that alleles are nearly in linkage equilibrium. Another approach, MPL, assumes a simple, additive fitness landscape, but allows for any level of correlation between alleles. We characterized differences in the distributions of fitness values inferred by each approach and in the ranks of fitness values that they assign to sequences across time. We find that in a large fraction of weeks the two methods are in good agreement as to their top-ranked sequences, \textit{i.e.} as to which sequences observed that week are most fit. We also find that agreement between the ranking of sequences varies with genetic unimodality in the population in a given week.&#xD.

Filtering coupled Wright-Fisher diffusions.

Coupled Wright-Fisher diffusions have been recently introduced to model the temporal evolution of finitely-many allele frequencies at several loci. These are vectors of multidimensional diffusions whose dynamics are weakly coupled among loci through interaction coefficients, which make the reproductive rates for each allele depend on its frequencies at several loci. Here we consider the problem of filtering a coupled Wright-Fisher diffusion with parent-independent mutation, when this is seen as an unobserved signal in a hidden Markov model. We assume individuals are sampled multinomially at discrete times from the underlying population, whose type configuration at the loci is described by the diffusion states, and adapt recently introduced duality methods to derive the filtering and smoothing distributions. These respectively provide the conditional distribution of the diffusion states given past data, and that conditional on the entire dataset, and are key to be able to perform parameter inference on models of this type. We show that for this model these distributions are countable mixtures of tilted products of Dirichlet kernels, and describe their mixing weights and how these can be updated sequentially. The evaluation of the weights involves the transition probabilities of the dual process, which are not available in closed form. We lay out pseudo codes for the implementation of the algorithms, discuss how to handle the unavailable quantities, and briefly illustrate the procedure with synthetic data.

A comprehensive study of common and rare genetic variants in spermatogenesis-related loci identifies new risk factors for idiopathic severe spermatogenic failure

Abstract STUDY QUESTION Can genome-wide genotyping data be analysed using a hypothesis-driven approach to enhance the understanding of the genetic basis of severe spermatogenic failure (SPGF) in male infertility? SUMMARY ANSWER Our findings revealed a significant association between SPGF and the SHOC1 gene, and identified three novel genes (PCSK4, AP3B1, and DLK1) along with 32 potentially pathogenic rare variants in 30 genes that contribute to this condition. WHAT IS KNOWN ALREADY SPGF is a major cause of male infertility, often with an unknown aetiology. SPGF can be due to either multifactorial causes, including both common genetic variants in multiple genes and environmental factors, or highly damaging rare variants. Next-generation sequencing (NGS) methods are useful for identifying rare mutations that explain monogenic forms of SPGF. Genome-wide association studies (GWAS) have become essential approaches for deciphering the intricate genetic landscape of complex diseases, offering a cost-effective and rapid means to genotype millions of genetic variants. Novel methods have demonstrated that GWAS datasets can be used to infer rare coding variants that are causal for male infertility phenotypes. However, this approach has not been previously applied to characterize the genetic component of a whole case-control cohort. STUDY DESIGN, SIZE, DURATION We employed a hypothesis-driven approach focusing on all genetic variation identified, using a GWAS platform and subsequent genotype imputation, encompassing over 6 million polymorphisms and a total of 1571 SPGF patients and 2431 controls. Both common (minor allele frequency, MAF &amp;gt; 0.01) and rare (MAF &amp;lt; 0.01) variants were investigated within a total of 1797 loci with a reported role in spermatogenesis. This gene panel was meticulously assembled through comprehensive searches in the literature and various databases focused on male infertility genetics. PARTICIPANTS/MATERIALS, SETTING, METHODS This study involved a European cohort using previously and newly generated data. Our analysis consisted of three independent methods: (1) variant-wise association analyses using logistic regression models, (2) gene-wise association analyses using combined multivariate and collapsing (CMC) burden tests, and (3) identification and characterisation of highly damaging rare coding variants showing homozygosity only in SPGF patients. MAIN RESULTS AND THE ROLE OF CHANCE The variant-wise analyses revealed an association between SPGF and SHOC1-rs12347237 (p = 4.15E-06, OR = 2.66), which was likely explained by an altered binding affinity of key transcription factors in regulatory regions and the disruptive effect of coding variants within the gene. Three additional genes (PCSK4, AP3B1, and DLK1) were identified as novel relevant players in human male infertility using the gene-wise burden test approach (p &amp;lt; 5.56E-04). Furthermore, we linked a total of 32 potentially pathogenic and recessive coding variants of the selected genes to 35 different cases. LARGE SCALE DATA Publicly available via GWAS catalog (Accession number: GCST90239721). LIMITATIONS, REASONS FOR CAUTION The analysis of low-frequency variants presents challenges in achieving sufficient statistical power to detect genetic associations. Consequently, independent studies with larger sample sizes are essential to replicate our results. Additionally, the specific roles of the identified variants in the pathogenic mechanisms of SPGF should be assessed through functional experiments. WIDER IMPLICATIONS OF THE FINDINGS Our findings highlight the benefit of using GWAS genotyping to screen for both common and rare variants potentially implicated in idiopathic cases of SPGF, whether due to complex or monogenic causes. The discovery of novel genetic risk factors for SPGF and the elucidation of the underlying genetic causes provide new perspectives for personalized medicine and reproductive counselling. STUDY FUNDING/COMPETING INTEREST(S) This work was supported by the Spanish Ministry of Science and Innovation through the Spanish National Plan for Scientific and Technical Research and Innovation (PID2020-120157RB-I00) and the Andalusian Government through the research projects of “Plan Andaluz de Investigación, Desarrollo e Innovación (PAIDI 2020)” (ref. PY20_00212) and “Proyectos de Investigación aplicada FEDER-UGR 2023” (ref. C-CTS-273-UGR23). SGM was funded by the previously mentioned projects (ref. PY20_00212 and PID2020-120157RB-I00). AGJ was funded by MCIN/AEI/10.13039/501100011033 and FSE “El FSE invierte en tu futuro” (grant ref. FPU20/02926). IPATIMUP integrates the i3S Research Unit, which is partially supported by the Portuguese Foundation for Science and Technology (FCT), financed by the European Social Funds (COMPETE-FEDER) and National Funds (projects PEstC/SAU/LA0003/2013 and POCI-01-0145-FEDER-007274). SS is supported by FCT funds (10.54499/DL57/2016/CP1363/CT0019), ToxOmics-Centre for Toxicogenomics and Human Health, Genetics, Oncology and Human Toxicology, and is also partially supported by the Portuguese Foundation for Science and Technology (UIDP/00009/2020 and UIDB/00009/2020). SLarriba received support from Instituto de Salud Carlos III (grant: DTS18/00101], co-funded by FEDER funds/European Regional Development Fund (ERDF)-a way to build Europe-), and from “Generalitat de Catalunya” (grant 2021SGR052). SLarriba is also sponsored by the “Researchers Consolidation Program” from the SNS-Dpt. Salut Generalitat de Catalunya (Exp. CES09/020). All authors declare no conflict of interest related to this study.

Abstract B064: Prevalence and clinical relevance of clonal hematopoiesis in metastatic urologic malignancies

Abstract Introduction: Clonal hematopoiesis (CH) is an age-related process whereby hematopoietic progenitor cells with specific mutations gain a proliferative advantage, resulting in populations that can be detected in blood. CH is a major confounder in plasma cell-free DNA (cfDNA) assays that do not profile patient-matched white blood cell (WBC) DNA to distinguish circulating tumor DNA (ctDNA) somatic mutations from those with hematopoietic origin. We aimed to characterize CH in patients with advanced urologic cancers and clarify the potential for CH to interfere with plasma-based tumor genotyping. Methods: We applied error-corrected targeted DNA sequencing (median 2200X) to matched cfDNA and WBC DNA from 301 patients with advanced urothelial cancer (UC) or renal cell carcinoma (RCC) enrolled in a provincial biobank, using a 57 gene panel capturing key CH and urologic cancer genes. CH mutations were defined as those detected in both WBC and cfDNA with a variant allele frequency (VAF) exceeding a 0.25% limit of detection and supported by at least 5 mutant reads. In contrast, ctDNA mutations were required to be exclusively detected in plasma, with a cfDNA VAF ≥5 times the VAF in WBC. Results: 73% (221/301) of patients had ≥1 CH mutation with a VAF above 0.25% (35% of patients had a variant above 2% VAF), and CH- patients were younger than CH+ (RCC: 56 vs 68.5, p&amp;lt;0.01; UC: 63 vs 72, p&amp;lt;0.01). There was no difference in CH prevalence by biological sex. There was no difference in overall survival according to CH status (UC: [CH+] 13 vs [CH-] 16 months, p=0.98; RCC: [CH+] 33 vs [CH-] 49 months, p=0.77), in contrast to the presence of ctDNA mutations which is an established prognostic factor linked with shorter overall survival (UC: [ctDNA+] 10 vs [ctDNA-] 23 months, p&amp;lt;0.01; RCC: [ctDNA+] 15 vs [ctDNA-] 49 months, p&amp;lt;0.01). CH+ patients carried CH mutations most commonly within hematologic malignancy-associated genes (e.g. DNMT3A [58%] and TET2 [26%]), although genes linked to DNA damage response such as TP53 (10%) and ATM (9%) were also mutated. PPM1D was mutated more frequently in UC compared to RCC within CH+ patients (UC: 22%, RCC: 9%, p&amp;lt;0.01), likely attributable to platinum chemotherapy exposure prior to blood collection in 50% of the UC cohort. Importantly, a notable proportion of mutations in UC- or RCC-relevant driver genes originated from CH rather than cancer, including 28% and 65% of mutations falling in TP53 and ATM, respectively. Finally, randomly downsampling sequencing reads from WBC DNA suggested that 500X coverage is sufficient to resolve 90% of CH events with &amp;gt;1% VAF. Conclusion: CH was highly prevalent in patients with advanced urologic malignancies but had no impact on survival in this heterogeneous real-world cohort. CH commonly affected clinically-relevant driver genes that are also altered in solid cancers, potentially causing false-positive genotyping and inflating tumor mutation burden estimates in plasma-only ctDNA assays. Incorporating WBC sequencing to liquid biopsy assays can help correctly identify tumor-originating mutations. Citation Format: Aslı D Munzur, Jack V W Bacon, Francine Fishbein, Gráinne Donnellan, Cecily Q Bernales, Karan Parekh, Gillian Vandekerkhove, David C Müller, Cameron Herberts, Lucia Nappi, Corinne Maurice-Dror, Kim N Chi, Bernhard J Eigl, Christian Kollmannsberger, Maryam Soleimani, Alexander W Wyatt. Prevalence and clinical relevance of clonal hematopoiesis in metastatic urologic malignancies [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr B064.

Abstract B043: Molecular profiling of serially collected cerebrospinal fluid uncovers therapy-resistant subclones in recurrent leptomeningeal metastatic disease

Abstract Leptomeningeal metastatic disease (LMD) is the spread of cancer cells to the cerebrospinal fluid (CSF)-filled spaces surrounding the central nervous system. After symptom onset, LMD patients survive for only weeks to months while enduring devastating neurologic symptoms. Current clinical tools to diagnose LMD, such as CSF cytology and magnetic resonance imaging, do not detect mutations susceptible to treatment, quantitatively monitor response to therapy, or identify mutation-based resistance mechanisms. Here, we sought to profile LMD using next-generation sequencing by studying CSF-derived cell-free DNA (CSF-cfDNA) from 9 breast cancer and 5 lung cancer patients with LMD and 4 LMD-negative controls. To conduct an unbiased search for somatic mutations, we used a custom-designed gene panel and our validated True2 sequencing workflow with a specificity of &amp;lt;1 false positive per 100kb panel positions and a 97.6% sensitivity for detecting single nucleotide variants (SNVs) with a variant allele frequency (VAF) ≥0.1%. Although CSF-cfDNA quantity ranged widely (&amp;lt;1 to 91 ng), genomic alterations were observed in 100% of LMD patients vs. none in the controls. In 13 patients with a SNV, an average of 4.5±3.3 variants were detected with a VAF ranging from 0.051% (HER2 p.S1151L associated with a 12-fold amplification and read depth of 25,542X) to 98.7% (PTEN p.Q733X associated with a loss of heterozygosity). Importantly, subclones in cancer driver genes were detected in 10 of 13 (76.9%) samples consistent with a high prevalence in LMD. Somatic mutations in genes common to both cancers were discovered (e.g., TP53, KMT2D, and EGFR), while other affected genes segregated based on cancer type (e.g., PIK3CA, ATM, and CDH1 in breast cancer; PTEN, MTOR and PDGFRA in lung cancer). An amplification or deletion was detected in 10 of 14 patients (71.4%), a subset of which segregated by cancer type (e.g., HER2 and ESR1 amplifications in breast cancer; CDKN2A and TP53 codeletions in lung cancer). Using serially collected CSF samples, we observed: (1) loss of subclones in response to therapy followed by resurgence at clinical recurrence showing suppression of disease but not eradication; (2) multiple subclones in the same gene supporting evidence of convergent evolution; and (3) emergence of new subclones at clinical recurrence consistent with mutation evolution in response to therapy. For example, in a breast cancer patient with a known HER2 amplification receiving intrathecal Trastuzumab, a previously absent HER2 p.V777L mutation was present at the end of therapy with a VAF of 18.6% which increased at clinical recurrence to 94.8% in association with a 2.4-fold HER2 amplification indicating the mutated HER2 subclone was already therapy-resistant. Our findings support a panel-based approach to detect and monitor LMD-derived somatic mutations using CSF-cfDNA. Moreover, the untargeted search for somatic mutations via True2 has the potential to guide personalized, adaptive therapeutics for LMD patients which may prove essential in improving patient survival. Citation Format: Yingqi Zhang, Rachna Malani, Charlie S Dean, Laura A Boatz, Brion E Harrison, Mei Wei, Wallace Akerley, Mary P Bronner, Hunter R Underhill. Molecular profiling of serially collected cerebrospinal fluid uncovers therapy-resistant subclones in recurrent leptomeningeal metastatic disease [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr B043.

Abstract A022: Novel technology affords real-time PCR the sensitivity required for minimal residual disease monitoring

Abstract Monitoring minimal residual disease (MRD) provides invaluable information for disease progression and therapeutic decision making. However, there are many challenges to reliably detect key mutations or biomarkers, such as the accessibility, turnaround time, and sensitivity of current methods. High sensitivity is crucial due to the limited DNA available in certain sample types such as liquid biopsy. Next generation sequencing (NGS) is capable of highly sensitive detection but is dependent on high sample input and read depth, which is not always feasible due to the variable nature of circulating tumor DNA (ctDNA) in blood or plasma. In addition, NGS is not an economical nor rapid option for recurrent longitudinal monitoring. Digital PCR (dPCR) offers the sensitivity required for MRD monitoring but has yet to be widely adopted for this application. While real-time and qPCR are commonly used in oncology research, RT-PCR does not have the sensitivity required for early detection of key MRD mutations. Here, we present novel patent-pending technology that enhances the sensitivity of RT-PCR, allowing this common platform to be used to reliably detect low abundant mutations. We performed comparisons between GT Molecular’s patent-pending technology paired with RT-PCR and commercially available research use only dPCR and NGS assays to assess the sensitivity this new technology provides for RT-PCR. Cell-free DNA (cfDNA) was extracted from real patient or contrived plasma samples using the QIAamp Circulating Nucleic Acid Kit and analyzed using RT-PCR and dPCR assays targeting KRAS or ESR1 mutations. Samples with KRAS or ESR1 mutations were tested at variant allele frequencies (VAFs) varying between 1%-0.01% to evaluate the ability of GT Molecular’s new technology to detect low abundant mutations using RT-PCR. KRAS and ESR1 mutations were reliably detected with RT-PCR at VAFs as low as 0.01%, demonstrating sensitivity comparable to dPCR. Samples demonstrating positivity for KRAS or ESR1 mutations via dPCR were also positive via RT-PCR. With this patent-pending technology, RT-PCR is now capable of achieving the sensitivity required for MRD monitoring, providing critical information from liquid biopsy samples at a fraction of the cost. Citation Format: Mary Stischer, Nick Allen, Stephanie Barbari, Sarah Kane. Novel technology affords real-time PCR the sensitivity required for minimal residual disease monitoring [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr A022.

Abstract PR009: Application of Liquid Biopsy-RECIST (LB-RECIST) criteria to track metastatic colorectal cancer (mCRC) molecular response in first-line (1L) setting: Findings from the PLATFORM-B study

Abstract Introduction: Circulating tumor DNA (ctDNA) analysis is a minimally invasive and reproducible method to explore tumor molecular biology. Recently, Gouda et al. proposed the LB-RECIST criteria to monitor molecular response, validating them on a heterogeneous metastatic population. However, data on mCRC are lacking. Here, we report the first attempt to apply LB-RECIST in the multicenter prospective PLATFORM-B study, which enrolled 130 mCRC patients (pts) receiving 1L standard therapy, with ctDNA analysis performed at different time points. Materials and Methods: Baseline (BL) and week 8 (W8) plasma samples were analyzed using a targeted NGS-based approach (Ion S5 system). Changes in ctDNA detectability were assessed to determine molecular response as per qualitative LB-RECIST criteria: Group1 (G1, detectable ctDNA at BL and W8); Group2 (G2, detectable BL and undetectable W8); Group3 (G3, undetectable BL and detectable W8); Group4 (G4, undetectable at BL and W8). Changes in aggregate variant allele frequency (VAF) - the sum of all VAFs of the studied genes in a sample - were used to assess quantitative LB-RECIST responses: ctDNA Complete Response (CCR, clearance after initial detectability), Partial Response (CPR, decrease &amp;gt;10%), Stable Disease (CSD, no change or decrease &amp;lt;10%), Progressive Disease (CPD, increase &amp;gt;10% or new mutations), and Non-measurable Disease (CND, undetectable at BL and W8). Molecular results were correlated with clinical outcomes. Statistically significant differences were assessed across different groups using 2-sided P values with an α ≤ .05 level of significance. Results: Overall, 79 pts had paired BL and W8 samples. Quantitative LB-RECIST identified 32 CCR, 18 CND, 20 CPR, 8 CPD, and 1 CSD cases. Qualitative LB-RECIST found 22 G1, 32 G2, 7 G3, and 18 G4 cases. Radiological RECIST identified 6 CR, 47 PR, 21 SD, and 5 PD. CCR showed the highest survival rates: with a median follow-up of 25 months (mo) (range: 1–49), median progression-free survival (mPFS) was not reached (NR) and median overall survival (mOS) was 41.8 mo. The CND group had similar rates (mPFS NR; mOS 41.1 mo). CPR and CPD had the poorest outcomes: for CPR, mPFS was 12.7 and mOS 16.4 mo; for CPD, mPFS and mOS were 11.9 and 25.5 mo, respectively. In the G1, G2, G3, and G4 groups, mPFS was 11.9, NR, 14.2, and NR, respectively, while mOS was 16.4, 41.8, 33.4, and 41.1 mo. Kaplan-Meier analysis of the quantitative groups showed statistically significant differences (P &amp;lt; .0001) for both PFS and OS curves. The same level of significance was found in the qualitative groups. Conclusion: LB- RECIST can be a rapid and reproducible method to assess molecular response in mCRC. However, concerns remain. The CND group (23% of cases) showed survival rates similar to the CCR group, as observed also between G4 and G2. These data suggest that absence of ctDNA at W8 is the best surrogate for OS. Moreover, CPD had higher OS rates than CPR, but similar PFS. Further investigations in larger populations are needed to refine these criteria and improve molecular response stratification Citation Format: Valentino Martelli, Joana Vidal, Maria Concepción Fernández-Rodríguez, Pilar García-Alfonso, David Páez, Joan Gibert, Jennifer Linares, Annarita Sibilio, Vicente Alonso, Maria Teresa Cano, Cristina Santos, Gema Duran, Elena Élez, José Luis Manzano, Rocio Garcia-Carbonero, Reyes Ferreiro, Ferran Losa, Estela Pineda, Javier Sastre, Auxiliadora Gomez-España, Rodrigo Toledo, Fernando Rivera, Beatriz Bellosillo, Josep Tabernero, Ramon Ramon Salazar, Enrique Aranda, Clara Montagut. Application of Liquid Biopsy-RECIST (LB-RECIST) criteria to track metastatic colorectal cancer (mCRC) molecular response in first-line (1L) setting: Findings from the PLATFORM-B study [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr PR009.

Abstract B025: Monitoring response to immunotherapy in lung cancer using cell-free DNA fragmentomes

Abstract BACKGROUND: Immune checkpoint inhibition (ICI) continues to be a promising area of research with treatments continuing to show efficacy in late-stage cancers. The detection of disease progression for patients undergoing treatment with this modality remains challenging however given the lack of reliable biomarkers for which to monitor disease. Current methods including imaging can be challenging to evaluate as they do not always capture the timing and nature of clinical responses. Liquid biopsies may offer a solution by allowing for minimally invasive tumor monitoring during therapy. We previously reported on the DELFI Tumor Fraction (DELFI-TF) algorithm, as a tumor- and mutation-naive approach to monitoring using cell-free DNA (cfDNA) fragmentomes (Alipanahi et al., AACR 2023). In this study we examine the performance of DELFI-TF in a cohort of patients with metastatic NSCLC treated with ICI. METHODS: Longitudinal blood samples (n=323) were collected from NSCLC patients (n=109) which received ICI therapy. Whole genome sequencing at low coverage (∼4x) was performed following cfDNA extraction. DELFI-TF scores, predicted using fragmentome features, were used to monitor changes in circulating tumor DNA (ctDNA) levels and associated variations in disease burden. Predictions from DELFI-TF were compared to maximum observed mutation allele frequencies (maxMAF) from a targeted sequencing gene panel for the same samples. In order to determine the prognostic performance of DELFI-TF we evaluated ctDNA changes at baseline and within 3 to 9 weeks of ICI initiation. The presence of undetectable DELFI-TF, defined as below the observed limit of blank (LOB) within 3 to 9 weeks of ICI initiation, indicated a molecular response (mR). In contrast, the persistence of DELFI-TF within this interval signified molecular disease progression (mPD). RESULTS: We observed a strong correlation between DELFI-TF predictions and maxMAF (r=0.93, p&amp;lt;0.001). Further, changes in ctDNA predictions from baseline to the first post-treatment timepoint showed a high correlation between DELFI-TF and maxMAF (r=0.94, p&amp;lt;0.001). Patients with a higher disease burden prior to immunotherapy, as predicted by DELFI-TF and maxMAF, had shorter overall survival (OS) compared to patients with a lower disease burden (DELFI-TF: 11.4 v 33.0 months, p=0.006; maxMAF: 7.82 vs low 33.0 months, log-rank p=0.001). Among 79 patients evaluable for molecular response, 28 (35%) were classified as mPD and 51 (65%) were classified in the mR category. Patients with mR attained longer progression free survival (PFS;16.04 v 2.70 months, log-rank p&amp;lt;0.001) compared to those in the mPD group. Furthermore, assessment of landmark PFS at 6 months revealed a significant association between ctDNA molecular response and durable clinical benefit (DCB; p &amp;lt; 0.001, Fisher’s exact test). CONCLUSIONS: DELFI-TF is a low-cost, tumor and mutation naive approach that accurately monitors longitudinal dynamic changes in tumor burden and captures durable therapeutic responses in patients with metastatic NSCLC receiving immunotherapy. Citation Format: Lavanya Sivapalan, Bahar Alipanahi, Jamie E Medina, Bridget Tripp, Zachary L Skidmore, Paola Ghanem, Gavin Pereira, Nisha Rao, Kavya Velliangiri, Caitlin Schonewolf, Noushin Niknafs, Bryan Chesnick, Jennifer Tom, Stephen Cristiano, Peter Bach, Nicholas C Dracopoli, Robert B Scharpf, Victor Velculescu, Lorenzo Rinaldi, Valsamo Anagnostou. Monitoring response to immunotherapy in lung cancer using cell-free DNA fragmentomes [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr B025.

Abstract B059: Assessment of ctDNA analysis methods using high purity ctDNA from the aqueous humor of retinoblastoma eyes

Abstract Background: Whole-genome sequencing (WGS) of cell-free DNA (cfDNA) paired with methods like ichorCNA and DELFI has become a valuable approach for detecting circulating tumor DNA (ctDNA). However, the mixture of ctDNA with non-tumor cfDNA in plasma complicates accurate validation of ctDNA detection methods. In contrast, aqueous humor (AH) from treatment-free retinoblastoma (RB) eyes is uniquely enriched in ctDNA, providing an optimal source for generating reference samples. This study leverages AH-derived biological ctDNA to evaluate two established ctDNA analysis methods. Methods: Biologically derived cfDNA mixtures were created by combining WGS reads from two AH samples with pooled reads from non-tumor blood samples (N=20). One AH sample (RB_023_OS) showed &amp;gt;99% tumor fraction (TFx) with ichorCNA, while the other (RB_025_OD) had confirmed presence of ctDNA based on &amp;gt;99% variant allele frequency (VAF) for an RB1 variant, despite being somatic copy number alteration (SCNA)-negative. Mixtures were prepared across a range of TFx values (0.01-0.5) and unique sequencing coverages (0.1x, 0.5x, 1x, and 2x). TFx was calculated using ichorCNA with standard parameters and optimized parameters for low TFx levels. We also tested an enrichment strategy targeting shorter ctDNA fragments (≤150 base pairs) and generated genome-wide fragmentation profiles using the DELFI method to assess short-to-long (S:L) fragment ratios across 5-Mb bins. Results: We evaluated the impact of sequencing coverage, parameter selection, and fragment size selection on TFx estimation by ichorCNA. Standard ichorCNA parameters demonstrated limited sensitivity, detecting ctDNA only in samples with TFx of 5-6% or higher. Increasing sequencing coverage did not substantially improve the limit of detection (LoD) or the accuracy of TFx quantification. Optimized parameters reduced the LoD to 3% TFx, but only in samples with ≥0.5x coverage, indicating a dependence on sequencing depth for accurate ctDNA detection and quantification. In RB_025_OD, where no SCNAs were present, ichorCNA failed to estimate the TFx despite an RB1 VAF exceeding 99%, highlighting the platform's reliance on SCNAs for accurate TFx detection. Short fragment enrichment (≤150 base pairs) significantly increased TFx values across all samples but could be mapped back to the original TFx values accurately using polynomial curve fitting (R-squared=0.97). Genome- wide fragmentation profiles showed increasing deviation from non-tumor controls as TFx increased, with SCNA-positive samples exhibiting greater deviations than SCNA-negative samples. Conclusions: These findings demonstrate the dependence on sequencing coverage and the presence of SCNAs by current ctDNA analysis methods, highlighting their limitations for accurately detecting ctDNA in challenging scenarios. Citation Format: Nikki Higa, Peter Kuhn, Jesse L Berry, Liya Xu. Assessment of ctDNA analysis methods using high purity ctDNA from the aqueous humor of retinoblastoma eyes [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr B059.

Abstract A017: High-throughput microfluidic enrichment of circulating tumor cells from entire diagnostic leukapheresis for comprehensive liquid biopsy

Abstract Circulating Tumor Cells (CTCs) in blood containing a complete set of single-cell analytes, including high-molecular-weight DNA, intact RNA, and both intracellular and cell-surface proteins serve as a promising tool in cancer diagnosis and prognosis through liquid biopsy. However, their clinical utility has been limited by their low blood concentration and inadequate enrichment technology. Diagnostic leukapheresis holds promise for isolating sufficient CTCs, but enriching ultra-rare cells from large, complex fluid volumes remains challenging. To address this, we developed the LPCTC-iChip, a high-throughput microfluidic device that uses high-flow channels and force-amplifying magnetic lenses to deplete hematopoietic cells, allowing tumor epitope-agnostic screening of massive blood volumes. Here, we present the first application of LPCTC-iChip to entire diagnostic leukapheresis products (leukopak) from seven patients with metastatic cancer, processing an average of 5.83 liters of blood per patient. Enumeration and morphology characterization of CTCs using multispectral immunofluorescence imaging identified high CTC yields - a mean of 10,057 CTCs per leukopak and showed notable intra- patient heterogeneity. Nuclear and cell diameter of CTC varies within individual patients, with 67% overlapping in size with patient-matched white blood cells. Paired single-cell DNA and RNA sequencing uncovered subclonal aneuploidy and distinct signaling pathways within CTCs with shared, clonal copy number variation (CNV) patterns. A subset of small aneuploid cells lacking epithelial markers was enriched for neuroendocrine characteristics in prostate cancer samples. The mutations found by FDA-approved genetic tests in either biopsied metastatic lesions or blood-based ctDNA sampling were reproduced by whole exome sequencing of a pool of CNV-confirmed CTCs. In addition, the sequencing revealed additional cancer-related high allele frequency variants that are not detectable with ctDNA or tissue needle biopsies. Overall, this high-throughput CTC enrichment from complete leukapheresis combined with multispectral imaging and single cell sequencing technology enables a comprehensive, cell-based liquid biopsy approach for early cancer detection and therapeutic responses monitoring. Citation Format: Avanish Mishra, Shih-Bo Huang, Taronish Dubash, Risa Burr, Jon F. Edd, Ben S. Wittner, Quinn E. Cunneely, Victor R. Putaturo, Akansha Deshpande, Ezgi Antmen, Kaustav A. Gopinathan, Keisuke Otani, Yoshiyuki Miyazawa, Ji Eun Kwak, Sara Y. Guay, Justin P. Kelly, John Walsh, Linda T. Nieman, Isabella Galler, PuiYee Chan, Michael S. Lawrence, Ryan J. Sullivan, Aditya Bardia, Douglas S. Micalizzi, Lecia V. Sequist, Richard J. Lee, Joseph W. Franses, David T. Ting, Patricia A. R. Brunker, Shyamala Maheswaran, David T. Miyamoto, Daniel A. Haber, Mehmet Toner. High-throughput microfluidic enrichment of circulating tumor cells from entire diagnostic leukapheresis for comprehensive liquid biopsy [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr A017.

Allele frequency impacts the cross-ancestry portability of gene expression prediction in lymphoblastoid cell lines.

Population-level genetic studies are overwhelmingly biased toward European ancestries. Transferring genetic predictions from European ancestries to other ancestries results in a substantial loss of accuracy. Yet, it remains unclear how much various genetic factors, such as causal effect differences, linkage disequilibrium (LD) differences, or allele frequency differences, contribute to the loss of prediction accuracy across ancestries. In this study, we used gene expression levels in lymphoblastoid cell lines to understand how much each genetic factor contributes to lowered portability of gene expression prediction from European to African ancestries. We found that cis-genetic effects on gene expression are highly similar between European and African individuals. However, we found that allele frequency differences of causal variants have a striking impact on prediction portability. For example, portability is reduced by more than 32% when the causal cis-variant is common (minor allele frequency, MAF >5%) in European samples (training population) but is rarer (MAF <5%) in African samples (prediction population). While large allele frequency differences can decrease portability through increasing LD differences, we also determined that causal allele frequency can significantly impact portability when the impact from LD is substantially controlled. This observation suggests that improving statistical fine-mapping alone does not overcome the loss of portability resulting from differences in causal allele frequency. We conclude that causal cis-eQTL effects are highly similar in European and African individuals, and allele frequency differences have a large impact on the accuracy of gene expression prediction.

A path integral approach for allele frequency dynamics under polygenic selection.

Many phenotypic traits have a polygenic genetic basis, making it challenging to learn their genetic architectures and predict individual phenotypes. One promising avenue to resolve the genetic basis of complex traits is through evolve-and-resequence experiments, in which laboratory populations are exposed to some selective pressure and trait-contributing loci are identified by extreme frequency changes over the course of the experiment. However, small laboratory populations will experience substantial random genetic drift, and it is difficult to determine whether selection played a role in a given allele frequency change. Predicting allele frequency changes under drift and selection, even for alleles contributing to simple, monogenic traits, has remained a challenging problem. Recently, there have been efforts to apply the path integral, a method borrowed from physics, to solve this problem. So far, this approach has been limited to genic selection, and is therefore inadequate to capture the complexity of quantitative, highly polygenic traits that are commonly studied. Here we extend one of these path integral methods, the perturbation approximation, to selection scenarios that are of interest to quantitative genetics. We derive analytic expressions for the transition probability (i.e., the probability that an allele will change in frequency from x to y in time t) of an allele contributing to a trait subject to stabilizing selection, as well as that of an allele contributing to a trait rapidly adapting to a new phenotypic optimum. We use these expressions to characterize the use of allele frequency change to test for selection, as well as explore optimal design choices for evolve-and-resequence experiments to uncover the genetic architecture of polygenic traits under selection.

Abstract 4148070: Clonal Hematopoeisis of Indeterminate Potential and Risk of Autopsy-defined Sudden Cardiac Death

Introduction: Clonal hematopoiesis of indeterminate potential (CHIP) has been associated with an increased risk of coronary artery disease (CAD) and cardiovascular (CV) mortality. Whether CHIP may affect myocardium beyond promoting CAD, or its potential association with sudden cardiac death (SCD), the most feared manifestation of CV disease, is unknown. Research Questions/Hypothesis: We hypothesize that CHIP-mutant macrophages infiltrate the myocardium and are associated with autopsy-defined arrhythmic death. Goals/Aims: (1) determine rate of CHIP at time of death in an unselected, countywide study of all incident sudden deaths; (2) evaluate whether CHIP-mutant macrophages infiltrate into myocardium; and (3) evaluate potential association of CHIP with autopsy-confirmed arrhythmic causes among countywide sudden deaths Methods/Approach: We used autopsy to adjudicate arrhythmic from non-arrhythmic (PE, stroke, overdose, tamponade) causes in 1,148 sudden deaths and 141 trauma control deaths in San Francisco County from 2011-23. DNA was extracted from frozen blood samples collected at time of death from 399 consented cases. Sequencing of 22 CHIP-associated genes was performed to ~2000x mean target coverage. All pathogenic/likely pathogenic CHIP variants at &gt;2% variant allele frequency (VAF) were considered CHIP+. DNA was then isolated from left ventricular (LV) tissue sampled at autopsy in all CHIP+ blood cases and sequenced as above. Results: Of 399 consented cases, 70 blood samples (17.5%) were CHIP+ (range 2%-39.6%; mean age 70.6 years vs. 58.1 years for CHIP-). The most frequent mutant genes were DNMT3A, TET2, and ASXL1. Proportion of arrhythmic causes among sudden deaths was similar for CHIP+ vs. CHIP- cases (36 of 70 [52%] vs. 171 of 329 [51%]). Sequencing of available LV tissue from 61 CHIP+ blood cases revealed 67% (n=41) harbored the same CHIP mutation identified in blood at &gt;0.2% VAF (range 0.2%-4.3%). Proportion of arrhythmic causes among sudden deaths was significantly higher in cases where both blood and LV were CHIP+ (24 of 41 [58.5%] vs. 5 of 19 [26.3%] CHIP+ blood/CHIP- LV, p=0.02). Conclusions: Prevalence of CHIP positivity at sudden death is similar to published studies and correlated with age. CHIP+ status in myocardial tissue but not peripheral blood was associated with arrhythmic causes of sudden death, suggesting a direct tissue effect of CHIP-mutant macrophages.

Abstract 4145859: Clonal Hematopoiesis of Indeterminate Potential Increases Mortality Risk in Coronary Artery Disease

Background: Clonal hematopoiesis of indeterminate potential (CHIP) has been associated with an increased incidence of common late-onset diseases including myocardial infarction. Research Question: Here we examined the impact of CHIP on mortality in patients with coronary artery disease (CAD) and further explored potential underlying mechanisms. Methods: CAD patients were examined for CHIP-defining mutations using deep DNA sequencing. Propensity score matching was used to compare CHIP CAD with non-CHIP CAD patients. Survival was analyzed using Kaplan-Meier curves and Cox proportional hazard models. Functional aspects were investigated at the protein and RNA level in cardiovascular relevant human postmortem tissue from the Munich cardIovaScular StudIes biObaNk (MISSION), in CAD patients from an independent cohort and in in-vitro macrophage models. Results: Within 7,933 CAD patients screened by deep-DNA-sequencing with a 13 gene panel, 2,002 carried at least one CHIP defining mutation with a variant allele frequency ≥2%. After 1:1 matching with non-CHIP carriers we revealed a significantly elevated 3-year mortality in the group of CHIP carriers [HR 1.48 (CI 95% 1.23-1.78); p&lt;0.0001)] as well as in carriers of mutations in one out of seven individual genes – ASXL1 , DNMT3A , PPM1D , SF3B1 , SRSF2 , TET2 , and U2AF1 . In human coronary atherosclerotic plaque we visualized CHIP-mutations within leukocytes. Further, within coronary artery plaque of deceased TET2 mutation carriers (n=26 vs. n=13 controls) we identified by proteomic profiling an upregulation of inflammatory and metabolic pathways, including enhanced lipid metabolism. In an independent cohort of CAD patients undergoing coronary bypass surgery, RNA sequencing data from monocyte-derived macrophages revealed in TET2 CHIP carriers associations with CAD complexity. TET2 -mutated macrophages studied in-vitro revealed an increased uptake of low-density lipoprotein (LDL) and oxidized LDL-cholesterol besides a known upregulation of inflammatory pathways. Conclusion: In summary, this study emphasizes CHIP as a significant prognostic factor in CAD patients, associated with increased complexity of CAD and altered gene expression within human coronary artery plaques. Mechanistically, TET2 mutations modify the behavior of CHIP-affected macrophages, leading to heightened inflammation and increased uptake of LDL-cholesterol.