Detection Of DNA Mutations Research Articles

Robustness and reliability of single-cell regulatory multi-omics with deep mitochondrial mutation profiling.

The detection of mitochondrial DNA (mtDNA) mutations in single cells holds considerable potential to define clonal relationships coupled with information on cell state in humans. Previous methods focused on higher heteroplasmy mutations that are limited in number and can be influenced by functional selection, introducing biases for lineage tracing. Although more challenging to detect, intermediate to low heteroplasmy mtDNA mutations are valuable due to their high diversity, abundance, and lower propensity to selection. To enhance mtDNA mutation detection and facilitate fine-scale lineage tracing, we developed the single-cell Regulatory multi-omics with Deep Mitochondrial mutation profiling (ReDeeM) approach, an integrated experimental and computational framework. Recently, some concerns have been raised about the analytical workflow in the ReDeeM framework. Specifically, it was noted that the mutations detected in a single molecule per cell are enriched on edges of mtDNA molecules, suggesting they resemble artifacts reported in other sequencing approaches. It was then proposed that all mutations found in one molecule per cell should be removed. We detail our error correction method, demonstrating that the observed edge mutations are distinct from previously reported sequencing artifacts. We further show that the proposed removal leads to massive elimination of bona fide and informative mutations. Indeed, mutations accumulating on edges impact a minority of all mutation calls (for example, in hematopoietic stem cells, the excess mutations on the edge account for only 4.3%-7.6% of the total). Recognizing the value of addressing edge mutations even after applying consensus correction, we provide an additional filtering option in the ReDeeM-R package. This approach effectively eliminates the position biases, leads to a mutational signature indistinguishable from bona fide mitochondrial mutations, and removes excess low molecule high connectedness mutations. Importantly, this option preserves the large majority of unique mutations identified by ReDeeM, maintaining the ability of ReDeeM to provide a more than 10-fold increase in variant detection compared to previous methods. Additionally, the cells remain well-connected. While there is room for further refinement in mutation calling strategies, the significant advances and biological insights provided by the ReDeeM framework are unique and remain intact. We hope that this detailed discussion and analysis enables the community to employ this approach and contribute to its further development.

MADDD-seq, a novel massively parallel sequencing tool for simultaneous detection of DNA damage and mutations.

Our genome is exposed to a wide variety of DNA-damaging agents. If left unrepaired, this damage can be converted into mutations that promote carcinogenesis or the development of genetically inherited diseases. As a result, researchers and clinicians require tools that can detect DNA damage and mutations with exceptional sensitivity. In this study, we describe a massively parallel sequencing tool termed Mutation And DNA Damage Detection-seq (MADDD-seq) that is capable of detecting O6-methyl guanine lesions and mutations simultaneously, with a single assay. To illustrate the dual capabilities of MADDD-seq, we treated WT and DNA repair deficient yeast cells with the DNA-damaging agent MNNG and tracked DNA lesions and mutations over a 24-htime period. This approach allowed us to identify thousands of DNA adducts and mutations in a single sequencing run and gain deep insight into the kinetics of DNA repair and mutagenesis.

Simultaneous detection of tumor-derived mutations in DNA and RNA extracted from a single circulating tumor cell based on droplet digital polymerase chain reaction.

e15053 Background: Androgen receptor splice variant 7 (AR-V7) is a constitutively activated isoform of AR and has been associated with resistance to AR-targeted therapies and development of metastatic castration-resistant prostate cancer. Confirmation of AR-V7 expression is important step in prostate cancer diagnosis and determining the appropriate drug, however, the detection of AR-V7 with tissues has limitations, such as the clinical challenge to poorly accessible tumor tissues before and after therapy. In addition, KRAS mutations serve as a prognostic biomarker for the administration of appropriate anti-cancer drugs in patients with various cancers, including non-small cell lung cancer (NSCLC). Here, we developed the process for the simultaneous detection of AR-V7 and KRAS mutation in CTCs using isolation with CytoGen’s Smart Biopsy™ System with droplet digital polymerase chain reaction (ddPCR) with high sensitivity and specificity, overcoming the limitations of tissue biopsy. Methods: ddPCR assays were performed to detect the KRAS G12C mutation using CTC gDNA and to evaluate the absolute levels of AR-V7 using CTC mRNA. Analytical validation was performed using NSCLC NCI-H358 and prostate cancer 22Rv1 cells (10 and 5 cells each), which harbor KRAS G12C and AR-V7, respectively, spiked into 5 mL of healthy blood to mimic CTC. CTCs were isolated using CytoGen's Smart Biopsy™ System. Oligo-dT magnetic beads were used to isolate mRNA from CTCs, and gDNA was isolated from the mRNA-depleted CTC lysates using the AllPrep DNA/mRNA Nano Kit. PCR and droplet reading for each assay were performed on the QX200 AutoDG Droplet Digital PCR System. Results: As a result of CTC KRAS G12C detection using the ddPCR™ KRAS G12/G13 Screening Kit, the mutated KRAS gene was successfully detected in as few as 5 NCI-H358 cells. In the results of CTC AR-V7 detection using the custom TaqMan ddPCR assay, transcripts (AR-V7 and HPRT1) were successfully detected even with only 5 22Rv1 cells. These results suggest that CytoGen's Smart Biopsy™ System could be applied to the simultaneous detection of DNA/RNA mutated genes in clinical NSCLC and prostate cancer patients with detection technology requiring high sensitivity and specificity. Conclusions: Accurately detecting multiple genetic mutations in CTCs is a challenging because CTCs are very rare in the blood. In this study, we developed the process to simultaneously extract and detect DNA mutations and RNA splicing variants in CTCs using CytoGen's Smart Biopsy™ System. This process, along with the results from our ongoing clinical trials, could provide information for the diagnosis and appropriate treatment of patients with various cancers, including NSCLC and prostate cancer.

CRISPR/Cas13a-based supersensitive circulating tumor DNA assay for detecting EGFR mutations in plasma

Despite recent technological advancements in cell tumor DNA (ctDNA) mutation detection, challenges persist in identifying low-frequency mutations due to inadequate sensitivity and coverage of current procedures. Herein, we introduce a super-sensitivity and specificity technique for detecting ctDNA mutations, named HiCASE. The method utilizes PCR-based CRISPR, coupled with the restriction enzyme. In this work, HiCASE focuses on testing a series of EGFR mutations to provide enhanced detection technology for non–small cell lung cancer (NSCLC), enabling a detection sensitivity of 0.01% with 40 ng cell free DNA standard. When applied to a panel of 140 plasma samples from 120 NSCLC patients, HiCASE exhibits 88.1% clinical sensitivity and 100% specificity with 40 μL of plasma, higher than ddPCR and Super-ARMS assay. In addition, HiCASE can also clearly distinguish T790M/C797S mutations in different positions at a 1% variant allele frequency, offering valuable guidance for drug utilization. Indeed, the established HiCASE assay shows potential for clinical applications.

Development of a nine-variant reference material panel to standardize cell-free DNA detection.

Detection of specific gene mutations in cell-free DNA (cfDNA) serves as a valuable cancer biomarker and is increasingly being explored as an appealing alternative to tissue-based methods. However, the lack of available reference materials poses challenges in accurately evaluating the performance of different assays. In this study, we present the development of a comprehensive reference material panel for cfDNA detection, encompassing nine hotspot mutations in KRAS/BRAF/EGFR/PIK3CA at three variant allele frequencies (VAFs), ranging from 0.33 to 23.9%. To mimic cfDNA, these reference materials were generated by enzymatically digesting cell-line DNA into approximately 154-bp to 173-bp fragments using a laboratory-developed reaction system. The VAFs for each variation were precisely determined through validated digital PCR assays with high accuracy. Furthermore, the reliability and applicability of this panel were confirmed through two independent NGS assays, yielding concordant results. Collectively, our findings suggest that this novel reference material panel holds great potential for validation, evaluation, and quality control processes associated with liquid biopsy assays.

Abstract A001: Urine-based liquid biopsy test for the detection of residual bladder cancer in radical cystectomy patients

Abstract One commonly employed management strategy for muscle-invasive bladder cancer (MIBC) is neoadjuvant chemotherapy followed by radical cystectomy (RC) and urinary diversion. Approximately 35% of patients achieve a pathologic complete response (pCR) when evaluated from RC specimens. RC with urinary diversion is a life-altering operation with high complication rates and therefore determining who are complete responders would improve quality of life and could decrease costs and still achieve a high cure rate via RC avoidance. Unfortunately, current methods cannot accurately identify complete responders. Our lab has developed a urine-based liquid biopsy test in which DNA isolated from urine and germline is isolated and subjected to panel exome sequencing for enumeration of somatic variants. This occurs under the premise that tumor DNA identified (as mutant alleles) is a marker of residual disease while the absence of tumor DNA (meaning the absence of mutant alleles) can indicate a pCR. A 60 gene panel was designed to be used for exonic sequencing of urinary DNA (uDNA) to identify mutations indicative of bladder cancer or therapeutic targets. We have shown that most mutations in tumor tissue (Mt) are detectable as mutations in urine (Mu). We also showed that the presence or absence of residual Mu after completion of chemotherapy strongly associates with residual disease or pCR at the time of RC, respectively. Therefore, this test could be used after neoadjuvant therapy to better identify patients for RC avoidance. We optimized this test by studying pre-analytical factors affecting the yield, purity, and integrity of uDNA and the quality of sequencing data using prospectively collected samples from patients with MIBC. We are also in the process of testing commercial and novel preservatives to optimize DNA integrity, yield, and mutation detection accuracy to enable temporary ambient temperature storage without compromising mutation detection, permitting shipping/centralized testing. Lastly, we are comparing two library preparation approaches. Study of these preanalytical factors has increased the number of Mu that can be detected and were applied to a cohort of patients who underwent bladder sparing curative-intent treatment in a prospective clinical trial. Studies described herein will enable liquid biopsy tests to accurately identify patients with pCR after neoadjuvant therapy and might safely avoid radical cystectomy. Citation Format: Philip H. Abbosh, Henkel Valentine, Rhea Arya. Urine-based liquid biopsy test for the detection of residual bladder cancer in radical cystectomy patients [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2024 May 17-20; Charlotte, NC. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(10_Suppl):Abstract nr A001.

Detection of Oncogene Hotspot Mutations in Female NSCLC Tumor DNA and Cell-Free DNA.

Non-small-cell lung cancer (NSCLC) is the most prevalent type of lung cancer, with extensively characterized mutational spectra. Several biomarkers (such as EGFR, BRAF, KRAS gene mutations, etc.) have emerged as predictive and prognostic markers for NSCLC. Unfortunately, the quality of the available tumor biopsy and/or cytology material is not always adequate to perform the necessary molecular testing, prompting the search for alternatives. Cell-free DNA (cfDNA) found in plasma is emerging as a highly promising avenue or a supplementary method for assessing the efficacy of cancer treatments. This is especially valuable in instances where conventional biopsy specimens, like formalin-fixed, paraffin-embedded (FFPE), or freshly frozen tumor tissues prove inadequate for conducting molecular pathology analyses subsequent to the initial diagnostic procedures. By leveraging cfDNA from plasma, clinicians gain an additional tool to gauge the effectiveness of cancer therapies, thereby enhancing their ability to optimize tailored treatment strategies. In this study, 51 Lithuanian females with NSCLC were analyzed, with adenocarcinoma being the predominant pathology diagnosis in 40 cases (78%). Target mutations were identified in 38 out of 51 patients (74.5%) in tumor tissue samples, while in plasma samples, they were identified in only 10 patients' samples (19.6%). Even though we did not have enough voluminous plasma samples in our study, gene mutations were detected in plasma from ten women, three of whom were diagnosed with early stages of lung cancer (stages I and II). For these patients, the following mutations were detected: deletion in exon 19 of the EGFR gene and single nucleotide polymorphisms in the TP53 and MET genes. All other women were diagnosed with stages III or IV of lung cancer. This indicates that the later stages of cancer contribute more cfDNA in plasma, making extraction less complicated.

Abstract PO3-16-05: Mitochondrial DNA mutation detection in tumors and circulating extracellular vesicles of triple negative breast cancer patients for biomarker development

Abstract Early detection of triple-negative breast cancer (TNBC) patients and their recurrence prediction are daunting tasks. We aim to develop mitochondrial DNA (mtDNA) based biomarkers to improve the management strategies of TNBC patients. We have undertaken mitochondrial genome (MG) enrichment and next-generation sequencing approaches to map the entire MG in 73 samples (64 tissues, 9 extracellular vesicle samples) from 32 highly-aggressive TNBC patients. Measurement of mtDNA and cardiolipin contents, in concert with NDUFB8 and SDHB proteins’ expression, was carried out in tumors and matched extracellular vesicles (EVs) of all the above TNBC patients. Overall, we identified 168 nonsynonymous mtDNA mutations, of which 73% (123/186) were coding and 27% (45/168) were non-coding. Twenty percent of mutations were nucleotide transversions. Respiratory complex I (RCI) appears to be the key target, which harbored 44% (74/168) of the overall mtDNA mutations. A panel of 11 hotspot mtDNA mutations was identified among 19-38% TNBCs, each of which was detectable in the matched EV samples with 82% specificity. Overall, 38% of the metastatic tumor-signature mtDNA mutations were detectable in matched EVs of the TNBC patients. An appreciable number of mtDNA mutations were homoplasmic (18%, 31/168), novel (14%, 23/168), and potentially pathogenic (9%, 15/168). The overall and RCI-specific mtDNA mutational load was recorded to be higher in African American (AA) compared to European American (EA) women with an exclusive abundance of respiratory complex (RC) protein NDUFB8 (RCI) and SDHB (RCII) therein. Increased mtDNA (p < 0.0001) content was measured in both tumors and matched EV samples along with an abundance of cardiolipin (p=0.0001) content in the EVs of the TNBC patients. Lethal tumor-signature mtDNA mutation detection and measurement of altered mtDNA and cardiolipin contents in the EVs at an early time point bear the potential to formulate noninvasive early detection and recurrence prediction strategies in TNBCs to improve the overall survival of the patients. Citation Format: Kunwar Vikramdeo, Shashi Anand, Sarabjeet Sudan, Paramahansa Pramanik, Seema Singh, Andrew Godwin, Ajay Singh, Santanu Dasgupta. Mitochondrial DNA mutation detection in tumors and circulating extracellular vesicles of triple negative breast cancer patients for biomarker development [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 PO3-16-05.

Performance characteristics of a polymerase chain reaction-based assay for the detection of EGFR mutations in plasma cell-free DNA from patients with non-small cell lung cancer using cell-free DNA collection tubes.

Survival rates in non-small cell lung cancer (NSCLC) are low. Detection of circulating tumor DNA in liquid biopsy (plasma) is increasingly used to identify targeted therapies for clinically actionable mutations, including EGFR mutations in NSCLC. The cobas® EGFR Mutation Test v2 (cobas EGFR test) is FDA-approved for EGFR mutation detection in tissue or liquid biopsy from NSCLC. Standard K2EDTA tubes require plasma separation from blood within 4 to 8 hours; however, Roche Cell-Free DNA (cfDNA) Collection Tubes (Roche cfDNA tube) enable whole blood stability for up to 7 days prior to plasma separation. This analysis assessed performance of Roche cfDNA tubes with the cobas EGFR test for the detection of EGFR mutations in plasma from healthy donors or patients with NSCLC. Overall, test performance was equally robust with either blood collection tube, eg, regarding limit of detection, linearity, and reproducibility, making Roche cfDNA tubes suitable for routine clinical laboratory use in this setting. Importantly, the Roche cfDNA tubes provided more flexibility for specimen handling versus K2EDTA tubes, eg, in terms of tube mixing, plasma separation, and sample stability, and do not require processing of blood within 8 hours thereby increasing the reach of plasma biopsies in NSCLC.

Abstract 6101: PLASMUT: An R Package for estimating the probability of tumor-specific mutations in cell-free DNA

Abstract Noninvasive approaches for detection of tumor-specific mutations in cell-free DNA (cfDNA) have the potential to track a patient’s response to treatment, enabling effective and timely decisions on therapy. However, mutations in cfDNA arising from clonal hematopoeisis (CH) are common and tumor biopsies for definitive identification of the origin of these mutations are not always available. Sequencing of matched cells from buffy coat and the absence of mutations in these cells has been used to rule-out white blood cell (WBC) mutations, but uneven sequencing depths between matched cfDNA and buffy coat and the fraction of mutant alleles are generally ignored by rule-based tests. A probabilistic approach that quantifies the evidence of tumor-derived mutations in cfDNA is needed. We developed a Bayesian framework to estimate the probability that a mutation identified in cfDNA is tumor specific. Our approach requires the number of reads with a mutant allele in plasma (yp) and WBCs (yw) and the corresponding number of total distinct reads at these locations. The posterior odds that a mutation is tumor-derived (S) versus CH or germline (H) is given by the ratio of the probabilities of observing the distinct reads given each model times the prior odds. Estimation of the Bayes factor is obtained by integrating over the unobserved mutant allele fractions in plasma and WBCs using Monte Carlo importance sampling. We applied this approach to 52 patients with initially unresectable colorectal cancer (CRC) liver metastases in the CAIRO5 clinical trial (NCT02162563), using ultra-deep targeted sequencing of cfDNA from plasma and matched WBCs. Among the CAIRO5 patients analyzed, we identified 95 mutations with moderate evidence of tumor-derived cfDNA mutations (Bayes factor > 10) and 19 mutations that were CH-derived (Bayes factor < 0.1). For a subset of 47 cfDNA mutations with no corresponding mutation identified by WBC sequencing, the evidence of tumor origin was highly variable (Bayes factor range: 0.03 to 5.6). While the standard rule-based approach identifies all of these mutations as tumor-derived, none of these mutations reach a moderate evidence cutpoint (all Bayes factors < 6). As a false positive would lead to identification of cfDNA mutations that do not track tumor burden, requiring even higher levels of evidence (Bayes factor > 99) for the selection of cfDNA mutations could be warranted, and still identifies one or more cfDNA mutations in 43 of the patients. This approach is implemented in the R package PLASMUT available from Bioconductor (doi:10.18129/B9.bioc.plasmut). We developed an approach that quantifies the evidence between two competing models for the origin of mutations in cfDNA. A cutpoint for determination of the probability of tumor-derived cfDNA mutations can be tailored to the disease application, balancing the potential benefits of noninvasive testing with the harms of false positives and negatives. Citation Format: Adith S. Arun, Jamie E. Medina, Stephen Cristiano, Daniel C. Bruhm, Remond J. Fijneman, Gerrit A. Meijer, Alessandro Leal, Victor E. Velculescu, Robert B. Scharpf. PLASMUT: An R Package for estimating the probability of tumor-specific mutations in cell-free DNA [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 6101.

Abstract 3701: Urine-based liquid biopsy test for the detection of residual bladder cancer in radical cystectomy patients

Abstract The gold standard therapy for muscle-invasive bladder cancer (MIBC) is neoadjuvant chemotherapy followed by radical cystectomy (RC) and urinary diversion. Some patients may have their disease completely eradicated by chemotherapy as ~35% of patients achieve a pathologic complete response (pCR) when evaluated from RC specimens. RC with urinary diversion is a life-altering operation with high complication rates and therefore determining who are complete responders would improve quality of life and could decrease costs and still achieve a high cure rate via RC avoidance. Unfortunately, current methods cannot accurately identify complete responders. Our lab has developed a urine-based liquid biopsy test in which DNA isolated from urine and germline is isolated and subjected to panel exome sequencing for enumeration of somatic variants. This occurs under the premise that tumor DNA identified (as mutant alleles) is a marker of residual disease while the absence of tumor DNA (meaning the absence of mutant alleles) can indicate a pCR. A 55 gene panel was designed to be used for exonic sequencing of urinary DNA (uDNA) to identify mutations indicative of bladder cancer or therapeutic targets. We have shown that most mutations in tumor tissue are detectable as mutations in urine (Mu). We also showed that the presence or absence of residual Mu after completion of chemotherapy strongly associates with residual disease or pCR at the time of RC, respectively. Therefore, this test could be used after neoadjuvant therapy to better identify patients for RC avoidance. To optimize this test, we are currently evaluating pre-analytical factors affecting the yield, purity, and integrity of uDNA and the quality of sequencing data using prospectively collected samples from patients with MIBC. We are also testing commercial and novel preservatives to identify one that maintains cell-free DNA integrity, yield, and mutation detection accuracy to enable temporary ambient temperature storage without compromising mutation detection, permitting shipping/centralized testing. Lastly, we are comparing two library preparation approaches: classical hybrid capture-based (Agilent SureSelect XTHS) and amplicon+hybrid capture-based (Agilent HaloPlexHS) to determine which performs the best for identifying the variants in a sample. Our results showed that both sequencing depth and the quality bases mapped was higher when utilizing the SureSelect XTHS kit as compared to HaloPlexHS using approximately the same number of reads. This improvement may also increase the power to detect rare alleles. Studies described herein will enable liquid biopsy tests to accurately identify patients with pCR after neoadjuvant therapy and might safely avoid radical cystectomy. Citation Format: Rhea Arya, Henkel Valentine, Uttam Satyal, Raju Chelluri, Mengmeng Li, Philip Abbosh. Urine-based liquid biopsy test for the detection of residual bladder cancer in radical cystectomy patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3701.

High-efficiency EGFR genotyping using cell-free DNA in bronchial washing fluid.

EGFR mutation testing is required for treatment of lung adenocarcinoma using epidermal growth factor receptor-tyrosine kinase inhibitor. However, the amounts of tumor tissue or tumor cells obtained by bronchoscopy are often insufficient. Bronchial washing fluid, obtained by lavage with saline after tumor biopsy or brushing, and the supernatant of bronchial washing fluid are thought to contain cell-free DNA that would be potentially applicable for EGFR testing. From among patients with suspected adenocarcinoma or non-small cell lung carcinoma diagnosed from biopsy or surgical specimens at the University of Tsukuba Hospital between 2015 and 2019, cell-free DNAs from 80 specimens of supernatant of bronchial washing fluid (50 with EGFR mutation and 30 with wild type EGFR) and 8 blood serum samples were examined for EGFR mutation using droplet digital PCR. Among the 50 patients harboring EGFR mutation, the rate of positivity for cell-free DNA extracted from supernatant of bronchial washing fluid was 80% (40/50). In nine of the EGFR mutation-positive cases, tumor cells were not detected by either biopsy or cytology, but the mutation was detected in four cases (4/9, 44%). Comparison of the cell-free DNA mutation detection rate between supernatant of bronchial washing fluid and blood serum in six cases showed that mutations were detected from the former in all cases (6/6, 100%), but from the latter in only one case (1/6, 17%). Using supernatant of bronchial washing fluid samples, the detection rate of EGFR mutation was high, and EGFR mutations were detectable even when no tumor cells had been detectable by biopsy or cytology. Supernatant of bronchial washing fluid might be an effective sample source for EGFR mutation testing.

Retracted: Detection and Significance of Cell-Free DNA Mutation in Pleural Effusion in Patients with Advanced NSCLC.

[This retracts the article DOI: 10.1155/2022/3112281.].

Detection of in vivo mutagenicity in rat liver samples using error-corrected sequencing techniques

BackgroundMutagenicity, the ability of chemical agents to cause mutations and potentially lead to cancer, is a critical aspect of substance safety assessment for protecting human health and the environment. Metabolic enzymes activate multiple mutagens in living organisms, thus in vivo animal models provide highly important information for evaluating mutagenicity in human. Rats are considered suitable models as they share a similar metabolic pathway with humans for processing toxic chemical and exhibit higher responsiveness to chemical carcinogens than mice. To assess mutagenicity in rats, transgenic rodents (TGRs) are widely used for in vivo gene mutation assays. However, such assays are labor-intensive and could only detect transgene mutations inserted into the genome. Therefore, introducing a technology to directly detect in vivo mutagenicity in rats would be necessary. The next-generation sequencing (NGS) based error-corrected sequencing technique is a promising approach for such purposes.ResultsWe investigated the applicability of paired-end and complementary consensus sequencing (PECC-Seq), an error-corrected sequencing technique, for detecting in vivo mutagenicity in the rat liver samples. PECC-Seq allows for the direct detection of ultra-rare somatic mutations in the genomic DNA without being constrained by the genomic locus, tissue, or organism. We tested PECC-Seq feasibility in rats treated with diethylnitrosamine (DEN), a mutagenic compound. Interestingly, the mutation and mutant frequencies between PECC-Seq and the TGR assay displayed a promising correlation. Our results also demonstrated that PECC-Seq could successfully detect the A:T > T:A mutation in rat liver samples, consistent with the TGR assay. Furthermore, we calculated the trinucleotide mutation frequency and proved that PECC-Seq accurately identified the DEN treatment-induced mutational signatures.ConclusionsOur study provides the first evidence of using PECC-Seq for in vivo mutagenicity detection in rat liver samples. This approach could provide a valuable alternative to conventional TGR assays as it is labor- and time-efficient and eliminates the need for transgenic rodents. Error-corrected sequencing techniques, such as PECC-Seq, represent promising approaches for enhancing mutagenicity assessment and advancing regulatory science.

Hairpin amplicon cleavage PCR (HAC-PCR): A novel, highly sensitive and cost-effective approach for detection of low-frequency DNA mutations

Highly sensitive detection of gene variants holds immense significance for cancer diagnosis and treatment. However, the presence of highly redundant wild DNA poses a substantial challenge when identifying low-frequency mutant circulating tumor DNA (ctDNA) in cancer cases. Here, a novel technique called hairpin amplicon cleavage PCR (HAC-PCR) for detecting low-level DNA mutations with exceptional sensitivity was presented. What sets HAC-PCR apart is its ability to achieve high sensitivity independently of strand-displacement, base modification, or the use of additional reagents. The discrimination between wild-type targets (WT) and mutant targets (MT) is significantly improved by introducing uniquely designed hairpin structures. These hairpins hinder the amplification of WT and are cleaved during the elongation of MT. For the first time, the unique mechanism of hairpin amplicon cleavage by Taq polymerase was exploited for mutation identification. The optimized HAC-PCR consistently demonstrates the capability to discriminate mutant DNA at levels as low as 0.01% variant allele fraction (VAF) for five mutation types. Furthermore, this impressive sensitivity of 0.01% VAF has also been achieved in a multiplex assay. When applied to the testing of tissue DNA from 24 patients with thyroid cancers and plasma ctDNA from 12 patients with non-small cell lung cancers (NSCLCs), HAC-PCR exhibited a 100% concordance rate with the existing commercial kit and droplet digital PCR (ddPCR). Therefore, HAC-PCR stands as a cost-effective and highly effective tool for variant identification in clinical testing.

B-246 The Evaluation of Actionable Mutations By Multiplex Analysis Using IntelliPlex® Lung Cancer Panel for Non-Small Cell Cancer Management

Abstract Background Understanding the genetic alterations which occur in genes associated with various cancers can improve therapy outcomes by promoting tailored treatments for each patient. Molecular profiling is therefore a crucial tool for precision medicine and enables for a personalized approach to cancer management. Current guidelines for molecular testing of Non-Small Cell Lung Cancer (NSCLC) patients involves treatment selection of a targeted Tyrosine Kinase Inhibitor based on the minimal testing of EGFR, ALK and ROS1 genes and strongly recommending the additional testing of KRAS, BRAF, MET, RET, NTRK and ERBB2 (Her2) genes to fully evaluate the patient’s mutational status. Method The IntelliPlex Lung Cancer Panel, based on PlexBio’s multiplexing πCode® technology, is an in vitro molecular amplification assay intended for the qualitative identification of 74 DNA mutations in the KRAS, NRAS, PIK3CA, BRAF, EGFR, HER2, MEK1 and AKT1 genes and 28 gene variants of the ALK, ROS1, RET, NTRK1 and MET genes. The assay is suitable for the use with formalin-fixed paraffin-embedded (FFPE) tumor tissues from NSCLC patients. The performance of IntelliPlex Lung Cancer Panel was evaluated on FFPE specimens by comparing the results with two commercially available molecular amplification assays. Discrepant samples were resolved by a corresponding digital PCR method kit. Results The IntelliPlex Lung Cancer Panel can detect DNA mutations at very low allele frequencies (as low as 0.1%) while only requiring DNA quantities of 10ng. Most RNA rearrangements are detected with 10 or more copies present in the sample while only low RNA input quantities (50 ng) are required. The IntelliPlex Lung Cancer Panel can analyze the mutational status of 102 targets in less than 5.5 h from sample to results and allows screening of up to 48 samples in parallel. Among 50 FFPE DNA specimens tested, the overall agreement for EGFR and KRAS mutations between IntelliPlex system and the comparison method were both found to be 98%. And the overall agreement after resolved the discrepancies by digital PCR was 100%. Conclusion The multiplex amplification and detection capabilities of the IntelliPlex Lung Cancer Panel provides a comprehensive and efficient way to evaluate the individual mutational status from lung cancer patients with high sensitivity and specificity. Accurate genetic analysis provides valuable information which can support precision medicine decisions for better cancer management. This can also be used further with non-invasive whole blood sampling (i.e., “liquid biopsy”) for subsequent treatment monitoring.

Detection of Cancer-Associated Gene Mutations in Urinary Cell-Free DNA among Prostate Cancer Patients in South Africa.

Prostate cancer (PCa) is the most common cause of cancer death among African men. The presence of tumor-specific variations in cell-free DNA (cfDNA), such as mutations, microsatellite instability, and DNA methylation, has been explored as a source of biomarkers for cancer diagnosis. In this study, we investigated the diagnostic role of cfDNA among South African PCa patients. We performed whole exome sequencing (WES) of urinary cfDNA. We identified a novel panel of 31 significantly deregulated somatic mutated genes between PCa and benign prostatic hyperplasia (BPH). Additionally, we performed whole-genome sequencing (WGS) on matching PCa and normal prostate tissue in an independent PCa cohort from South Africa. Our results suggest that the mutations are of germline origin as they were also found in the normal prostate tissue. In conclusion, our study contributes to the knowledge of cfDNA as a biomarker for diagnosing PCa in the South African population.

Detection of low-frequency mutations in clinical samples by increasing mutation abundance via the excision of wild-type sequences.

The efficiency of DNA-enrichment techniques is often insufficient to detect mutations that occur at low frequencies. Here we report a DNA-excision method for the detection of low-frequency mutations in genomic DNA and in circulating cell-free DNA at single-nucleotide resolution. The method is based on a competitive DNA-binding-and-digestion mechanism, effected by deoxyribonuclease I (DNase) guided by single-stranded phosphorothioated DNA (sgDNase), for the removal of wild-type DNA strands. The sgDNase can be designed against any wild-type DNA sequences, allowing for the uniform enrichment of all the mutations within the target-binding region of single-stranded phosphorothioated DNA at mild-temperature conditions. Pretreatment with sgDNase enriches all mutant strands with initial frequencies down to 0.01% and leads to high discrimination factors for all types of single-nucleotide mismatch in multiple sequence contexts, as we show for the identification of low-abundance mutations in samples of blood or tissue from patients with cancer. The method can be coupled with next-generation sequencing, droplet digital polymerase chain reaction, Sanger sequencing, fluorescent-probe-based assays and other mutation-detection methods.

Accuracy of genome-wide mutational analysis for tumor-informed ctDNA-guided MRD monitoring in bladder cancer.

4589 Background: Neoadjuvant chemotherapy (NAC) followed by radical cystectomy (RC) is the standard management of localized muscle-invasive bladder cancer (MIBC). However, about 45% of patients develop metastases within 2 years after RC. Biomarkers for treatment efficacy evaluation and early detection of minimal residual disease (MRD) are needed for earlier treatment initiation and monitoring of treatment response. Tumor-informed detection of mutations in cell-free DNA (cfDNA) has shown promising results to monitor MRD. However, the low tumor fraction after surgery and limited input material obtained from a typical plasma sample limits the probability of detecting low metastatic burden scenarios. Here we implemented and applied a whole-genome sequencing (WGS) approach to monitor circulating tumor DNA (ctDNA) for sensitive ctDNA detection. Methods: 110 MIBC patients undergoing NAC and RC were enrolled. cfDNA was extracted from ~1mL plasma (n=978) and procured from longitudinal plasma sampling during NAC and pre-RC and post-RC. WGS of tumor/germline pairs (30x/20x) and plasma cfDNA (>20x) was performed, facilitating detection of genome-wide genomic alterations and quantification of ctDNA using the C2inform method. Results: For each patient we developed a tumor-informed WGS model by integrating genome-wide mutation and copy number variation data coupled with advanced signal processing and AI-based error suppression. Patient-specific somatic variant patterns were used for detection and measuring ctDNA levels in low-input blood samples by WGS. Post-RC ctDNA analysis identified patients with recurrence with 78% sensitivity and 95% specificity and with a median lead time over radiographic imaging of 118 days (full follow-up included). When restricting follow-up time to 12 months following the latest ctDNA test, a sensitivity of 86% and specificity of 94% was achieved. ctDNA status was associated with recurrence-free (p<0.0001) and overall survival (p<0.0001). Furthermore, ctDNA clearance during NAC was also associated with recurrence-free survival (p=0.0051). The APOBEC associated signatures SBS2 and SBS13 were identified as the primary contributors to the mutational landscape of the primary tumors and TP53, KMT2D, PIK3CA, RB1 and KDM6A were the main affected driver genes. Half of the tumors displayed whole genome doubling. Moreover, our analysis of plasma samples after treatment showed the presence of chemotherapy-induced mutational signatures not present in the primary tumor, along with increases in copy numbers on chromosomes 19q and 20, and a focal amplification of the FGFR3 gene on chromosome 4. Conclusions: Our results highlight the clinical potential of personalized genome-wide mutation integration as an ultra-sensitive, non-invasive method for MRD detection and treatment response monitoring.

KRAS Mutation Analysis Using Cell-free DNA of Pancreatic Cancer.

Detection of pancreatic cancer using small samples of the pancreas obtained by endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) remains a challenge. The purpose of this study was to investigate whether the detection of KRAS mutations in cell-free DNA (cfDNA) extracted from supernatants of liquid-based fixed cytology (LBC) specimens obtained using EUS-FNA in solid pancreatic cancer can be an auxiliary test for differential diagnosis. The purpose of this study was to investigate whether the detection of KRAS mutations in cell-free DNA (cfDNA) extracted from supernatants of liquid-based fixed cytology (LBC) specimens obtained using EUS-FNA in solid pancreatic cancer can be an auxiliary test for differential diagnosis. This was a single-institution cohort study that included 50 patients with pancreatic lesions. cfDNA was isolated from the supernatant of fixed LBC samples, and KRAS mutation status was compared between cfDNA samples and FFPE small fragment tissues. Of the 50 cfDNA samples, 84% (42/50) were valid. KRAS mutations were detected in 57.1% (24/42) of the 42 valid samples. The sensitivity, specificity, and accuracy of KRAS mutation detection using cfDNA samples in the pancreatic lesions were 63.2% (24/38), 100.0% (4/4), and 66.7% (28/42), respectively. KRAS mutation status between FFPE small tissues and cfDNA samples were comparable. Gene mutation analysis using cfDNA from the supernatant of fixed LBC samples is an effective ancillary diagnostic tool for pancreatic cancer.