DNA Analysis Research Articles

Evaluation of Different Swab Wetting Chemicals Affecting the Yield of DNA Obtained From Biological Evidence on Cartridge Casings

Cartridge casings made from transition metals can be examined ballistically and also serve as significant evidence by containing touch DNA. However, the success rate of profiles obtained from this type of evidence is generally low. To enhance the success of DNA profiling from suspects' biological evidence, using swabs moistened with chemicals can be beneficial. Swabs are typically moistened with water, whose hypotonic nature disrupts cell integrity, causing the release of DNA. However, water is not the only agent used for moistening swabs; various buffer solutions are also utilized. The ability of swabs to transfer touch DNA depends on the type of buffer solution used. Sodium dodecyl sulfate (SDS), a strong anionic detergent, denatures non-covalently linked secondary and tertiary structures to increase the release of bound DNA. Another buffer solution used for swab moistening is Te+4 buffer, which contains EDTA and Tris. EDTA chelates metal ions, inactivating enzymes that could potentially damage DNA, while Tris adjusts the pH to an optimal level. This study aims to compare the effectiveness of microfibre and cotton swabs moistened with SDS, Te+4 buffer, and water in recovering genetic material from blood and epithelial cells deposited on brass cartridge casings. The study also evaluates the impact of firing on the quality of DNA profiles. Among the profiles obtained, the swabs moistened with SDS produced the highest rate of complete profiles at 44%, while the lowest rate of complete profiles at 22% was observed with swabs moistened with Te+4 buffer. SDS is particularly advantageous over water when used on casings with epithelial cells. Microfibre swabs are more effective in eliminating degradative factors caused by firing, thus enhancing profiling success. Comparisons of the RFU values indicate that casings yield lower values compared to cartridges, supporting the negative impact of the high heat, pressure, and residues generated during firearm discharge.

Testing the informativeness of Y-STR and mitochondrial DNA control region markers in an attempt to predict ancestry of World War II victims from Slovenian mass grave.

Identification of human remains is a challenge in forensic genetics without relatives or personal items available. In Slovenia, a Konfin II mass grave from the Second World War (WWII) was found, containing skeletal remains of 65 victims. The archival documents detailing victims' information describe 45 persons of which 33 could be considered Germanic and 12 Slavic. This study aims to check for concordance between the victim list and actual victims found by using uniparental markers to differentiate between Slavic and non-Slavic origin by attempting to infer ancestry by analyzing the control region (CR) of mitochondrial DNA (mtDNA) and Y-chromosomal STRs. Diaphyses of femurs were used as a DNA source. Next Generation Sequencing (NGS) technology was used for mtDNA- namely HID Ion Chef™ Instrument, Precision ID mtDNA Control Region Panel, and Ion GeneStudio™ S5 System. For the Y-chromosome, PowerPlex® Y23 System (Promega) kit and SeqStudio™ for human identification (HID) were used. European DNA Profiling mtDNA Population Database (EMPOP) and Y-Chromosome STR Haplotype Reference Database (YHRD) were searched for haplotype matches. Closest haplogroups were predicted using EMPOP, Y-DNA Haplogroup Predictor- NevGen, and Whit Athey's Haplogroup Predictor. Despite mitotypes being more diverse than Y-haplotypes, the Y-haplotypes had more database matches and more unequivocal differentiation between populations. 16 victims could be considered Slavic, 15 non-Slavic, and the remaining 34 had a rather scarce informativeness- either unclear or not providing any match. To address ancestry inference more comprehensively, analysis of autosomal ancestry informative markers as well as expansion on haploid markers will be conducted in future research.

Personalized Nutrition Plan Based on DNA

This project explores the concept of personalized nutrition by using DNA analysis to create customized diet plans tailored to an individual's genetic profile. As people increasingly seek healthier lifestyles, generic diet recommendations often fall short because they do not account for the unique genetic factors that influence how we process different nutrients. By examining an individual's DNA, we can gain insights into how their body metabolizes carbohydrates, fats, and proteins, as well as identify potential food sensitivities, nutrient deficiencies, and predispositions to certain health conditions. This approach allows for the development of highly personalized nutrition plans designed to optimize health, support weight management, and reduce the risk of chronic diseases such as diabetes, heart disease, and obesity. Ultimately, this project aims to empower individuals to make more informed dietary choices based on their genetic makeup, leading to better long-term health outcomes.

The Genomic SSR Millets Database (GSMDB): enhancing genetic resources for sustainable agriculture.

The global population surge demands increased food production and nutrient-rich options to combat rising food insecurity. Climate-resilient crops are vital, with millets emerging as superfoods due to nutritional richness and stress tolerance. Given limited genomic information, a comprehensive genetic resource is crucial to advance millet research. Whole-genome sequencing provides an unprecedented opportunity, and molecular genetic methodologies, particularly simple sequence repeats (SSRs), play a pivotal role in DNA fingerprinting, constructing linkage maps, and conducting population genetic studies. SSRs are composed of repetitive DNA sequences where one to six nucleotides are repeated in tandem and distributed throughout the genome. Different millet species exhibit genomic variations attributed to the presence of SSRs. While SSRs have been identified in a few millet species, the existing information only covers some of the sequenced genomes. Moreover, there is an absence of complete gene annotation and visualization features for SSRs. Addressing this disparity and leveraging the de-novo millet genome assembly available from the NCBI, we have developed the Genomic SSR Millets Database (GSMDB; https://bioinfo.icgeb.res.in/gsmdb/). This open-access repository provides a web-based tool offering search functionalities and comprehensive details on 6.747645 million SSRs mined from the genomic sequences of seven millet species. The database, featuring unrestricted public access and JBrowse visualization, is a pioneering resource for the research community dedicated to advancing millet cultivars and related species. GSMDB holds immense potential to support myriad studies, including genetic diversity assessments, genetic mapping, marker-assisted selection, and comparative population investigations aiming to facilitate the millet breeding programs geared toward ensuring global food security. Database URL: https://bioinfo.icgeb.res.in/gsmdb/.

Recent advancements in meat traceability, authenticity verification, and voluntary certification systems

The growing demand for transparency in the food industry has led to significant advancements in meat traceability. Ensuring the authenticity and origin of meat products is critical for consumer trust, public health, and compliance with regulations. This paper reviews recent innovations in meat traceability, with a focus on blockchain technology as a novel approach to ensuring traceability. Additionally, advanced methods for verifying meat authenticity and origin, such as isotope fingerprinting, DNA analysis, and spectroscopic methods, are discussed. The role of voluntary certification schemes in enhancing traceability and authenticity verification in the meat industry is also explored. The findings highlight the importance of integrating cutting-edge technologies and certification schemes to build a robust and transparent meat supply chain.

Sensitive and reliable detection of KIT p.D816V mutation in decalcified archival bone marrow trephines.

The majority of mastocytosis cases are characterized by an activating mutation in the KIT gene in codon 816. The detection of this alteration is of importance for proper diagnostic workup. Therefore, reliable and sensitive methods for the detection of KIT Codon 816 hotspot mutations in various types of patient samples are required. Since mutated cancer genes are often overexpressed, we evaluated the feasibility and sensitivity of KIT p.D816V detection by analysing mRNA/cDNA instead of genomic DNA. From 80 bone marrow trephines harboring a KIT p.D816 mutation, seven were only mutated by mRNA/cDNA pyrosequencing and 11 only by digital PCR analysis of genomic DNA. These results clearly demonstrate that detection of clinically relevant mutations in mRNA extracted from routinely processed decalcified archival bone marrow trephines is not only possible in a reliable fashion but under many circumstances advantageous. This enables the direct correlation of genomic data with high-quality morphological evaluation.

Gender differences in periodontal pocket microflora in patients with chronic generalized periodontitis

Background: Periodontal disease, the primary cause of tooth loss, is a critical public health concern with a considerable impact on quality of life. It is of interest to explore gender differences in the course of inflammatory periodontal diseases, as well as the prospects of their personalized treatment and prevention in males and females. Aim: To assess the dental health and quantitative composition of periodontal pocket microflora in periodontitis patients, taking into account gender differences. Materials and Methods: The study included 39 patients with moderate chronic generalized periodontitis aged 40–60 years. The diagnosis was made using both primary and secondary dental diagnostic tools. The study had two groups: males (41%, n=16) and females (59%, n=23). The following parameters were assessed: oral hygiene index (OHI-S), severity of gingival inflammation (PMA), and quantitative composition of periodontal pocket microflora (DENTOSCREEN, “Lytech” Co. Ltd., Russia), taking into account gender differences. The study findings were statistically processed using Microsoft Office Excel 2016 with Data Analysis and AtteStat 12.0.5 add-ins. The Shapiro–Wilk test was used. The data are presented as median and interquartile range: Me [Q1; Q3]. The frequency of microorganisms is presented as percentage of the mean and standard error of the mean. The confidence interval was calculated using the Wald method, according to the formula for proportions and frequencies. For small values, the Wald method with Agresti–Coull correction was used. Samples were compared using the following tests: the Mann–Whitney U test was used for quantitative data and Fisher’s angular transformation (φ*emp) for frequency (nominal) attributes. Significant differences were defined as a probability 95% (p 0.05). Results: The quantitative composition of periodontal pocket microflora in patients with moderate periodontitis revealed significant gender differences, as demonstrated by an increase in the count of Fusobacterium nucleatum and Prevotella intermedia, as well as the frequency of Porphyromonas gingivalis, in males compared to females (φ*emp=1.736; p 0.05). Dental health indices confirmed the hypothesis of significant differences between males and females in terms of plaque index (OHI-S) and РМА. In males, poor oral hygiene was reported in 93.7% cases, which was significantly higher (φ*emp=2.04; p 0.05) than in females (69.6%). Moderate gingival inflammation was more prevalent in males, while mild gingival inflammation was more prevalent in females (p 0.05). Conclusion: The study findings emphasize the importance of oral hygiene in the prevention of chronic periodontitis and can be used to improve complex treatment algorithms for patients with periodontal diseases. This includes more frequent dental check-ups and professional oral care sessions, as well as oral hygiene training and monitoring, in males compared to females. Diagnosis by polymerase chain reaction allows for the identification and quantitative determination of DNA from periodontopathogenic bacteria at low concentrations, as well as the justification of treatment algorithms and prediction of treatment outcomes.

Field Trials of an Autonomous eDNA Sampler in Lotic Waters.

Environmental DNA (eDNA) analysis has become a transformative technology, but sample collection methods lack standardization and sampling at effective frequencies requires considerable field effort. Autonomous eDNA samplers that can sample water at high frequencies offer potential solutions to these problems. We present results from four case studies using a prototype autonomous eDNA sampler as part of the U.S. Geological Survey's Rapid Environmental eDNA Assessment and Deployment Initiative & Network (READI-Net) project. These case studies involved short-term deployments of an eDNA autosampler (Smith-Root) across a range of riverine habitats with the objectives of (a) identifying what insights could be gained from high-frequency autosampling and (b) benchmarking these autosamples against manually collected samples. The high frequency autosampling revealed high temporal variability of eDNA concentrations and provided valuable insights about eDNA associations with environmental covariates, such as discharge and turbidity. Benchmarking assessments indicated autosamples had similar detection rates to manual samples and obtained similar or greater eDNA quantities. We did find minimal carryover contamination in autosampler field controls. We conclude that eDNA autosamplers have potential to improve freshwater biosurveillance by reducing logistical sampling barriers, standardizing collection methods, and clarifying the influence of environmental covariates on eDNA results.

Structural insights into thyroid hormone receptors.

Thyroid hormone receptors (TRs) are essential components of the endocrine system, mediating the cellular effects of thyroid hormones. The two TR genes, THRA and THRB, encode four isoforms, with TRα1 and TRβ1 being the most prevalent. TRs are ligand-dependent transcription factors and members of the nuclear receptor superfamily, indispensable for human growth, development and metabolism. Dysfunctional TR signaling can lead to conditions like resistance to thyroid hormone (RTH) syndrome, thyroid cancer, and metabolic disorders. Structurally, TRs comprise several domains: a variable N-terminal domain, a conserved DNA-binding domain, and a ligand-binding domain that mediates interaction with hormones and transcriptional coregulators. TRs predominantly function as heterodimers with the retinoid X receptor (RXR), binding to thyroid hormone response elements (TREs) in target genes to regulate their transcription. This review examines the structural studies on TRs, primarily performed through X-ray crystallography, that have provided detailed insights into TR functions, including DNA recognition, ligand binding, and coregulator interactions. We also discuss how these findings have deepened our understanding of TR mechanisms and contributed to the interpretation of pathogenic mutations.

Opportunistic post mortem scavenging by the terrestrial flatworm Platydemus manokwari.

Post mortem damage by predators varies with geography, climate, and location of cadavers. Frequently encountered facultatively parasitic terrestrial organisms include fly larvae (maggots), ants and beetles. This report describes for the first time opportunistic post mortem damage caused by the terrestrial flatworm Platydemus manokwari de Beauchamp,1963 (New Guinea Flatworm) (phylum Platyhelminthes: order Tricladida: suborder Continenticola: family Geoplanidae) to exposed skin of a body located in a tropical urban location. Several flatworms were attached to the body associated with areas of skin loss. Microscopy showed epidermal damage with no vital reaction and the presence of PAS-positive granules consistent with flatworm secretions. No human DNA profile was recovered with genetic testing of two of the flatworms. This case extends the range of potential post mortem predators to include flatworms, albeit opportunistically.

Abstract PR011: Advance prostate cancer detection through epigenomic profiling of cell-free DNA

Abstract Introduction: Metastatic castration-resistant prostate cancer (mCRPC) is a heterogeneous disease which can be classified into clinically relevant subtypes based on the expression of genes, such as the androgen receptor (AR) and neuroendocrine markers. Neuroendocrine prostate cancer (NEPC), characterized by gain of stem-like and neuroendocrine features and lack of AR expression is a clinically aggressive variant. Due to the lack of adequate biomarkers, NEPC is usually detected at a very advanced stage. There is mounting evidence that molecular subtype changes seen in NEPC are enforced by widespread epigenetic alterations, in particular DNA methylation changes. In this study, we aim to devise a novel DNA methylation-based assay for molecular subtyping and disease monitoring from cell-free DNA (cfDNA). Methods: We analyzed genome wide methylation patterns in 56 prostate cancer patient-derived xenograft (PDX) and 128 mCRPC tumors using array- and sequencing-based assays. We integrated DNA methylation at promoters, gene bodies and transcription factor binding site (TFBS) to determine the landscape of methylation alterations at key lineage specific genes. Using whole genome methylation derived from tissue with matched expression data we developed a deep learning framework to predict gene expression directly from tissue or cfDNA. Using key marker genes, the model was used to discern tumor molecular phenotypes from tissue and cfDNA in three independent cohorts of mCRPC patients using whole genome bisulfite sequencing and low-pass Enzymatic Methyl-Seq (EM-seq). Results: We observed a tight association between promoter, gene body and TFBS methylation with gene expression. Inferring gene expression from methylation for lineage specific markers such as AR, KLK3, ASCL1, INSM1, SRRM4 and DLL3 we classified molecular subtypes from both tissue and cfDNA. Additionally, for AR and ASCL1, we identified core sets of TFBSs whose differential methylation allowed for accurate assay-independent molecular subtype quantification. Applying the optimized quantitative model to mCRPC patients who underwent comprehensive tissue sampling by rapid autopsy we observed accurate subtype classification from both tissue samples and cfDNA for all cases. A similar analytical performance was observed in additional clinical mCRPC cohorts with cfDNA. Conclusion: Whole-genome methylation analysis of cfDNA allows for the prediction of gene expression patterns in tumor tissues, enabling non-invasive tumor subclassification and assessment of therapeutic targets. Citation Format: Mohamed Adil, Brian Hanratty, Pallabi Mustafi, Chitvan Mittal, Helen Richards, Ilsa Coleman, Radhika Patel, Anna-Lisa Doebley, Robert Patton, Eden Cruikshank, Patricia Galipeau, Ruth Dumpit, Martine Roudier, Jin-Yih Low, Navonil Sarkar, Robert Montgomery, Eva Corey, Colm Morrissey, Peter Nelson, Gavin Ha, Michael Haffner. Advance prostate cancer detection through epigenomic profiling of cell-free DNA [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 PR011.

Abstract B035: Detection and monitoring of translocation renal cell carcinoma via epigenomic profiling of cell-free DNA

Abstract We evaluated the ability of plasma chromatin immunoprecipitation and sequencing (ChIP-seq) and methylated DNA immunoprecipitation sequencing (MeDIP-seq) to detect translocation renal cell carcinoma (tRCC), to distinguish tRCC from clear cell renal cell carcinoma (ccRCC), and to monitor the disease evolution. tRCC is an aggressive subtype of kidney cancer usually driven by a fusion involving the TFE3 gene. Due to histologic overlap with other subtypes of RCC, tRCC is frequently misclassified. Methods for accurate diagnosis and detection of this molecularly distinct entity are therefore a pressing need. Most cell-free DNA (cfDNA) technologies are genomic-based and not optimized for tRCC as molecular fusions are not easily detected in circulating tumor DNA (ctDNA), and 50% of tRCC cases do not have other alteration. Epigenomic profiling of ctDNA via ChIP-seq has recently emerged as a powerful tool to detect and molecularly subtype cancers and may offer a more sensitive and specific detection of fusion- driven tumors. We first identified differentially methylated regions (DMRs) and regulatory elements (REs) specific to tRCC vs. ccRCC via MeDIP-seq and H3K4me3/H3K27Ac ChIP-Seq, of 4 tRCC and 5 ccRCC cell lines. We also identified TFE3 transcription factor binding sites (TFBS) via TFE3 ChIP-seq in 3 tRCC cell lines. We collected 30 plasma samples from metastatic patients with tRCC, 12 with ccRCC and 9 healthy individuals. Ultra low pass whole genome sequencing (ulpWGS) was performed to infer ctDNA fraction (TF), and cfMeDIP-seq, H3K4me3/H3K27ac cfChIP-seq for epigenomic profiling. Signal at tRCC-specific regions derived from cell lines profiling was aggregated for each mark and normalized to common active REs/methylated regions and used to discriminate tRCC vs. ccRCC vs. healthy plasma. Classification performance was evaluated using the area under the receiver operating characteristic (AUROC) curve. Overall 10/30 tRCC and 5/12 ccRCC samples had >3% TF by ulpWGS. H3K4me3 cfChIP-seq signal was significantly higher in all tRCC samples compared to healthy plasma (AUROC=1) at tRCC-specific REs and TFE3 TFBS. Furthermore, H3K27ac and H3K4me3 cfChIP-seq signal in plasma were also significantly higher at tRCC-specific REs and TFE3 TFBS in tRCC samples compared to ccRCC (AUROC=0.86-0.89). A cfDNA-based classifier combining the four scores distinguished tRCC from ccRCC with an AUROC=0.94. MeDIP-seq could not discriminate between tRCC and ccRCC. Moreover, three patients had multiple time points, and the integrated cfChIP H3K4me3/H3K27Ac score was able to accurately monitor the evolution of the disease. In conclusion, although a majority of tRCC plasma samples profiled had TF <3%, all could be distinguished from healthy samples on the basis of cfChIP. The combined score of H3K27ac and H3K4me3 cfChIP-Seq was also discriminatory for tRCC vs. ccRCC. Epigenomic profiling of cfDNA appears as a powerful tool for both detection of tRCC and discrimination from ccRCC/healthy plasma, with potential implications for diagnosis, disease monitoring and guiding therapy. Citation Format: Simon Garinet, Karl Semaan, John Caniff, Noa Phillips, Jacob Berchuck, Jiao Li, Kevin Lyons, Ananthan Sadagopan, Brad Fortunato, Mary Lee, Jack Horst, Rachel Trowbridge, Ji-Heui Seo, Matthew Freedman, Toni Choueiri, Sylvan Baca, Srinivas Viswanathan. Detection and monitoring of translocation renal cell carcinoma via epigenomic profiling of cell-free DNA [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 B035.

Abstract A057: Cancer-induced epigenomic changes in circulating immune cells enable breast, colorectal and lung cancer detection

Abstract Background: Epigenomic changes via the measurement of 5-hydroxymethylation cytosine (5hmC) in DNA have been shown to enable detection in a variety of cancers including pancreatic and ovarian cancer in plasma-derived cell-free DNA (cfDNA). Here, we investigated whether blood-based 5hmC profiling of genomic DNA (gDNA) derived from cells of the buffy coat (BC) possess signals that enable the detection of breast, colorectal and lung cancers. Method: Blood samples collected in Streck tubes from cancer patients or non-cancer controls were processed to obtain buffy coat and plasma samples. gDNA and cfDNA was isolated from buffy coat and plasma, respectively. gDNA or cfDNA was then subjected to 5hmC enrichment by click chemistry followed by preparation of sequencing libraries. Sequencing was performed to obtain genome-wide 5hmC profiles of buffy coat gDNA and cfDNA samples. Results: Differential hydroxymethylation analysis of BC gDNA revealed increased 5hmC over genes associated with myeloid lineage in breast, colorectal and lung cancers relative to non-cancer controls. Flow cytometry analysis confirmed increases in myeloid cells in cancer samples relative to non-cancer controls. 5hmC patterns in BC gDNA showed a larger number of cancer-associated changes compared to cfDNA, in particular this was found in early-stage disease. 5hmC features from BC gDNA or plasma-derived cfDNA were used to build prediction models that yielded cancer classification with overall sensitivity of 51.3% and 52.6% at 98% specificity, for BC or cfDNA, respectively. For early-stage cancer, the sensitivity at 98% specificity was 46.5% for the BC and 28.2% for cfDNA model. A final prediction model incorporating features from both tissue sources achieved superior classification (65.8% sensitivity) at 98% specificity, compared with using features from either tissue source alone, particularly for early-stage cancer which achieved 53.5% sensitivity of cancer detection. Conclusion: Our results demonstrate that cancer impacts the 5hmC profiles of buffy coat cells which can be leveraged for cancer detection using blood. Citation Format: Gulfem D Guler, Giuliana P Mognol, Yuhong Ning, Carolina Fraire, David Haan, Michael Kesling, Roger Malta, Melissa Peters, Anna Bergamaschi, Tierney Phillips, Shimul Chowdhury, Wayne Volkmuth, Samuel Levy. Cancer-induced epigenomic changes in circulating immune cells enable breast, colorectal and lung cancer detection [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 A057.

Abstract A036: Next gen liquid biopsy: Comprehensive analysis from a single tube of blood

Abstract Introduction There remains a largely untapped potential for utilizing liquid biopsy as a non- invasive, real-time window into tumor biology. In this study, we report on the use of the high- multiplex protein quantitation capability of the OrionTM spatial biology platform (RareCyte®, Inc) and cell picking capability of the CyteFinder® rare cell platform, to perform a comprehensive liquid biopsy analysis of whole blood that provides a deeper understanding of samples. This novel technology was used to characterize circulating tumor cells (CTCs), perform immune profiling of white blood cells (WBCs), and perform targeted mutation profiling of CTCs and cell-free DNA (cfDNA). All of these analytes can be measured from one 7.5 mL draw of blood. This array of tests was applied to cancer samples to demonstrate clinical feasibility. MethodsBlood samples were collected into AccuCyte® blood collection tubes. Buffy coats were processed to microscope slides using AccuCyte® blood processing kits. Slides were fixed and stained with an 18-plex immuno-oncology panel and imaged using the Orion Spatial Biology platform to evaluate protein biomarker expression on CTCs and to profile WBCs. Individual CTCs and WBCs from clinical samples were isolated using the CytePicker® for downstream molecular analysis. cfDNA was isolated from plasma collected during the AccuCyte process. Picked CTCs, WBCs, and cfDNA were sequenced using the CleanPlex® OncoZoom® Cancer Hotspot Kit. ResultsCancer patient blood was tested with a high-plex immunofluorescence assay using the Orion spatial biology system. CTCs were identified and phenotypically characterized in multiple cancer types. On the same slides, immunophenotyping of WBCs was performed. T- cell, B-cell, and macrophage populations were quantified. Additional sub-populations of T-cells (regulatory and memory) and proliferating cells (Ki67+) were enumerated. Expression of checkpoint-specific markers PD-L1 and PD-1 was measured on CTCs and WBCs. Sequencing results from individual picked CTCs showed evidence of minor clones representative of tumor heterogeneity that were not detectable in the cfDNA analysis. Conclusions This novel application of a high-plex spatial biology imaging system to liquid biopsies, in conjunction with the proven capabilities of rare cell detection and genomic analysis of single cells and cfDNA, has the potential to revolutionize liquid biopsy testing. From a single blood sample, this approach allows one to ask important questions about the complex interactions between the immune system and the tumor and to follow these interactions longitudinally in real time over the course of the disease. The goal is to leverage this unprecedented depth of analysis to improve patient outcomes and better understand the biological complexity of disease. Future directions of this work are to study rare types of WBCs that indicate immune activation and response to the tumor, and the detection and longitudinal monitoring of cell therapies such as CAR T cells after infusion. Citation Format: Arturo B Ramirez, Jon Ladd, Erin Bayer, Brock Bartels, Rachel Ponting, Arista Tischner. Next gen liquid biopsy: Comprehensive analysis from a single tube of blood [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 A036.

Comparison of Universal mtDNA Primers in Species Identification of Animals in a Sample with Severely Degraded DNA

Analysis of mitochondrial DNA, specifically the cytochrome b gene (cyt b), has become an essential tool for species identification. In the case of degraded samples, in which DNA is fractionated, universal primers, which are highly effective at amplifying the target region, are necessary. The material analysed in this study was a keychain made of bone, which was secured at a border crossing due to the suspicion that it was made of ivory. Due to processing of the bone and the likelihood of DNA degradation, five pairs of universal primers with different product lengths (from 148 to 990 base pairs) were used for species identification. Fragments of mtDNA from the cyt b and the 12S rRNA and 16S rRNA subunits were analysed. The analysis showed that only one pair of primers (L15601/H15748) enabled identification of the species, which is very common in samples with highly degraded DNA. The material was bone tissue belonging to the species Bos taurus (cattle). Species identification by molecular methods is extremely important in analysis of material when the species cannot be identified on the basis of morphological characteristics.

Detection of Brain-Derived Cell-Free DNA in Plasma

Background: Neuronal loss is a major pathological feature of neurodegenerative diseases. The analysis of plasma cell-free DNA (cfDNA) is an emerging approach to track cell death events in a minimally invasive way and from inaccessible areas of the body, such as the brain. Previous studies showed that DNA methylation (DNAm) profiles can be used to map the tissue of origin of cfDNA and to identify molecules released from the brain upon cell death. The aim of the present study is to contribute to this research field, presenting the development and validation of an assay for the detection of brain-derived cfDNA (bcfDNA). Methods: To identify CpG sites with brain-specific DNAm, we compared brain and non-brain tissues for their chromatin state profiles and genome-wide DNAm data, available in public datasets. The selected target genomic regions were experimentally validated by bisulfite sequencing on DNA extracted from 44 different autoptic tissues, including multiple brain regions. Sequencing data were analysed to identify brain-specific epihaplotypes. The developed assay was tested in plasma cfDNA from patients with immune effector cell-associated neurotoxicity syndrome (ICANS) following chimeric antigen receptor T (CAR-T) therapy. Results: We validated five genomic regions with brain-specific DNAm (four hypomethylated and one hypermethylated in the brain). DNAm analysis of the selected genomic regions in plasma samples from CAR-T patients revealed higher levels of bcfDNA in participants with ongoing neurotoxicity syndrome. Conclusions: We developed an assay for the analysis of bcfDNA in plasma. The assay is a promising tool for the early detection of neuronal loss in neurodegenerative diseases.

Abstract B073: Leveraging micronuclei DNA from erythrocytes for early detection of hepatocellular carcinoma

Abstract Background: Hepatocellular carcinoma (HCC) is the most common form of liver cancer and accounts for ∼90% of cases globally. Detection of early-stage HCC is vital as it allows for potentially curative treatment, yet remains a significant challenge. Micronuclei (MN) are a hallmark of genomic instability. An increased frequency of MN is commonly observed in tumor cells as well as other somatic cells, including erythrocytes, in cancer patients. Here, we have established a technique (WO2021/228246 A1), for the isolation and analysis of micronuclei DNA (MN-DNA) in erythrocytes from peripheral blood. Significant changes in read densities at specific genomic locations within MN-DNA were identified in patients, termed as tumor- associated MN-DNA (taMN-DNA) features. This study evaluates the potential of these taMN- DNA features for early-stage HCC detection across human and murine model. Methods: To explore the potential of MN-DNA in cancer detection, we first collected 1-2 mL of whole blood from HDs (N = 53) and HCC patients (N = 53). MN-DNA was isolated and purified from erythrocytes, followed by whole-genome sequencing. Participants were randomly divided into training, test cohorts in an 8:2 ratio. We applied machine learning algorithms to leverage these features for cancer detection in the human model. Additionally, to investigate the formation of taMN-DNA features, we employed a well-established mouse model that mimics HCC development. Mice were injected with the genotoxic agent diethylnitrosamine (DEN) 2 weeks after birth, and developed malignant macroscopic liver nodules at approximately 40 weeks of age. Results: In the human cohort, we enrolled 106 participants, with more than half of the HCC cases were diagnosed at early-stage (stage 0-II). The cancer detection model utilizing taMN- DNA features achieved an overall accuracy of 86.3%, with a sensitivity of 81.8% and specificity of 90.9%. For early-stage, the model demonstrated sensitivities of 54.5% at a specificity of 90.9%. In the DEN-induced HCC mouse model, unsupervised hierarchical clustering based on these selected taMN-DNA features clearly separated WT and tumor mice into two distinct groups. Notably, mice that were not tumorigenic at 36 weeks post-treatment in the DEN-treated group fell into the WT group when classified by the same taMN-DNA features, indicating that the formation of taMN-DNA signatures is associated with the presence of tumors specifically. Conclusions: This pilot study highlights the potential of MN-DNA as a promising tool for early HCC detection. Leveraging these taMN-DNA features can accurately distinguish early-stage HCC patients from HDs with high sensitivity. In both human and murine models, there is a significant relationship between these signatures and tumor presence, suggesting that taMN- DNA features might reflect the diseased state across mammalian species. Research sponsor: Timing Biotech. Citation Format: Hui Lin, Haobo Sun, Xingyun Yao, Xiaoxiao Fan, Fei Meng, Honghao Liang, Xiaofei Gao. Leveraging micronuclei DNA from erythrocytes for early detection of hepatocellular carcinoma [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 B073.

Abstract A024: Evaluating the effect of plasma-related preanalytical variables on the robustness of the liquid biopsy early CRC detection Signal-C assay

Abstract Liquid biopsies and cell-free DNA (cfDNA) analysis have emerged as novel non-invasive diagnostic tools in oncology. Analysis of cfDNA methylation status has been proposed as promising approach for several diagnostic purposes, and robust methylation-detecting technologies are of primary importance for the clinical implementation of novel diagnostic assays. Signal-C is an accurate NGS-based methyl-sensitive assay detecting colorectal cancer (CRC) currently under clinical validation. In this study we assessed the influence of different preanalytical variables influencing plasma stability, as the storage time of plasma samples and the tube type used for blood collection, on Signal-C assay results to determine its robustness. 958 samples with 3ml to 4ml plasma were used for cfDNA extraction and run through Signal-C assay following UDX protocol. In brief, 8 to 20ng cfDNA were used as an input for methylation- specific library preparation, which was run according to manufacturer’s instructions. Hybrid- capture was performed to enrich libraries for genomic regions included in Signal-C assay and sequenced on Illumina Novaseq machines. Relevant sequencing QC metrics, such as depth filtered, read yield from raw to filtered and PCR duplicate proportion were calculated, together with cancer predictions according to Signal-C test. Pearson coefficient and Pearson's Chi- squared test with Yates' continuity correction have been used to assess correlations, and Wilcoxon test has been used to measure the significance of the differences between quality metrics with respect to the tube type. We have analyzed the influence of different plasma-related preanalytical variables on sequencing metrics and cancer predictions of Signal-C assay. Plasma age, calculated as the time elapsed between blood collection and cfDNA quantification, shows a small negative correlation with YIELD_RAW_FILTERED (R = - 0.08, p=0.013) and a small positive correlation with PCR_DUP_PROP (R = 0.098, p=0.0024), while it doesn't influence DEPTH_FILTERED values (p>0.05). Use of different blood collection tubes (EDTA or Streck tubes) influenced in a small but significative manner the YIELD_RAW_FILTERED and the PCR_DUP_PROP (p<0.001), while it doesn’t affect the DEPTH_FILTERED values (p>0.05). Despite the sequencing QC metrics being subject to small but statistically significant differences, the overall cancer classification performance of Signal-C assay was not affected by the changes in plasma age (Pearson's product-moment correlation, sensitivity p=0.87, specificity p=0.091) nor in tube type used (Pearson's Chi-squared test with Yates' continuity correction, p=0.219). Here we evaluated the effect of plasma’s storage time and the type of blood collection tube on Signal-C assay performance. While signal processing features show small differences between the tested variables, this does not affect overall clinical performance of the test, supporting its robustness. Additional studies to analyze the effect of other preanalytical variables on Signal-C performance are underway to establish its robustness. Citation Format: Fernando Trincado Alonso, Pol Canal Noguer, Kristi Kruusmaa, Arianna Bertossi. Evaluating the effect of plasma-related preanalytical variables on the robustness of the liquid biopsy early CRC detection Signal-C assay [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 A024.

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 >10%), Stable Disease (CSD, no change or decrease <10%), Progressive Disease (CPD, increase >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 < .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 B011: Circulating tumor DNA profiling in colorectal cancer to detect guideline-based targeted mutations at a Quebec health care center

Abstract Liquid biopsy is an emerging tool in oncology, providing minimally invasive detection of tumor- specific DNA in body fluids, known as circulating tumor DNA (ctDNA). Colorectal cancer (CRC) has shown a high rate of shedding of ctDNA, making it a promising prognostic and possibly predictive biomarker in the personalized management of patients with CRC. Therefore, the half-life of ctDNA in circulation is estimated to be between 16 minutes and several hours, indicating a need for rapid processing. This study aimed to explore the concordance between the mutational profiles in tumor tissue and ctDNA from plasma obtained using EDTA tubes as well as the correlation between ctDNA and disease stage, to evaluate the potential of implementing liquid biopsy testing in CRC cases, following biobank standards. In this retrospective cohort, 58 patients with CRC stage T1 to T4 were enrolled at the Research Institute McGill University Health Centre (RI-MUHC). Tumor tissue was obtained through surgical resection or biopsies of formalin-fixed paraffin-embedded tissue, and blood samples were drawn pre-surgery using EDTA tubes. Cell-free DNA (cfDNA) was extracted from 1-2mL plasma samples using QIAamp Circulating Nucleic Acids. Next-Generation Sequencing using a 52-gene (AmpliSeq for Illumina Focus Panel) was performed to identify the tumor mutations and subsequently droplet digital PCR (ddPCR) was performed to identify the plasma mutations. Among the 58 patients, the mean age was 62 years (range 29-86), and 29 were women. Forty- one patients had left-sided tumor. At least 1 tumor-specific mutation was detected in all tissue tumor samples. Overall, the most frequently identified mutations in this cohort were: BRAF V600E (22.4%), KRAS G12D (12.1%), KRAS G12V and KRAS G13D (8.6%). Cell-free DNA was obtained from 32 patients and showed a mean concentration of 975 ng/mL (range 400- 1,995) for stage T1, 584 ng/mL (range 228-1,300) for stage T2, 1,127 ng/mL (range 249-4,160) for stage T3 and 2,083 ng/mL (range 860-3,680) for stage T4. ddPCR revealed mutation concordance between ctDNA and matched tumor tissue of 28.1%. When tumors were stratified by stage, the concordance was 0% (n=0/3) in patients with T1 tumors, 16.7% (n=1/6) in patients with T2 tumors, 25% (n=5/20) in patients with T3 tumors and 100% (3/3) in patients with T4 tumors. This study showed that ctDNA detection of mutations has excellent concordance in stage T4 tumors, highlighting the potential of ctDNA in advanced stage disease. However, in T1-3 tumor stages the concordance was low, showing the importance of preanalytical steps such as the use of cell-free DNA preservative tubes for sensitive and accurate detection of ctDNA in patients with earlier stage disease. Citation Format: Monyse de Nobrega, Kyle Dickinson, Saba Alsaddah, Alexandra Bartolomucci, Tadhg Ferrier, Anne Mahalia Olivier Mahalia Olivier, Mina Farag, Gertuda Evaristo, Sophie Camilleri-Broet, Andrea Gomez, Thupten Tsering, Lawrence Lee, Pierre Olivier Fiset, Julia V. Burnier. Circulating tumor DNA profiling in colorectal cancer to detect guideline-based targeted mutations at a Quebec health care center [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 B011.