DNA Analysis Research Articles

Exploring DNA Analysis Methods and Genetic Research Applications in Low and Middle-Income Nations: A Study of Sri Lanka

Using Sri Lanka as a case study, the paper explores how DNA analysis has transformed genetic research, particularly in low and middle-income countries (LMICs). It discusses various DNA analyzing techniques, from traditional methods like Sanger sequencing to advanced techniques such as next-generation sequencing (NGS) and CRISPR-Cas9, highlighting their applications in disease research, population genetics, and forensic science. Sri Lanka's advancements in genetic research, including DNA sequencing, typing, and recent developments in X-chromosome-based DNA typing, are emphasized. The paper also examines challenges and opportunities in LMICs regarding genetic research and underscores the importance of DNA analysis in advancing personalized medicine and understanding genetic diversity. Additionally, it discusses Sri Lanka's efforts in education and training in molecular biology. It explores the country's rich genetic diversity and demographic history, focusing on ethnic studies and historical interactions among different population groups. Overall, the paper highlights the significance of DNA analysis in genetic research and its potential implications for LMICs. Sri Lanka is a notable example of progress in the field.

Unlocking the potential of protein-derived peptides to target G-quadruplex DNA: from recognition to anticancer activity.

Noncanonical nucleic acid structures, particularly G-quadruplexes, have garnered significant attention as potential therapeutic targets in cancer treatment. Here, the recognition of G-quadruplex DNA by peptides derived from the Rap1 protein is explored, with the aim of developing novel peptide-based G-quadruplex ligands with enhanced selectivity and anticancer activity. Biophysical techniques were employed to assess the interaction of a peptide derived from the G-quadruplex-binding domain of the protein with various biologically relevant G-quadruplex structures. Through alanine scanning mutagenesis, key amino acids crucial for G-quadruplex recognition were identified, leading to the discovery of two peptides with improved G-quadruplex-binding properties. However, despite their in vitro efficacy, these peptides showed limited cell penetration and anticancer activity. To overcome this challenge, cell-penetrating peptide (CPP)-conjugated derivatives were designed, some of which exhibited significant cytotoxic effects on cancer cells. Interestingly, selected CPP-conjugated peptides exerted potent anticancer activity across various tumour types via a G-quadruplex-dependent mechanism. These findings underscore the potential of peptide-based G-quadruplex ligands in cancer therapy and pave the way for the development of novel therapeutic strategies targeting these DNA structures.

Phosphorothioate DNA modification by BREX Type 4 systems in the human gut microbiome.

Among dozens of microbial DNA modifications regulating gene expression and host defense, phosphorothioation (PT) is the only known backbone modification, with sulfur inserted at a non-bridging oxygen by dnd and ssp gene families. Here we explored the distribution of PT genes in 13,663 human gut microbiome genomes, finding that 6.3% possessed dnd or ssp genes predominantly in Bacillota, Bacteroidota, and Pseudomonadota. This analysis uncovered several putative new PT synthesis systems, including Type 4 Bacteriophage Exclusion (BREX) brx genes, which were genetically validated in Bacteroides salyersiae. Mass spectrometric analysis of DNA from 226 gut microbiome isolates possessing dnd, ssp, and brx genes revealed 8 PT dinucleotide settings confirmed in 6 consensus sequences by PT-specific DNA sequencing. Genomic analysis showed PT enrichment in rRNA genes and depletion at gene boundaries. These results illustrate the power of the microbiome for discovering prokaryotic epigenetics and the widespread distribution of oxidation-sensitive PTs in gut microbes.

Pattern and Dynamics of Genetic Stock of Freshwater Fishes Using Various Molecular Markers for Their Conservation Management Concerns in the Indian Riverine System

India has a world's richest, most abundant and most promising inland fisheries resources with variety fishes in tributaries, streams, canals, lakes, ponds, and reservoirs. There are several significant river systems in India, including the Cauvery, Tapi, Narmada, Krishna, Indus, Brahmaputra, Ganga, Mahanadi, and Godavari which showing shrinkage of fish fauna because of environmental disturbance, human interference and human threats. In order to conserve the fish diversity, the molecular markers are helpful in determining genetic diversity, gene polymorphism and gene flows from generation to generation. The topic experienced a surge in interest due to the introduction of strong statistical analysis tools and the accessibility of DNA fingerprinting, DNA sequencing techniques and consequently population genetic studies. Some molecular markers, Microsatellite markers, RAPD, Allozymes and mitochondrial (cox1, cytob, ATPase6/8) implications have been discussed in this review which will provide an overview that have been used by scientists to studied population genetic structure and genetic variations at various levels.

Burial of two closely related infants under a “dragon stone” from prehistoric Armenia

“Dragon stones” are prehistoric basalt stelae carved with animal imagery found in Armenia and surrounding regions. These monuments have a complex history of use and reuse across millennia, and the original date of creation is still a matter of debate. In this article, we present a unique dragon stone context excavated at the site of Lchashen, Armenia, where a three-and-a-half-meter high basalt stela with an image of a sacrificed bovid was found above a burial dating to the 16th century BC. The burial stands out among hundreds from this site as the only one in connection with a “dragon stone”, and one of very few containing the remains of newborn babies. Furthermore, our analyses of ancient DNA extracted from the well-preserved skeletal remains of two 0–2-month-old individuals showed them to be second-degree related females with identical mitochondrial sequences of the haplogroup U5a1a1 lineage, thus indicating that the infants are closely related. Additionally, we assessed that the buried individuals displayed genetic ancestry profiles similar to other Bronze Age individuals from the region.

Clinical utility of comprehensive liquid molecular profiling in patients with advanced endometrial cancer.

Molecular characterization has significantly improved the management of advanced endometrial cancer (EC). It distinguishes four molecular subclasses associated with prognosis and personalized therapeutic strategies. This study assesses the clinical utility of cell-free DNA (cfDNA) profiling in EC to identify targetable alterations. Women with metastatic or recurrent EC were prospectively recruited within the framework of the STING trial (NCT04932525), during which cfDNA was analyzed. Genomic alterations were identified with the FoundationOne CDx assay. Each molecular report underwent review by a molecular tumor board. Alterations were categorized via the European Society of Medical Oncology Scale for Clinical Actionability of Molecular Targets (ESCAT). A total of 61 patients were enrolled. The median age was 66.9 years, with 43% presenting frontline metastatic disease. All histologic subgroups were represented. Notably, 89% of patients yielded informative cfDNA analysis. Six tumors were classified with deficient mismatch repair/microsatellite instability (11%) and 37 as TP53 gene mutant (67%), and 12 had nonspecific molecular profiles (22%). Molecular classification based on liquid biopsy showed 87.5% accuracy in correlating with tissue results. Moreover, 65% of cases exhibited ≥1 actionable alteration, including 25% ESCAT I alterations and 13% ESCAT II alterations. Consequently, 16% of patients received a molecularly matched therapy, and presented with a 56% response rate and median progression-free survival of 7.7 months. cfDNA sequencing in EC is a feasible approach that produces informative results in 89% of cases and accurately categorizes patients into the main molecular subclasses. It also reveals multiple actionable alterations, which offers the potential for personalized therapeutic strategies.

Identification of genes related to PD-L1 expression in non-small cell lung cancer (NSCLC) tumor samples by GFPrint, a machine learning framework (MLF).

e13600 Background: The advent of drugs targeting the PD-1/PD-L1 axis has revolutionized NSCLC treatment. Although promising, individual responses to these therapies can vary among individuals, and research is ongoing to better understand the mechanisms involved in PD1/PD-L1 expression to optimize treatment protocols, and identify which patients are most likely to benefit from these therapies. In this study, Genetic Fingerprint (GFPrint), a MLF, was employed to identify mutations in genes linked to PD-L1 expression in NSCLC tumor samples. Methods: Whole exome sequencing (WES) was run on samples from biopsies of 46 NSCLC patients (histologic subtypes: squamous-cell carcinoma and adenocarcinoma). WES data were encoded using GFPrint, a MLF that generates virtual representations of the tumor exomes in a latent space (LS) defined by the genetic features of the samples. Clusters of patients were identified in such LS based on the genetic similarities of their tumors. Statistical univariate analysis was run to analyze differences in the distribution among both clusters of clinical (age, smoking, and metastatic status) and pathological (histotype and PD-L1 expression) features. The mutational burden (MB) of each cluster was also examined by pathway enrichment analysis (PEA). Results: Two main clusters were identified, C0 (n=16) and C1 (n=17), with 13 unassigned patients. Main differences between clusters were related to histology and PD-L1 expression. C0 included significantly less patients with adenocarcinoma (10/16; 62.5%) compared to CL1 (17/17; 100%) (p-value=.01). In addition, C0 presented more patients expressing PD-L1 (9/16; 56.2%) compared to C1 (3/17; 17.6%) (p-value=.04). Of interest, PD-L1 positivity was significantly higher (p value=.03) in adenocarcinomas of C0 patients (6/10; 60.0%) than those of C1 (3/17; 17.6%). Moreover, C0 adenocarcinomas presented higher levels of PD-L1 expression (6.3+/-9.7 %) than those of C1 (2.3+/-7.5 %) (p value=.06). MB analysis revealed that 1,357 genes were mutated exclusively in C0 patients, and PEA allowed the identification of 3 enriched cancer-related KEGG pathways: HIF-1 signaling, Ca2+ signaling and actin cytoskeleton. Selection of non-silent mutations in these pathways enabled the identification of 10 genes, mostly included in the HIF-1 signaling pathway, with previous data suggesting their involvement in PD-L1 regulation and expression: PIK3R1, HK1, NOS2, NRAS, BRAF, ITGA1, ITGA9, EGLN1, EGLN2 and AKT1, being the first 6 the only ones found in adenocarcinomas. Conclusions: The identified genes deserve further studies to validate them as possible targets of pharmacological intervention concomitant to PD-1/PD-L1 blockade, and also as potential biomarkers for patients’ selection to improve the efficiency of immunotherapy in NSCLC treatment.

Exploring health disparities in prostate cancer through circulating tumor DNA analysis.

1603 Background: African American men (AAM) suffer from the highest rates of prostate cancer in the world. In addition, AAM experience the highest rates of aggressive prostate cancer and prostate cancer-specific mortality of any ethnic group in the United States [1, 2]. The reasons behind the increased burden of prostate cancer poor outcomes in AAM remain unresolved. A large proportion of variation in prostate cancer mortality remains unexplained solely by epidemiologic risk factors. We investigated genomic differences between AAM and Caucasian men with metastatic castration resistant prostate cancer (mCRPC) utilizing circulating tumor DNA (ctDNA) analysis. Methods: AAM and Caucasian men with mCRPC were enrolled in this study. We collected and analyzed 88 plasma samples, including 66 from Caucasian and 22 from African American patients, with no significant age difference between the groups. The targeted sequencing libraries were prepared following the Agilent SureSelect XT HS2 target enrichment workflow coupled with the SureSelect custom prostate cancer panel (Agilent, Inc.). The final libraries were sequenced on an Illumina NextSeq 2000 to generate 150 bp paired-end reads. Results: A total of 88 men with mCRPC were enrolled. About 60% of African American patients exhibited organ metastasis at the time of diagnosis, while this proportion was 36% for Caucasian patients and they primarily reported metastasis during relapse. Analyzing 1824 variants in 138 genes meeting inclusion criteria, 281 significant mutations across 88 genes revealed racial differences. Notably, 29% of these variants were somatic. Approximately 60 cancer-related pathways, tissues, and diseases were identified from these datasets, encompassing 59 of our significant genes. Among these associated genes, the prevalence of significant variants, higher in AAM, exceeds that in Caucasians in 76% of cases. The top genes that have more contribution in considering pathways are shown (Table). Conclusions: Genomic variations were observed in ctDNA analysis between African American and Caucasian patients diagnosed with metastatic castration-resistant prostate cancer. Our findings highlight the potential inclusion of ctDNA analysis in the clinical assessment of these patients, offering valuable insights for informed therapeutic decision-making. The top genes with the most contribution to prostate-related pathways. [Table: see text]

The Development of Non-natural Type Nucleoside to Stabilize Triplex DNA Formation against CG and TA Inversion Site.

Based on the sequence-specific recognition of target duplex DNA by triplexforming oligonucleotides (TFOs) at the major groove side, the antigene strategy has been exploited as a gene-targeting tool with considerable attention. Triplex DNA is formed via the specific base triplets by the Hoogsteen or reverse Hoogsteen hydrogen bond interaction between TFOs and the homo-purine strand from the target duplex DNA, leading to the established sequence-specificity. However, the presence of inversion sites, which are known as non-natural nucleosides that can form satisfactory interactions with 2'- deoxythymidine (dT) and 2'-deoxycytidine (dC) in TA and CG base pairs in the target homo-purine DNA sequences, drastically restricts the formation of classically stable base triplets and even the triplex DNA. Therefore, the design of non-natural type nucleosides, which can effectively recognize CG or/and TA inversion sites with satisfactory selectivity, should be of great significance to expanding the triplex-forming sequence. Here, this review mainly provides a comprehensive review of the current development of novel nonnatural nucleosides to recognize CG or/and TA inversion sites in triplex DNA formation against double-strand DNA (dsDNA).

Plasma circulating tumor DNA in patients with cutaneous squamous cell carcinoma: Initial experience.

e15034 Background: Circulating tumor DNA (ctDNA) has emerged as a potential noninvasive biomarker to assess tumor burden throughout cancer treatment and to aid in clinical decision-making. However, data on the use of ctDNA in cutaneous squamous cell carcinoma (cSCC) is currently lacking. In this study, we report our first experience with ctDNA in patients with cSCC. Methods: In this IRB-approved study at a tertiary care center, an initial cohort of patients with intermediate to high-risk cSCC had tissue and plasma sent for ctDNA analysis using Natera®’s proprietary bioinformatics pipeline. This cohort included 21 patients treated from 2023 to the present. Initial tissue samples were obtained through biopsy (i.e., excisional, punch, or shave), Mohs surgery, or surgical resection. Some patients had additional plasma sample(s) drawn later during or after the treatment course. Patients either received a “positive,” “positive below analytical range,” or “negative” result or had ‘insufficient sample’ to receive a result based on Natera®’s analysis. All patients were treated curatively with surgical resection with or without adjuvant radiation as per the standard of care. The assessment of gross disease was determined based on whether the patient had disease present on imaging or physical exam at the time of the plasma draw. A comparison of ctDNA results and the presence of gross disease was done using Fisher’s exact test. Results: Of the samples submitted for Natera® ctDNA analysis, 18/21 had sufficient tissue for analysis of tumor DNA. All 3 tumors with insufficient tissue were from biopsies (6/9 sufficient), while 12/12 of those from Mohs or surgical resection had sufficient tissue. The average tumor volume in these patients with insufficient samples for tumor analysis was relatively low (0.35 cm3) compared to those that resulted (17-30 cm3). 7/9 (78%) patients with gross disease had a positive ctDNA result, which was significantly greater than those with no gross disease (1/9 patients, 11%) (p = 0.004). Three patients in our cohort with an initially positive ctDNA result had multiple plasma draws, with all having a later draw demonstrating a negative ctDNA result after having surgery and/or radiation. Conclusions: Circulating tumor DNA is a reliable marker for patients with cSCC with a high tumor burden and is feasible in those with adequate tissue. However, in patients where it is only possible to obtain a small volume of tumor tissue and/or low tumor burden, ctDNA may be a less reliable biomarker. [Table: see text]

The real-world impact of next-generation sequencing in advanced stages of solid tumor malignancies.

e23066 Background: Next-generation sequencing (NGS) is a revolutionary technology that allows for DNA and RNA genomic analysis of malignant tumors via tissue or blood sample to aid in diagnosis and treatment while providing predictive and prognostic data about cancer patients. 75.6% of US oncologists use NGS in advanced cancer patients. As there are now site-agnostic approvals of certain targeted therapies, ASCO published a provisional clinical opinion stating that there is a rationale for genomic testing of all solid tumors. This retrospective study evaluates the impact of NGS testing in advanced solid tumor patients in a community based cancer center in Florida. Methods: Patients were diagnosed with stage 3 or 4 solid malignancy between January 1st, 2019 - December 31st, 2022. 496 patients were reviewed of which 269 patients were included in the study as they underwent NGS testing with either tissue (70.6%), blood (25.3%) or both (4.1%). Results: Median age of the study population was 71% with 87% of patients being insured with Medicare. 52.4% of patients were male. Overall, 67.7% of patients had biomarkers identified, 47.2% had FDA approved targeted therapies, and 77.3% had clinical trials identified. NGS testing impacted first line therapy in only 10% of patients where a matched therapy was initiated as first line treatment. Median time from initial diagnosis to NGS result was 53 days while the median time from diagnosis to initiation of first line of therapy was 47.5 days. In the entire study population, 14.1% never received therapy and 40.9% of patients passed. Of the 110 patients who passed, median overall survival in patients who had a biomarker identified through NGS testing was 6 months compared to 7 months in patients without a biomarker. Rates of biomarker identification and availability of FDA approved therapy were similar between deceased patients (67.3% and 45.5%) and those who were alive at time of analysis (63.1% and 48.4%). Conclusions: NGS testing has become part of routine oncologic care in patients with advanced solid malignancies, however in only 10% of cases did it impact first line therapy. Though currently NGS testing has a limited role in the initial management of advanced solid tumors, it is imperative that this technology continues to be used and developed as it leads to hypothesis generating and clinically relevant questions in discovery of oncogenic drivers, targets for novel therapeutics, and mechanisms of resistance to therapies. [Table: see text]

Molecular characteristics of dedifferentiated liposarcoma with broad rhabdomyosarcomatous differentiation.

e23530 Background: Dedifferentiated liposarcoma (DDLPS) with broad rhabdomyosarcomatous differentiation has a worse prognosis compared with other types of DDLPS. Therefore, it is of importance to recognize its molecular characteristics in order to investigate individualized treatment strategies. Methods: We conduct next-generation sequencing (NGS) including DNA and RNA sequencing analysis in 5 DDLPS patients with broad rhabdomyosarcomatous differentiation and perform comparative analysis between well-differentiated and dedifferentiated components from the patients. Results: In the ten samples, several somatic copy-number variations (CNVs) including the gain of 12q are considered as frequent molecular alterations. IGFN1, FGFR4, BLM, RICTOR, RUNX1 and TOP1 mutation is the high-frequency single-nucleotide variations (SNVs). Novel BEST3/CD63/LGR5/CENPL-MDM2 and ERBB3-TAFA2 fusion genes are identified in this subtype of tumor. The comparative analysis between well-differentiated and dedifferentiated components recognized two categories of molecular variations: shared variations, such as FGFR4 and BLM mutation, ERBB3-TAFA2 fusion, and MYC amplification that are associated with tumorigenesis, and dedifferentiated-specific variations including GRIN2A, FOXP1 and TP53 mutation, CERS4-PTPRB and TRIO-TERT fusion, and ESR1 amplification, which are related with malignant progression. After surgery, the patient received adjuvant therapy. The mean follow-up of the patients was 30 months, and the mean recurrence-free survival (RFS) was 9.6 months. Conclusions: This NGS analysis disclosed the possible progression mechanisms and therapeutic targets in DDLPS with broad rhabdomyosarcomatous differentiation and provided insights contributing to the refinement of management in this tumor. [Table: see text]

Analytical and clinical validation of a personalized, tumor-informed circulating tumor DNA assay for minimal residual disease detection.

e14545 Background: The detection of minimal residual disease (MRD) through circulating tumor DNA (ctDNA) analysis provides an advanced molecular method for early identification of surgical patients at high risk of recurrence. In this study, we present the development of mClear, a personalized, tumor-informed next-generation sequencing (NGS) assay for MRD detection. Furthermore, we report the analytical and clinical validation processes conducted to evaluate the performance of the MRD assay. Methods: mClear is designed to assess MRD in patients with solid tumors. Whole exome sequencing (WES) was performed on tumor tissues and matched peripheral blood samples to detect somatic variants comprehensively. Personalized MRD assays were carefully designed by selecting up to 40 prioritized variants, including single nucleotide variants (SNVs), small insertions and deletions (InDels), and fusions while ensuring coverage of 21 additional key driver genes. For analytical validation, we utilized a pan-cancer MRD ctDNA quality control product (LDT-999, LDT Bioscience), characterized by known variant allele frequency (VAF) gradients of 0%, 0.005%, 0.05%, and 0.5%, confirmed through droplet digital PCR (ddPCR). We conducted 18 replicate tests at VAF gradients of 0.5% and 0.05%, 23 replicates at the 0.005% VAF gradient, and 16 replicates at the 0% VAF gradient. For clinical validation, preoperative and postoperative blood samples from a cohort of 264 pan-cancer patients were analyzed. ctDNA collected for MRD detection underwent ultra-deep next-generation sequencing (100,000× average depth of coverage). The library preparation process commenced with an input of 30ng DNA. Results: At VAF gradients of 0.5% and 0.05%, the assay achieved an excellent sensitivity of 100%, and at VAF gradients of 0.005% and 0%, it demonstrated a sensitivity of 95.7% and a specificity of 100%. Preoperative ctDNA was detected in 86.7% (72/83) of the pan-cancer patients with 71.4% (5/7), 90.9% (10/11), 85.5% (47/55), and 100.0% (10/10) in pathological stage (pStage)I, pStage II, pStage III, and pStage IV, respectively. In the pan-cancer cohort, the positive rate of longitudinal MRD was 35.7% (75/210) with 17.8% (21/118), 50.0% (13/26), 55.1% (27/49), and 82.4% (14/17) in pStage I, II, III, and IV, respectively. In four patients who relapsed, MRD detection during surveillance preceded radiological recurrence by a lead time of 1-8 months. Conclusions: Our personalized MRD assay demonstrated excellent analytical sensitivity and specificity with a limit of detection (LoD) of 0.005% VAF at the sample level. Furthermore, it displayed high clinical sensitivity for the detection of ctDNA in the pan-cancer cohort, highlighting its potential for monitoring of recurrence.

Preliminary results correlating post-operative ctDNA status with disease-free survival in patients with stage II (high risk)/III colorectal cancer in the BNT000-001 epidemiology study.

3526 Background: Despite standard-of-care surgical resection and adjuvant chemotherapy (ACT), disease recurrence in patients (pts) with stage II or III colorectal cancer (CRC) remains high. Circulating tumor DNA (ctDNA) analysis post-surgery with curative intent may help to identify pts with minimal residual disease at risk for recurrence. We report preliminary results of the BNT000-001 study exploring the prevalence and prognostic value of ctDNA positivity post-surgery in pts with stage II (high-risk) / III CRC. Methods: The BNT000-001 (NCT04813627) epidemiological study is enrolling pts in US, Germany (including screening through the Colopredict registry study), Spain, and Belgium with resected high-risk (per NCCN guidelines) stage II / III CRC for longitudinal follow up of their ctDNA status post-surgery and ACT. The primary objective of the study is to estimate the prevalence of ctDNA positivity after surgery. Other objectives include evaluation of the prognostic value of ctDNA and identification of ctDNA positive pts who might be candidates for the interventional clinical trial BNT122-01 (NCT04486378). Disease-free survival (DFS) was measured from the post-surgery visit to disease recurrence/death and analyzed for all eligible pts who remain on study. ctDNA was analyzed using the laboratory developed AVENIO Oncology Assays (AOA) Surveillance test and reported as detected (“positive”), not detected (“negative”), or no result (“non-evaluable”). Results: 1094 pts have been screened, of which 874 pts were analyzed for post-surgery ctDNA status (data cutoff 15 Dec 2023). 142 pts were non-evaluable, resulting in 732 pts with defined ctDNA status. Prevalence of post-operative ctDNA positivity was 12.3% (90*/732). 87.7% (642/732) of pts were ctDNA negative. 826 pts were followed for the prognostic value of post-surgery ctDNA-positivity, excluding 48 ctDNA positive pts who transferred to the BNT122-01 treatment study. At a median follow-up time of 25.9 months, the median DFS of ctDNA positive pts was 12.16 months vs. median DFS not reached for ctDNA negative pts. The 12-month DFS rate was 55% for ctDNA positive vs. 92% for ctDNA negative pts. Of note, our analyses support the identification of ctDNA negative T4N2 pts as a high-risk group for recurrence. Post-surgery ctDNA positivity was associated with significantly increased risk for disease recurrence or death, with a hazard ratio of 10.2. Conclusions: This study supports existing evidence of the prognostic value of post-operative ctDNA in CRC pts. In ctDNA positive pts, the 12-month DFS rate was almost halved (55% vs. 92% in ctDNA negative). Clinical trial information: NCT04813627 . [Table: see text]

Comparative genomic profiling of circulating tumor DNA and paired formalin-fixed paraffin-embedded (FFPE) DNA from Indian patients with breast cancer.

e14554 Background: Breast cancer (BC) is the most frequent malignancy and a leading cause of mortality in women worldwide. Although most BCs are treated with curative intent, 30-40 % patients experience disease progression or metastatic relapse. Considering high heterogeneity of BC, mutational profiling of solid biopsy may not be adequate for informed treatment decisions in all cases. Liquid biopsy based on circulating tumor DNA (ctDNA) analysis is emerging as an equivalent and alternative to tissue biopsy in clinical settings. Though extremely critical for management of BC, liquid biopsy is not a standard of care in India. Here we compared comprehensive genomic profiles of ctDNA and paired biopsy samples to determine the efficacy of ctDNA assay in detecting actionable mutational targets. Methods: Retrospectively, genomic DNA and matched ctDNA was isolated from both FFPE and blood samples of14 BC patients. The patients were confirmative with pathological findings, having metastatic infiltrating ductal carcinoma, or triple negative breast cancers. Genome-wide HRD, TMB and MSI scores based on the OncoIndx comprehensive gene panel (CGP) was determined. OncoIndx CGP targets 1080 cancer related genes along with 47 HRR genes and 1600 MSI signature sites. Libraries were prepared using a hybridization-capture method based on custom-designed OncoIndx 1080 CGP and sequenced (depth 5500 X) on Illumina Nextseq 2000 in a pair-end mode (150 x2). Variant calling was performed using a proprietary bioinformatics pipeline iCare. Results: All patients were diagnosed with metastatic infiltrating ductal carcinoma, or triple negative breast cancers. From the patient cohort, microsatellite instability (MSI) from ctDNA showed 100% concordance with FFPE DNA while tumor mutation burden (TMB) and homologous recombination deficiency (HRD) scores showed 71.4 % and 64.2 % concordance with FFPE DNA respectively. At the individual gene mutation level, the concordance TP53, BRCA1/2, PI3K family of genes, ESR1, PTEN, ERBB2, PALB2, and BRAF genes between ctDNA and FFPE DNA was 54.7%, 92.8%, 54.7%, 78.5%, 92.8%, 100%, 100%, and 100% respectively. Many important genomic alterations including PIK3CA:p.L334H, PIK3CA:p.E81K, ESR1:p.Q375L, BRCA2:p.X3040_splice, PTEN:p.D19H were exclusive to ctDNA thereby denoting potential tumor heterogeneities which could be missed from FFPE DNA. Conclusions: Our results suggest a high correlation between ctDNA and FFPE DNA detected by OncoIndx test in metastatic or triple negative breast cancer patients of Indian ethnicity. Although not a part of standard of care, liquid biopsy could be of significant potential for management of BC in the standard clinical settings. However, a thorough investigation of cancer stage, and clinical presentations are necessary to carefully evaluate the selection of ctDNA or FFPE DNA-based genomic profiling.

Assessing eDNA capture method from aquatic environment to optimise recovery of human mt-eDNA

Previous studies have shown that environmental DNA (eDNA) from human sources can be recovered from natural bodies of water, and the generation of DNA profiles from such environmental samples may assist in forensic investigations. However, fundamental knowledge gaps exist around the factors influencing the probability of detecting human eDNA and the design of optimal sampling protocols. One of these is understanding the particle sizes eDNA signals are most strongly associated with and the most appropriate filter size needed for efficiently capturing eDNA particles. This study assessed the amount of mitochondrial eDNA associated with different particle sizes from human blood and skin cells recovered from freshwater samples. Samples (300 mL) were taken from experimental 10 L tanks of freshwater spiked with 50 µL of human blood or skin cells deposited by vigorously rubbing hands together for two minutes in freshwater. Subsamples were collected by passing 250 mL of experimental water sample through six different filter pore sizes (from 0.1 to 8 µm). This process was repeated at four time intervals after spiking over 72 hours to assess if the particle size of the amount of eDNA recovered changes as the eDNA degrades. Using a human-specific quantitative polymerase chain reaction (qPCR) assay targeting the HV1 mitochondrial gene region, the total amount of mitochondrial eDNA associated with different particle size fractions was determined. In the case of human blood, at 0 h, the 0.45 µm filter pore size captured the greatest amount of mitochondrial eDNA, capturing 42 % of the eDNA detected. The pattern then changed after 48 h, with the 5 µm filter pore size capturing the greatest amount of eDNA (67 %), and 81 % of eDNA at 72 h. Notably, a ten-fold dilution proved to be a valuable strategy for enhancing eDNA recovery from the 8 µm filter at all time points, primarily due to the PCR inhibition observed in hemoglobin. For human skin cells, the greatest amounts of eDNA were recovered from the 8 µm filter pore size and were consistent through time (capturing 37 %, 56 %, and 88 % of eDNA at 0 hours, 48 hours, and 72 hours respectively). There is a clear variation in the amount of eDNA recovered between different cell types, and in some forensic scenarios, there is likely to be a mix of cell types present. These results suggest it would be best to use a 5 µm filter pore size to capture human blood and an 8 µm filter pore size to capture human skin cells to maximize DNA recovery from freshwater samples. Depending on the cell type contributing to the eDNA, a combination of different filter pore sizes may be employed to optimize the recovery of human DNA from water samples. This study provides the groundwork for optimizing a strategy for the efficient recovery of human eDNA from aquatic environments, paving the way for its broader application in forensic and environmental sciences.

Circulating tumor DNA analysis guiding adjuvant therapy in stage II colon cancer: Overall survival and updated 5-year results from the randomized DYNAMIC trial.

108 Background: Previous results of the DYNAMIC study demonstrated that a ctDNA-guided approach versus standard management in stage II colon cancer (CC) reduced adjuvant chemotherapy (ACT) use without compromising 2-year recurrence-free survival (RFS). MMR status defines two distinct subsets of stage II CC. Here, we report the impact of ctDNA burden, end of ACT (EOT) ctDNA, and updated survival data including overall survival (OS). Methods: DYNAMIC is a multi-center randomized phase II trial. Eligible patients (pts) had resected stage II CC and were suitable for ACT. Pts were randomly assigned 2:1 to ctDNA-guided management or standard management (clinician-guided based on conventional criteria). For ctDNA-guided management, a ctDNA-positive result at 4 or 7 weeks after surgery with a tumor-informed assay prompted oxaliplatin-based or fluoropyrimidine ACT; ctDNA-negative pts were not treated. Between Aug 2015 and Aug 2019, 302 received ctDNA-guided and 153 standard management. The primary endpoint was RFS, with a non-inferiority margin of 8.5%. Pre-specified key secondary endpoints were ACT use and OS, with an additional secondary endpoint of ctDNA clearance rate. Results: With a median follow-up of 59.6 months (IQR 55.0–61.5), 5-year RFS were 88% and 87% with ctDNA-guided and standard management, respectively (difference 1.1%, 95% confidence interval, -5.8% to 8.0%). 5-year OS for ctDNA-guided treatment was 93.8% and standard management 93.3% (HR 1.05; 95% CI, 0.47 to 2.37; P = 0.887). 5-year OS was significantly worse in treated ctDNA-positive versus untreated ctDNA-negative pts (85.6% vs 95.3%, HR 3.30; 95% CI, 1.02 to 9.05; P = 0.014). The 5-year OS for ctDNA-negative T3 and T4 disease were 96.0% and 90.6%, respectively (HR 2.45; 95% CI, 0.65 to 9.25; P = 0.171). For treated ctDNA-positive pts, ctDNA clearance was observed at EOT in 35/40 (87.5%). The 5-year RFS for EOT ctDNA clearance vs ctDNA persistence were 85.2% and 20.0%, respectively (HR 15.4; 95% CI, 3.91 to 61.0; P < 0.001). Pts with ≥ 0.38 (the median) mutant tumor molecules (MTM/mL) had a lower ctDNA clearance rate and worse RFS than pts with < 0.38 MTM/mL (ctDNA clearance 75% vs 100%, P = 0.047; 5-year RFS 58.9% vs 95.2%, HR 10.62, P = 0.005). Post-op ctDNA was detected in 5/59 (8.5%) of dMMR and 40/235 (17%) of pMMR cases. In an exploratory analysis, ctDNA clearance was observed in 3/4 (75%) and 32/36 (89%) of dMMR and pMMR cases, respectively. Conclusions: Mature outcome data confirms the previous finding of non-inferiority of RFS with a ctDNA-guided approach to ACT for stage II CC. For ctDNA-positive pts, the post-surgery mutation burden provides additional prognostic information, as does the EOT ctDNA result. Additional data is needed to define any differential impact of ACT by MMR status. This data supports a role for ctDNA analysis, including serial sampling, in the management of stage II CC. Clinical trial information: ACTRN12615000381583 .

MSK-ACCESS powered with SOPHiA DDM: Performance analysis of a decentralized MSK-ACCESS solution.

e15063 Background: Blood plasma cell-free DNA (cfDNA) testing of cancer patients allows for genomic profiling when tissue sampling is too invasive or of insufficient quality. This approach has demonstrated clinical utility to identify treatment targets, screen for resistance and monitor disease burden. MSK-ACCESS (Memorial Sloan Kettering – Analysis of Circulating cfDNA to Evaluate Somatic Status) is a validated, high sensitivity cfDNA assay employed at MSK to identify biologically actionable tumor alterations in clinical care. SOPHiA GENETICS is developing a decentralized solution of this assay that incorporates major features of MSK-ACCESS such as molecular barcoding, duplex-based variant calling, the inclusion of tumor-informed prior knowledge and identification of somatic alterations through the removal of both germline and clonal hematopoiesis (CH) variants. Here, we present the results of a performance study obtained during product development. Methods: A cohort of 24 plasma cfDNA and matched white blood cell (WBC) DNA samples were collected from patients with solid tumors and processed at MSK according to the validated standard operating procedures and bioinformatic analyses. In parallel, for the same set of materials libraries were constructed at SOPHiA GENETICS using the proprietary CUMIN molecular identifiers, captured with probes targeting the updated MSK-ACCESS v2 set of 146 genes and sequenced on an Illumina NovaSeq 6000. Variants were called using a duplex-aware approach by SOPHiA GENETICS and germline and CH variants were identified and filtered by paired cfDNA – WBC DNA analysis. Accuracy of the decentralized “MSK-ACCESS powered with SOPHiA DDM” solution was assessed through concordance to the orthogonal results obtained by MSK. Results: A total of 104 somatic SNV/Indels were detected with variant allele fractions (VAF) ranging from 0.1% to 97.0% with a median of 5 variants per sample. The positive percent agreement (PPA) for all variants above the limit of detection of the original MSK-ACCESS assay was 98.9% with 99.98% specificity. For the subset of prior-knowledge variants PPA was 95.6% down to 0.1% VAF. A total of additional 59 variants with ≤ 15% VAF were found both in cfDNA and WBC and assigned as CH-derived. Importantly, these occurred not only in CH-associated genes but also in common oncogenes such as BRCA1/2 and ERBB2, highlighting the utility of matched WBC analysis to correctly identify somatic variants. Conclusions: The results demonstrate that the decentralized “MSK-ACCESS powered with SOPHiA DDM” solution exhibits a high degree of agreement to the results achieved by MSK. Further studies will be conducted to expand the analytical performance evaluation.

Unveiling the genetic structure of pig population in a Himalayan state Uttarakhand through microsatellite and mitochondrial DNA analyses.

This study traced the maternal lineage of the domestic swine populations using mitochondrial DNA control region markers and genetic diversity using microsatellite markers in Uttarakhand, an Indian state situated at the foothills of the world's youngest (geo-dynamically sensitive) mountain system, "the Himalayas". Analysis of 68 maternally unrelated individuals revealed 20 haplotypes. The maternal signature of the Pacific, Southeast Asian, European, and ubiquitously distributed Chinese haplotypes was present in Uttarakhand's domestic pig population. The D3 haplotype reported in wild pigs from North India was also identified in 47 domestic samples. A unique gene pool, UKD (Uttarakhand Domestic), as another lineage specific to this region has been proposed. Genotypes were analyzed, using 13 sets of microsatellite markers. The observed (Ho) and expected (He) heterozygosities were 0.83 ± 0.02 and 0.84 ± 0.01, respectively. The average polymorphic information content value of 0.83 ± 0.01 indicated the high informativeness of the marker. The overall mean FIS value for all the microsatellite markers was low (F = 0.04, P < 0.01). Seven loci deviated from Hardy-Weinberg equilibrium (HWE) at a significant level (p < 0.05). Two clusters were identified, indicating overlapping populations. These results suggested that though belonging to different maternal lineages, the traditional management practices in Uttarakhand have allowed for genetic mixing and the sharing of genetic material among pig populations. It could contribute to increased genetic diversity but might also result in the loss of distinct genetic characteristics or breed purity of the local breeds if not carefully managed.

Kiwifruit (Actinidia deliciosa) aqueous extract improves hyperglycemia, testicular inflammation, apoptosis, and tissue structure induced by Streptozotocin via oxidative stress inhibition

Diabetes mellitus (DM) is a well-known hyperglycemic metabolic condition identified by oxidative stress and biological function disruption. Kiwifruit is a valuable source of polyphenols and vitamin C with great antioxidant, nutritional, and health-promoting effects. Therefore, this study was initiated to explore the antioxidant and anti-hyperglycemic effects of kiwifruit aqueous extract (KFE) against oxidative injury and testis dysfunction in rats with diabetes. Twenty-four male Wistar Albino rats (160–170 g) were divided into four groups: Group 1 served as the control, Group 2 supplemented orally with kiwifruit extract (KFE; 1 g/kg/day) for one month, Group 3 was treated with a single streptozotocin dose (STZ; 50 mg/kg ip), and Group 4 where the diabetic rats were administered with KFE, respectively. According to the results, the GC-MS analysis of KFE revealed several main components with strong antioxidant properties. In diabetic rats, lipid peroxidation and hyperglycemia were accompanied by perturbations in hormone levels and sperm characteristics. Antioxidant enzymes, glutathione content, aminotransferase, phosphatase activities, and protein content were decreased. Furthermore, histology, immunohistochemical PCNA expression, and histochemical analysis of collagen, DNA, RNA, and total protein. were altered in rat testis sections, supporting the changes in biochemistry. Furthermore, diabetic rats supplemented with KFE manifested considerable amendment in all the tested parameters besides improved tissue structure and gene expressions (NF-kB, p53, IL-1β, Bax, IL-10, and Bcl2) relative to the diabetic group. In conclusion, KFE has beneficial effects as it can improve glucose levels and testis function, so it might be used as a complementary therapy in DM.