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.

Safety and immunological effects of ablative radiotherapy followed by pembrolizumab in patients with advanced adrenocortical carcinoma.

TPS4630 Background: Adrenocortical carcinoma (ACC) is a rare and aggressive cancer with poor prognosis. While pembrolizumab is a NCCN recommended treatment, our prior study showed no benefit in patients with liver metastases (NCT02673333).1 Preclinical evidence suggests combining liver-directed radiation therapy (RT) with pembrolizumab might enhance anti-tumor immunity.2 Our current study will assess the safety and describe preliminary efficacy data for ablative RT to symptomatic liver metastases followed by treatment with pembrolizumab in patients with advanced ACC. Methods: This is a single-center pilot study. Main inclusion criteria include: adult patients with metastatic ACC with symptomatic liver metastases, presence of extrahepatic metastases, ECOG 0-1. Ablative RT in 5 or 10 once daily fractions will be delivered over 1 to 2 weeks; pembrolizumab treatment (200mg Q3W) will begin one week after RT completion. The primary objective is to evaluate the safety of treatment with ablative RT followed by pembrolizumab. The study will include a 6 patient safety lead-in, with 10 to 12 patients enrolled in total. The secondary endpoint, efficacy, is defined as the objective response (partial and complete response) according to RECIST v1.1, in the hepatic and extrahepatic disease. Correlative objectives include evaluation of the innate and adaptive immune response during treatment through analysis of pre- and on-treatment blood and tumor tissue samples, circulating tumor (ct) DNA analyses, and PD-L1 and next-generation sequencing testing in pre-treatment tumor tissue samples to evaluate for biomarkers of response and resistance. This trial began enrollment in 9/27/2023, with 3 patients enrolled to date.

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).

Genome-Scale Multimodal Analysis of Cell-Free DNA Whole-Methylome Sequencing for Noninvasive Esophageal Cancer Detection.

Simultaneous profiling of cell-free DNA (cfDNA) methylation and fragmentation features to improve the performance of cfDNA-based cancer detection is technically challenging. We developed a method to comprehensively analyze multimodal cfDNA genomic features for more sensitive esophageal squamous cell carcinoma (ESCC) detection. Enzymatic conversion-mediated whole-methylome sequencing was applied to plasma cfDNA samples extracted from 168 patients with ESCC and 251 noncancer controls. ESCC characteristic cfDNA methylation, fragmentation, and copy number signatures were analyzed both across the genome and at accessible cis-regulatory DNA elements. To distinguish ESCC from noncancer samples, a first-layer classifier was developed for each feature type, the prediction results of which were incorporated to construct the second-layer ensemble model. ESCC plasma genome displayed global hypomethylation, altered fragmentation size, and chromosomal copy number alteration. Methylation and fragmentation changes at cancer tissue-specific accessible cis-regulatory DNA elements were also observed in ESCC plasma. By integrating multimodal genomic features for ESCC detection, the ensemble model showed improved performance over individual modalities. In the training cohort with a specificity of 99.2%, the detection sensitivity was 81.0% for all stages and 70.0% for stage 0-II. Consistent performance was observed in the test cohort with a specificity of 98.4%, an all-stage sensitivity of 79.8%, and a stage 0-II sensitivity of 69.0%. The performance of the classifier was associated with the disease stage, irrespective of clinical covariates. This study comprehensively profiles the epigenomic landscape of ESCC plasma and provides a novel noninvasive and sensitive ESCC detection approach with genome-scale multimodal analysis.

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]

A phase II study evaluating the effect of IMNN-001 on second-look laparoscopy (SLL) when administered in combination with bevacizumab (BEV) and neoadjuvant chemotherapy (NACT) in patients newly diagnosed with advanced epithelial ovarian cancer (EOC).

TPS5633 Background: IMNN-001, an interleukin-12 (IL-12) DNA plasmid formulated with a DNA carrier, polyethyleneglycol-polyethyleneimine cholesterol lipopolymer, has been shown to exhibit a favorable benefit/risk ratio when administered intraperitoneally (IP), in combination with intravenous platinum-taxane chemotherapy, up to doses of 100 mg/m2 in women with advanced EOC in earlier Phase I & II studies. The 201-21-202 study (NCT05739981) is a multi-center, randomized, open-label phase I/II study which hypothesizes the addition of IMNN-001 to neoadjuvant & adjuvant carboplatin-paclitaxel and bevacizumab (chemo + BEV) reduces the risk of histologically documented minimal residual disease (MRD) as determined by SLL by 50%. Methods: Patients with newly diagnosed stage III and IV high grade serous EOC who are candidates for NACT will be enrolled into the trial. Initial safety lead-in will evaluate at least 6 patients randomized to the experimental arm. Upon confirmation of safety by the DSMB, a target of ~50 patients will be randomized 1:1 to receive either chemo + BEV with or without IMNN-001. Patients receive at least 4 cycles of NACT followed by interval cytoreductive surgery (ICS), and 3 additional adjuvant cycles of study treatments will be administered. BEV will be included with each cycle except cycle 1, the last cycle of NACT immediately preceding ICS, and the first cycle of adjuvant chemotherapy. In the experimental arm, IMNN-001 will be administered on C1D15 and continue weekly though the last cycle of adjuvant therapy. Following adjuvant therapy, all patients will undergo SLL with peritoneal washing and multiple biopsies. Pathologic detection of residual tumor in any biopsies or peritoneal cytology constitutes presence of MRD. All patients will receive maintenance therapy with Olaparib + BEV, IMNN-001 + BEV, or BEV alone based on their homologous recombination deficiency status and treatment randomization. Translational:All subjects will undergo biospecimen collection and organoid preparation prior to treatment, at the time of ICS and SLL for comprehensive longitudinal genomic, circulating tumor DNA, immune, and microbiome profiling. Subjects in the experimental arm will also undergo peritoneal lavage and collection of fluid and cells for assessment of the peritoneal tumor environment. Endpoints: The primary endpoint is the rate of MRD at SLL. Secondary endpoints include progression-free survival, overall survival, objective response rate, chemotherapy response score, surgical response, and serologic response. All patients will be evaluated for safety. Status: The study is actively enrolling and is in the safety lead-in portion. We plan to enlist approximately 4 sites in USA. Clinical trial information: NCT05739981 .

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]

Longitudinal circulating tumor DNA (ctDNA) analysis during treatment (Tx) of locally advanced resectable (LAR) gastric or gastroesophageal junction (G/GEJ) adenocarcinoma (ADENOCA): The PLAGAST prospective biomarker study.

Longitudinal circulating tumor DNA (ctDNA) analysis during treatment (Tx) of locally advanced resectable (LAR) gastric or gastroesophageal junction (G/GEJ) adenocarcinoma (ADENOCA): The PLAGAST prospective biomarker study.

Neoadjuvant dalpiciclib combined with aromatase inhibitors in stage I-III HER2 negative luminal B breast cancer (DANCER): Preliminary findings from an ongoing phase II study.

e12622 Background: CDK4/6 inhibitors, in combination with endocrine therapy, have shown efficacy as neoadjuvant treatment in HR+/HER2- breast cancer (BC). This study investigates the efficacy and safety of dalpiciclib, a CDK4/6 inhibitor, combined with aromatase inhibitors (AI) as a neoadjuvant treatment for luminal B BC (defined as ER+/HER2- BC, with either Ki67≥20% and/or PR<20%). Methods: This single-arm, single-center, phase II study (NCT05640778) enrolled patients aged 18-75 with clinical stage I-III luminal B BC, primary tumor size ≥2 cm, and ECOG PS 0-1. Eligible patients, regardless of menopausal status, received AI therapy (either anastrozole 1 mg daily or letrozole 2.5 mg daily) combined with dalpiciclib (150 mg daily on days 1-21) starting from cycle 1 day 1 (C1D1), for four 28-day cycles, followed by surgery. Pre-/peri-menopausal patients received AI therapy 14 days prior to the first dose of dalpiciclib (C0D1-C0D14), concurrent with a GnRH agonist administered every 28 days from C0D1. Ki67 levels were assessed through serial biopsies at baseline, C1D15, and surgery. MRI assessments were performed at baseline, C2D28, and before surgery per RECIST v1.1. Serial plasma samples were collected for circulating tumor DNA (ctDNA) analysis. The primary endpoint was the complete cell cycle arrest (CCCA: Ki67≤2.7%) rate at 2 weeks (C1D15). Results: From Oct 2022 to Dec 2023, 23 patients with a median age of 52 years (range 38-73) were enrolled and received study treatment. Of these patients, 11 (47.8%) were pre-/peri-menopausal. Sixteen (69.6%) were diagnosed with stage II and the remaining had stage III disease. High Ki67 expression (≥20%) was observed in 21 patients (91.3%). As of Jan 7, 2024, all patients underwent the C1D15 Ki67 evaluation and the CCCA rate was 82.6% (95% CI: 60.5%-94.3%). The ctDNA concentration at C2D28 and post-surgery was significantly higher in patients who did not achieve CCCA compared to those who achieved CCCA ( P = 0.003 and 0.011, respectively). Ki67 expression decreased by a geometric mean of 93.1% (95% CI:76.0%-98.8%) from baseline to C1D15. Out of 20 patients who completed 2 cycles of treatment and received an MRI scan, 12 achieved a partial response, yielding an ORR of 60.0% at C2D28. Among 17 who completed all neoadjuvant therapy and underwent surgery, the ORR before surgery was 82.4% with 14 partial responses; none demonstrated residual cancer burden (RCB) 0/1, seven exhibited RCB 2, and 10 had RCB 3; one reached breast pathological complete response (ypTisN1). Grade ≥3 treatment-emergent adverse events included neutrophil count decreased (16, 69.6%), white blood cell decreased (9, 39.1%), and oral mucositis (1, 4.3%). Conclusions: Dalpiciclib plus anastrozole or letrozole exhibited biological and clinical activity with manageable toxicities in neoadjuvant treatment for stage I-III luminal B/HER2- BC. Clinical trial information: NCT05640778 .

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]

A randomized, open-label phase II/III efficacy and safety study of atezolizumab in combination with FLOT versus FLOT alone in patients with gastric cancer and adenocarcinoma of the oesophagogastric junction and high immune responsiveness: The IKF633/DANTE trial, a trial of AIO in collaboration with SAKK.

TPS4184 Background: Despite recent advances, long-term survival of patients with locally advanced, operable esophagogastric adenocarcinoma (EGA) is below 50%. Immune checkpoint inhibitors in the neoadjuvant setting of this tumor type have lately been shown to increase pathological responses in all comers (IKF633/DANTE pII, KEYNOTE-585, Matterhorn), but the effect on survival is still unknown. The IKF633/DANTE pIII trial evaluates the efficacy and clinical activity of atezolizumab (ATZ) with perioperative FLOT in patients (pts) with locally advanced, resectable EGA considered having an immune-responsive profile. Methods: IKF633/DANTE is conducted as a randomized, multinational, multicenter, prospective, investigator-initiated, open label phase II/III trial. For the phase III portion, pts with histologically confirmed EGA of a clinical stage ≥cT2 and/or cN+ without evidence of distant metastases and with at least one immune response criterium (MSI, PD-L1 CPS≥1, TMB ≥10/MB or EBV+) are eligible. Pts are stratified acc. to nodal stage (cN- vs. cN+), tumor site (GEJ type I vs. GEJ type II/III vs. gastric) and PD-L1 expression levels (CPS≥5 vs. CPS<5). Pts are randomized 1:1 to receive 4 pre- and post-operative cycles FLOT/ATZ, followed by 8 cycles ATZ maintenance (Arm A) or 2x4 perioperative cycles FLOT alone (Arm B). In total, 556 pts will be enrolled, thereof 379 pts in phase III. This estimates to a recruitment time of 30 months plus 18 months follow-up for 80% statistical power and a significance level of p<0.05 (two-sided log rank test). The primary endpoint is event-free survival (EFS) with a target hazard ratio of 0.72, reflecting a clinically relevant improvement to 41.65 months in arm A. Main secondary endpoints are pCR, overall survival (OS) also in high immune-responsive subgroups (CPS ≥5/ CPS≥ 10/ MSI), R0 resection rate and safety/tolerability. In addition, a prospective biomarker study with serial circulating tumor DNA (ctDNA) analysis accompanying therapy is performed to explore the prognostic impact of ctDNA on efficacy and survival parameters, as well as on relapse incidence. Recruitment of phase III started in Aug 2023 and is still ongoing. Clinical trial information: NCT03421288 .

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]