Integrated approach to generate artificial samples with low tumor fraction for somatic variant calling benchmarking.

BACKGROUND: High-throughput sequencing (HTS) has become the gold standard approach for variant analysis in cancer research. However, somatic variants may occur at low fractions due to contamination from normal cells or tumor heterogeneity; this poses a significant challenge for standard HTS analysis pipelines. The problem is exacerbated in scenarios with minimal tumor DNA, such as circulating tumor DNA in plasma. Assessing sensitivity and detection of HTS approaches in such cases is paramount, but time-consuming and expensive: specialized experimental protocols and a sufficient quantity of samples are required for processing and analysis. To overcome these limitations, we propose a new computational approach specifically designed for the generation of artificial datasets suitable for this task, simulating ultra-deep targeted sequencing data with low-fraction variants and demonstrating their effectiveness in benchmarking low-fraction variant calling. RESULTS: Our approach enables the generation of artificial raw reads that mimic real data without relying on pre-existing data by using NEAT, a fine-grained read simulator that generates artificial datasets using models learned from multiple different datasets. Then, it incorporates low-fraction variants to simulate somatic mutations in samples with minimal tumor DNA content. To prove the suitability of the created artificial datasets for low-fraction variant calling benchmarking, we used them as ground truth to evaluate the performance of widely-used variant calling algorithms: they allowed us to define tuned parameter values of major variant callers, considerably improving their detection of very low-fraction variants. CONCLUSIONS: Our findings highlight both the pivotal role of our approach in creating adequate artificial datasets with low tumor fraction, facilitating rapid prototyping and benchmarking of algorithms for such dataset type, as well as the important need of advancing low-fraction variant calling techniques.

Tumor extrachromosomal DNA: Biogenesis and recent advances in the field.

Extrachromosomal DNA (ecDNA) is a self-replicating circular DNA originating from the chromosomal genome and exists outside the chromosome. It contains specific gene sequences and non-coding regions that regulate transcription. Recent studies have demonstrated that ecDNA is present in various malignant tumors. Malignant tumor development and poor prognosis may depend on ecDNA's distinctive ring structure, which assists in amplifying oncogenes. During cell division, an uneven distribution of ecDNA significantly enhances tumor cells' heterogeneity, allowing tumor cells to adapt to changes in the tumor microenvironment and making them more resistant to treatments. The application of ecDNA as a cancer biomarker and therapeutic target holds great potential. This article examines the latest advancements in this area and discusses the potential clinical applications of ecDNA.

Lung cancer biomarkers: Raising the clinical value of the classical and the new ones.

Blood-based diagnostics for lung cancer support the diagnosis, estimation of prognosis, prediction, and monitoring of therapy response in lung cancer patients. The clinical utility of serum tumor markers has considerably increased due to developments in serum protein tumor markers analytics and clinical biomarker studies, the exploration of preanalytical and influencing conditions, the interpretation of biomarker combinations and individual biomarker kinetics, as well as the implementation of biostatistical models. In addition, circulating tumor DNA (ctDNA) and other liquid biopsy markers are playing an increasingly prominent role in the molecular tumor characterization and the monitoring of tumor evolution over time. Thus, modern lung cancer biomarkers may considerably contribute to an individualized companion diagnostics and provide a sensitive guidance for patients throughout the course of their disease. In this special edition on Tumor Markers in Lung Cancer, experts summarize recent developments in clinical laboratory diagnostics of lung cancer and give an outlook on future challenges and opportunities.

Analytical validation of NeXT Personal®, an ultra-sensitive personalized circulating tumor DNA assay.

We describe the analytical validation of NeXT Personal®, an ultra-sensitive, tumor-informed circulating tumor DNA (ctDNA) assay for detecting residual disease, monitoring therapy response, and detecting recurrence in patients diagnosed with solid tumor cancers. NeXT Personal uses whole genome sequencing of tumor and matched normal samples combined with advanced analytics to accurately identify up to ~1,800 somatic variants specific to the patient's tumor. A personalized panel is created, targeting these variants and then used to sequence cell-free DNA extracted from patient plasma samples for ultra-sensitive detection of ctDNA. The NeXT Personal analytical validation is based on panels designed from tumor and matched normal samples from two cell lines, and from 123 patients across nine cancer types. Analytical measurements demonstrated a detection threshold of 1.67 parts per million (PPM) with a limit of detection at 95% (LOD95) of 3.45 PPM. NeXT Personal showed linearity over a range of 0.8 to 300,000 PPM (Pearson correlation coefficient = 0.9998). Precision varied from a coefficient of variation of 12.8% to 3.6% over a range of 25 to 25,000 PPM. The assay targets 99.9% specificity, with this validation study measuring 100% specificity and in silico methods giving us a confidence interval of 99.92 to 100%. In summary, this study demonstrates NeXT Personal as an ultra-sensitive, highly quantitative and robust ctDNA assay that can be used to detect residual disease, monitor treatment response, and detect recurrence in patients.

Circulating Tumor DNA (ctDNA) and Its Role in Gynecologic Malignancies.

OPINION STATEMENT: Circulating tumor DNA (ctDNA) refers to small fragments of DNA released into the bloodstream by cancer cells. It is obtained through "liquid biopsy;" which most commonly refers to plasma or blood samples, but can be obtained from a number of bodily fluids including ascitic fluid, saliva, and even urine and stool. ctDNA is detected via polymerase chain reaction (PCR) or next-generation sequencing (NGS). The DNA from these samples is analyzed for the detection of point mutations, copy-number alterations, gene fusion, and DNA methylation. These results have the potential for use in cancer diagnosis, determining prognosis, targeting gene-specific therapies, and monitoring for/predicting disease recurrence and response to treatment. ctDNA offers an alternative to tissue biopsy; it is less invasive and can be monitored serially over time without multiple procedures. Moreover it may have the ability to detect disease recurrence or predict behavior in a way that solid tissue biopsies, tumor marker surveillance, and imaging cannot. Recent explosion in interest in ctDNA shows promising developments for widespread adoption of these techniques in cancer care. However, the use of ctDNA in diagnosis and treatment of gynecologic malignancies is currently limited, compared to adoption in other solid-organ tumors such as breast and colorectal cancers. Compared to other cancer types, there appear to be fewer comprehensive studies and clinical validations specifically focusing on the use of ctDNA in gynecologic cancers. More research is needed in this area to advance the potential for use of ctDNA in ovarian, endometrial, and cervical cancers before this can be routinely adopted to improve care for patients with gynecologic malignancies.

Red blood cells function as reservoirs of tumor DNA.

Novel screening techniques for early detection of lung cancer are urgently needed. Profiling circulating tumor cell-free DNA (ctDNA) has emerged as a promising tool for biopsy-free tumor genotyping. However, both the scarcity and short half-life of ctDNA substantially limit the sensitivity and clinical utility of ctDNA detection methodologies. Our discovery that red blood cells (RBCs) sequester mitochondrial DNA opens a new avenue for detecting circulating nucleic acids, as RBCs represent an unrecognized reservoir of circulating nucleic acid. Here, we show that RBCs acquire tumor DNA following coculture with lung cancer cell lines harboring Kirsten rat sarcoma viral oncogene homolog (KRAS) and epidermal growth factor receptor (EGFR) mutations. RBC-bound tumor DNA is detectable in patients with early-stage non-small cell lung cancer (NSCLC) but not in healthy controls by qPCR. Our results collectively uncover a previously unrecognized yet easily accessible reservoir of tumor DNA, offering a promising foundation for future RBC-based tumor diagnostics.NEW & NOTEWORTHY We present a novel method for lung cancer detection by revealing RBCs as a reservoir for tumor DNA, overcoming the limitations of current circulating tumor ctDNA methodologies. By demonstrating that RBCs can capture tumor DNA, including critical mutations found in lung cancer, we provide a promising, biopsy-free avenue for early cancer diagnostics. This discovery opens up exciting possibilities for developing RBC-based diagnostic tools, significantly enhancing the sensitivity and clinical utility of noninvasive cancer detection.

Application of plasma circulating KRAS mutations as a predictive biomarker for targeted treatment of pancreatic cancer.

Kirsten rat sarcoma viral oncogene homolog (KRAS) mutations in circulating tumor deoxyribonucleic acid (ctDNA) have been reported as representative noninvasive prognostic markers for pancreatic ductal adenocarcinoma (PDAC). Here, we aimed to evaluate single KRAS mutations as prognostic and predictive biomarkers, with an emphasis on potential therapeutic approaches to PDAC. A total of 128 patients were analyzed for multiple or single KRAS mutations (G12A, G12C, G12D, G12R, G12S, G12V, and G13D) in their tumors and plasma using droplet digital polymerase chain reaction (ddPCR). Overall, KRAS mutations were detected by multiplex ddPCR in 119 (93%) of tumor DNA and 68 (53.1%) of ctDNA, with a concordance rate of 80% between plasma ctDNA and tumor DNA in the metastatic stage, which was higher than the 44% in the resectable stage. Moreover, the prognostic prediction of both overall survival (OS) and progression-free survival (PFS) was more relevant using plasma ctDNA than tumor DNA. Further, we evaluated the selective tumor-suppressive efficacy of the KRAS G12C inhibitor sotorasib in a patient-derived organoid (PDO) from a KRAS G12C-mutated patient using a patient-derived xenograft (PDX) model. Sotorasib showed selective inhibition in vitro and in vivo with altered tumor microenvironment, including fibroblasts and macrophages. Collectively, screening for KRAS single mutations in plasma ctDNA and the use of preclinical models of PDO and PDX with genetic mutations would impact precision medicine in the context of PDAC.

Analytical evaluation of circulating tumor DNA sequencing assays.

In China, circulating tumor DNA analysis is widely used and numerous assays are available. Systematic evaluation to help users make informed selections is needed. Nine circulating tumor DNA assays, including one benchmark assay, were evaluated using 23 contrived reference samples. There were two sample types (cell-free DNA and plasma samples), three circulating tumor DNA inputs (low, < 20 ng; medium, 20-50 ng; high, > 50 ng), two variant allele frequency ranges (low, 0.1-0.5%; intermediate, 0.5-2.5%), and four variant types (single nucleotide, insertion/deletion, structural, and copy number). Sensitivity, specificity, reproducibility, and all processes from cell-free DNA extraction to bioinformatics analysis were assessed. The test assays were generally comparable or superior to the benchmark assay, demonstrating high analytical sensitivity. Variations in circulating tumor DNA extraction and quantification efficiency, sensitivity, and reproducibility were observed, particularly at lower inputs. These findings will guide circulating tumor DNA assay choice for research and clinical studies, allowing consideration of multiple technical parameters.

Clinical Application of Liquid Biopsy in Pancreatic Cancer: A Narrative Review.

Pancreatic ductal adenocarcinoma contributes significantly to global cancer-related deaths, featuring only a 10% survival rate over five years. The quest for novel tumor markers is critical to facilitate early diagnosis and tailor treatment strategies for this disease, which is key to improving patient outcomes. In pancreatic ductal adenocarcinoma, these markers have been demonstrated to play a crucial role in early identification, continuous monitoring, and prediction of its prognosis and have led to better patient outcomes. Nowadays, biopsy specimens serve to ascertain diagnosis and determine tumor type. However, liquid biopsies present distinct advantages over conventional biopsy techniques. They offer a noninvasive, easily administered procedure, delivering insights into the tumor's status and facilitating real-time monitoring. Liquid biopsies encompass a variety of elements, such as circulating tumor cells, circulating tumor DNA, extracellular vesicles, microRNAs, circulating RNA, tumor platelets, and tumor endothelial cells. This review aims to provide an overview of the clinical applications of liquid biopsy as a technique in the management of pancreatic cancer.

Circulating Tumor DNA in the Immediate Postoperative Setting.

BACKGROUND: Circulating tumor DNA (ctDNA) has emerged as an accurate real-time biomarker of disease status across many solid tumor types. Most studies evaluating the utility of ctDNA have focused on time points weeks to months after surgery, which, for many cancer types, is significantly later than decision-making time points for adjuvant treatment. In this systematic review, we summarize the state of the literature on the feasibility of using ctDNA as a biomarker in the immediate postoperative period. METHODS: We performed a systematic review evaluating the early kinetics, defined here as 3 days of ctDNA in patients who underwent curative-intent surgery. RESULTS: Among the 2057 studies identified, eight cohort studies met the criteria for evaluation. Across six different cancer types, all studies showed an increased risk of cancer recurrence in patients with detectable ctDNA in the immediate postoperative period. CONCLUSION: While ctDNA clearance kinetics appear to vary based on tumor type, across all studies detectable ctDNA after surgery was predictive of recurrence, suggesting early postoperative time points could be feasibly used for determining minimal residual disease. However, larger studies need to be performed to better understand the precise kinetics of ctDNA clearance across different cancer types as well as to determine optimal postoperative time points.

Plasma cell-free DNA in canine lymphoma patients as a novel material for genotyping.

Canine lymphoma is a disease with high morbidity and poor long-term prognosis, despite a high response rate to chemotherapy. In this study, we focused on liquid biopsy, in which small amounts of substances from body fluids were analysed, to determine whether cell-free DNA (cfDNA) in the plasma can be used as a biomarker for lymphoma in dogs. We found that 23 patients with lymphoma had significantly higher cfDNA concentrations than the 12 healthy dogs (median 2360 ng/mL versus 299 ng/mL, p < .0001). Polymerase chain reaction for antigen receptor rearrangement (PARR) was also employed using cfDNA from the lymphoma group to investigate whether cfDNA could be used for the detection of genetic clonality of lymphomas, as well as the genomic DNA (gDNA) extracted from an original lesion in each case. The correlation of the PARR results between cfDNA and gDNA was observed in 100% of B-cell lymphomas (10/10), 77.8% of T-cell lymphomas (7/9), and 100% of other types of lymphomas (4/4), respectively. These results indicate that plasma cfDNA levels are increasing in canine lymphoma patients, that cfDNA concentration can be a novel diagnostic tool, and that it can be used as a diagnostic tool for PARR.

Tumor-Informed Circulating Tumor DNA for Minimal Residual Disease Detection in the Management of Colorectal Cancer.

PURPOSE: Recurrence after curative-intent treatment occurs in 20%-50% of patients with stage II-IV colorectal cancer (CRC), underscoring the need for early detection of minimal residual disease (MRD) using circulating tumor DNA (ctDNA). Here, we examined the pattern of use of a tumor-informed ctDNA assay in CRC MRD monitoring in routine clinical practice at Mayo Clinic, Rochester. METHODS: We conducted a retrospective analysis of health records of patients with CRC who had at least one tumor-informed ctDNA assay from May 2019 through July 1, 2022. Recurrence was defined as radiographic evidence of disease. Descriptive characteristics of the cohort, ctDNA results, and subsequent interventions were recorded. RESULTS: Of the 120 patients included, the median age at diagnosis was 67 years, 46% were female, and 94% were White. At diagnosis, 10 patients had stage I, 23 stage II, 60 stage III, and 25 stage IV disease. Of 476 ctDNA assays performed, 70% were performed in patients who had recurrent disease most commonly to monitor the effectiveness of therapeutic interventions and 16% resulted in a change in clinical decision making. There were 110 recurrences identified in 62 patients, as some patients experienced more than one recurrence over time. Compared with serum carcinoembryonic antigen levels, ctDNA results correlated better with radiologic imaging. CONCLUSION: Routine ctDNA monitoring for MRD detection has been adopted in clinical practice; however, 84% of ctDNA assays performed did not result in a change in clinical management. This suggests the need for further clinical research data to guide routine clinical use of ctDNA MRD testing in CRC.

Unraveling the potential clinical utility of circulating tumor DNA detection in colorectal cancer-evaluation in a nationwide Danish cohort.

BACKGROUND: Increasingly, circulating tumor DNA (ctDNA) is proposed as a tool for minimal residual disease (MRD) assessment. Digital PCR (dPCR) offers low analysis costs and turnaround times of less than a day, making it ripe for clinical implementation. Here, we used tumor-informed dPCR for ctDNA detection in a large colorectal cancer (CRC) cohort to evaluate the potential for post-operative risk assessment and serial monitoring, and how the metastatic site may impact ctDNA detection. Additionally, we assessed how altering the ctDNA-calling algorithm could customize performance for different clinical settings. PATIENTS AND METHODS: Stage II-III CRC patients (N = 851) treated with a curative intent were recruited. Based on whole-exome sequencing on matched tumor and germline DNA, a mutational target was selected for dPCR analysis. Plasma samples (8 ml) were collected within 60 days after operation and-for a patient subset (n = 246)-every 3-4 months for up to 36 months. Single-target dPCR was used for ctDNA detection. RESULTS: Both post-operative and serial ctDNA detection were prognostic of recurrence [hazard ratio (HR) = 11.3, 95% confidence interval (CI) 7.8-16.4, P < 0.001; HR = 30.7, 95% CI 20.2-46.7, P < 0.001], with a cumulative ctDNA detection rate of 87% at the end of sample collection in recurrence patients. The ctDNA growth rate was prognostic of survival (HR = 2.6, 95% CI 1.5-4.4, P = 0.001). In recurrence patients, post-operative ctDNA detection was challenging for lung metastases (4/21 detected) and peritoneal metastases (2/10 detected). By modifying the cut-off for calling a sample ctDNA positive, we were able to adjust the sensitivity and specificity of our test for different clinical contexts. CONCLUSIONS: The presented results from 851 stage II-III CRC patients demonstrate that our personalized dPCR approach effectively detects MRD after operation and shows promise for serial ctDNA detection for recurrence surveillance. The ability to adjust sensitivity and specificity shows exciting potential to customize the ctDNA caller for specific clinical settings.

Revolutionizing Non-Small Cell Lung Cancer Diagnosis: Ultra-High-Sensitive ctDNA Analysis for Detecting Hotspot Mutations with Long-term Stored Plasma.

PURPOSE: Circulating cell-free DNA (cfDNA) has great potential in clinical oncology. The prognostic and predictive values of cfDNA in non-small cell lung cancer (NSCLC) have been reported, with epidermal growth factor receptor (EGFR), KRAS, and BRAF mutations in tumor-derived cfDNAs acting as biomarkers during the early stages of tumor progression and recurrence. However, extremely low tumor-derived DNA rates hinder cfDNA application. We developed an ultra-high-sensitivity lung version 1 (ULV1) panel targeting BRAF, KRAS, and EGFR hotspot mutations using small amounts of cfDNA, allowing for semi-quantitative analysis with excellent limit-of-detection (0.05%). MATERIALS AND METHODS: Mutation analysis was performed on cfDNAs extracted from the plasma of 104 patients with NSCLC by using the ULV1 panel and targeted next-generation sequencing (CT-ULTRA), followed by comparison analysis of mutation patterns previously screened using matched tumor tissue DNA. RESULTS: The ULV1 panel demonstrated robust selective amplification of mutant alleles, enabling the detection of mutations with a high degree of analytical sensitivity (limit-of-detection, 0.025%-0.1%) and specificity (87.9%-100%). Applying ULV1 to NSCLC cfDNA revealed 51.1% (23/45) samples with EGFR mutations, increasing with tumor stage: 8.33% (stage I) to 78.26% (stage IV). Semi-quantitative analysis proved effective for low-mutation-fraction clinical samples. Comparative analysis with PANAMutyper EGFR exhibited substantial concordance (κ=0.84). CONCLUSION: Good detection sensitivity (~80%) was observed despite the limited volume (1 mL) and long-term storage (12-50 months) of plasma used and is expected to increase with high cfDNA inputs. Thus, the ULV1 panel is a fast and cost-effective method for early diagnosis, treatment selection, and clinical follow-up of patients with NSCLC.

A comparative study on ctDNA and tumor DNA mutations in lung cancer and benign cases with a high number of CTCs and CTECs.

BACKGROUND: Liquid biopsy provides a non-invasive approach that enables detecting circulating tumor DNA (ctDNA) and circulating tumor cells (CTCs) using blood specimens and theoretically benefits early finding primary tumor or monitoring treatment response as well as tumor recurrence. Despite many studies on these novel biomarkers, their clinical relevance remains controversial. This study aims to investigate the correlation between ctDNA, CTCs, and circulating tumor-derived endothelial cells (CTECs)  while also evaluating whether mutation profiling in ctDNA is consistent with that in tumor tissue from lung cancer patients. These findings will help the evaluation and utilization of these approaches in clinical practice. METHODS: 104 participants (49 with lung cancer and 31 with benign lesions) underwent CTCs and CTECs detection using integrating subtraction enrichment and immunostaining-fluorescence in situ hybridization (SE-iFISH) strategy. The circulating cell-free DNA (cfDNA) concentration was measured and the mutational profiles of ctDNA were examined by Roche AVENIO ctDNA Expanded Kit (targeted total of 77 genes) by next generation sequencing (NGS) in 28 patients (20 with lung cancer and 8 with benign lesions) with highest numbers of CTCs and CTECs. Mutation validation in matched tumor tissue DNA was then performed in 9 patients with ctDNA mutations using a customized xGen pan-solid tumor kit (targeted total of 474 genes) by NGS. RESULTS: The sensitivity and specificity of total number of CTCs and CTECs for the diagnosis of NSCLC were 67.3% and 77.6% [AUC (95%CI): 0.815 (0.722-0.907)], 83.9% and 77.4% [AUC (95%CI): 0.739 (0.618-0.860)]. The concentration of cfDNA in plasma was statistically correlated with the size of the primary tumor (r = 0.430, P = 0.022) and CYFRA 21-1 (r = 0.411, P = 0.041), but not with the numbers of CTCs and CTECs. In this study, mutations were found to be poorly consistent between ctDNA and tumor DNA (tDNA) in patients, even when numerous CTCs and CTECs were present. CONCLUSION: Detection of CTCs and CTECs could be the potential adjunct tool for the early finding of lung cancer. The cfDNA levels are associated with the tumor burden, rather than the CTCs or CTECs counts. Moreover, the poorly consistent mutations between ctDNA and tDNA require further exploration.

E74-like Factor 5 Promoter Methylation in Circulating Tumor DNA as a Potential Prognostic Marker in Breast Cancer Patients.

BACKGROUND: Epigenetic alternations, such as DNA methylation, play a crucial role in breast tumor initiation and progression. The identification of noninvasive prognostic biomarkers has great importance in cancer management. Methylated cell-free DNA (cfDNA), circulating in the blood as a convenient tumor-associated DNA marker, can be used as a minimally invasive cancer biomarker. This study aimed to evaluate the promoter methylation status of E74-like factor 5 (ELF5) tumor suppressor gene in both tumors and plasma cell-free DNA of 80 breast cancer patients, compared with normal controls. METHODS: Plasma cfDNA concentrations were measured using quantitative real-time PCR, and methylation pattern in the ELF5 gene promoter region was performed using methylation-specific polymerase chain reaction (MS-PCR) technique. RESULTS: The data revealed a statistically significant increase in cfDNA concentrations in breast cancer patients, particularly in those with higher stages of the disease, triple-negative status, and metastasis (p<0.001). ELF5 promoter region hypermethylation was observed in 70% of breast cancer patients in both plasma cfDNA and tumor tissues. Notably, all patients with lymph node involvement and distant metastatic exhibited promoter hypermethylation in the ELF5 gene. CONCLUSION: Our findings suggest that ELF5 promoter methylation in circulating DNA could serve as a potential non-invasive prognostic molecular marker in breast cancer patients. However, further studies are warranted to evaluate its diagnostic value.

Liquid-based biomarkers in breast cancer: looking beyond the blood.

In recent decades, using circulating tumor cell (CTC), circulating tumor DNA (ctDNA), circulating tumor RNA (ctRNA), exosomes and etc. as liquid biomarkers has received enormous attention in various tumors, including breast cancer (BC). To date, efforts in the area of liquid biopsy predominantly focus on the analysis of blood-based markers. It is worth noting that the identifications of markers from non-blood sources provide unique advantages beyond the blood and these alternative sources may be of great significance in offering supplementary information in certain settings. Here, we outline the latest advances in the analysis of non-blood biomarkers, predominantly including urine, saliva, cerebrospinal fluid, pleural fluid, stool and etc. The unique advantages of such testings, their current limitations and the appropriate use of non-blood assays and blood assays in different settings are further discussed. Finally, we propose to highlight the challenges of these alternative assays from basic to clinical implementation and explore the areas where more investigations are warranted to elucidate its potential utility.

Sequencing paired tumor DNA and white blood cells improves circulating tumor DNA tracking and detects pathogenic germline variants in localized colon cancer.

BACKGROUND: In the setting of localized colon cancer (CC), circulating tumor DNA (ctDNA) monitoring in plasma has shown potential for detecting minimal residual disease (MRD) and predicting a higher risk of recurrence. With the tumor-only sequencing approach, however, germline variants may be misidentified as somatic variations, precluding the possibility of tracking in up to 11% of patients due to a lack of known somatic mutations. In this study, we assess the potential value of adding white blood cells (WBCs) to tumor tissue sequencing to enhance the accuracy of sequencing results. PATIENTS AND METHODS: A total of 148 patients diagnosed with localized CC were prospectively recruited at the Hospital Clínico Universitario in Valencia (Spain). Employing a custom 29-gene panel, sequencing was conducted on tumor tissue, plasma and corresponding WBCs. Droplet digital PCR and amplicon-based NGS were performed on plasma samples post-surgery to track MRD. Oncogenic somatic variants were identified by annotating with COSMIC, OncoKB and an internal repository of pathogenic mutations database. A variant prioritization analysis, mainly characterized by the match of oncogenic mutations with the evidence levels defined in OncoKB, was carried out to select specific targeted therapies. RESULTS: Utilizing paired tumor and WBCs sequencing, we identified somatic mutations in all patients (100%) within our cohort, compared to 89% using only tumor tissue. Consequently, the top 10 most frequently mutated genes for plasma monitoring were altered. The sequencing of WBCs identified 9% of patients with pathogenic mutations in the germline, with APC and TP53 being the most frequently mutated genes. Additionally, mutations in genes related to clonal hematopoiesis of indeterminate potential were detected in 27% of the cohort, with TP53, KRAS, and KMT2C being the most frequently altered genes. There were no observed differences in the sensitivity of monitoring MRD using ddPCR or amplicon-based NGS (p = 1). Ultimately, 41% of the patients harbored potentially targetable alterations at diagnosis. CONCLUSION: The germline testing method not only enhanced sequencing results and raised the proportion of patients eligible for plasma monitoring, but also uncovered the existence of pathogenic germline variations, thereby aiding in the identification of patients at a higher risk of hereditary cancer syndromes.

Preoperative ctDNA Levels Are Associated With Poor Overall Survival in Patients With Ovarian Cancer.

BACKGROUND/AIM: Circulating tumor DNA (ctDNA), which is shed from cancer cells into the bloodstream, offers a potential minimally invasive approach for cancer diagnosis and monitoring. This research aimed to assess the preoperative ctDNA levels in ovarian tumors patients' plasma and establish correlations with clinicopathological parameters and patient prognosis. PATIENTS AND METHODS: Tumor DNA was extracted from ovarian tumor tissue from 41 patients. Targeted sequencing using a panel of 127 genes recurrently mutated in cancer was performed to identify candidate somatic mutations in the tumor DNA. SAGAsafe digital PCR (dPCR) assays targeting the candidate mutations were used to measure ctDNA levels in patient plasma samples, obtained prior to surgery, to evaluate ctDNA levels in terms of mutant copy number/ml and variant allele frequency. RESULTS: Somatic mutations were found in 24 tumor samples, 17 of which were from ovarian cancer patients. The most frequently mutated gene was TP53. Preoperative plasma ctDNA levels were detected in 14 of the 24 patients. With higher stage, plasma ctDNA mutant concentration increased (p for trend <0.001). The overall survival of cancer patients with more than 10 ctDNA mutant copies/ml in plasma was significantly worse (p=0.008). CONCLUSION: Pre-operative ctDNA measurement in ovarian cancer patients' plasma holds promise as a predictive biomarker for tumor staging and prognosis.

Completing a genomic characterisation of microscopic tumour samples with copy number.

BACKGROUND: Genomic insights in settings where tumour sample sizes are limited to just hundreds or even tens of cells hold great clinical potential, but also present significant technical challenges. We previously developed the DigiPico sequencing platform to accurately identify somatic mutations from such samples. RESULTS: Here, we complete this genomic characterisation with copy number. We present a novel protocol, PicoCNV, to call allele-specific somatic copy number alterations from picogram quantities of tumour DNA. We find that PicoCNV provides exactly accurate copy number in 84% of the genome for even the smallest samples, and demonstrate its clinical potential in maintenance therapy. CONCLUSIONS: PicoCNV complements our existing platform, allowing for accurate and comprehensive genomic characterisations of cancers in settings where only microscopic samples are available.