LABS: linear amplification-based bisulfite sequencing for ultrasensitive cancer detection from cell-free DNA.

Methylation-based liquid biopsies show promises in detecting cancer using circulating cell-free DNA; however, current limitations impede clinical application. Most assays necessitate substantial DNA inputs, posing challenges. Additionally, underrepresented tumor DNA fragments may go undetected during exponential amplification steps of traditional sequencing methods. Here, we report linear amplification-based bisulfite sequencing (LABS), enabling linear amplification of bisulfite-treated DNA fragments in a genome-wide, unbiased fashion, detecting cancer abnormalities with sub-nanogram inputs. Applying LABS to 100 patient samples revealed cancer-specific patterns, copy number alterations, and enhanced cancer detection accuracy by identifying tissue-of-origin and immune cell composition.

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.

Combined DNA Analysis from Stool and Blood Samples Improves Tumor Tracking and Assessment of Clonal Heterogeneity in Localized Rectal Cancer Patients.

Objectives: In this study, stool samples were evaluated for tumor mutation analysis via a targeted next generation sequencing (NGS) approach in a small patient cohort suffering from localized rectal cancer. Introduction: Colorectal cancer (CRC) causes the second highest cancer-related death rate worldwide. Thus, improvements in disease assessment and monitoring that may facilitate treatment allocation and allow organ-sparing "watch-and-wait" treatment strategies are highly relevant for a significant number of CRC patients. Methods: Stool-based results were compared with mutation profiles derived from liquid biopsies and the gold standard procedure of tumor biopsy from the same patients. A workflow was established that enables the detection of de-novo tumor mutations in stool samples of CRC patients via ultra-sensitive cell-free tumor DNA target enrichment. Results: Notably, only a 19% overall concordance was found in mutational profiles across the compared sample specimens of stool, tumor, and liquid biopsies. Conclusion: Based on these results, the analysis of stool and liquid biopsy samples can provide important additional information on tumor heterogeneity and potentially on the assessment of minimal residual disease and clonal tumor evolution.

Tumor- and Fibroblast-Derived Cell-Free DNAs Differently Affect the Progression of B16 Melanoma In Vitro and In Vivo.

It is widely postulated that the majority of pathologically elevated extracellular or cell-free DNA (cfDNA) in cancer originates from tumor cells; however, evidence has emerged regarding the significant contributions of other cells from the tumor microenvironment. Here, the effect of cfDNA originating from murine B16 melanoma cells and L929 fibroblasts on B16 cells was investigated. It was found that cfDNAL929 increased the viability and migration properties of B16 cells in vitro and their invasiveness in vivo. In contrast, cfDNAB16 exhibited a negative effect on B16 cells, reducing their viability and migration in vitro, which in vivo led to decreased tumor size and metastasis number. It was shown that cell treatment with both cfDNAs resulted in an increase in the expression of genes encoding DNases and the oncogenes Braf, Kras, and Myc. cfDNAL929-treated cells were shown to experience oxidative stress. Gene expression changes in the case of cfDNAB16 treatment are well correlated with the observed decrease in proliferation and migration of B16 cells. The obtained data may indicate the possible involvement of fibroblast DNA in the tumor microenvironment in tumor progression and, potentially, in the formation of new tumor foci due to the transformation of normal cells.

DNA Quantity and Quality Comparisons between Cryopreserved and FFPE Tumors from Matched Pan-Cancer Samples.

Personalized cancer care requires molecular characterization of neoplasms. While the research community accepts frozen tissues as the gold standard analyte for molecular assays, the source of tissue for testing in clinical cancer care comes almost universally from formalin-fixed, paraffin-embedded tissue (FFPE). As newer technologies emerge for DNA characterization that requires higher molecular weight DNA, it was necessary to compare the quality of DNA in terms of DNA length between FFPE and cryopreserved samples. We hypothesized that cryopreserved samples would yield higher quantity and superior quality DNA compared to FFPE samples. We analyzed DNA metrics by performing a head-to-head comparison between FFPE and cryopreserved samples from 38 human tumors representing various cancer types. DNA quantity and purity were measured by UV spectrophotometry, and DNA from cryopreserved tissue demonstrated a 4.2-fold increase in DNA yield per mg of tissue (p-value < 0.001). DNA quality was measured on a fragment microelectrophoresis analyzer, and again, DNA from cryopreserved tissue demonstrated a 223% increase in the DNA quality number and a 9-fold increase in DNA fragments > 40,000 bp (p-value < 0.0001). DNA from the cryopreserved tissues was superior to the DNA from FFPE samples in terms of DNA yield and quality.

Microsatellite Instability Detection in Cancer: A Multiplex qPCR Approach that Obviates the Need for Matching Normal Samples.

BACKGROUND: Microsatellite instability (MSI) indicates DNA mismatch repair deficiency in certain types of cancer, such as colorectal cancer. The current gold standard technique, PCR-capillary electrophoresis (CE), requires matching normal samples and specialized instrumentation. We developed VarTrace, a rapid and low-cost quantitative PCR (qPCR) assay, to evaluate MSI using solely the tumor sample DNA, obviating the requirement for matching normal samples. METHODS: One hundred and one formalin-fixed paraffin-embedded (FFPE) tumor samples were tested using VarTrace and compared with the Promega OncoMate assay utilizing PCR-CE. Tumor percentage limit of detection was evaluated on contrived samples derived from clinical high MSI (MSI-H) samples. Analytical sensitivity, specificity, limit of detection, and input requirements were assessed using synthetic commercial reference standards. RESULTS: VarTrace successfully analyzed all 101 clinical FFPE samples, demonstrating 100% sensitivity and 98% specificity compared to OncoMate. It detected MSI-H with 97% accuracy down to 10% tumor. Analytical studies using synthetic samples showed a limit of detection of 5% variant allele frequency and a limit of input of 0.5 ng. CONCLUSIONS: This study validates VarTrace as a swift, accurate, and economical assay for MSI detection in samples with low tumor percentages without the need for matching normal DNA. VarTrace's capacity for highly sensitive MSI analysis holds potential for enhancing the efficiency of clinical work flows and broadening the availability of this test.

Improving diagnostic accuracy of identifying gastric cancer patients with peritoneal metastases: tumor-guided cell-free DNA analysis of peritoneal fluid.

Detection of peritoneal dissemination (PD) in gastric cancer (GC) patients remains challenging. The feasibility of tumor-guided cell-free DNA (cfDNA) detection in prospectively collected peritoneal fluid (ascites and peritoneal lavage) was investigated and compared to conventional cytology in 28 patients. Besides conventional cytology, next generation sequencing was performed on primary tumor DNA and cell-free DNA from peritoneal fluid. Patients were retrospectively grouped into: a positive group (with PD) and a negative group (without PD). Detectable mutations were found in the primary tumor of 68% (n = 19). Sensitivity of PD detection by tumor-guided cfDNA analysis was 91%, compared to 64% by conventional cytology. Within the positive group (n = 11), tumor-guided cfDNA was detected in all patients with ascites samples (4/4, 100%) and in 86% (6/7) of the lavage samples, opposed to 4/4 (100%) patients with ascites and 43% (3/7) with lavage by conventional cytology. Within the negative group (n = 8), conventional cytology was negative for all samples. In two patients, tumor-guided cfDNA was detected in peritoneal lavage fluid. Interestingly, these 2 patients developed PD within 6 months, suggesting a prognostic value of tumor-guided cfDNA detection. This study showed that tumor-guided cfDNA detection in peritoneal fluids of GC patients is feasible and superior to conventional cytology in detecting PD.

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.

Tumor DNA sampling from aqueous humor in retinoblastoma - A report from South Asia.

PURPOSE: Retinoblastoma (RB) is the most common intraocular tumor in pediatric age group. The role of genetics has been explored in predicting survival prognosis, but its role in predicting globe salvage remains largely unexplored. We hereby aim to isolate cell-free DNA (cfDNA) from aqueous humor (AH) in RB eyes and validate its use for genetic studies. METHODS: AH was obtained from 26 eyes undergoing enucleation (arm A) or intravitreal chemotherapy (arm B). Isolation of cfDNA was done using QIAamp ® Circulating Nucleic Acid kit, and the cfDNA was utilized for targeted sequencing of RB1 gene. RESULTS: We could isolate cfDNA in all eyes (72% unilateral and 28% bilateral) with a distribution peak between 140 and 160 bp and a mean concentration of 27.75 ng/µl for arm A and 14 ng/µl for arm B. Targeted sequencing done on four samples showed RB1 gene mutations, namely, inframe deletion (c. 78-80del, p.Pro29del), start-loss mutation (c.1A>T, p.Met1?), nonsense mutations (c.2236G>T, p.Glu746Ter), (c.1659T>A, p.Cys553Ter), and (c.2065C>T, p.Gln689Ter), and novel missense mutations (c.672C>A, p.Asp224Glu) and c.692C>T (p.Pro231Leu). Genetic profile of cfDNA extracted from AH and genomic DNA from the tumor tissue was comparable. CONCLUSION: Our study supports the previous reports that AH may be used as a source of tumor-derived cfDNA. This is the first report from South Asia on isolation and genetic analysis of cfDNA from AH of RB eyes and, therefore, a big step forward in paving the role of tumor genetics in RB. Further studies are required to elucidate concordance between the tumor and AH genetic profile.

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.

ctDNA transiting into urine is ultrashort and facilitates noninvasive liquid biopsy of HPV+ oropharyngeal cancer.

BACKGROUNDTransrenal cell-free tumor DNA (TR-ctDNA), which transits from the bloodstream into urine, has the potential to enable noninvasive cancer detection for a wide variety of nonurologic cancer types.MethodsUsing whole-genome sequencing, we discovered that urine TR-ctDNA fragments across multiple cancer types are predominantly ultrashort (<50 bp) and, therefore, likely to be missed by conventional ctDNA assays. We developed an ultrashort droplet digital PCR assay to detect TR-ctDNA originating from HPV-associated oropharyngeal squamous cell carcinoma (HPV+ OPSCC) and confirmed that assaying ultrashort DNA is critical for sensitive cancer detection from urine samples.ResultsTR-ctDNA was concordant with plasma ctDNA for cancer detection in patients with HPV+ OPSCC. As proof of concept for using urine TR-ctDNA for posttreatment surveillance, in a small longitudinal case series, TR-ctDNA showed promise for noninvasive detection of recurrence of HPV+ OPSCC.ConclusionOur data indicate that focusing on ultrashort fragments of TR-ctDNA will be important for realizing the full potential of urine-based cancer diagnostics. This has implications for urine-based detection of a wide variety of cancer types and for facilitating access to care through at-home specimen collections.FundingNIH grants R33 CA229023, R21 CA225493; NIH/National Cancer Institute grants U01 CA183848, R01 CA184153, and P30CA046592; American Cancer Society RSG-18-062-01-TBG; American Cancer Society Mission Boost grant MBGI-22-056-01-MBG; and the A. Alfred Taubman Medical Research Institute.

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.

Characterization and mitigation of artifacts derived from NGS library preparation due to structure-specific sequences in the human genome.

BACKGROUND: Hybridization capture-based targeted next generation sequencing (NGS) is gaining importance in routine cancer clinical practice. DNA library preparation is a fundamental step to produce high-quality sequencing data. Numerous unexpected, low variant allele frequency calls were observed in libraries using sonication fragmentation and enzymatic fragmentation. In this study, we investigated the characteristics of the artifact reads induced by sonication and enzymatic fragmentation. We also developed a bioinformatic algorithm to filter these sequencing errors. RESULTS: We used pairwise comparisons of somatic single nucleotide variants (SNVs) and insertions and deletions (indels) of the same tumor DNA samples prepared using both ultrasonic and enzymatic fragmentation protocols. Our analysis revealed that the number of artifact variants was significantly greater in the samples generated using enzymatic fragmentation than using sonication. Most of the artifacts derived from the sonication-treated libraries were chimeric artifact reads containing both cis- and trans-inverted repeat sequences of the genomic DNA. In contrast, chimeric artifact reads of endonuclease-treated libraries contained palindromic sequences with mismatched bases. Based on these distinctive features, we proposed a mechanistic hypothesis model, PDSM (pairing of partial single strands derived from a similar molecule), by which these sequencing errors derive from ultrasonication and enzymatic fragmentation library preparation. We developed a bioinformatic algorithm to generate a custom mutation "blacklist" in the BED region to reduce errors in downstream analyses. CONCLUSIONS: We first proposed a mechanistic hypothesis model (PDSM) of sequencing errors caused by specific structures of inverted repeat sequences and palindromic sequences in the natural genome. This new hypothesis predicts the existence of chimeric reads that could not be explained by previous models, and provides a new direction for further improving NGS analysis accuracy. A bioinformatic algorithm, ArtifactsFinder, was developed and used to reduce the sequencing errors in libraries produced using sonication and enzymatic fragmentation.