Isolation Of Cell-free DNA Research Articles

Isolation and Characterization of Cell-Free DNA from Cerebral Organoids.

Early detection of neurological conditions is critical for timely diagnosis and treatment. Identifying cellular-level changes is essential for implementing therapeutic interventions prior to symptomatic disease onset. However, monitoring brain tissue directly through biopsies is invasive and poses a high risk. Bodily fluids such as blood or cerebrospinal fluid contain information in many forms, including proteins and nucleic acids. In particular, cell-free DNA (cfDNA) has potential as a versatile neurological biomarker. Yet, our knowledge of cfDNA released by brain tissue and how cfDNA changes in response to deleterious events within the brain is incomplete. Mapping changes in cfDNA to specific cellular events is difficult in vivo, wherein many tissues contribute to circulating cfDNA. Organoids are tractable systems for examining specific changes consistently in a human background. However, few studies have investigated cfDNA released from organoids. Here, we examined cfDNA isolated from cerebral organoids. We found that cerebral organoids release quantities of cfDNA sufficient for downstream analysis with droplet-digital PCR and whole-genome sequencing. Further, gene ontology analysis of genes aligning with sequenced cfDNA fragments revealed associations with terms related to neurodevelopment and autism spectrum disorder. We conclude that cerebral organoids hold promise as tools for the discovery of cfDNA biomarkers related to neurodevelopmental and neurological disorders.

Developing tardigrade-inspired material: Track membranes functionalized with Dsup protein for cell-free DNA isolation.

When developing functionalized biomaterials, the proteins from extremophilic organisms, in particular unique tardigrade disordered proteins, are of great value. The damage suppressor protein (Dsup), initially discovered in the tardigrade Ramazzottius varieornatus and found to be an efficient DNA protector under oxidative and irradiation stress, has been hypothesized to possess a good potential for the development of the material, which can isolate cell-free DNA. With this in mind, DNA-nonadsorbing polyethylene terephthalate track membranes have been functionalized using the Dsup protein via covalent bonding with glutaraldehyde. The filtration experiments have verified the ability of track membranes with the immobilized Dsup protein to adsorb cell-free DNA, with an accumulation capacity of 70 ± 19 mg m-2. The resulting track membrane-based biomaterial might be used in various devices for filtration and separation of cell-free DNA molecules from biological solutions and environmental samples, and also for their accumulation, storage, and further manipulation.

Abstract 5040: Evaluation of commercially available cell free DNA extraction kits using biosynthetic reference materials

Abstract Extraction of cell free DNA (cfDNA) from a patient’s blood sample is a crucial preanalytic step of the liquid biopsy process. Whether testing is being performed for early disease diagnosis, therapy selection, or disease and treatment surveillance, the input material must be of sufficient quantity as well as high quality to return informative data. In addition, accurate capture of the full cfDNA content from a sample is necessary to evaluate tumor growth or recurrence. Fragmentomics is an emerging field that can indicate tumor origin among other information, and again, accurate and precise cfDNA content is critical. The cfDNA content in patient plasma is highly variable, and sample mass is quite limited compared to that of resected tumors. Healthy donor plasma, though abundant, varies in cfDNA levels batch to batch and should not contain any somatic variants relevant to liquid biopsy assay development or validation. Despite the challenges associated with cfDNA isolation and ctDNA analysis there are few reference materials available to optimize laboratory process for these preanalytic challenges. Here we evaluate various commercially available extraction kits using novel Seraseq® cfDNA Extraction Reference Materials. Genomic DNA from the GM24385 cell line was blended with 4 biosynthetic DNA EGFR variants targeted at 1% allele frequency and verified using the Bio-Rad QX-200 Droplet Digital PCR (ddPCR) System. The mixed DNA was fragmented and size selected to create ctDNA-like size profiles. The ctDNA was mixed into a synthetic plasma matrix to simulate native cfDNA samples and diluted to three distinct concentrations (20, 50, and 80 ng/mL). Verification of concentration was performed using the QIAGEN QIAamp Circulating Nucleic Acid Kit for extraction. The final materials were also extracted with other kits: the Maxwell RSC ccfDNA Plasma Kit, the MagMAX Cell-Free DNA Isolation kit, the Zymo MAGicBead™ cfDNA Isolation Kit, and the Takara NucleoSnap cfDNA Kit. Yields were measured with Qubit dsDNA BR assay and EGFR variants were measured using Bio-Rad ddPCR. Other sites evaluated the materials as well, using various kits. The kits showed variable yields, but there was a clear difference between the cfDNA extracted from each reference material correlating with the intended increasing concentration. The EGFR variants were all present at ~1% regardless of the cfDNA concentration and the kit used for extraction. Seraseq ctDNA Plasma Reference Materials serve to evaluate extraction of cfDNA in liquid biopsy testing workflows without having to source donor plasma, which is limited and variable in cfDNA content. The consistency and availability of these biosynthetic materials allow for optimization without risking patient samples. Citation Format: Dana Ruminski Lowe, Sanchita Jamindar, Andrew Anfora, David Merriam, Catherine Huang, Russell Garlick, Bharathi Anekella. Evaluation of commercially available cell free DNA extraction kits using biosynthetic reference materials [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5040.

Cell-Free Microbial DNA Analysis: Effects of Blood Plasma and Serum Quantity, Biobanking Protocols, and Isolation Kits.

Recent studies highlight the presence of bacterial sequences in the human blood, suggesting potential clinical significance for circulating microbial signatures. These sequences could presumably serve in the diagnosis, prediction, or monitoring of various health conditions. Ensuring the similarity of samples before bacterial analysis is crucial, especially when combining samples from different biobanks prepared under varying conditions (such as different DNA extraction kits, centrifugation conditions, blood collection tubes, etc.). In this study, we aimed to analyze the impact of different sample collection and nucleic acid extraction criteria (blood collection tube, centrifugation, input volume, and DNA extraction kit) on circulating bacterial composition. Blood samples from four healthy individuals were collected into three different sample collection tubes: K2EDTA plasma tube, sodium citrate plasma tube, and gel tube for blood serum. Tubes were centrifugated at standard and double centrifugation conditions. DNA extraction was performed using 100, 200, and 500 μL plasma/serum input volumes. DNA extraction was performed using three different isolation kits: Norgen plasma/serum cell-free circulating DNA purification micro kit, Applied Biosystems MagMAX cell-free DNA isolation kit, and Qiagen QIAamp MinElute cell-free circulating DNA mini kit. All samples were subjected to 16S rRNA V1-V2 library preparation and sequencing. In total, 216 DNA and 18 water control samples were included in the study. According to PERMANOVA, PCoA, Mann-Whitney, and FDR tests the effect of the DNA extraction kit on the microbiota composition was the greatest, whereas the type of blood collection tube, centrifugation type, and sample input volume for the extraction had minor effects. Samples extracted with the Norgen DNA extraction kit were enriched with Gram-negative bacteria, whereas samples extracted with the Qiagen and MagMAX kits were enriched with Gram-positive bacteria. Bacterial profiles of samples prepared with the Qiagen and MagMAX DNA extraction kits were more similar, whereas samples prepared with the Norgen DNA extraction kit were significantly different from other groups.

Fragmentomics of cell-free DNA from targeted panels in genitourinary malignancies.

199 Background: The detection of genomic alterations in cancer is critical for identifying clinically actionable alterations for treatment decisions. Tumor samples historically have been required, but obtaining tissue for molecular profiling is not always feasible, especially in the metastatic setting. The isolation and analysis of cell-free DNA (cfDNA) including circulating tumor DNA (ctDNA) via blood-based “liquid” biopsies offers non-invasive sampling of the tumor. Recently, fragmentation patterns of cfDNA (i.e. “fragmentomics”) have emerged as a method for inferring epigenomic and transcriptomic information. However, these analyses use whole-genome sequencing, which lacks the necessary depth to cost-effectively assess genomic alterations, limiting the application of these techniques clinically. Methods: We developed a novel cfDNA fragmentomics machine learning approach for standard targeted cancer gene panels in order to identify genitourinary (GU) and non-GU cancers from two independent metastatic cancer cohorts: a published cohort from GRAIL (prostate cancer, non-GU cancers, normal samples, N=198) and an expanded institutional cohort from the University of Wisconsin (prostate adenocarcinoma, neuroendocrine prostate cancer (NEPC), renal cell carcinoma (RCC), bladder cancer, non-GU cancers, normal samples, n = 431). Results: In the GRAIL cohort, 10-fold cross-validation AUCs were 0.987 for identifying prostate cancer, 1.00 for normal samples, and ranged from 0.922-0.958 for the non-GU cancers. In the UW cohort, 10-fold cross-validation AUCs were 0.950 for bladder cancer, 0.982 for prostate cancer, 0.993 for NEPC, 0.925 for RCC, and 0.980 for normal samples, and ranged from 0.874-0.954 for non-GU cancers. Our assay can sensitively detect and accurately distinguish GU and non-GU malignancies despite a median ctDNA fraction of only 0.076 in the GRAIL cohort, and a median ctDNA fraction of only 0.022 in the UW cohort. Conclusions: Our enhanced machine learning approach for examining fragmentomics in standard cancer gene cfDNA panels unlocks the potential for these established assays to be utilized to answer critical clinical questions beyond somatic variant identification. The excellent performance of our innovative approach even in samples with low ctDNA fractions suggest potential applications such as multi-cancer early detection and minimal residual disease monitoring. This framework could dramatically expand the potential of already existing clinically used assays and minimizes barriers for continued biomarker development.

Abstract A022: Dissecting patterns of small cell lung cancer evolution using deep whole genome sequencing of circulating tumor DNA

Abstract Background: Small cell lung cancer (SCLC) is the most lethal form of lung cancer. A key driver of this near universal lethality is SCLC’s recalcitrance to therapy. The standard of care for SCLC is a chemotherapy doublet of etoposide and cis/carboplatin in combination with immunotherapy (EP+IO). While most tumors respond to these therapies, >80% of SCLCs progress within one year of treatment. Additionally, the aggressive clinical course of SCLC has historically precluded collection of patient-matched treatment naïve and recurrent tumor tissue traditionally required to study evolution. In lieu of tissue, several groups have used circulating tumor DNA (ctDNA) to study SCLC evolution. However, these studies used targeted sequencing panels that analyze <0.04% of the SCLC genome. Using new methods to comprehensively trace how SCLCs rapidly evolve to therapy resistance is a significant unmet need. Methods: Medical records were reviewed to identify treatment naïve SCLC patients treated at MD Anderson that had both pre- and post-EP+IO liquid biopsies collected under IRB approved protocols. ctDNA was isolated from plasma using a MiniMax High Efficiency Cell-Free DNA Isolation Kit (Apostle Bio). Germline DNA was extracted from buffy coat samples using a QIAamp DNA Blood Midi Kit (Qiagen). Deep whole genome sequencing (WGS) (>100X) of ctDNA and WGS of germline DNA (>60X) was performed using an Illumina NovaSeq X sequencer. Reads were aligned to the hg38 reference genome using BWA-mem. Somatic mutations were called using MuTect2, and clonal and subclonal copy number alterations were identified using Battenberg. DPClust was used to reconstruct cancer cell populations, leveraging mutation and copy number calls. Results: We identified several deleterious genomic alterations impacting TP53 and RB1 tumor suppressor genes, known genetic drivers of SCLC development. We also detected recurrent, whole chromosome arm loss of heterozygosity events impacting many chromosomes in treatment naïve samples, some of which are known to be present in >80% of SCLCs. Additionally, we found that both treatment naïve and recurrent SCLCs were composed of multiple subclones, each defined by dozens to thousands of unique somatic mutations. COSMIC signature analysis identified high activity of tobacco carcinogens, APOBEC, and replicative-based processes across ctDNA samples. We also identified emergence of novel subclones carrying unique copy number alterations following EP+IO recurrence. Importantly, the affected regions encode key effectors that regulate anti-tumor immune responses and cell survival following DNA damage, both of which are intimately related to the mechanism of action of EP+IO. Conclusions: Our data shows that deep WGS of ctDNA recapitulates known genetics of SCLC and allows for sensitive characterization of therapy resistant clones. Using this method, we have captured snapshots of both intrinsic and acquired resistance mechanisms to frontline EP+IO. Deep WGS of ctDNA is a promising approach to comprehensively dissect routes of SCLC evolution and therapy resistance. Citation Format: Benjamin B. Morris, Zhihui Zhang, Simon Heeke, Kyle Concannon, Bingan Zhang, Waree Rinsurongkawong, Vadeerat Rinsurongkawong, J. Jack Lee, Jianjun Zhang, Don L. Gibbons, Ara A. Vaporciyan, Carl M. Gay, Hai T. Tran, John V. Heymach, Lauren A. Byers, Peter Van Loo. Dissecting patterns of small cell lung cancer evolution using deep whole genome sequencing of circulating tumor DNA [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Translating Cancer Evolution and Data Science: The Next Frontier; 2023 Dec 3-6; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_2):Abstract nr A022.

Optimization of a Protocol for Isolating Cell-free DNA From Cerebrospinal Fluid.

A standardized protocol for the isolation of cell-free DNA (cfDNA) from cerebrospinal fluid (CSF) is lacking. Therefore, we established a cfDNA isolation protocol optimized for clinical CSF specimens, integrating acceptable modifications and using artificial CSF generated from remnant CSF spiked with reference cell-free tumor DNA (ctDNA). We compared the isolation yields of in vitro diagnostic (IVD)-certified column-based (CB) and magnetic bead-based (MB) isolation. Furthermore, we modified both methods, including pre- and post-elution steps. To confirm ctDNA integrity and quantify the variant allele frequency after isolation, we performed droplet digital PCR (ddPCR) targeting IDH1 R132C in the reference ctDNA. MB isolation had a higher yield than CB isolation (P<0.0001), and post-isolation vacuum increased the final concentration in both methods, with little effect on cfDNA integrity. Our study provides a protocol to maximize CSF-ctDNA concentrations in IVD testing and future studies.

BIOM-58. ZERO-WASTE MOLECULAR DIAGNOSTICS FROM BIOPSY NEEDLE WASHES

Abstract BACKGROUND Brain tumor classification has increasingly integrated molecular diagnostics into official criteria. However, biopsy tissue is limited due to the small quantity acquired in eloquent areas of the brain and is, therefore, usually earmarked for histology. We have identified that the fluid used to wash biopsy needles contains excess tumor material that can be diagnostically useful. Characterization of this non-zero-sum waste material provides an opportunity for improvement in the diagnosis and treatment for brain tumor patients. METHODS For suspected or established high grade brain tumor biopsies (n = 37 across a multi-institutional cohort at submission), neurosurgeons were instructed to save fluid used to wash biopsy needles. Samples were processed and characterized in a variety of methods to establish diagnostic utility including (1) cell-free DNA isolation and targeted panel sequencing (n = 13), (2) isothermal amplification of cell-free DNA from raw wash water for rapid diagnostics (n = 2), (3) cell-line generation (n = 24) and, (4) cell-free mutant protein capture and quantification (n = 9). RESULTS (1) 13 wash samples were considered for cf-tDNA extraction and Illumina TruSight Oncology 500 panel sequencing (with extracted DNA ranging from 20ng-40ug). In 2 returned cases (11 pending), concordance was 100% with clinical targeted panel sequencing where panels overlapped. (2) One case suspected of harboring H3K27M was subjected to targeted, loop-mediated isothermal amplification and Oxford Nanopore real-time sequencing, confirming an H3.3K27M diagnosis (11.8% allele fraction) within 1hr35min of receipt of sample from the operating room. (3) 5 cases resulted in primary cell culture generation (4) A novel single-molecule mutant protein quantification technique was performed on 9 cases. RESULTS from all analyses and concordance with outstanding sequencing will be presented. CONCLUSION We demonstrate that biopsy byproducts contain enough high-quality tumor-derived material to perform a variety of useful diagnostics with the potential to have immediate translational impact on standard of care.

Impact of Cell-Debris and Room-Temperature Storage on Urine Circulating Tumor DNA from Hepatocellular Carcinoma

This study evaluated the impact of cell debris and 7-day room temperature storage on the quality and yield of transrenal DNA. Archived urine specimens collected from five hepatocellular carcinoma (HCC) patients and two pregnant women carrying male fetuses were used to assess the impact of cell debris on urine cell-free DNA (ucfDNA) isolation as measured by quantitative PCR for Y-chromosome DNA, or HCC-associated mutation and methylation markers, and by capillary electrophoresis. Prospectively collected urine from 21 HCC patients was aliquoted after collection for paired immediate freezing versus 7-day room temperature storage followed by freezing for further analysis. Cell debris contained more Y-chromosome DNA than supernatant in three of the six urine specimens tested from pregnant women, suggesting that cell debris can be associated with 20.6% to 84.9% of transrenal ucfDNA. Ninety-five percent (20 of 21) of frozen and room temperature urine pairs had overlapping DNA size distribution. ucfDNA quantity determined by quantitative PCR for TP53, CTNNB1, TERT, and HCC-associated urine circulating tumor DNA markers were statistically similar between room temperature and frozen samples. This suggests no significant difference in DNA degradation between the groups. The association of transrenal ucfDNA with cell debris and HCC circulating DNA stability at room temperature is significant to further the understanding of transrenal circulating tumor DNA pre-analytical handling for HCC screening.

830P Isolation of cell-free DNA of patients with mucosa-associated lymphoid tissue (MALT) lymphoma

830P Isolation of cell-free DNA of patients with mucosa-associated lymphoid tissue (MALT) lymphoma

Integrating extracellular vesicle and circulating cell-free DNA analysis using a single plasma aliquot improves the detection of HER2 positivity in breast cancer patients.

Multi-analyte liquid biopsies represent an emerging opportunity for non-invasive cancer assessment. We developed ONCE (One Aliquot for Circulating Elements), an approach for the isolation of extracellular vesicles (EV) and cell-free DNA (cfDNA) from a single aliquot of blood. We assessed ONCE performance to classify HER2-positive early-stage breast cancer (BrCa) patients by combining EV-associated RNA (EV-RNA) and cfDNA signals on n=64 healthy donors (HD) and non-metastatic BrCa patients. Specifically, we isolated EV-enriched samples by a charge-based (CB) method and investigated EV-RNA and cfDNA by next-generation sequencing (NGS) and by digital droplet PCR (ddPCR). Sequencing of cfDNA and EV-RNA from HER2- and HER2+ patients demonstrated concordance with in situ molecular analyses of matched tissues. Combined analysis of the two circulating analytes by ddPCR showed increased sensitivity in ERBB2/HER2 detection compared to single nucleic acid components. Multi-analyte liquid biopsy prediction performance was comparable to tissue-based sequencing results from TCGA. Also, imaging flow cytometry analysis revealed HER2 protein on the surface of EV isolated from the HER2+ BrCa plasma, thus corroborating the potential relevance of studying EV as companion analyte to cfDNA. This data confirms the relevance of combining cfDNA and EV-RNA for HER2 cancer assessment and supports ONCE as a valuable tool for multi-analytes liquid biopsies' clinical implementation.

Fragmentomic analysis of circulating tumor DNA-targeted cancer panels

The isolation of cell-free DNA (cfDNA) from the bloodstream can be used to detect and analyze somatic alterations in circulating tumor DNA (ctDNA), and multiple cfDNA-targeted sequencing panels are now commercially available for Food and Drug Administration (FDA)-approved biomarker indications to guide treatment. More recently, cfDNA fragmentation patterns have emerged as a tool to infer epigenomic and transcriptomic information. However, most of these analyses used whole-genome sequencing, which is insufficient to identify FDA-approved biomarker indications in a cost-effective manner. We used machine learning models of fragmentation patterns at the first coding exon in standard targeted cancer gene cfDNA sequencing panels to distinguish between cancer and non-cancer patients, as well as the specific tumor type and subtype. We assessed this approach in two independent cohorts: a published cohort from GRAIL (breast, lung, and prostate cancers, non-cancer, n = 198) and an institutional cohort from the University of Wisconsin (UW; breast, lung, prostate, bladder cancers, n= 320). Each cohort was split 70%/30% into training and validation sets. In the UW cohort, training cross-validated accuracy was 82.1%, and accuracy in the independent validation cohort was 86.6% despite a median ctDNA fraction of only 0.06. In the GRAIL cohort, to assess how this approach performs in very low ctDNA fractions, training and independent validation were split based on ctDNA fraction. Training cross-validated accuracy was 80.6%, and accuracy in the independent validation cohort was 76.3%. In the validation cohort where the ctDNA fractions were all <0.05 and as low as 0.0003, the cancer versus non-cancer area under the curve was 0.99. To our knowledge, this is the first study to demonstrate that sequencing from targeted cfDNA panels can be utilized to analyze fragmentation patterns to classify cancer types, dramatically expanding the potential capabilities of existing clinically used panels at minimal additional cost.

Circulating tumor DNA for monitoring colorectal cancer: A prospective observational study to assess the presence of methylated SEPT9 and VIM promoter genes and its role as a biomarker in colorectal cancer management.

Methylation status of Septin9 (SEPT9) and vimentin (VIM) genes in circulating tumor DNA of colorectal cancer (CRC) patients is a promising bio-marker for the early detection of CRC. The aim of the present study was to identify the methylation status in promoter regions of the SEPT9 and VIM genes in a cohort of Indian patients with biopsy proven colorectal cancer. Forty-five consecutive patients of colorectal cancer were recruited. 10 mL venous samples were collected from each patient and processed for isolation of cell-free DNA, bisulfite conversion of cell-free DNA, polymerase chain reaction (PCR) amplification and detection of SEPT9 and VIM genes. Partial methylation in vimentin was present in 42.22% of the patients and 57.78% showed no methylation and none of the tumors had complete methylation. Only three (6.66%) patients showed complete methylation patterns in SEPT9 and the remaining 42 (93.33%) tumors showed partial methylation. Considering the two genes together, only three (6.66%) out of 45 showed complete methylation. The association of methylation patterns in both genes (complete, partial, and no methylation) with sex, age, T stage, N stage, M stage, CEA, histology, and location (right or left colon) were explored and none of these parameters were statistically significant. In our study, only 6.66% CRC patients showed hypermethylation and there was no association of methylation patterns in the both genes (complete, partial, and no methylation) with any of the parameters like age, sex, TNM stage, CEA, and histology.

Analysis of Plasma Circulating Tumor DNA in Borderline Resectable Pancreatic Cancer Treated with Neoadjuvant Modified FOLFIRINOX: Clinical Relevance of DNA Damage Repair Gene Alteration Detection.

There are no reliable biomarkers to guide treatment for patients with borderline resectable pancreatic cancer (BRPC) in the neoadjuvant setting. We used plasma circulating tumor DNA (ctDNA) sequencing to search biomarkers for patients with BRPC receiving neoadjuvant mFOLFIRINOX in our phase 2 clinical trial (NCT02749136). Among the 44 patients enrolled in the trial, patients with plasma ctDNA sequencing at baseline or post-operation were included in this analysis. Plasma cell-free DNA isolation and sequencing were performed using the Guardant 360 assay. Detection of genomic alterations, including DNA damage repair (DDR) genes, were examined for correlations with survival. Among the 44 patients, 28 patients had ctDNA sequencing data qualified for the analysis and were included in this study. Among the 25 patients with baseline plasma ctDNA data, 10 patients (40%) had alterations of DDR genes detected at baseline, inclu-ding ATM, BRCA1, BRCA2 and MLH1, and showed significantly better progression-free survival than those without such DDR gene alterations detected (median, 26.6 vs. 13.5 months; log-rank p=0.004). Patients with somatic KRAS mutations detected at baseline (n=6) had significantly worse overall survival (median, 8.5 months vs. not applicable; log-rank p=0.003) than those without. Among 13 patients with post-operative plasma ctDNA data, eight patients (61.5%) had detectable somatic alterations. Detection of DDR gene mutations from plasma ctDNA at baseline was associated with better survival outcomes of pati-ents with borderline resectable pancreatic ductal adenocarcinoma treated with neoadjuvant mFOLFIRINOX and may be a prognostic biomarker.

Isolation of salivary cell-free DNA for cancer detection.

Saliva is an emerging source of disease biomarkers, particularly for cancers of the head and neck. Although analysis of cell-free DNA (cfDNA) in saliva holds promise as a liquid biopsy for cancer detection, currently there are no standardized methodologies for the collection and isolation of saliva for the purposes of studying DNA. Here, we evaluated various saliva collection receptacles and DNA purification techniques, comparing DNA quantity, fragment size, source, and stability. Then, using our optimized techniques, we tested the ability to detect human papillomavirus (HPV) DNA- a bona fide cancer biomarker in a subset of head and neck cancers- from patient saliva samples. For saliva collection, we found that the Oragene OG-600 receptacle yielded the highest concentration of total salivary DNA as well as short fragments <300 bp corresponding to mononucleosomal cell-free DNA. Moreover, these short fragments were stabilized beyond 48 hours after collection in contrast to other saliva collection receptacles. For DNA purification from saliva, the QIAamp Circulating Nucleic Acid kit yielded the highest concentration of mononucleosome-sized DNA fragments. Freeze-thaw of saliva samples did not affect DNA yield or fragment size distribution. Salivary DNA isolated from the OG-600 receptacle was found to be composed of both single and double-stranded DNA, including mitochondrial and microbial sources. While levels of nuclear DNA were consistent over time, levels of mitochondrial and microbial DNA were more variable and increased 48 hours after collection. Finally, we found that HPV DNA was stable in OG-600 receptacles, was reliably detected within the saliva of patients with HPV-positive head and neck cancer, and was abundant among mononucleosome-sized cell-free DNA fragments. Our studies have defined optimal techniques for isolating DNA from saliva that will contribute to future applications in liquid biopsy-based cancer detection.

Analysis of plasma circulating tumor DNA in borderline resectable pancreatic ductal adenocarcinoma treated with neoadjuvant modified FOLFIRINOX: Clinical relevance of DNA damage repair gene alterations.

737 Background: There is no reliable biomarker to guide treatment for patient with borderline resectable pancreatic ductal adenocarcinoma (PDAC) treated with neoadjuvant modified (m)FOLFIRINOX. We investigated the role of plasma circulating tumor DNA (ctDNA) sequencing to discover potential biomarkers for patients treated with neoadjuvant mFOLFIRINOX in our phase 2 clinical trial (ClinicalTrials.gov identifier: NCT02749136). Methods: Among 44 patients enrolled in the trial, patients with plasma ctDNA analysis performed at baseline or post-operative period were included in the analysis. Plasma cell-free DNA isolation and sequencing of ctDNA were performed using the Guardant 360 assay. Association of clinical outcomes with germline or somatic mutations of DNA damage repair (DDR) genes was analyzed. Somatic alterations other than DDR genes were also analyzed to find potential biomarkers of neoadjuvant mFOLFIRINOX. Progression free-survival (PFS) was defined as time from the initiation of mFOLFIRINOX to disease progression or any cause of death. Overall survival (OS) was defined as the time from the initiation of mFOLFIRINOX to any cause of death. Results: Among 44 patients, 28 patients had ctDNA sequencing data qualified for the analysis and were included in this study (15 patients with baseline data only, 3 patients with post-operative data only, and 10 patients with both baseline and post-operative data). Median age was 59 (range, 41-73) years and 15 patients (53.6%) were male. Among 25 patients with baseline plasma ctDNA data, 11 patients (44%) had somatic or germline alteration of DDR genes detected at baseline including ATM, BRCA1, BRCA2 and MLH1, and showed significantly better PFS than those without such DDR gene alterations (median 26.6 vs. 13.5 months, log-rank p = 0.004). Although there was a trend for better OS in patients with DDR gene alterations, this was not statistically significant (log-rank p = 0.29). The most observed baseline somatic alteration was TP53 (n = 7, 28%), followed by KRAS (n = 6, 24%). Patients with somatic KRAS mutation at baseline had significantly worse OS (median 8.5 months vs. not applicable, log-rank p = 0.003) than those without. Among 13 patients with post-operative plasma ctDNA data, 8 patients (61.5%) had detectable somatic alterations. Conclusions: Detection of germline or somatic mutations of DDR genes from plasma ctDNA at baseline was associated with better survival outcomes of patients with borderline resectable PDAC treated with neoadjuvant mFOLFIRINOX. Detection of DDR gene alterations from plasma ctDNA may have a potential role as biomarker for prognosis in patients with borderline resectable PDAC treated with mFOLFIRINOX.

A new and improved method of library preparation for non-invasive prenatal testing: plasma to library express technology.

This study aims to develop a novel library preparation method, plasma to library express technology (PLET), to construct next-generation sequencing (NGS) libraries directly from plasma without cell-free DNA (cfDNA) isolation. Peripheral blood samples (600) were obtained from a retrospective cohort of 300 pregnant women prior to invasive diagnostic testing. The samples were subsequently distributed between library preparation methodologies, with 300 samples prepared by PLET and 300 by conventional methods for non-invasive prenatal testing (NIPT) to screen for common trisomies using low-pass whole genome next generation sequencing. NIPT conducted on PLET libraries demonstrated comparable metrics to libraries prepared using conventional methods, including 100% sensitivity and specificity. Our study demonstrates the potential utility of PLET in the clinical setting and highlights its significant advantages, including dramatically reduced process complexity and markedly decreased turnaround time.

Exosomes Derived from Post-Surgical Drain Fluid of HPV+ Oropharyngeal Cancer Patients Contain HPV DNA and can Induce E6 mRNA Transcription in HPV-Negative Cells

<h3>Purpose/Objective(s)</h3> Human papillomavirus (HPV) infection predisposes to oropharyngeal squamous cell carcinoma (OPC). Exosomes are a component of cell-free DNAs that are actively shed by tumor and immune cells. The role of exosomes in cancer metastasis and in HPV-induced carcinogenesis remains unknown. We hypothesize that exosomes derived from surgical drain fluid (SDF) in HPV+ OPC patients contain HPV viral DNA that can be transmitted directly into HPV-negative cells, resulting in transcription of viral oncogenes. <h3>Materials/Methods</h3> Following surgical neck dissection, SDF samples were collected and processed from 21 HPV+ and 5 HPV- OPC patients (discovery cohort). Exosomes were isolated from SDFs using either the Qiagen exoEasy kit or the SBI SEC EV isolation kit. Exosome structure was characterized by transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA). Immunoblotting was performed to verify exosome-specific biomarkers. Exosomes were pretreated with Dnase (NEB), then exosomal DNA was extracted using a cell-free DNA isolation kit (Qiagen). HPV16 copy number was determined by TaqMan quantitative PCR. Validation was then performed using SDF from a separate cohort of 10 HPV+ OPC patients. To examine transcription of exosome-derived HPV DNA, exosomes were isolated from 3 HPV+ SDF and 1 HPV- SDF. After incubating these exosomes with two HPV- cell lines (SCC22A and SCC25), we isolated total cell line RNA, and performed reverse transcriptase PCR (RT-PCR). <h3>Results</h3> TEM and NTA results revealed rounded structures with a size of 70-150 nm, respectively. Immunoblots detected CD63, CD9, and TSG101 in the protein lysates, indicating the vesicles isolated from SDF were indeed exosomes. In the discovery cohort, HPV16 DNA was detected in SDF exosomes isolated from 81% (n = 17) of patients with HPV+ OPC, while SDF-derived exosomes from all (n=5) HPV- patients had undetectable HPV16 DNA. Importantly, paired comparison of HPV16 levels revealed a significant proportion (46.7±34.4%) of HPV16 cell-free DNA in SDF was contained within exosomes. Results from our validation cohort showed that all the exosome samples (100%) isolated from SDF from 10 HPV+ OPC patients contained HPV DNA, regardless of the exosome isolation method; 24.4±14.4% of HPV16 cell-free DNA was localized in exosomes. Furthermore, an <i>in vitro</i> cell culture assay showed that exosomal HPV16 DNA can undergo uptake by SCC22A and SCC25 cells. RT-PCR of RNA from SCC22A cells incubated with HPV DNA-containing exosomes revealed that exosomal HPV16 DNA can be transcribed into HPV16 E6 mRNA in HPV- recipient cells. <h3>Conclusion</h3> SDF, a novel post-surgical biofluid analyte, is enriched for HPV-containing exosomes from HPV+ OPC patients. Exosomes from these HPV+ OPC patients can transmit viral oncogenes to HPV-negative cells, suggesting a novel mode of virus-mediated spread of disease. Furthermore, exosomal HPV DNA can mediate HPV16 E6 mRNA transcription in HPV- recipient cells, indicating its potential role in OPC tumorigenesis.

Isolation and Quantification of Plasma Cell-Free DNA Using Different Manual and Automated Methods.

Plasma cell-free DNA (cfDNA) originates from various tissues and cell types and can enable minimally invasive diagnosis, treatment and monitoring of cancer and other diseases. Proper extraction of cfDNA is critical to obtain optimal yields and purity. The goal of this study was to compare the performance of six commercial cfDNA kits to extract pure, high-quality cfDNA from human plasma samples and evaluate the quantity and size profiles of cfDNA extracts—among them, two spin-column based, three magnetic bead-based and two automatic magnetic bead-based methods. Significant differences were observed in the yield of DNA among the different extraction kits (up to 4.3 times), as measured by the Qubit Fluorometer and Bioanalyzer. All kits isolated mostly small fragments corresponding to mono-nucleosomal sizes. The highest yield and reproducibility were obtained by the manual QIAamp Circulating Nucleic Acid Kit and automated MagNA Pure Total NA Isolation Kit. The results highlight the importance of standardizing preanalytical conditions depending on the requirements of the downstream applications.

Human papillomavirus DNA resides in surgical drain fluid exosomes from HPV+ oropharyngeal squamous cell carcinoma patients and can be spread to neighboring HPV-negative cells.

e18050 Background: Human papillomavirus (HPV) is the primary driver of oropharyngeal squamous cell carcinoma (OPC) in the US. Previous studies have investigated the role of plasma and saliva-based cell-free HPV DNA in OPC, yet the pathophysiological impact of exosomes in HPV+ OPC remains unknown. Exosomes are nanometer-sized membrane vesicles of endocytic origin carrying DNA, RNA and proteins, and have been shown to exist in blood, plasma, and in urine. We hypothesize that serosanguinous fluid drained from the postoperative bed, which we call “surgical drain fluid” (SDF) also contains exosomes. We further hypothesize that exosomes derived from SDF in HPV+ OPC patients contain HPV DNA that can be directly transmitted into HPV-negative cells. Methods: Twenty-six 5-mL SDF samples were collected following surgical neck dissections directly from the surgical drain; 21 from HPV+ and 5 from HPV- OPC patients. Exosomes were isolated from SDF using the Qiagen exoEasy kit. Exosome structure was characterized by transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA). Western blotting (WB) targeted known exosomal surface markers. Exosomes were pretreated with DNase (New England Biolabs) to cleave the surface DNA, then exosomal DNA was extracted using a cell-free DNA isolation kit (Sigma). TaqMan real-time PCR was employed to detect HPV16 copy numbers. Lastly, exosomes isolated from 3 HPV+ patients’ SDF and 1 HPV- patient’s SDF were incubated with HPV- cell lines, SCC22A and SCC25, in order to query exosome-mediated HPV transmission. Results: TEM identified exosomes as round/oval-shaped vesicles measuring between 70 and 150 nm. NTA enumerated an average of 1.5 x 1011 particles per mL of SDF. WB further confirmed exosomal identity with strong CD63, CD9, and TSG101 signal detected. Among SDF exosomes from HPV+ OPC, 81% (n = 17) had detectable HPV16 DNA by real-time PCR, while all HPV- SDF exosomes (n = 5) showed undetectable HPV16. Paired comparison of HPV16 levels within exosomes to total HPV16 in SDF revealed an average of 90% of HPV16 in SDF was contained within exosomes. In an in vitro assay, SCC22A and SCC25 cells treated with HPV16 + exosomes became real-time PCR-positive for HPV16, while cells treated with HPV16- exosomes remained HPV16 negative. Conclusions: This study demonstrates that SDF, a novel post-surgical biofluid analyte, is enriched for HPV-containing exosomes in HPV+ OPC patients. We also found that exosomes from HPV+ OPC patients can transmit HPV to HPV-negative cells, suggesting a novel mode of virus-mediated spread of disease.