cfDNA Yield Research Articles

Bronchoalveolar Lavage Fluid (BALF) as a Compartment for Donor-Derived Cell-Free DNA (dd-cfDNA) Assessment in Lung Recipients

<h3>Purpose</h3> Circulating dd-cfDNA has been proposed as a biomarker to discriminate acute lung rejection. It is unknown whether BALF may represent a more sensitive compartment for dd-cfDNA analysis in lung recipients. Additionally, the relative contribution of the recipient or donor to the BALF cfDNA content remains uncertain. We sought to understand the feasibility of distinguishing dd-cfDNA in the BALF and determine the greatest contributor of BALF cfDNA. <h3>Methods</h3> The cohort was drawn from the Clinical Trials in Organ Transplantation-20 study and comprised 126 first adult lung recipients with paired BALF and plasma samples (n=320 each). Total cfDNA was isolated from ∼1mL of sample using automated nucleic acid purification. The % cfDNA contributed by the donor vs. recipient was distinguished in plasma and BALF samples using a next generation sequencing (NGS) assay that measures single nucleotide polymorphisms (SNPs) followed by bioinformatics algorithms. Because these algorithms were developed on plasma, for each BALF sample the SNP typing from the paired plasma sample was used to verify the accuracy of the donor vs. recipient cfDNA contribution in the BALF. <h3>Results</h3> Applying standard QC filters, 168 BALF samples "passed" and generated a dd-cfDNA result while 96 "failed" and did not generate a dd-cfDNA result. 45 samples yielded insufficient total cfDNA to support NGS while 11 generated a potentially invalid dd-cfDNA result. To understand reasons underlying inability to estimate BALF dd-cfDNA, we compared the volume of BALF used for total cfDNA isolation and the total cfDNA yield from the BALF in passed vs. failed samples. The BALF volume was similar between these groups, however the total cfDNA yield was notably less among samples that failed (median total cfDNA 0.38ng/uL in failed vs. 1.42ng/uL in passed samples). Considering only passed BALF samples, the median (25^th, 75^th percentile) fraction of cfDNA contributed by the donor was 6.95% (2.40%, 14.00%), with the remainder contributed by the recipient. <h3>Conclusion</h3> Our data point to challenges in consistently generating dd-cfDNA data from the BALF and suggest the dilute nature of BALF may be a contributing factor. Additionally, these results indicate that the recipient, rather than the donor, is the largest contributor to the BALF cfDNA content potentially reflecting intragraft recipient immune cells.

Circulating Cell-Free DNA-Based Comprehensive Molecular Analysis of Biliary Tract Cancers Using Next-Generation Sequencing

Simple SummaryIn the era of personalized oncology, next-generation sequencing plays an important role in identifying mutations that may predict the molecular pathomechanism and manage biliary tract cancers (BTC) therapy. The peripheral blood of cancer patients represents variable amounts of cell-free DNA (cfDNA) released from the tumor. Tumor-derived cfDNA in BTCs also allows the effective monitoring of the molecular genetic profile and the response to chemotherapy. Our study aimed to identify genetic aberrations in cell-free and matched tumor DNA in BTCs. We assume that the efficacy of the LB-based sequencing provides a novel perspective for BTCs therapy.Biliary tract cancer (BTC) is a rare malignancy with a long disease course and an overall poor prognosis. Despite multiple chemotherapy agents, there is no defined second-line treatment opportunity for advanced BTCs. In the era of precision oncology, NGS plays an important role in identifying mutations that may predict the molecular pathomechanism and manage the BTC therapy. The peripheral blood liquid biopsy (LB) of cancer patients represents variable amounts of cell-free DNA (cfDNA) released from tumor foci of any anatomical location. Our study aimed to identify somatic mutations and tumor variant burden (TVB) in cell-free and matched tumor DNA. We found a positive correlation between the estimated tumor volume and cfDNA yield (r = 0.9326, p < 0.0001). Comparing tissue and LB results, similar TVB was observed. SNVs were proven in 84% of the cases, while in two cases, only the LB sample was informative for molecular analysis. The most important aberrations in BTCs, such as FGFR2, IDH1, IDH2, KRAS, and TP53, could be detected in matched LB samples. Our prospective study demonstrates a minimally invasive testing approach to identify molecular genetic alterations in cholangiocarcinoma and gallbladder cancers. Clinical applications of cfDNA reflect by capturing the outstanding spatial tumor heterogeneity and guarantee novel aspects for the precision oncology treatment.

Circulating Cell-Free DNA Yield and Circulating-Tumor DNA Quantity from Liquid Biopsies of 12 139 Cancer Patients.

The amounts of circulating cell-free DNA (cfDNA) and circulating-tumor DNA (ctDNA) present in peripheral blood liquid biopsies can vary due to preanalytic/analytic variables. In this study, we examined the impact of patient age, sex, stage, and tumor type on cfDNA yield, ctDNA fraction, and estimated ctDNA quantity from a large cohort of clinical liquid biopsy samples. We performed a retrospective analysis of 12 139 consecutive samples received for liquid biopsy (FoundationOne® Liquid) clinical testing. Significant differences in both cfDNA yield and estimated ctDNA quantity were observed based on the underlying tumor type that initiated the liquid biopsy analysis and the stage of the patient (P < 0.001). In addition, significant differences in ctDNA quantity were present based in both the patient age and sex (P < 0.001). Importantly, we saw a significantly higher success rate of issuing a clinically useful report in patients with higher levels of cfDNA yield and ctDNA quantity (P < 0.001). In this study, we show that ctDNA quantity varied significantly based on patient age, sex, stage, and tumor type, which could offer an explanation as to why certain liquid biopsy specimens are more likely to fail sequencing or provide clinically meaningful results. In addition, this could affect future clinical decisions on the blood sample volumes required to allow successful liquid biopsy testing.

Optimization of Preanalytical Variables for cfDNA Processing and Detection of ctDNA in Archival Plasma Samples.

DNA released from cells into the peripheral blood is known as cell-free DNA (cfDNA), representing a promising noninvasive source of biomarkers that could be utilized to manage Diffuse Large B-Cell Lymphoma (DLBCL), among other diseases. The procedure for purification and handling of cfDNA is not yet standardized, and various preanalytical variables may affect the yield and analysis of cfDNA, including the purification kits, blood collection tubes, and centrifugation regime. Therefore, we aimed to investigate the impact of these preanalytical variables on the yield of cfDNA by comparing three different purification kits DNeasy Blood & Tissue Kit (Qiagen), QIAamp Circulating Nucleic Acid Kit (Qiagen), and Quick-cfDNA Serum & Plasma Kit (Zymo Research). Two blood collection tubes (BCTs), EDTA-K2 and Cell-Free DNA (Streck), stored at four different time points before plasma was separated and cfDNA purified, were compared, and for EDTA tubes, two centrifugation regimes at 2000 × g and 3000 × g were tested. Additionally, we have tested the utility of long-term archival blood samples from DLBCL patients to detect circulating tumor DNA (ctDNA). We observed a higher cfDNA yield using the QIAamp Circulating Nucleic Acid Kit (Qiagen) purification kit, as well as a higher cfDNA yield when blood samples were collected in EDTA BCTs, with a centrifuge regime at 2000 × g. Moreover, ctDNA detection was feasible from archival plasma samples with a median storage time of nine years.

Abstract 572: The liquid biopsy Guardant360 CDx has robust performance at low inputs allowing for high rate of returning patient results

Abstract Background Liquid biopsy tests capable of tumor profiling with NGS have recently received FDA approval for companion diagnostic treatment selection and tumor profiling, demonstrating the clinical validity of the identification of druggable variants with such information. As the amount of cfDNA per volume of plasma varies greatly from patient to patient, and whole blood/plasma volumes from advanced cancer patients are often limited, it would be advantageous for a liquid biopsy to be able to operate with minimal input, providing robust results across a broad spectrum of blood sample volumes and resultant cfDNA input. In this study, we evaluate the performance of Guardant360 CDx with as little as 3 mL whole blood and 5 ng cfDNA input. Methods The primary concern with low input like 3 mL whole blood is excess preservative in a blood collection tube (BCT) relative to whole blood. In a simulated short draw experiment, the same advanced cancer patient provided whole blood in BCTs with standard, elevated (2X), and high (3.3X) preservatives. We evaluated the molecule coverage across the panel at key exons and positions of interest, across genes evaluated for copy number amplifications, and at frequent fusion gene breakpoint positions. Precision and limit of detection (LoD) have been demonstrated at 5 ng input (P200010B) the lowest allowable input. For clinical utility of a liquid biopsy with low cfDNA input, we also assessed the cfDNA input of 87,305 samples tested with Guardant360 CDx or LDT from a single 10mL BCT. Results and Discussion Differences in molecule coverage between the high preservative and standard preservative were found to fall within expected technical variation (the median of log10 differences in exon-level DNA molecule coverage between short draw and reference conditions fell within +/- 0.125), indicating no meaningful loss of sensitivity for detecting somatic variants. Among the 87,305 clinical samples tested by Guardant360 CDx or LDT, median cfDNA yield was 25ng, with 98.5% yielding at least 5 ng, 88.4% at least 10ng, 73.5% at least 15 ng, and 17.9% at least 60ng. Clinically actionable biomarkers are detectable in low input samples. Of the Guardant360 CDx samples yielding 5-15ng cfDNA input, an average of 0.64 clinically significant variants was reported per sample. With the robust analytical performance demonstrated at low inputs, Guardant360 CDx (a 5ng cutoff) may provide tumor profiling results to guide therapy decisions for about 34% more patients compared to some liquid biopsies that use 2 BCTs for minimal 30ng cfDNA yield (a 15 ng cutoff). Retrospective studies for pharma research are often performed with as little as 2mL of plasma, meaning Guardant360 CDx can return results on 88.4% of such samples whereas other technologies may only be able to address 17.9% (extrapolated from the cfDNA yield distribution for 10mL whole blood yielding 4mL plasma). Citation Format: Aaron O. Richardson, Aaron Hardin, Soni Shukla, Jing Zhao, Catalin Barbacioru, Hilda Beas, Carin Espenschied, Adrian Bubie, Jamie Hutchins, Han-Yu Chuang, Tingting Jiang, Darya Chudova. The liquid biopsy Guardant360 CDx has robust performance at low inputs allowing for high rate of returning patient results [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 572.

Abstract 575: Enhancing cell-free DNA mutation detection with novel liquid-phase extraction

Abstract Background: Low yields of extracted cell-free DNA (cfDNA) from plasma limit further development of liquid biopsies for early-stage cancer diagnostics and cancer screening applications. Conventional nucleic acid extraction methods have limitations in capturing low levels of cfDNA and risk capturing high levels of high molecular weight contaminating genomic DNA. We developed a liquid-phase based DNA extraction method (PHASIFY™ ENRICH; Phase Scientific International Ltd., Kwun Tong, Hong Kong) that leverages aqueous two-phase systems (ATPS) to purify, concentrate, and enrich for cfDNA to improve mutant signal detection. Methods: The PHASIFY ATPS consists of unique formulations that are optimized to drive cfDNA into one liquid phase and plasma contaminants into the other, achieving simultaneous target isolation and concentration. In a previous study using the PHASIFY technique, we demonstrated improved cfDNA and mutant copy yield over solid-phase methods. We further developed an additional size-selection feature (PHASIFY ENRICH) to remove high molecular weight genomic DNA (&amp;gt;500 bp) and to reduce signal noise for molecular diagnostics. We compared cfDNA recovery and mutation detection from plasma samples extracted with the industry standard QIAamp Circulating Nucleic Acid kit (QCNA; Qiagen, Hilden, Germany) and PHASIFY ENRICH cfDNA extraction kit. According to the respective kit protocols, the cfDNA was extracted from plasma from patients with advanced cancers, including those known to shed low amounts of cfDNA into circulation (e.g., low grade serious ovarian, appendiceal, thyroid, and pancreatic cancer), with known tissue mutation status. Total DNA recovery was determined with the Quant-iT PicoGreen dsDNA Assay Kit. Mutation status was determined with probe-based Droplet Digital PCR (ddPCR; Bio-Rad, Hercules, CA). In a follow up study, we tested a similar cohort of plasma samples that were previously tested negative for cfDNA mutation status when using QCNA cfDNA extraction, despite known positive mutation status from corresponding tumor tissue genotyping. Results: In the head-to-head study against QCNA, 57 plasma samples from 23 unique patients with diverse advanced cancers with known tissue mutation status were used for cfDNA extraction. The PHASIFY ENRICH method resulted in a 153% increase in median mutant copy recovery compared to QCNA (P = 0.01 Wilcoxon Signed-Rank). In the follow up study, 47 plasma samples from 31 patients with diverse advanced cancers and prior cfDNA negative mutation status with tissue positive mutation status were used for extraction. Of the 47samples extracted by PHASIFY ENRICH, 9 samples resulted in a conversion to positive cfDNA mutation status compared to no mutant signals when previously using QCNA. Conclusion: Our results indicate that PHASIFY ENRICH provides improved cfDNA mutation detection by increasing cfDNA recovery and reducing contaminating genomic DNA background. Citation Format: Filip Janku, Helen Huang, David Y. Pereira, Masae Kobayashi, Steven G. Call, Kristen T. Woodbury, Felix Chao, Daniel R. Marshak, Ricky Y. Chiu. Enhancing cell-free DNA mutation detection with novel liquid-phase extraction [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 575.

Cell-free DNA From Pleural Effusion Samples: Is It Right for Molecular Testing in Lung Adenocarcinoma?

Pathogenic molecular features gained specific significance in therapeutic decisions in lung carcinoma in the past decade. Initial and follow up genetic testing requres appropriate amounts and quality of tumor derived DNA, but tumor sampling, especially for disease monitoring is generally limited. Further to the peripheral blood (PB), samples from pleural fluid, accumulating in diverse lung processes might serve as an alternative source for cell-free DNA (cfDNA) for genetic profiling. In our study, cfDNA isolated from the pleural effusion and from the PB, and genomic DNA (gDNA) obtained from tissue/cellular samples were analyzed and compared from altogether 65 patients with pulmonary disease, including 36 lung adenocarcinomas. The quantity of effusion cfDNA yield appeared to be significantly higher compared to that from simultaneously collected PB plasma (23.2 vs. 4.8 ng/μl, p < 0.05). Gene mutations could be safely demonstrated from the effusion cfDNA fraction obtained from adenocarcinoma patients, 3/36 EGFR, 9/36 KRAS and 1/36 BRAF gene variants were detected. In this series, 9/13 samples showed an effusion+/plasma-mutational status, while only 1/13 samples presented with the opposite findings (effusion-/plasma+). gDNA analysis from sediment cell blocks from the identical effusion sample was surprisingly ineffective for lung adenocarcinoma profiling due to the low DNA yield. In conclusion, the cell free supernatant of pleural effusions appears to concentrate cancer derived cfDNA and seems to be particularly suitable for serial genotyping of pulmonary adenocarcinoma.

A Standardized Protocol for Donor-Derived Cell-Free DNA Quantification in the Diagnosis of Allograft Injury

Purpose There is an increasing interest in the use of donor-derived cfDNA (dd-cfDNA) in transplantation for non-invasive diagnosis of allograft injury. We hereby describe a non-commercial routine procedure for dd-cfDNA detection and quantification using digital droplet PCR and target-specific preamplification of a SNP-panel of 35 SNP-assays. The method is based on the difference in genotype between recipients and donors. After establishing our protocol, it has been used in a prospective study following recipients for 12 months after heart transplantation (HTx), and we here briefly describe our protocol. Methods A total of 728 samples from 67 HTx patients have been retrieved and analyzed. The recipients were genotyped preoperatively with respect to the SNP-panel. Upon transplantation, the donor genotype was interrogated with respect to the homozygous alleles found in the recipient. After HTx, blood samples were collected in cell free DNA collection tubes and delivered within the same day to the hospital laboratory. Plasma was separated by centrifugation at 2000g and 16000g, respectively. Isolated plasma was stored at -80C or used immediately for cfDNA extraction. Quality controls were performed by the 4200 Tape Station. The same primers were used for preamplification and downstream PCR. Non-template controls were included in all preamplification steps. The amount of dd-cfDNA was calculated as a fraction of the total cfDNA. Results The variation of extracted cfDNA yield was 10-130 ng/ml plasma. The 50-800 bp sized cfDNA constitutes 90% of the extracted samples, indicating a low background. An average of 2-5 informative SNPS assays for each transplant recipient were used for dd-cfDNA quantification with a detection rate down to 0.01% dd-cfDNA. Elevation of dd-cfDNA was seen not only in conjunction with cellular rejection but also during infection and in episodes of heart failure Conclusion Samples from HTx recipients demonstrated low levels of dd-cfDNA in consecutive samples during optimal treatment in non-eventful cases. Elevation of dd-cfDNA was seen not only during rejection but also in other conditions. This non-invasive method is relatively cheap, enables large sample yields by preamplification, has a fast turn-around time (48 hours) and is thus useful for clinical monitoring of HTx patients. However, it needs to be carefully related to clinical events.

Comprehensive genomic profiling (CGP) utilizing cell-free DNA (cfDNA) in patients (pts) with pancreatic ductal adenocarcinoma (PDAC).

421 Background: PDAC has a propensity for early systemic dissemination and most pts with primary tumors radiographically confined to the pancreas harbor micro-metastases. cfDNA based CGP offers a non-invasive mechanism to identify actionable genomic targets in PDAC and account for serial clonal evolution in response to therapeutic selection pressure. We interrogated the Foundation Medicine database to characterize cfDNA based CGP in pts with PDAC. Methods: We performed a retrospective analysis of pts with PDAC who underwent cfDNA based CGP between May 2016 and February 2020. cfDNA based CGP was done utilizing either the FoundationOne Liquid (F1-Liquid) or the Foundation-ACT (F-ACT) testing platform. Samples were interrogated for exonic and select intronic regions of cancer-related genes for both F1-Liquid (Exons: 70 genes; Select Introns: 7 genes) and F-ACT (Exons: 59 genes; Select Introns: 6 genes). Variant zygosity and somatic/germline status (SGZ) for short variant mutations was computationally predicted without matched normal tissue using an investigational method. A Gaussian Mixture Model (GMM) was used to identify latent molecular classes in samples harboring somatic, pathogenic variants (n=613); the Bayesian Information Criteria (BIC) was used for model selection. Results: We identified 1,009 pts with cfDNA based CGP - median age was 65, 55% were male, and median cfDNA concentration was 24 ng/µL. cfDNA yield had an influence on the detection of somatic alterations (p &lt;0.001). 613/1009 (60.8%) of pts had at least one somatic alteration detected on cfDNA based CGP. TP53 (53.7%), KRAS (40.3%) and CDKN2A (6.5%) were the most frequently altered genes (Table1). Homologous Recombination DNA Damage Repair (HR-DDR) gene alterations [somatic and/or germline BRCA2, ATM, and CHEK2 alterations], were detected in 12.3% (124/1009) pts – 11.4% (46/403) in KRAS mutated pts and 12.9% (78/606) in KRAS wildtype (WT) pts. Fusions were detected only in KRAS WT pts (1.5%, 6/403). Pts with cfDNA based CGP could be classified into 4 latent classes – class 1 ( KRAS - 100%, TP53 - 100% and CDKN2A – 33.3%), class 2 ( KRAS - 65.7%), class 3 ( KRAS – 68.2%, CDKN2A/B – 7.7% ) and class 4 ( KRAS – 38.5%, TP53 - 26.7%). Conclusions: The putative role of cfDNA based CGP in monitoring therapeutic response and clonal evolution in pts with PDAC may be predicated on cfDNA yield. In clinical scenarios where pre-treatment tissue availability is limited, cfDNA based CGP may be have utility in leveraging the predictive value of somatic and/or germline HR-DDR gene alterations. The prognostic and/or predictive relevance of the four latent classes needs validation in clinically annotated data sets. [Table: see text]

Correlation between comprehensive genomic profiling (CGP) utilizing tissue-based testing (T-CGP) and cell-free DNA (cfDNA) in patients (pts) with pancreatic ductal adenocarcinoma (PDAC).

422 Background: Early systemic dissemination is a salient feature of PDAC and the ability to reliably detect as well as monitor cfDNA based alterations may have predictive and/or prognostic value. However, a clear correlation between the clonality of the primary tumor and micro-metastatic disease has not been established in PDAC. We examined the correlation between T-CGP and cfDNA based CGP in pts with PDAC. Methods: We performed a retrospective analysis of PDAC pts who had both T-CGP (October 2010 – March 2020) and cfDNA based CGP (May 2016 – March 2020), with an average of 645 days between tests (Range: 21 – 2101 days) For T-CGP, exonic regions and select intronic regions of cancer related genes were sequenced to a high uniform depth (&gt;500X median coverage) using FoundationOne Heme (Exons: 315 genes; Select Introns: 28 genes) and FoundationOne CDx (Exons: 310 genes; Select Introns: 34 genes). cfDNA based CGP, was performed using either FoundationOne Liquid (Exons: 70 genes; Select Introns: 7 genes) or Foundation-ACT (Exons: 59 genes; Select Introns: 6 genes) platform. Results: Eighty-one pts were identified with both T-CGP and cfDNA based CGP - median age was 61 years, 59.3% (48/81) were male and 66.2% (49/74) had reported metastatic disease. The median tumor DNA content available for T-CGP analysis was 112.3 ng. The median cfDNA concentration was 1.7 ng/µL, and the number of reported ct-DNA based somatic alterations correlated significantly with the concentration of ct-DNA (Spearman’s R = 0.19, p &lt; 10-8). 75/81 (92.6%) and 41/81 (50.6%) of pts had at least one pathogenic somatic alteration detected by T-CGP and cfDNA based CGP, respectively. KRAS, TP53 and CDKN2A were the most frequently altered genes in both T-CGP and cfDNA based CGP (Table1). Homologous Recombination DNA Damage Repair (HR-DDR) gene alterations [ BRCA2, ATM, and CHEK2 alterations] were detected in 7.4% (6/81) of T-CGP and 7.4% (6/81) cfDNA based CGP, respectively. Two BRAF fusions were detected from samples tested with T-CGP, however these were not detected with the cfDNA assay. Conversely, an ALK-EML4 fusion was detected using both platforms. Conclusions: The concordance between T-CGP and cfDNA based CGP in HR-DDR gene alterations may be indicative of the germline status of some of these alterations; nevertheless, it suggests the predictive utility of cfDNA based CGP in PDAC. The differential prevalence of other gene alterations between the two modalities is likely reflective of the cfDNA yield available for CGP and to a lesser extent, clonal evolution. cfDNA based CGP holds promise as a complimentary predictive tool to T-CGP. [Table: see text]

Qualitative and quantitative comparison of cell-free DNA and cell-free fetal DNA isolation by four (semi-)automated extraction methods: impact in two clinical applications: chimerism quantification and noninvasive prenatal diagnosis

BackgroundNon-invasive molecular analysis of cell-free DNA (cfDNA) became a sensitive biomarker for monitoring organ transplantation or for detection of fetal DNA (cffDNA) in noninvasive prenatal test. In this study, we compared the efficiencies of four (semi)-automated cfDNA isolation instruments using their respective isolation kit: MagNA Pure 24 (Roche®), IDEAL (IDSolution®), LABTurbo 24 (Taigen®) and Chemagic 360 (Perkin Elmer®). The cfDNA was isolated from 5 plasma samples and the Rhesus D (RhD)-cffDNA from 5 maternal plasmas. The cfDNA were quantified by digital droplet PCR (ddPCR), BIABooster system and QUBIT fluorometer. The cfDNA fragment size profiles were assessed by BIABooster system. Chimerism were quantified by home-made ddPCR and Devyser NGS kit. RhD-cffDNA in maternal plasma were detected between weeks 14 and 24 of amenorrhea using free DNA Fetal RHD Kit® (Biorad®).ResultsStatistical tests have shown differences in DNA yield depending on the isolation procedure and quantification method used. Magna Pure isolates smaller cfDNA fragment size than other extraction methods (90% ± 9% vs. 74% ± 8%; p = 0.009). Chimerism was only reliable from LABTurbo 24 extractions using the NGS but not with ddPCR whatever extraction methods. RhD-cffDNA were detected by all isolation methods, although IDEAL and LABTurbo 24 systems seemed more efficient.ConclusionsThis comparative study showed a dependency of cfDNA yield depending on isolation procedure and quantification method used. In total, these results suggest that the choice of pre-analytical isolation systems needs to be carefully validated in routine clinical practice.

Harmonizing Cell-Free DNA Collection and Processing Practices through Evidence-Based Guidance.

Circulating cell-free DNA (cfDNA) is rapidly transitioning from discovery research to an important tool in clinical decision making. However, the lack of harmonization of preanalytic practices across institutions may compromise the reproducibility of cfDNA-derived data and hamper advancements in cfDNA testing in the clinic. Differences in cellular genomic contamination, cfDNA yield, integrity, and fragment length have been attributed to different collection tube types and anticoagulants, processing delays and temperatures, tube agitation, centrifugation protocols and speeds, plasma storage duration and temperature, the number of freeze-thaw events, and cfDNA extraction and quantification methods, all of which can also ultimately impact subsequent downstream analysis. Thus, there is a pressing need for widely applicable standards tailored for cfDNA analysis that include all preanalytic steps from blood draw to analysis. The NCI's Biorepositories and Biospecimen Research Branch has developed cfDNA-specific guidelines that are based upon published evidence and have been vetted by a panel of internationally recognized experts in the field. The guidelines include optimal procedures as well as acceptable alternatives to facilitate the generation of evidence-based protocols by individual laboratories and institutions. The aim of the document, which is entitled "Biospecimen Evidence-based Best Practices for Cell-free DNA: Biospecimen Collection and Processing," is to improve the accuracy of cfDNA analysis in both basic research and the clinic by improving and harmonizing practices across institutions.

Abstract A18: Plasma circulating tumor DNA is scarce and confounded by clonal hematopoiesis in metastatic renal cell carcinoma

Abstract Despite the recent implementation of novel targeted therapies, the 5-year survival rate for metastatic renal cell carcinoma (mRCC) remains dismal. Prognostic and predictive biomarkers are urgently required to avoid unnecessary patient morbidity and financial toxicity. Exploratory studies leveraging archival primary tumor tissue are considered suboptimal in this setting due to the considerable heterogeneity of metastatic lesions. Circulating tumor DNA (ctDNA) permits the noninvasive characterization of metastatic cancers using a simple blood draw. In this study, we sought to establish the utility of a ctDNA-based assay as a tool to profile the somatic genome of patients with mRCC. We collected whole blood from 55 progressing mRCC patients. All patients were systemic therapy naïve at the time of sample collection. Plasma cell-free DNA (cfDNA) and matched leukocyte DNA were subjected to targeted sequencing across 981 cancer-associated genes. Matched tumor tissue from 14 patients was also analyzed. The median cfDNA sequencing depth for this cohort was 938x. 33% of patients had evidence for RCC-derived ctDNA above 1% of total cfDNA; this was significantly lower than prostate or bladder cancer patients analyzed using the same approach. Among ctDNA-positive patients, ctDNA fraction averaged only 3.9% and showed no association with clinical variables or cfDNA yield. In these patients, the most commonly mutated genes were VHL, BAP1, and PBRM1, and matched tissue concordance was 77%. Evidence of somatic expansions unrelated to RCC, such as clonal hematopoiesis of indeterminate potential (CHIP), was detected in 43% of patients. Pathogenic germline mutations in DNA repair genes were detected in 11% of patients. Patients with ctDNA above 1% had shorter overall survival and progression-free survival on first-line therapy. Patients with evidence of CHIP but not ctDNA had an intermediate prognosis compared to ctDNA-positive and ctDNA-negative patients. CfDNA sequencing enables characterization of the somatic RCC genome in only a minority of metastatic RCC patients. Due to low ctDNA abundance and presence of non-RCC derived somatic clones in circulation, cfDNA sequencing may not be a simple pan-patient alternative to tissue biopsy in metastatic RCC. Citation Format: Jack V. W. Bacon, Matti Annala, Maryam Soleimani, Jean-Michel Lavoie, Alan I. So, Martin E. Gleave, Ladan Fazli, Kim N. Chi, Christian K. Kollmannsberger, Alexander W. Wyatt, Lucia Nappi. Plasma circulating tumor DNA is scarce and confounded by clonal hematopoiesis in metastatic renal cell carcinoma [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr A18.

Abstract B03: Urine cell-free DNA (cfDNA) concentration and stability test for future clinical use

Abstract Introduction: Urine was shown to be a liquid biopsy source with high sensitivity for cfDNA mutation detection in both early- and late-stage cancer patients, outperforming plasma cfDNA and tissue samples in some patients. In addition, obtaining a urine sample is completely noninvasive. Limitations of using urine samples in the clinical setting include their unknown nucleic acid stability and the large total volume of one deposit, and therefore the presence of highly diluted cfDNA. Urine volume concentration, cfDNA stability, cfDNA quantity, and detection of wild-type (wt) EGFR alleles were explored to determine the future suitability of urine cfDNA as a diagnostic or prognostic biosource. Methods: First-void urine samples, with a higher fraction of nucleic acids due to overnight concentration, were collected from 5 healthy donors and 5 cancer patients. Cell-free urine samples were concentrated using Amicon filter tubes to determine differences in cfDNA concentration. In addition, we tested the ability to detect wild-type EGFR exon 19 in cfDNA from concentrated and unconcentrated urine samples using a peptide nucleic acid (PNA) Taqman Assay. Hereafter, stability of urine cfDNA was explored using variable storage temperatures and times and addition of a urine cfDNA preservative directly after sample collection. cfDNA was extracted using the automated QIAsymphony system with the DSP virus/pathogen kit and quantified using the Qubit dsDNA HS Assay in all experiments. Results: Amicon filter-based concentration of urine samples from 15 mL to 1.2 mL yielded a two-fold increase in cfDNA concentration. Importantly, while wild-type EGFR could not be detected in first-void urine using a PNA Taqman Assay, detection was possible using concentrated urine samples. Therefore, in all following experiments, samples were concentrated unless specified otherwise. Stability tests indicated that storage temperature matters when samples are not processed within two hours, with nearly doubled cfDNA concentrations found in samples stored at four degrees for 26 hours, compared to room temperature (RT). Addition of a cfDNA preservative yielded a six-fold higher cfDNA concentration after 26 hours of storage at RT, compared to samples without a preservative added. For this reason, we tested the use of only cfDNA preservative without concentrating the sample and found a four-fold increase in cfDNA concentration when adding only preservative, versus a 15-fold increase when using both after sample storage at RT for 24 hours. Conclusion: Patient urine samples should be either concentrated and processed within two hours, or a urine cfDNA preservative should be added directly after sample collection to prevent cfDNA degradation. Depending on the downstream application, samples where preservative was added may be concentrated to enhance the cfDNA yield, for example, when downstream NGS analysis will be performed. Citation Format: Jillian W. P. Bracht, Niki Karachaliou, Jordi Berenguer, Manuel Fernandez-Bruno, Mónica Garzón, Ana Gimenez-Capitan, Clara Mayo-de-las-Casas, Miguel Angel Molina-Vila, Rafael Rosell. Urine cell-free DNA (cfDNA) concentration and stability test for future clinical use [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr B03.

Abstract A58: Advancing blood biopsy through the canine comparative model

Abstract Pet dogs are a powerful spontaneous model for human cancers. Dogs develop the same cancers that humans do, with striking similarities at the clinical, histopathologic, and genomic levels. These cancers have an accelerated clinical course compared with their human counterparts, and canine patients undergo many of the same interventions and treatments as human patients. The dog model thus offers the opportunity to implement rapid clinical trials informative for human medicine. We are developing liquid biopsy in dogs to further refine its clinical use by addressing questions that are difficult to assess in human patients. We have four ongoing pilot studies examining several different aspects of blood biopsy in canine patients. (1) We characterized the yield and tumor fraction of cell-free DNA (cfDNA) in nine canine cancer histologies using prospectively collected and banked plasma samples. (2) We tested the correlation between large-scale somatic copy number aberrations (SCNAs) in 24 osteosarcoma tumor samples and matched plasma samples and are now preparing to perform whole-exome sequencing (WES) of these samples to assess the concordance of SNVs and INDELs. (3) We investigated the use of cfDNA as a biomarker for response to therapy in four dogs with multicentric lymphoma treated with L-asparaginase via longitudinal blood biopsy. (4) We are optimizing phlebotomy techniques and characterizing short-term cfDNA dynamics by testing the effect of blood draw site and time of day on yield and tumor fraction. Samples from three sites (jugular, saphenous, and cephalic veins) were collected in a cohort of ten patients, and collection of samples at three time points throughout the day in a separate cohort is ongoing. (1) The majority (93%) of plasma samples yielded or were projected to yield sufficient DNA (&amp;gt;2ng) for ultra-low-pass whole-genome sequencing, and 37% had a tumor fraction ≥10%, allowing for WES given sufficient input DNA. (2) We found a strong correlation (Spearman coefficient of 0.74) between SCNAs in 13 tumor/plasma pairs with tumor fraction ≥10%. We are moving forward with WES of these cfDNA samples to examine simple somatic mutations. (3) Longitudinal cfDNA yield and tumor fraction correlated with response to therapy in three of four lymphoma patients. (4) Sample collection and sequencing are under way for the studies on the effect of blood draw site and time of day. Our studies demonstrated the feasibility of blood biopsy in dogs, the high concordance between cfDNA and tumor samples, and suggested that blood biopsy is a promising method for monitoring response to therapy. Emerging results will help to optimize phlebotomy protocols and characterize cfDNA dynamics over short time scales, as well as assessing in more detail the concordance between cfDNA and matched tumor samples. Further development of blood biopsy in dogs has the potential to improve and accelerate clinical application of this technology in human medicine and to facilitate further high-impact comparative translational studies in the dog model. Citation Format: Kate Megquier, Kan Xiong, Heather L. Gardner, Justin Rhoades, Viktor Adalsteinsson, Cheryl A. London, Elinor K. Karlsson. Advancing blood biopsy through the canine comparative model [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr A58.

Impact of long-term storage of plasma and cell-free DNA on measured DNA quantity and fetal fraction.

Optimal sample storage conditions are essential for non-invasive prenatal testing of cell-free fetal and total DNA. We investigated the effect of long-term storage of plasma samples and extracted cfDNA using qPCR. Fetal and total cfDNA yield and fetal fraction were calculated before and after storage of plasma for 0-6years at -25°C. Dilution experiments were performed to investigate PCR inhibition. Extraction with or without proteinase K was used to examine protein dissociation. Storage of extracted cfDNA was investigated by testing aliquots immediately, and after 18months and 3years of storage at -25°C. We observed a marked increase in the levels of amplifiable fetal and total DNA in plasma stored for 2-3years, and fetal fraction was slightly decreased after 3years of storage. cfDNA detection was independent of proteinase K during DNA extraction in plasma samples stored >2years, indicating a loss of proteins from DNA over time, which was likely to account for the observed increase in DNA yields. Measured fetal and total DNA quantities, as well as fetal fraction, increased in stored, extracted cfDNA. Fetal and total cell-free DNA is readily detectable in plasma after long-term storage at -25°C. However, substantial variation in measured DNA quantities and fetal fraction means caution may be required when using stored plasma and extracted cfDNA for test development or validation purposes.

Comparison of Four Commercial Kits for Isolation of Urinary Cell-Free DNA and Sample Storage Conditions.

Urinary cell-free DNA (cfDNA) is an attractive body fluid for liquid biopsy. In this study, we compared the efficiencies of four commercial kits for urinary cell-free DNA (cfDNA) isolation and of various sample storage conditions. Urinary cfDNA was isolated from 10 healthy individuals using four commercial kits: QIAamp Circulating Nucleic Acid Kit (QC; Qiagen), MagMAX™ Cell-Free DNA Isolation Kit (MM; Applied Biosystems), Urine Cell-Free Circulating DNA Purification Midi Kit (NU; Norgen Biotek), and Quick-DNA™ Urine Kit (ZQ; Zymo Research). To assess the isolation efficiency, an Agilent 2100 Bioanalyzer with High Sensitivity DNA chips was used, and cfDNA yield was defined as the amount of cfDNA obtained from 1 mL of urine. MM and QC provided the highest cfDNA yield in the 50–300 bp range, and MM and NU gave the highest cfDNA yield in the 50–100 bp range. In particular, the NU kit was efficient for isolation of more fragmented cfDNA in the range of 50–100 bp with the lowest cellular genomic DNA contamination. ZQ had the best cost-efficiency for isolating the same amount of urinary cfDNA. Samples stored at −70 °C with the addition of 10 mM EDTA resulted in the highest cfDNA yield 3 months after sample collection.

Abstract P5-06-28: Optimization and validation of PIK3CA mutation detection with droplet digital PCR in liquid biopsies of patients with metastatic breast cancer

Abstract Background Approximately 40% of oestrogen receptor positive (ER+) metastatic breast cancer harbour PIK3CA hotspot mutations, leading to an over-activated PI3K pathway. The PI3Kα-selective inhibitor, alpelisib, is FDA approved and currently available in early access program for patients with ER+ HER2-negative metastatic disease, if an activating PIK3CA mutation is confirmed. Mutational analysis on liquid biopsies may increase the number of eligible patients given the challenges of tissue procurement in metastatic setting. We optimized and validated ddPCR assays for PIK3CA hotspot mutations in cell-free DNA (cfDNA) of metastatic breast cancer patients. Patients and Methods We prospectively collected two blood samples (cfDNA Collection tubes, Roche Diagnostics) of 20 metastatic breast cancer patients at progression (N=12) or during therapy (N=8). PIK3CA mutation status was previously demonstrated by Next Generation Sequencing (NGS) performed in 4 patients on primary tumor tissue and in 16 patients on metastatic tumor tissue; 8 patients tested positive and 12 negative. Tumors were ER+ HER2-negative, triple-negative and HER2-positive in 14, 5 and one patients, respectively. After plasma-isolation, cfDNA was manually extracted with Cobas® cfDNA Sample Preparation Kit (2mL plasma) and semi-automatically with Maxwell® RSC ccfDNA Plasma Kit (2mL and 4mL plasma). Inter-run variability, intra-run variability, precision and robustness of the assays, and concordance between NGS and ddPCR for the detection of PIK3CA hotspot mutations on tumor tissue and in plasma, respectively, were assessed. Results All 20 samples were successfully processed with ddPCR. The highest cfDNA yield was obtained by Maxwell 4mL extraction method (median: 0.483 ng/µL; range: 0.140 - 10.500 ng/µL). The per-mutation sensitivity and specificity was 87.5% and 95.8%, respectively. Two samples showed discordant results between PIK3CA mutations detected by NGS on tumor tissue and by ddPCR in plasma. One sample tested positive for p.(H1047R) on tumor tissue, and for p.(E542K), p.(E545K) and p.(H1047R) in cfDNA. The other switched between p.(E545K) mutation on tumor tissue and p.(E542K) in cfDNA. Tumor tissue of samples for which the results of the NGS test were discordant with the ddPCR test on plasma, was tested again using ddPCR on the tissue. This still showed discordances in PIK3CA mutations which can be explained by tumor heterogeneity and the lower detection limit of ddPCR versus NGS. Conclusion Detection of PIK3CA hotspot mutations with ddPCR in cfDNA is feasible. We observed good concordance between NGS and ddPCR for the detection of PIK3CA hotspot mutations on tumor tissue and in plasma, respectively. In 10% of cases, discordant PIK3CA mutation status in tissue versus plasma was detected. Further investigation of different metastatic lesions in these cases is ongoing. Citation Format: Laurence Slembrouck, Demi Renders, Sara Vander Borght, Patrick Neven, Giuseppe Floris, Lien Spans, Hans Wildiers, Kevin Punie, Ann Smeets, Ines Nevelsteen, Ignace Vergote, Adriaan Vanderstichele, Isabelle Vanden Bempt. Optimization and validation of PIK3CA mutation detection with droplet digital PCR in liquid biopsies of patients with metastatic breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P5-06-28.

Effects of Quantification Methods, Isolation Kits, Plasma Biobanking, and Hemolysis on Cell-Free DNA Analysis in Plasma.

Cell-free DNA (cfDNA) has become a promising noninvasive clinical marker widely studied in early disease detection, monitoring, and therapy selection. However, there is lack of data on a number of cfDNA-associated procedural features such as blood plasma biobanking conditions, isolation, and quantification methods that should be taken into account as they can affect downstream applications. In this study cfDNA from 125 plasma samples from healthy individuals were isolated using three different commercial kits (bead and vacuum based). Yield of cfDNA, distribution of cfDNA fragments and absolute amount of miR-223 were estimated. Moreover, the impact of different plasma biobanking conditions and hemolytic plasma were evaluated. In conclusion, results showed that quantification method (fluorescence or microcapillary electrophoresis based) has a major impact in estimating cfDNA amount. Samples isolated by QIAamp showed a higher amount of larger (around 300 bp) DNA fragments and miRNA yield, suggesting possible applications for multiomics approach. On the other hand, the highest cfDNA yield was obtained in samples isolated by the MagMAX Isolation Kit. This kit also showed lowest coefficient of variation and low miRNA yield. Plasma storage conditions and hemolysis affected performance of isolation kits differently.

Cell-free DNA analysis in healthy individuals by next-generation sequencing: a proof of concept and technical validation study

Pre-symptomatic screening of genetic alterations might help identify subpopulations of individuals that could enter into early access prevention programs. Since liquid biopsy is minimally invasive it can be used for longitudinal studies in healthy volunteers to monitor events of progression from normal tissue to pre-cancerous and cancerous condition. Yet, cell-free DNA (cfDNA) analysis in healthy individuals comes with substantial challenges such as the lack of large cohort studies addressing the impact of mutations in healthy individuals or the low abundance of cfDNA in plasma. In this study, we aimed to investigate the technical feasibility of cfDNA analysis in a collection of 114 clinically healthy individuals. We first addressed the impact of pre-analytical factors such as cfDNA yield and quality on sequencing performance and compared healthy to cancer donor samples. We then confirmed the validity of our testing strategy by evaluating the mutational status concordance in matched tissue and plasma specimens collected from cancer patients. Finally, we screened our group of healthy donors for genetic alterations, comparing individuals who did not develop any tumor to patients who developed either a benign neoplasm or cancer during 1–10 years of follow-up time. To conclude, we have established a rapid and reliable liquid biopsy workflow that allowed us to study genomic alterations with a limit of detection as low as 0.08% of variant allelic frequency in healthy individuals. We detected pathogenic cancer mutations in four healthy donors that later developed a benign neoplasm or invasive breast cancer up to 10 years after blood collection. Even though larger prospective studies are needed to address the specificity and sensitivity of liquid biopsy as a clinical tool for early cancer detection, systematic screening of healthy individuals will help understanding early events of tumor formation.