The emerging role of cell-free DNA as a molecular marker for cancer management

The application of circulating tumor cell and cell-free DNA liquid biopsies in ovarian cancer

The emergence of targeted therapies has transformed ovarian cancer treatment. However, biomarker profiling for precision medicine is limited by access to quality, tumour-enriched tissue samples. The use of cell-free DNA (cfDNA) in ascites presents a potential solution to this challenge. In this study, next-generation sequencing was performed on ascites-derived cfDNA samples (26 samples from 15 human participants with ovarian cancer), with matched DNA from ascites-derived tumour cells (n = 5) and archived formalin-fixed paraffin-embedded (FFPE) tissue (n = 5). Similar tumour purity and variant detection were achieved with cfDNA compared to FFPE and ascites cell DNA. Analysis of large-scale genomic alterations, loss of heterozygosity and tumour mutation burden identified six cases of high genomic instability (including four with pathogenic BRCA1 and BRCA2 mutations). Copy number profiles and subclone prevalence changed between sequential ascites samples, particularly in a case where deletions and chromothripsis in Chr17p13.1 and Chr8q resulted in changes in clinically relevant TP53 and MYC variants over time. Ascites cfDNA identified clinically actionable information, concordant to tissue biopsies, enabling opportunistic molecular profiling. This advocates for analysis of ascites cfDNA in lieu of accessing tumour tissue via biopsy.

Background The fecal immunochemical test (FIT) is widely used in population based colorectal cancer (CRC) screening programs. Recently, circulating cell-free DNA (cfDNA) analyses have emerged as a new avenue for early cancer detection. The performance of cfDNA methods in comparison with FIT is currently unknown. The present study compared pre-operative cfDNA analyses to FIT for detection of patients with CRC who participated in a population-based screening program. Methods In the Dutch national CRC screening program, individuals aged 55-75 years are biennially invited to perform a single FIT. The database of the Dutch CRC screening program was queried to identify individuals with FIT data who also participated in two cfDNA studies, PLCRC-MEDOCC and PLCRC-PROVENC3 (AACR abstract C. Rubio Alarcón). Sensitivities with 95% confidence intervals (CI) for detecting individuals with CRC were determined for FIT, tumor-informed cfDNA analyses, as well as both tests combined. For FIT, sensitivities were determined at 20 µg and 47 µg hemoglobin (Hb)/g feces positivity cut-offs as these represent the most commonly used international and Dutch cut-offs, respectively. Liquid biopsy analyses were performed using next generation sequencing of cfDNA. Tumor-specific alterations were identified through parallel next generation sequence analysis of resected tumor tissues. Results The query identified 120 individuals with stage I (n=3, 2.5%), stage II (n= 57, 47.5%) and stage III (n=60, 50.0%) CRC who had participated in the Dutch CRC screening program and either PLCRC-MEDOCC or PLCRC-PROVENC3. All FIT samples were collected at a median time of 38 days before diagnosis (IQR 27-112 days). FIT sensitivity for detection of individuals with CRC was 83.3% (100/120 CRCs, 95% CI 75.4-89.5%) at a 20 µg Hb/g feces cut-off and 80.0% (96/120, 95% CI 71.7-86.7%) at a 47 µg Hb/g feces cut-off. Tumor-informed cfDNA analyses detected 75.8% (91/120, 95% CI 67.2-83.2%) of individuals with CRC in this population. The combination of cfDNA analyses and and FIT (20 µg Hb/g feces cut-off) identified almost all individuals with CRCs (119/120, sensitivity 99.2%, 95% CI 97.0-100%). The individual who was missed had a cancer that was a T3N0 microsatellite stable moderately differentiated adenocarcinoma in the ascending colon. Discussion While both FIT and cfDNA analyses identified most CRC cases (75-83%) in this series, both tests also demonstrated a substantial level of complementarity, indicating that in principle combining FIT with cell-free DNA testing would allow to increase sensitivity of CRC screening. Since the cell-free DNA tests used here are tumor-informed, which in real life screening practice is not feasible, it remains to be determined to what extent this result can be reached using non-tumor-informed approaches. Citation Format: Pieter Henk Abraham Wisse, Carmen Rubio-Alarcon, Suzanna J. Schraa, Adria C. Mosquera, Mark Sausen, Remond J. Fijneman, Geraldine R. Vink, Meike de Wit, Jillian Phallen, Victor E. Velculescu, Beatriz Carvalho, Gerrit A. Meijer. Comparison of FIT and cell-free DNA analyses for detection of individuals with colorectal cancer in population based screening [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 6079.

Somatic cloning is emerging as a new biotechnology by which the opportunities arising from the advances in molecular genetics and genome analysis can be implemented in animal breeding. Significant improvements have been made in SCNT protocols in the past years which now allow to embarking on practical applications. The main areas of application of SCNT are: Reproductive cloning, therapeutic cloning and basic research. A great application potential of SCNT based cloning is the production of genetically modified (transgenic) animals. Somatic cell nuclear transfer based transgenic animal production has significant advances over the previously employed microinjection of foreign DNA into pronuclei of zygotes. This cell based transgenesis is compatible with gene targeting and allows both, the addition of a specific gene and the deletion of an endogenous gene. Efficient transgenic animal production provides numerous opportunities for agriculture and biomedicine. Regulatory agencies around the world have agreed that food derived from cloned animals and their offspring is safe and there is no scientific basis for questioning this. Commercial application of somatic cloning within the EU is via the Novel Food regulation EC No. 258/97. Somatic cloning raises novel questions regarding the ethical and moral status of animals and their welfare which has prompted a controversial discussion in Europe which has not yet been resolved.

The clinical landscape of cell-free DNA alterations in 1671 patients with advanced biliary tract cancer

In singleton pregnancies, the use of cell-free DNA (cfDNA) analysis as a screening test for common fetal trisomies has spread worldwide though we still lack sufficient data for its use in triplet pregnancies. The objective of this study is to assess the performance of cfDNA testing in detecting fetal aneuploidies in triplet pregnancies as a first-tier test. We performed a retrospective cohort study including data from pregnant women with a triplet pregnancy who underwent cfDNA testing between May 1, 2017, and January 15, 2020. cfDNA was obtained by massive parallel sequencing (VeriSeq NIPT solution; Illumina®). The objectives of the study were to assess the diagnostic performance of cfDNA testing for trisomy 21 (T21) (primary outcome), trisomy 18 (T18) and 13 (secondary outcomes). During the study period, cfDNA testing was performed in 255 women with triplet pregnancy, of which 165 (64.7%) had a neonatal outcome available. Three tests were positive for T21, one of which was confirmed by an antenatal karyotype, and the other was confirmed at birth. The third case did not undergo an invasive procedure and was not confirmed at birth (false positive). In one case, cfDNA testing was positive for T18 and was confirmed by an antenatal karyotype. There were no cases of trisomy 13 in the cohort. The no-call rate was 2.4% at first sampling. Fifty-eight (22.7%) women had embryo reduction, which in 40 (69%) of whom was performed after the cfDNA test result. cfDNA testing could be offered as primary screening for main fetal aneuploidies in triplet pregnancies after provision of appropriate patient information.

Non-small cell lung cancer (NSCLC) is difficult to treat when metastasis has occurred. This study explores the use of cell-free DNA in the clinical management of NSCLC patients who have Kirsten rat sarcoma viral oncogene homolog (KRAS)-positive mutations and as a marker for prognosis. Peripheral blood collected from advanced NSCLC patients was examined with digital droplet polymerase chain reaction and ultraviolet spectrometry. KRAS mutations were analyzed and quantitated. The specificity and sensitivity of the proposed assay was computed by associating the results with tumor tissue specimens. Comparison against different sub-groups of patients with different metastatic sites and healthy volunteers were made. Patients were subsequently followed up and survival analysis was conducted. Among the 186 patients recruited, 150 had concordant KRAS mutational profiles using cell-free DNA with tumor tissues. The assay sensitivity and specificity were 80.6% and 100%, respectively. For the 150 patients with concordant results, the range of cell-free DNA quantities in peripheral blood was 5.3 to 115 ng. Among the patient groups with different metastatic sites, we observed that patients with bone metastasis had higher concentrations of cell-free DNA. Survival analysis showed that these patients had worse survival outcome. Patients with higher KRAS counts in peripheral blood also had worse outcome. The use of cell-free DNA presents opportunities for risk stratification of patients and possibly aids in the clinical management of the disease. In the current study for NSCLC, patients with bone metastases showed higher cell-free DNA concentrations. Quantitating the concentrations of cell-free DNA presents a noninvasive biomarker capable of prognostic utility.

Background: The Y537S and D538G mutations in ESR1 are recurrent alterations in metastatic breast cancer (MBC) that promote ligand-independent receptor activation and resistance to estrogen deprivation therapy in laboratory models. The clinical prevalence of these mutations is not well established, but has been reported to be ∼10% by tumor sequencing. Cell free DNA (cfDNA) analysis typically reflects tumor-derived genetic alterations and can be used to characterize a population of patients at a common time point. We hypothesized that cfDNA could be used to detect ESR1 mutation at entry and its clinical impact in a large subset from BOLERO-2 that randomized patients with MBC to Exemestane (EXE) or EXE plus Everolimus (EVE). Methods: Patients with ER+/HER2– MBC with prior exposure to at least 1 non-steroidal aromatase inhibitor (NSAI) were enrolled in BOLERO-2. cfDNA was extracted from 560 baseline plasma samples using QIAamp Circulating DNA kit or QIAsymphony DSP Virus/Pathogen kit. Samples were analyzed by droplet digital PCR for Y537S and D538G. Cox-proportional hazards model was used to assess progression free survival (PFS) in patient subgroups defined by each ESR1 mutation, and the prognostic effect of each ESR1 mutation on overall survival (OS). Results: Of 541 evaluable patients (74.7% of study population), 156 (28.8%) had mutation in ESR1 in D538G (21.1%) and/or Y537S (13.3%) with 30 samples having both mutations. Sequencing of 302 archival tumor specimens (244 primary and 57 metastases) from this study only yielded 4 instances of D538G (1.3%) and 1 Y537S (0.3%). In the overall population, both mutations were poor prognostic factors associated with shorter OS (Table 1). Table 1AlterationNEventsMedian OS (95%CI)HR (95%CI)WT38521732.1 (28.1-36.4) D538G835726.0 (19.2-32.4)1.25 (1.02-1.54)Y537S423020.0 (13.0-29.3)2.31 (1.34-3.97)Double mt302415.2 (10.9-27.4)1.77 (1.31-2.39) PFS results were different for the two mutations. D538G but not Y537S mutation was associated with a shorter PFS with EXE compared to wild type (WT), (hazard ratios, D538G: 1.44 [95%CIs, 1.04-1.99] and Y537S: 0.92 [95%CIs, 0.44-1.93]). The D538G mutant group derived a similar benefit as WT from the addition of EVE to EXE, whereas the Y537S group did not (Table 2). Table 2AlterationGroupNEventsMedian PFS (95%CI)HR (95%CI)WTEXE1281163.9 (2.8-4.2)0.4 (0.31-0.51) EXE/EVE2571728.5 (6.9-9.9) D538GEXE24222.7 (1.4-2.8)0.34 (0.2-0.57) EXE/EVE59455.8 (4.2-8.4) Y537SEXE21164.1 (1.4-6.7)0.98 (0.49-1.94) EXE/EVE21194.2 (1.4-5.4) Conclusions: cfDNA analysis identifies a high rate of the Y537S and D538G ESR1 mutations in ER+ NSAI-treated MBC. As the two mutations may only represent 50-60% of all activating ESR1 mutations, the clinical prevalence of ESR1 mutations in ER+ MBC may be much higher than previously reported. Both mutations appear to be associated with a more aggressive disease biology. Interestingly, these two activating mutations appear to have differential effects on EXE and EVE sensitivity, highlighting new areas for research in ER biology. Citation Format: Chandarlapaty S, Sung P, Chen D, He W, Samoila A, You D, Bhatt T, Patel P, Voi M, Gnant M, Hortobagyi G, Baselga J, Moynahan ME. cfDNA analysis from BOLERO-2 plasma samples identifies a high rate of ESR1 mutations: Exploratory analysis for prognostic and predictive correlation of mutations reveals different efficacy outcomes of endocrine therapy–based regimens. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr S2-07.

Background Precision-medicine initiatives are driven by the molecular analysis of tumor samples (fresh or FFPE material). Such an approach is limited by the availability of the tumor material and the challenges related to on-purpose tumor biopsies. A very appealing alternative to advance precision-medicine initiatives is the development of liquid biopsies using cfDNA. Methods We investigated the use of NGS on tumor biopsy and plasma, and evaluated the consistency between the tissue biopsy (tDNA) and cfDNA analysis on a prospective cohort of patients with metastatic or locally advanced solid tumors enrolled in the MOSCATO 01 trial (NCT01566019). Blood samples were collected at inclusion before tumor biopsy and cfDNA was extracted from 500μl plasma. Hot-spot mutations from 50 genes were screened with Ampliseq CHP2 panel (IonTorrent, Life Technologies, Dramstadt). Only variants reported by the Torrent Suite Variant Caller v4.2 were retained for the analysis. Paired results in tumor and plasma were described using Cohen's Kappa agreement coefficient (κ). Results From November 2011 to May 2014, among the 516 patients enrolled in the MOSCATO 01 trial, 190 patients (37%) were analyzed for tDNA and cfDNA. In addition, cfDNA was evaluated in 43 patients for whom no tumor analysis was performed because of low cellularity (< 10% of tumor cells). Patient characteristics were as follows: median age at biopsy: 57 years (range, 18-78); main tumor types: lung (19%), ENT (14%), colorectal (10%), breast (10%); median of 3 previous lines of treatment. Overall, 325 mutations were identified in the tDNA of 184 patients: 146 mutations were identified both in tumor and plasma and 179 in tumor, but not in plasma. 15 mutations were only found in cfDNA, thereby providing additional information. The κ.was 59% (95%CI, 0.54-0.64). The sensibility of using NGS for 50 targeted hot-spot genes analysis in cfDNA compared to tDNA was 44.9% and the specificity was 99.8%, with a positive predictive value of 90.7% and negative predictive value of 98.1%. The ten most frequent pathogenic mutations found in the tDNA or cfDNA (>5 cases each) included KRAS (33 cases), PIK3CA (22 cases) and TP53 genes (22 cases). When considering only these ten most frequent mutations, 77 mutations were identified in 71 patients: 39 in tumor and plasma, 36 in tumor, but not in plasma and 2 mutations only in plasma, with a κ of 66% (95%CI, 56-76%). The p.H1047R PIK3CA mutation, was found only in tumor (5 cases); for the other nine mutations, the κ coefficient varied from 56% to 89%, with a median of 75%. The cfDNA analysis of the 43 patients without tDNA analysis revealed at least one mutation in 24 patients (56%), including 11 pathogenic variants of therapeutic interest. Conclusion The analysis of cfDNA using NGS represents an attractive and noninvasive alternative to tumor biopsies, and can be used as a surrogate method to screen for mutations. Further prospective validation is warranted. Citation Format: Ecaterina Ileana, Cécile Jovelet, Marie-Cécile Le Deley, Christophe Massard, Nelly Motté, Antoine Hollebecque, Amélie Boichard, Charles Ferté, Sophie Postel-Vinay, Silvia Rosellini, Maud Ngo-Camus, Thierry De Baere, Philippe Vielh, Catherine Richon, Mélanie Laporte, Siham Gouissem, Yohann Loriot, Rastilav Bahleda, Anas Gazzah, Andrea Varga, Gilles Vassal, Alexander Eggermont, Fabrice André, Jean-Charles Soria, Ludovic Lacroix. Circulating cell-free tumor DNA (cfDNA) analysis of 50-genes by next-generation sequencing (NGS) in the prospective MOSCATO trial. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2401. doi:10.1158/1538-7445.AM2015-2401

This was a randomized controlled trial to compare risk assessment by first-trimester combined screening (FTCS) with an approach that combines a detailed ultrasound examination at 11-13 weeks' gestation and cell-free DNA (cfDNA) analysis. Pregnant women with a normal first-trimester ultrasound examination at 11-13 weeks' gestation (fetal nuchal translucency (NT)≤ 3.5 mm and no fetal defects) were randomized into one of two groups. In the first group, risk of aneuploidy was assessed using FTCS based on the most recent UK Fetal Medicine Foundation algorithm. In the second group, risk assessment was based on ultrasound findings and cfDNA analysis. An additional tube of blood was collected for FTCS in case the cfDNA analysis was uninformative. Primary outcome was false-positive rate in screening for trisomy 21. A case was considered false positive if the karyotype was not trisomy 21 and if the risk for trisomy 21 was >1:100, irrespective of the method of risk calculation. Results were compared using 95% CIs using the Clopper-Pearson method. Between October 2015 and December 2016, 1518 women with singleton pregnancy underwent first-trimester screening. Thirty-one (2.0%) pregnancies were not eligible for randomization due to increased NT (> 3.5 mm) and/or fetal defect. After exclusion of women who declined randomization (n= 87) and cases of fetal death and loss to follow-up (n= 24), 688 pregnancies were randomized into the FTCS arm and 688 into the ultrasound + cfDNA analysis arm. There were no differences in maternal and gestational age, maternal weight and BMI, ethnicity, use of assisted reproduction and cigarette smoking between the two arms. In the ultrasound + cfDNA analysis arm, median risk for trisomy 21 was 1 in 10 000. None of the cases had a risk above 1: 100 (95% CI, 0.0-0.5%). In the FTCS arm, the median risk for trisomy 21 was 1 in 3787 and in 17 cases, the risk was higher than 1:100, which corresponds to 2.5% (95% CI, 1.5-3.9%) of the FTCS study-arm population. Our study has shown that first-trimester risk assessment for trisomy 21 that includes a detailed ultrasound examination as well as NT measurement and is followed by cfDNA testing is associated with a significant reduction in the false-positive rate compared with FTCS. This approach obviates the need for maternal serum free β-human chorionic gonadotropin and pregnancy-associated plasma protein-A in screening for fetal aneuploidy. Copyright © 2017 ISUOG. Published by John Wiley & Sons Ltd.

12046Background: Next-generation sequencing of cell-free DNA (cfDNA) can assess presence of somatic genomic alterations in patients with cancer without an invasive biopsy. Results may guide therape...

Background. Despite great advances in the treatment of burn patients, useful prognostic markers are sparse. During the past years there has been increasing interest in circulating plasma cell free DNA as a potential marker for tissue injury. We have developed a rapid direct fluorescent assay for cell free DNA quantification that allows obtaining accurate, fast, and inexpensive measurements. Objective. To use this technique for measuring plasma cell free DNA levels in burn patients and to further explore the use of cell free DNA as a potential marker of patient outcome in burns. Methods. Cell free DNA levels obtained from 14 burn victims within 6 hours of injury and 14 healthy controls were quantified by a direct rapid fluorometric assay. Results. Patient admission cell free DNA levels were significantly elevated compared with that of controls (1797 ± 1523 ng/mL versus 374 ± 245 ng/mL, P = 0.004). There are statistically significant correlations between cell free DNA admission levels and burn degree (Spearman's correlation = 0.78, P = 0.001), total body surface area (Spearman's correlation = 0.61, P = 0.02), and total burn volume (Spearman's correlation = 0.64, P = 0.014). Conclusions. Admission cell free DNA levels can serve as a prognostic factor in burns and future routine use can be made possible by use of our direct rapid fluorometric assay.

We aimed to identify the most relevant cost-effectiveness threshold of first-trimester Down syndrome (DS) maternal serum screening (T21T1) for the use of cell-free DNA (cfDNA) as a second-tier test in the French context. A cost-effectiveness analysis was performed on 108,121 singleton pregnancies using a simulation model. The threshold of T21T1 screening was ranged from 1/51 to 1/1,000 in steps of 1/50. The most relevant threshold was based on cost-effectiveness ratio (CER; costs =direct medical costs after T21T1 screening/ effectiveness =number of DS cases identified). In the sample, 161cases of DS were identified. At the threshold of ≥1/50, 47.2% of total DS cases were diagnosed. In the simulation model, for a threshold ≥ 1/250, 73.9% of total DS cases were diagnosed, for ≥ 1/500, 78.8% and for ≥ 1/1,000, only two additional cases were diagnosed. The slope of the cost increase was slight from threshold ≥ 1/250 (978,634€), then steep up to 1/500 (1,966,576€) and increased exponentially to 1/1,000 (3,980,216€). The CER was 38,560 for a threshold ≥ 1/500. The most cost-effective threshold for cfDNA as a second-tier test seems to be ≥1/500. For higher thresholds, costs increase dramatically for only a few additional cases of DS identified.

Simple SummaryThe aim of this review is to evaluate the present status of the use of cell-free DNA and its fraction of circulating tumor DNA (ctDNA) because this year July 2022, an ESMO guideline was published regarding the application of ctDNA in patient care. In the near future the data obtained from ctDNA may routinely be used for finding minimal residual disease, detecting relapse, determining either the unknown primary tumor, or the site of metastases. It can also be used for deciding upon the appropriate efficiency of the therapy and/or emerging resistance to the therapy. Therefore, clinicians should be aware of the potentials and the limitations of the assays. Of course, several open questions are still under research and as a result, cfDNA and ctDNA testing are not part of routine care yet.The aim of this review is to evaluate the present status of the use of cell-free DNA and its fraction of circulating tumor DNA (ctDNA) because this year July 2022, an ESMO guideline was published regarding the application of ctDNA in patient care. This review is for clinical oncologists to explain the concept, the terms used, the pros and cons of ctDNA; thus, the technical aspects of the different platforms are not reviewed in detail, but we try to help in navigating the current knowledge in liquid biopsy. Since the validated and adequately sensitive ctDNA assays have utility in identifying actionable mutations to direct targeted therapy, ctDNA may be used for this soon in routine clinical practice and in other different areas as well. The cfDNA fragments can be obtained by liquid biopsy and can be used for diagnosis, prognosis, and selecting among treatment options in cancer patients. A great proportion of cfDNA comes from normal cells of the body or from food uptake. Only a small part (<1%) of it is related to tumors, originating from primary tumors, metastatic sites, or circulating tumor cells (CTCs). Soon the data obtained from ctDNA may routinely be used for finding minimal residual disease, detecting relapse, and determining the sites of metastases. It might also be used for deciding appropriate therapy, and/or emerging resistance to the therapy and the data analysis of ctDNA may be combined with imaging or other markers. However, to achieve this goal, further clinical validations are inevitable. As a result, clinicians should be aware of the limitations of the assays. Of course, several open questions are still under research and because of it cfDNA and ctDNA testing are not part of routine care yet.

Circulating free DNA in plasma or serum as biomarker of carcinogenesis: Practical aspects and biological significance