Cell-free DNA Concentration Research Articles

Prognostic Impact of Pre-Treatment Cell-Free DNA Concentration in Newly Diagnosed Peripheral T-Cell Lymphomas

Peripheral T-cell lymphomas (PTCLs) are a heterogeneous group of non-Hodgkin lymphomas with poor prognosis. Although some risk factors associated with worse survival are known, there are no reliable predictive biomarkers of response. Considering the prognostic relevance of cell-free DNA (cfDNA) in B-cell lymphomas, in this work we aimed to explore its role in 75 patients (pts) enrolled in the PTCL13 prospective study (NCT02223208), including newly diagnosed Peripheral T-cell Lymphoma, Not Otherwise specified (PTCL-NOS, n=30), nodular T Follicular Helper Lymphoma (nTFHL, n=26) and Anaplastic Lymphoma Kinase negative Anaplastic Large Cell Lymphoma (ALK-ALCL, n=19). Cell-free DNA was extracted using the Maxwell® RSC LV ccfDNA Kit (Promega) from plasma samples. Pre-treatment cfDNA concentration had a median value of 19.1 ng/mL of plasma (range: 2.27-1082 ng/mL) and was significantly increased in the presence of high-risk clinical parameters, including disease-related risk factors (increased lactate dehydrogenase levels, p<0.0001; presence of extranodal disease p=0.04), prognostic scores used in PTCLs (International Prognostic Index >2, p<0.0001; Prognostic Index for T-cell lymphoma >1, p=0.0006), and patient condition (poor performance status, p=0.0003), compared with cfDNA concentration in low-risk categories. On the other hand, no association was detected between age, Ann Arbor stage, bone marrow involvement and cfDNA levels. In addition, higher baseline cfDNA concentration was found in pts who progressed compared with those who achieved a response at the end of 6 chemotherapy cycles (p= 0.0303). Importantly, pre-treatment cfDNA level had a negative impact on both progression-free survival (PFS, Univariate Cox linear model, p=0.0001) and overall survival (OS, Univariate Cox nonlinear model, p=0.0105). Multivariate analysis confirmed a significant influence of cfDNA on PFS [Multivariate Cox Model, HR 1.34, 95% Confidence Interval (CI) 1.14-1.58, p= 0.0004 and HR 1.28, 95% CI 1.11-1.48, p= 0.0006] greater than that of IPI and PIT scores, LDH, performance status, and extranodal disease. Furthermore, to identify a specific pre-treatment cfDNA concentration associated with a worse prognosis, an analysis by receiver operating characteristic (ROC) curve was performed by dividing the values according to the death event. In doing so, a cut-off of 42.78 ng/mL was identified (Youden's J statistics) above which PFS and OS are significantly reduced (log-rank test p= 0.0177 and < 0.0001, Figure 1 and 2 respectively). Among the 21 pts with cfDNA>42.78 ng/mL, 8 were PTCL-NOS, 4 nTFHL and 9 ALK- ALCL, with nTFHL being significantly less represented than ALK-ALCL (p=0.043). Multivariate analysis confirmed a significant effect on survival when cfDNA above or below 42.78 ng/mL were tested with IPI, LDH and ECOG (HR 2.76, 95% CI 1.24-6.16, p= 0.0128 for PFS; HR 9.45, 95% CI 3.10-28.81, p=0.0001 for OS). When tested together with PIT, ECOG and extranodal disease, both cfDNA and PIT score were associated with OS (HR 9.30, 95% CI 3.22-26.87, p<0.0001 and HR 0.34, 95% CI 0.12-0.94, p=0.037 respectively), while no significant correlations with PFS were found (cfDNA≥ vs <42.78 ng/mL, HR 1.83, 95% CI 0.86-3.91, p= 0.1178). Moreover, sequencing of cfDNA and diagnostic biopsy-extracted DNA was performed in an exploratory cohort of 25 pts using a target custom panel of 60 genes. We identified lymphoma mutations in 18 plasma samples (number of mutations in pts with at least a mutation: median=2, range 1-5) and in 20 biopsy samples (median=2, range 1-6). Mutations identified in biopsy were confirmed in coupled plasma samples in 74.4% cases. In conclusion, our data demonstrate that pre-treatment cfDNA concentration has a strong prognostic impact in PTCLs and represents a superior predictor of shorter survival, compared to canonical parameters. Despite the fact that sequencing is feasible as demonstrated in our preliminary analysis, and is crucial to identify molecular alterations, its applicability in routine clinical practice is limited. On the other hand, cfDNA concentration is an inexpensive biomarker that can quickly provide prognostic information in the real-world scenario. Its influence on survival in the context of a prospective study where PTCLs were homogeneously treated identifies cfDNA as an easily assessable biomarker that could become part of those to be measured in newly diagnosed PTCLs.

Combinations of plasma cfDNA concentration, integrity and tumor markers are promising biomarkers for early diagnosis of non-small cell lung cancer

BackgroundCirculating cell-free DNA (cfDNA) concentration and integrity as noninvasive biomarkers play an important role in cancer diagnosis, prognosis and therapy monitoring. However, few studies have been conducted on the combination of plasma cfDNA concentration, integrity and tumor markers (CEA, CA125, NSE and CYFRA21-1) for cancer detection. Thus, the purpose of this study was to investigate the diagnostic value of combining plasma cfDNA concentration, integrity and tumor markers in early detection of non-small cell lung cancer (NSCLC). MethodsPlasma cfDNA concentration from 50 healthy controls and 84 NSCLC patients were assessed by quantitative real-time PCR of ALU repeated sequence. Plasma cfDNA integrity was calculated as the ratio of long to short fragments (ALU115/60). ResultsPlasma cfDNA concentration (ALU60 and ALU115) and integrity ALU115/60 were significantly higher in NSCLC patients with stage III/IV than in healthy controls (p = 0.0002, p < 0.0001, and p = 0.0093, respectively). The receiver operating characteristic (ROC) curve for discriminating NSCLC patients from healthy controls had an area under the curve (AUC) of 0.936 (95 % CI, 0.939–0.996). Moreover, the combination of plasma cfDNA concentration, integrity and tumor markers (CEA, CA125, NSE and CYFRA21-1) had higher diagnostic performance than either plasma cfDNA concentration alone, integrity alone or tumor markers alone, with sensitivity, specificity and AUC value of 94.05%, 90.00% and 0.968, respectively. These results demonstrated that the combination of plasma cfDNA concentration, integrity and tumor markers could significantly improve the diagnostic accuracy of NSCLC. ConclusionCombination of plasma cfDNA concentration, integrity and tumor markers is a promising biomarker for early diagnosis of NSCLC.

Relationship between plasma circulating cell-free DNA concentration and treatment outcomes including prognosis in patients with advanced non-small cell lung cancer

BackgroundSome research found that elevated plasma cell-free DNA (cfDNA) concentrations and poor prognosis are associated in non-small cell lung cancer (NSCLC). However, more studies need to be carried out to verify this conclusion. Therefore, this study investigated the relationship between cfDNA concentration and treatment outcomes including prognosis in patients with advanced NSCLC.MethodsWe retrospectively collected medical records and cfDNA data from 160 patients with advanced NSCLC. Progression-free survival (PFS) were calculated using the Kaplan-Meier method and were compared between groups using the log rank test. Cox regression analysis was used for estimating the independent predictors of PFS. And we used logistic regression to evaluate the relationship between baseline biomarkers and efficacy. In our study, BT1 cfDNA, BT2 cfDNA, and BT3 cfDNA were defined as cfDNA concentration before the first treatment (baseline cfDNA concentration), cfDNA concentration before the second treatment, and cfDNA concentration before the third treatment, respectively.ResultsPatients with low cfDNA (BT1 cfDNA < 15 (ng/mL)) were reported a significantly prolonged median progression-free survival (mPFS) compared with patients with patients with high cfDNA (BT1 cfDNA ≥ 15(ng/mL)) (mPFS: 14.6 vs. 8.3 months, P = 0.002), as well as patients with neutrophil/lymphocyte ratio (NLR)<2.98 (mPFS: 13.1 vs. 7.9 months, P = 0.023). In addition, Cox proportional hazards regression analysis identified independent indicators associated with PFS including BT1 cfDNA ≥ 15 (ng/mL), NLR ≥ 2.98 and extrapulmonary metastasis. The best cut-off value for BT3 cfDNA for predicting disease progression is 41.46 (ng/mL) (Area Under the Curve (AUC): 0.652, 95%CI: 0.516–0.788), achieving 90.7% sensitivity and 37.5% specificity for the prediction of disease progression. BT3 cfDNA (OR = 6.08, 95% CI: 1.94–19.57, P = 0.002) was an independent factor for disease progression in patients with advanced NSCLC.ConclusionsBT1 cfDNA may be a biomarker to assess the prognosis of advanced NSCLC. Patients with advanced NSCLC with lower cfDNA and NLR before treatment had a better prognosis. Increased BT3 cfDNA concentration was an independent factor of disease progression in advanced NSCLC patients. These findings may assist in identifying high-risk patients and guiding treatment strategies.

Maternal, neonatal, pregnancy outcome characteristics of pregnant women with high plasma cell-free DNA concentration in non-invasive prenatal screening: a retrospective analysis.

Cell-free DNA (cfDNA) is a useful biomarker in various clinical contexts. Herein, we aimed to identify maternal characteristics and pregnancy outcomes associated with a failed NIPS test due to high cfDNA concentrations. A retrospective study of cases with high plasma cfDNA concentration in pregnant women in which NIPS test was performed (from 174,318 cases). We reported the detection of 126 cases (118 with complete clinical information) in which the high amount of cfDNA did not allow the performance of NIPS and study the possible causes of this result. 622 (0.35%) of 174,318 pregnant women had failed the NIPS test, including 126 (20.3%) cases with high plasma cfDNA concentrations. The failed NIPS due to high plasma cfDNA concentrations was associated with maternal diseases and treatment with low-molecular-weight heparin (LMWH). Further follow-up of the 118 pregnant women in the case group revealed that the pregnancy outcomes included 31 premature deliveries, 21 abortions. The cfDNA concentrations of pregnant women with preterm deliveries were 1.15 (0.89, 1.84), which differed significantly from those who had full-term deliveries. Among pregnant women with high cfDNA concentrations, systemic autoimmune diseases, pregnancy complications and LMWH were associated with increased incidence of failed NIPS test. High maternal cfDNA concentrations may not be associated with chromosomal abnormalities in the fetus. However, they should be alerted to the possibility of preterm births and stillbirths. Further clinical studies on pregnant women with high cfDNA concentrations are required.

Liquid biopsy: circulating tumor DNA monitors neoadjuvant chemotherapy response and prognosis in stage II/III gastric cancer.

A good response to neoadjuvant chemotherapy (NACT) is strongly associated with a higher curative resection rate and favorable outcomes for patients with gastric cancer (GC). We examined the utility of serial circulating tumor DNA (ctDNA) testing for monitoring NACT response and prognosis in stage II-III GC. Seventy-nine patients were enrolled to receive two cycles of NACT following gastrectomy with D2-lymphadenectomy. Plasma at baseline, post-NACT, and after surgery, and tissue at pretreatment and surgery were collected. We used a 425-gene panel to detect genomic alterations (GAs). Results show that the mean cell-free DNA concentration of patients with clinical stage III was significantly higher than patients with stage II (15.43 ng·mL-1 vs 14.40 ng·mL-1 ). After receiving NACT and surgery, the overall detection rate of ctDNA gradually reduced (59.5%, 50.8%, and 47.4% for baseline, post-NACT, and postsurgery). The maximum variant allele frequency (max-VAF) and the number of GAs decreased from 0.50% to 0.08% and from 2.9 to 1.7 after NACT. For patients with a partial response after NACT, the max-VAF and the number of GAs declined significantly, but they increased for patients with progressive disease. Patients with detectable ctDNA at baseline, after NACT, or after surgery have a worse overall survival (OS) than patients with undetectable ctDNA. The estimated 3-year OS was 73% for the post-NACT ctDNA-negative patients and 34% for ctDNA-positive. Patients with perpetual negative ctDNA before and after NACT have the best prognosis. In conclusion, ctDNA was proposed as a potential biomarker to predict prognosis and monitor the NACT response for stage II-III GC patients.

Exploring the Characteristics of Circulating Tumor DNA in Pt1a Clear Cell Renal Cell Carcinoma: A Pilot Study.

Circulating tumor DNA (ctDNA) is a promising biomarker for clear cell renal cell carcinoma (ccRCC); however, its characteristics in small renal masses of ccRCC remain unclear. In this pilot study, we explored the characteristics of ctDNA in pT1a ccRCC. Plasma samples were collected preoperatively from 53 patients with pT1a ccRCC. The ctDNA of pT1a ccRCC was profiled using next-generation sequencing and compared with that of higher-stage ccRCC. The association of ctDNA in pT1a ccRCC with clinicopathological features was investigated. The positive relationship of mutations between ctDNA and matched tissues was evaluated. In pT1a ccRCC, the ctDNA detection rate, cell-free DNA concentration, and median variant allele frequency were 20.8%, 5.8 ng/mL, and 0.38%, respectively, which were significantly lower than those in metastatic ccRCC. The ctDNA gene proportions in pT1a samples differed from those in metastatic ccRCC samples. The relationships between ctDNA and tumor size, tumor grade, and patient age were not elucidated. The positive concordance between ctDNA and matched tissues was poor for pT1a ccRCC. Strategies are needed to increase sensitivity while eliminating noise caused by clonal hematopoiesis to increase the clinical utility of ctDNA analysis in small renal masses of ccRCC.

Size and Concentration of Cell-Free DNA Measured Directly from Blood Plasma, without Prior DNA Extraction.

Cell-free DNA in human blood plasma (cfDNA) is now widely used and studied as a biomarker for several physiological and pathological situations. In addition to genetic and epigenetic alterations that provide information about the presence and the nature of non-constitutive DNA in the body, cfDNA concentration and size distribution may potentially be independent biomarkers suitable for monitoring at-risk patients and therapy efficacy. Here, we describe a simple, in-line, method, which measures cfDNA concentration and size distribution from only a few microliters of plasma without the need to extract and/or concentrate the DNA prior to the analysis. This method is based on a dual hydrodynamic and electrokinetic actuation, adapted for samples containing salts and proteins such as biological fluids. The method provides analytical performances equivalent to those obtained after purification and concentration of cfDNA, with a precision of ∼1% for size features and of 10-20% for the concentrations of the different size fractions. We show that concentration and size distribution of cfDNA analyzed from plasma can differentiate advanced lung cancer patients from healthy controls. This simple and cost-effective method should facilitate further investigations into the potential clinical usefulness of cfDNA size profiling.

Clinical validity of oncogenic driver genes detected from circulating tumor DNA in the blood of lung cancer patients.

This study aimed to evaluate the concordance of oncogenic driver mutations between tumor tissues and circulating tumor DNA (ctDNA) in patients with lung cancer. In addition, this study attempted to reveal the clinical utility of ctDNA in lung cancer treatment. Recurrent or metastatic non-small cell lung cancer (NSCLC) patients were prospectively enrolled in this study. Tumor tissue and serial blood samples were obtained from newly diagnosed patients (Cohort A) or patients treated with targeted therapy (Cohort B) and targeted gene panel sequencing was conducted to identify tumor mutational profiles. At the time of diagnosis, patients in Cohort A with a high cell-free DNA (cfDNA) concentration had poorer overall survival than those with a low cfDNA concentration. The sensitivity and precision of ctDNA analysis in pre-treatment patients compared with those of tissue sequencing were 58.4% and 61.5%, respectively. Known lung cancer-associated variants of oncogenic driver genes, including EGFR and KRAS, and tumor suppressor genes, including TP53 and APC, were frequently detected in the ctDNA of the patients (76.9%). An association between smoking and TP53 mutation status was observed in both tissues and ctDNA (P=0.005 and 0.037, respectively). In addition, the EGFR T790M resistance mutation was detected solely from the ctDNA of two patients after treatment with an EGFR tyrosine kinase inhibitor. ctDNA may be a reliable prognostic biomarker with an additional role in treating patients with lung cancer. Further analyses are necessary to understand the properties of ctDNA and widen its clinical use.

Short-length DNA fragments in plasma to predict clinical outcome in unresected glioblastoma patients.

2025 Background: Early prediction of therapeutic response is an important issue in glioblastoma patients. Cell-free DNA (cfDNA) concentration in plasma shown potential prognostic value. cfDNA release is influenced by non-tumor parameters. Short-length cfDNA (slDNA) may better reflect tumor evolution. The aim was to investigate plasmatic slDNA as prognostic marker in unresected glioblastoma patients. Methods: An ancillary study of a prospective trial was conducted (GLIOPLAK trial). Patients had newly diagnosed and histomolecular confirmed glioblastoma IDH wild-type (WHO 2021 classification). Patients with resected tumor were excluded. All patients underwent radiotherapy/temozolomide (RT/TMZ) schedule after biopsy. Plasmas were collected at three times during first-line treatment: before (pre-), after (post-) RT and at the time of disease progression/relapse. Cell-free DNA (cfDNA) was extracted from plasma using the QIAamp circulating nucleic acid kit (Qiagen). Short-length DNA (slDNA) fragments (equal or lower than 250 bp length) concentration was calculated based on AUC after automated electrophoresis using 4200 TapeStation System. Primary objective was to investigate the impact on survival of slDNA during RT/TMZ phase. Secondary objectives were to explore the association between tumor volume, corticosteroid exposition and slDNA. Results: Thirty-eight patients were included: median age was 63 [interquartile range 55-66], 84.2% (n=32) had Karnofsky index higher than 80% at baseline and median overall survival (OS) was 13.3 months [CI 95% 11.5; 15.5]. slDNA was detected in 69.4% at the pre-RT time (mean 722.3 pg/ml ±sd 1257.7) and in 83.3% at the post-RT-time (686.1 pg/ml ±1217.1). One third of the patients experienced a slDNA decrease between pre- and post-RT time. Presence of slDNA at the pre-RT time was associated with improved OS: median of 11.7 months [9.8-19.6] in the slDNA(+) group (n=25) versus 8.8months [5.5-NA] in the slDNA(-) group (n=11), HR 0.309 [0.133-0.716], log rank p=0.004. Decrease of slDNA concentration was also associated with better outcome compared to stability or increase of slDNA between pre- and post-RT: respectively median OS 14.1 vs 10.1 months, 0.424 [0.197-0.909], p=0.02. Corticosteroid exposition and dose were not correlated to slDNA concentration at pre-RT or slDNA variation (respectively correlation coefficient 0.081 and 0.047). At pre-RT, presence of slDNA was independent of tumor volume: mean 265.0 cm3 ±84.7 in slDNA(+) group vs 237.7 cm3 ±115.7 in slDNA(-) group, p=0.492. Total circulating cfDNA concentration did not influence OS in our cohort. Conclusions: slDNA during the RT/TMZ phase is an independent prognostic marker in unresected glioblastoma patients. The role of slDNA as a biomarker for adaptative treatment after RT as well as non-invasive molecular characterization tool can be explored in dedicated trial. Clinical trial information: NCT02617745 .

092 Plasma cell-free DNA concentration increases during luteolysis in beef cows

092 Plasma cell-free DNA concentration increases during luteolysis in beef cows

Abstract 1044: Cell-free DNA concentration as a prognostic biomarker in patients with non-small cell lung cancer under immunotherapy treatment

Abstract The irruption of the immunotherapy for the treatment of non-small cell lung cancer (NSCLC) based on Immune Checkpoint Inhibitors (ICI) PD-1 and PD-L1 inhibitors is considered a treatment revolution. However, only a small percentage of patients benefit with ICI treatment over the long term, and precise biomarkers that can recognize these individuals before or early during treatment have so far eluded. PD-L1 expression and tumor mutational burden (TMB) are the most well studied biomarkers for predicting response to PD- (L)1 blockade-based ICI prior to treatment. TMB is still being clinically assessed whereas PD-L1 has several drawbacks for prediction of persistent benefit. Our aim is to evaluate the basal state and dynamic changes of cell-free DNA (cfDNA) concentration to predict and monitor response in NSCLC patients starting ICI. A total of 248 cfDNA concentration measurements were performed from 87 NSCLC patients. The quantification was done before the start of the treatment, at the second ICI cycle, after 6 and 12 months in treatment, and at progression if it was within the first 12 months. Quality and quantity of the cfDNA was assessed using Qubit High Sensitivity and Bioanalyzer 2100. We first explored the association with response of the basal cell-free DNA concentration using Mann-Whitney-Wilcoxon test. The longitudinal analysis between different time points was tested with Wilcoxon signed-rank tests. Response was ascertained using RECIST parameters at 3, 6, and 12 months. We evaluated the utility of the cfDNA concentration as a prognostic factor using Mantel-Cox test. The response association results indicate that early and long-term response is associated with lower levels of basal cfDNA (p&amp;lt;0.002, 3 months; p&amp;lt;0.001, 6 months; p&amp;lt;0.05, 12 months). The longitudinal analysis could also detect a decrease in cfDNA from the second cycle to the month 6 of treatment in responders (p&amp;lt;0.05), and an increase in non-responders (p&amp;lt;0.05). To identify a prognostic concentration threshold, statistical stratification of cfDNA levels based on the selected maximum rank method of cfDNA levels was used to group them into low and high concentration. Patients with high basal cfDNA concentrations showed both worse FPS (log-rank 3.4x10-03, Medians: Low 12.57 [8.36 - 18.46] High 5.50 [3.03 - 10.06]) and OS (log-rank 3.2x10-05, Medians: Low 21.26 [15.33 - 33.96] High 5.45 [3.10 - 15.66]). With these results, we provide proof-of-principle that specifically detecting tumor cfDNA by PCR or NGS methods would not be necessary to predict the long-term clinical benefit to ICI in NSCLC patients. Moreover, concentration cut-offs can be defined to categorize the patients in potential responders and non-responders before the start of the treatment. This would constitute a cost-effective and easily implementable prognostic tool that could be particularly useful for avoiding loss of treatment windows of opportunities. Citation Format: Isabel Barragan, Elisabeth Perez-Ruiz, Juan Luis Onieva, Maria Garrido-Ramos, Beatriz Martinez-Galvez, Jaime Dubbelman, Emilio Alba, Juan Zafra, Manuel Cobo, Javier Oliver, Antonio Rueda-Dominguez. Cell-free DNA concentration as a prognostic biomarker in patients with non-small cell lung cancer under immunotherapy treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1044.

Analysis of cell-free DNA concentration, fragmentation patterns and TP53 gene expression in mammary tumor-bearing dogs: A pilot study.

Liquid biopsy based on the analysis of circulating cell-free DNA (cfDNA), as well as on detection of point mutations by digital droplet PCR (ddPCR), has revolutionized the research in oncology. In recent years, this technique has been pioneering in veterinary medicine since it is a minimally invasive approach with very promising results for characterization of tumors. The aim of this study was, firstly, to analyze the concentration and the fragmentation pattern of cfDNA of dogs with mammary tumors (n = 36) and healthy dogs (n = 5) and its correlation with clinicopathological data. Secondly, analysis of TP53 gene expression and the point mutation in the codon 245 were performed in cfDNA and in tumor tissues to assess their potential as plasma biomarkers. Our results highlighted that those dogs with worse clinicopathological characteristics (simple or undifferentiated carcinomas, higher histological grade and presence of peritumoral inflammation) shown higher cfDNA concentration and higher concentrations of short-fragments (<190 bp) than healthy dogs. In addition, although no detection of the point mutation in codon 245 of TP53 gene could be detected neither in plasma nor tumor tissue, an increased TP53 expression was detected in animals with tumors bearing malignant characteristics. Finally, a high concordance with TP53 gene expression in plasma and tumor tissue and cfDNA concentration was also found. The results derived from this work confirm the valuable potential of cfDNA and its fragments, as well as the analysis of TP53 expression in plasma as useful liquid biomarkers for clinical application in veterinary oncology.

Perioperative ctDNA-Based MRD Detection in NSCLC-Letter.

We read with great interest the article by Xia and colleagues reporting circulating tumor DNA (ctDNA) as biomarker for early detection of molecular residual disease (MRD) and predictor for relapse after surgery in patients with non–small cell lung cancer in a prospective multicenter cohort named LUNGCA-1 (1).LUNGCA-1 was designed to predict prognosis by perioperative ctDNA detection, including preoperative, 3 days postoperative and 1 month postoperative ctDNA. The number of patients in prognosis analysis is 330 (Fig. 1), whereas Supplementary Table S5 shows only 329 patients with at least one postoperative ctDNA sample. In LUNGCA-1, some patients tested postoperative ctDNA before or without adjuvant therapies, while others tested postoperative ctDNA after adjuvant therapies. We questioned the trial design because the authors mixed different aims: MRD detection at a specific postoperative timepoint for recurrence prediction; MRD detection at both preadjuvant/postadjuvant therapies timepoints for evaluation of the outcome of adjuvant therapies. The authors could separate different aims into different trials or do statistics separately in preadjuvant/postadjuvant groups.The authors defined “MRD positive” for patients with detectable ctDNA at 3 days and/or 1 month postoperatively. We noticed that 5 of 26 patients with MRD positive (19.2%) had ctDNA positive only in the detection at postoperative 3 days and their ctDNA detection was negative before and 1 month after surgery. Therefore, we consider the issue of timepoint setting for postoperative blood collection. Diehl and colleagues reported that ctDNA increased after surgery, most likely due to release of ctDNA from damaged tissue (2). Thus, it may be more reasonable to collect blood at a later timepoint after surgery from 10 days to 16 weeks (3). As reported by Chaudhuri and colleagues (4) and Moding and colleagues (5), blood sample should be collected within 4 months for ctDNA detection after surgery or completing radiation and chemotherapy. In this article, the cell-free DNA concentration at 3 days after surgery is also significantly higher than before and 1 month after surgery (Supplementary Table S1), suggesting a more reasonable manner to collect blood for a longer period after surgery. Also, whether the 5 patients with ctDNA positive for only 3 days after surgery relapsed or not was not mentioned.Chaudhuri and colleagues (4) reported that concentration of ctDNA should be correlated with stage and tumor volume, which the authors did not mention. Furthermore, how to determine ctDNA status is positive or negative needs to be clarified. In addition, the criteria for judging the patient's disease relapse were not mentioned in the text or Supplementary Materials and Methods.See the Response, p. 1156No disclosures were disclosed.We thank the grant from the Fundamental Research Funds for the Central Universities in China (4206-413100049).

An all-in-one strategy for bisulfite-free DNA methylation detection by temperature-programmed enzymatic reactions

The fragmentation and low concentration of cell-free DNA (cfDNA) pose higher challenges for the cfDNA methylation detection technologies. Conventional bisulfite conversion-based methods are inadequate for cfDNA methylation analysis due to cumbersome operation and exacerbating cfDNA degradation. Herein, we proposed temperature-programmed enzymatic reactions for cfDNA methylation analysis in a single tube. Endonuclease was used to mildly recognize DNA methylation to avoid the degradation of cfDNA. And two stages of amplification reactions significantly improved the detection sensitivity for GC-rich sequence. With vimentin as the target, the detection sensitivity was 10 copies of methylated DNA. Meanwhile, the proposed method can accurately quantify the methylation level of target sequence from 1000-fold of unmethylated DNA background. Further, the methylated vimentin gene in 20 clinical plasma samples was successfully detected. The results shown significant differences in methylation levels of the vimentin gene between healthy volunteers and colorectal cancer patients. These results lead us to believe that the proposed method has great application potential for DNA methylation analysis as a complement to bisulfite conversion-based methods.

Preoperative cell-free DNA concentration in plasma as a diagnostic and prognostic biomarker of clear cell renal cell carcinoma.

Assessment of renal tumour masses is based on conventional imaging studies (computer tomography or magnetic resonance), which does not allow characterisation of the histopathological type. Moreover, the prediction of prognosis in localised and metastatic renal cell carcinoma requires improvement as well. Analysis of circulating free DNA (cfDNA) in blood is one of the variants of liquid biopsy that may improve diagnostics and prognosis issues of patients with renal tumour masses suspected to be renal cell carcinoma. The aim of the study was to assess the diagnostic and prognostic role of preoperative cfDNA concentration in the plasma samples of clear cell renal cell carcinoma (ccRCC) patients. The preoperative plasma cfDNA concentration was assessed in ccRCC patients (n = 46) and healthy individuals (control group) (n = 17). The circulating free DNA concentration was reflected by the 90 bp DNA fragments determined by real-time polymerase chain reaction. The median cfDNA concentration was significantly higher in ccRCC patients (n = 46) compared to the control g roup (n = 17) (2588 ±2554 copies/ml vs. 960 ±490 copies/ml, p < 0.01). In multivariate analysis, the preoperative plasma cfDNA concentration was the significant factor increasing the probability of ccRCC detection (OR: 1.003; 95% CI: 1.001-1.005). The median cfDNA concentration depended on the stage of ccRCC; it was higher in metastatic ccRCC patients (n = 11) compared to non-metastatic ccRCC patients (n = 35) (3619 ±4059 copies/ml vs. 2473 ±1378 copies/ml, p < 0.03). Kaplan-Meier survival analysis demon-strated that patients with high cfDNA values (above 2913 copies/ml) had significantly worse cancer-specific survival (HR: 4.5; 95% CI: 1.3-16.9, log-rank Mantel-Cox test p = 0.015). Preoperative plasma cfDNA concentration has diagnostic and prognostic potential in ccRCC pa-tients.

Potential clinical utility of liquid biopsy in early-stage non-small cell lung cancer

BackgroundLiquid biopsy has been widely researched for early diagnosis, prognostication and disease monitoring in lung cancer, but there is a need to investigate its clinical utility for early-stage non-small cell lung cancer (NSCLC).MethodsWe performed a meta-analysis and systematic review to evaluate diagnostic and prognostic values of liquid biopsy for early-stage NSCLC, regarding the common biomarkers, circulating tumor cells, circulating tumor DNA (ctDNA), methylation signatures, and microRNAs. Cochrane Library, PubMed, EMBASE databases, ClinicalTrials.gov, and reference lists were searched for eligible studies since inception to 17 May 2022. Sensitivity, specificity and area under the curve (AUC) were assessed for diagnostic values. Hazard ratio (HR) with a 95% confidence interval (CI) was extracted from the recurrence-free survival (RFS) and overall survival (OS) plots for prognostic analysis. Also, potential predictive values and treatment response evaluation were further investigated.ResultsIn this meta-analysis, there were 34 studies eligible for diagnostic assessment and 21 for prognostic analysis. The estimated diagnostic values of biomarkers for early-stage NSCLC with AUCs ranged from 0.84 to 0.87. The factors TNM stage I, T1 stage, N0 stage, adenocarcinoma, young age, and nonsmoking contributed to a lower tumor burden, with a median cell-free DNA concentration of 8.64 ng/ml. For prognostic analysis, the presence of molecular residual disease (MRD) detection was a strong predictor of disease relapse (RFS, HR, 4.95; 95% CI, 3.06–8.02; p < 0.001) and inferior OS (HR, 3.93; 95% CI, 1.97–7.83; p < 0.001), with average lead time of 179 ± 74 days between molecular recurrence and radiographic progression. Predictive values analysis showed adjuvant therapy significantly benefited the RFS of MRD + patients (HR, 0.27; p < 0.001), while an opposite tendency was detected for MRD − patients (HR, 1.51; p = 0.19). For treatment response evaluation, a strong correlation between pathological response and ctDNA clearance was detected, and both were associated with longer survival after neoadjuvant therapy.ConclusionsIn conclusion, our study indicated liquid biopsy could reliably facilitate more precision and effective management of early-stage NSCLC. Improvement of liquid biopsy techniques and detection approaches and platforms is still needed, and higher-quality trials are required to provide more rigorous evidence prior to their routine clinical application.

Finding a Needle in Haystack: Digital-PCR As a Sensitive Tool for MRD Detection in the Circulating-Tumor DNA Samples in Hodgkin Lymphoma

Introduction: Hodgkin lymphoma (HL) is the most frequent malignancy in young adults. The high probability of the cure is redeemed by both acute and long-term treatment sequelae. Balancing toxicity and efficacy requires highly reliable decision-making tools. Current treatment tailoring is based on the positron-emission tomography (PET/CT) assessment. The way how to improve the sensitivity of imaging methods could be a detection of minimal residual disease (MRD) in the circulating-tumor DNA (ctDNA) samples. The standard of the detection method is a next-generation sequencing (NGS), which covers a large spectrum of mutations, but is time-consuming, costly, and requires a significant concentration of ctDNA. The new digital PCR (dPCR) is a faster, cheaper method, which needs a lower amount of ctDNA. Aim: To validate the reliability of the dPCR for target-mutations of STAT6 and XPO1 genes detection in the peripheral blood ctDNA of patients with Hodgkin lymphoma Methods: CtDNA samples were obtained at the time of diagnosis/relapse, at the time of PET/CT assessment (interim, final), and during follow-up - every 3-6 months. CfDNA was extracted from peripheral blood plasma using the QiaAmp Circulating nucleic acid kit (Qiagen). Specific NGS panel covering coding sequences (including UTRs) of 13 selected genes (B2M, CD36, CIITA, GNA13, HIST1H1E, ITPKB, NFKBIE, PTPN1, SOCS1, SPEN, STAT6, TNFAIP3, and XPO1) was designed. For library preparation, we used SureSelect XT HS2 technology (Agilent Technologies) based on "target enrichment" with molecular barcodes. Sequencing was performed on a NovaSeq6000 (Illumina). Data were analyzed with the SureCall software (Agilent Technologies) with the sensitivity of 1.0% VAF. The detected variants were annotated using COSMIC, dbSNP, Ensembl, and ClinVar. Selected variants STAT6 p.N417Y and XPO1 p.E571K were further monitored by dPCR (QIAcuity Digital PCR System; Qiagen) with the sensitivity of 0.1% VAF. For dPCR experiments, we used a QIAcuityTM probe PCR kit (Qiagen) and nanoplate QIAcuity Nanoplate 26K 24-well (Qiagen). Primers and probes were taken and modified according to Camus et al., 2016 (EXPO1 p.E571K) and Spina et al., 2018 (STAT6 p.N417Y). All pts were analyzed with the same Siemens Biograph mCT scanner and results were independently reviewed by two physicians blinded for clinical outcome. Results: We have analyzed ctDNA using an NGS panel in 48 pts with CHL (44 at diagnosis, 4 in relapse). Mutations were detected in 22/48 (46%) pts. The most frequently mutated genes were STAT6 (12/48 pts), TNFAIP3 (10/48 pts), XPO1 (7/48 pts), and SOCS1 (7/26 pts). Frameshift deletions prevailed in TNFAIP3 and SOCS1 genes. Hotspot mutations in the STAT6 (p.N417Y, in five pts) and XPO1 (p.E571K, in six pts) genes we used as target mutations for dPCR cohort. The first date of ctDNA analysis was the first CHL diagnosis in ten and 1st relapse in one case. This dPCR-cohort (n=11) consists of 9 females and 2 males with a median age at the time of diagnosis of 40 yrs, with disease subtypes of nodular sclerosis (8), mixed-cellularity (1), lymphocyte-rich (1), and CHL unclassified (1). Mean VAF at diagnosis was 9.14% (0.76-25.0), mean cell-free DNA concentration 1.47 ng/mL (0.76-2.88). Three of the pts were considered to have intermediate and eight advanced cHL stages according to the GHSG system. Pts have received ABVD (n=5) or BEACOPPesc (n=5) or platinum-based+BEAM200 (n=1) protocols. Interim PET-2 was positive in 1 pt (stable disease), and final PET was positive in 2 (both PD). dPCR captured mutation in all three PETpos (0.12, 0.86, and 2.22%) cases, whereas was negative (<0.1% VAF) in all PETneg cases. During follow-up, two patients relapsed (both with detectable mutation, illustrative case is a Figure 1), in one case (pt during the BV post-ASCT maintenance) we observed transient dPCR positivity without evidence of relapse. Conclusion: A combination of the NGS (large-scale mutation screening) at the diagnosis with rapid and sensitive mutation monitoring using dPCR at the time of the treatment-tailoring or follow-up could improve decision-making accuracy in the routine care of Hodgkin lymphoma patients. Further analyses are needed to directly compare the sensitivity of the NGS and dPCR methods. Acknowledgement: Supported by MZCR-RVO (FNOL, 00098892) and AZV NU22-03-0018 grants. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Uncovering Novel Prognostic Factors of Sudden Sensorineural Hearing Loss by Whole-Genome Sequencing of Cell-Free DNA.

Sudden sensorineural hearing loss is a common disease with several etiologic hypotheses, such as infection, vascular occlusion, inflammation, oxidative stress, etc. Studies have reported that the concentration of cell-free DNA in plasma will elevate in these situations. Former studies have reported that the whole-genome sequencing of cell-free DNA has high accuracy and sensitivity in inferring gene expressions. In this study, we plan to use the whole-genome sequencing of cell-free DNA to uncover novel prognostic factors of sudden sensorineural hearing loss and provide new insight into the clinical application of cell-free DNA. In this study, 84 sudden sensorineural hearing loss patients (47 in recovery group and 37 in no-recovery group) were enrolled. After whole-genome sequencing of the cell-free DNA, the protein-protein interaction network was constructed using the differentially expressed genes. Multinomial logistics regression analysis was used to analyze the prognostic factors of hearing improvement. In this study, we found distinct patterns of expressed and unexpressed genes in cell-free DNA sequence read depth coverage in sudden sensorineural hearing loss patients. The top centrality hub genes IGF1, NOTCH1, APOE, FAM3C, RPS6KB1, and RELB were identified from the protein-protein interaction network. Multinomial logistics regression analysis demonstrated that the coverage patterns of 3 key differentially expressed genes (NOTCH1, APOE, and RELB) are significantly different in sudden sensorineural hearing loss with and without hearing recovery. The cell-free DNA could have more applications in diverse diseases, and the coverage patterns of 3 differentially expressed genes (NOTCH1, APOE, and RELB) are independent prognostic factors of sudden sensorineural hearing loss. Their expression levels may play a critical role in the hearing improvement of sudden sensorineural hearing loss patients.

250 (PB-074) Poster - Cell-free DNA concentration in patients with different molecular subtypes of breast cancer

Background: Breast cancer is the most common form of cancer diagnosed in women worldwide, and is one of the leading causes of death among women in Greece. Elevated levels of ccfDNA in the blood of breast cancer patients has been demonstrated in a few studies in the past. As the scope of breast cancer intratumour genetic heterogeneity unravels, the development of robust and standardized methods for the assessment of circulating biomarkers like ccfDNA will be essential for the realization of the potentials of personalized medicine. The aim of this study is to correlate the patient’s ccfDNA levels with the different molecular subtypes of the disease.

A standardised methodology for the extraction and quantification of cell-free DNA in cerebrospinal fluid and application to evaluation of Alzheimer’s disease and brain cancers

Cerebrospinal fluid (CSF) is a source of diagnostic biomarkers for a range of neurological conditions. Cell-free DNA (cfDNA) is detected in CSF and differences in the concentration of cell-free mitochondrial DNA have been reported in studies of neurodegenerative disorders including Alzheimer’s disease (AD). However, the influence of pre-analytical steps has not been investigated for cfDNA in CSF and there is no standardised approach for quantification of total cfDNA (copies of nuclear genome or mitochondria-derived gene targets). In this study, the suitability of four extraction methods was evaluated: QIAamp Circulating Nucleic Acid (Qiagen), Quick-cfDNA Serum & Plasma (Zymo), NucleoSnap® DNA Plasma (Macherey-Nagel) and Plasma/Serum Circulating DNA Purification Mini (Norgen) kits, for cfDNA extraction from CSF of controls and AD dementia patients, utilising a spike-in control for extraction efficiency and fragment size. One of the optimal extraction methods was applied to a comparison of cfDNA concentrations in CSF from control subjects, AD dementia and primary and secondary brain tumour patients. Extraction efficiency based on spike-in recovery was similar in all three groups whilst both endogenous mitochondrial and nucleus-derived cfDNA was significantly higher in CSF from cancer patients compared to control and AD groups, which typically contained < 100 genome copies/mL. This study shows that it is feasible to measure low concentration nuclear and mitochondrial gene targets in CSF and that normalisation of extraction yield can help control pre-analytical variability influencing biomarker measurements.