Presence Of ctDNA Research Articles

Multiomic, plasma-only ctDNA NGS assay for minimal residual disease (MRD) detection in solid tumors.

3045 Background: Detection of minimal residual disease (MRD) by circulating tumor DNA (ctDNA) post-curative intent treatment is predictive of recurrence in many solid tumors. Due to biological challenges with low ctDNA shed in early-stage disease and potential to detect non-tumor derived alterations in plasma (e.g. CHIP), most ctDNA MRD assays are dependent on a priori knowledge of genomic alterations from tumor tissue to achieve high sensitivity and specificity. Tissue-dependent methods limit the clinical application of a MRD assay, especially in cancer types where tissue biopsy is challenging, neoadjuvant therapy (NAT) is standard of care, and/or rapid turnaround times are needed for clinical decision making. We previously validated an assay (Guardant Reveal) that combines somatic and epigenomic analysis to detect ctDNA from early-stage colorectal tumors without tumor tissue or peripheral blood cells. Here we describe the expansion of this assay to detect MRD across multiple tumor types. Methods: Cell-free DNA (cfDNA) fragments are extracted from patient plasma, partitioned based on methylation fraction, enriched using a panel to target informative genomic and epigenomic regions, barcoded, and pooled for sequencing. Methylation status is determined non-destructively and with minimal loss of molecules, allowing sensitive genomic and epigenomic analysis of the same cfDNA fragments. A single assay with a total panel size of 5.3 Mb was developed for MRD analysis in multiple cancer types, including CRC, Lung and Bladder. A “ctDNA detected” result is defined by the de novo identification of tumor-derived somatic variants and/or the observation of a tumor-specific methylation profile exceeding predefined thresholds. Results: The assay performance was tested using 163 pre-treatment clinical samples from patients with early-stage non small cell lung cancer (NSCLC) and bladder cancer and 133 self-declared healthy donors. Sensitivity for pre-treatment detection in NSCLC was 68.9% at 95% specificity (20/42 Stage I: 47.6%, 25/30 Stage II: 83.3%, 19/21 Stage III: 90.5%). Sensitivity for bladder cancer was 44.2% at 95% specificity (13/43 NMIBC: 30.2%, 18/27 MIBC: 66.7%). Additional development from larger cohorts and other tumor types is ongoing and data will be presented as available. Conclusions: Using cancer-specific genomic and epigenomic signals combined with learning-based classifiers, we developed a highly specific method for detecting the presence of ctDNA in early-stage NSCLC and bladder cancer patients from plasma without the need for tumor tissue but with comparable sensitivities to tissue-dependent approaches. A plasma-only MRD assay will allow for greater clinical impact by overcoming the challenges of tissue procurement, particularly following NAT, and enabling faster time to results.

Synthesis, Crystal Structure, Spectroscopic and Antimicrobial Properties of Ruthenium Complexes of Vinyl Imidazole and 4-Ethylaminomethyl Pyridine Ligands

Ruthenium complexes of vinylimidazole (VIMD) and 4-ethylaminomethyl pyridine (EMP) ligands were synthesized and characterized by XRD and spectroscopic methods. The binding of these Ru(IIII) complexes with calf thymus-DNA has been studied by UV-visible spectroscopic and electrochemical studies. A prominent increase in the intensity of fluorescence spectra of these complexes with CT-DNA was observed and a distinct UV-visible spectral shift in presence of CT-DNA is probably due to an interaction of these complexes with CT-DNA. The evidence of DNA binding has been found from the change in the intensity of fluorescence and UV visible spectra. Also, the electron transfer ability of these complexes is very important in order to rationalize their activity towards the biological system therefore, we have studied the electrochemical studies of these complexes by using cyclic voltammetry. From this study, it is possible to draw some ideas on electrochemical potentials of the complexes relevant to biological reduction possibly Ru(III)/Ru(II). Both complexes were also tested for antimicrobial activity against bacterial strain and responses well as antimicrobial agent.

True conversions from RAS mutant to RAS wild-type in circulating tumor DNA from metastatic colorectal cancer patients as assessed by methylation and mutational signature.

The paucity of targeted treatments available in patients with RAS mutant colorectal cancers contributes to the poor prognosis of this patient group compared to those with RAS wild-type disease. Recent liquid biopsy-driven studies have demonstrated that RAS mutant clones might disappear in plasma during the clonal evolution of the disease, opening new unforeseen perspectives for EGFR blockade in these patients. Nevertheless, the lack of detection of RAS mutations in plasma might depend on the low amount of released circulating tumor DNA (ctDNA), making it necessary a more accurate selection of patients with true RAS mutation conversions. In this liquid biopsy-based study, we assessed RAS mutational status in initially RAS-mutant patients at the time of progressive disease from any line of therapy and investigated the incidence of true conversions to plasma RAS wild-type, comparing a colon cancer specific methylation profile with a mutational signature of ctDNA. Globally, considering either mutational panel or methylation profile as reliable tests to confirm or exclude the presence of ctDNA, the percentage of "true RAS converters" was 37.5%. In our series we observed a trend toward a better PFS in patients who received anti-EGFR as second or subsequent treatment lines compared to those who did not.

Role of ctDNA to predict risk of recurrence following potentially curative resection of biliary tract and pancreatic malignancies.

336 Background: The potential role of ctDNA to identify residual disease after potentially curative resection has been suggested in some malignancies; its role in resected pancreatico(P)-biliary(B) malignancies is unknown. Methods: Patients diagnosed with PB malignancies underwent molecular profiling (ctDNA) using FoundationMedicine Liquid (72 cancer-related genes) following potentially curative resection. Baseline patient characteristics and molecular profiling outcomes, including mutant allele frequency (MAF) for pathological alterations were extracted. Primary objective: prevalence of ctDNA identification and its correlation with recurrence (relapse-free survival (RFS) and relapse rate). Results: Total of 11 individuals had ctDNA analysed following potentially curative resection for PB malignancies: 8 B (4 extra-hepatic cholangiocarcinoma (eCCA), 2 ampulla, 1 intrahepatic cholangiocarcinoma (iCCA), 1 gallbladder cancer (GBC)) and 3 P. Baseline characteristics: 6 female (54.55%), median age 71.59 years (range 39.98-81.19). Most were pT2 (45.45%), pN0 (54.55%) and R0 (63.64%). Following surgery, 6 patients were started on adjuvant chemotherapy; at the end of follow-up (data cut-off 25/6/2020; median follow-up 11.15 months (range 5.45-13.52); 5 relapsed (45.45%) and 2 died (18.18%). Estimated median RFS was 11.43 months (95% CI 2.28-not reached); median overall survival was not reached. No sample failed ctDNA analysis; presence of ctDNA was identified in 3/11 (27.27%) of the samples; 2 and 1 samples had 2 and 1 pathological alterations identified, respectively: ALK fusion (1 sample; GBC), TP53 mutation (2 samples; eCCA and GBC), CHEK2 mutation (1 sample; pancreas), IDH2 mutation (1 sample; eCCA). Mean maximum MAF was 1.47 (2 in biliary; 0.43 in pancreas). Variants of unknown significance were identified in 72.73% of the samples (87.5% in B; 33.33% in P; p-value 0.152). None of the baseline characteristics explored correlated with presence of ctDNA. There was a trend towards increased relapse risk in the patients with ctDNA present following potentially curative surgery; Cox regression for RFS [HR 2.64 (95% CI 0.36-19.31); median RFS 11.44 months (95% CI 2.28-not reached) vs 10.87 (95% CI 2.21-not reached)]; relapse rate 37.5% (ctDNA absent) vs 66.67% (ctDNA present); statistical significance was not reached (p-value 0.340 and p-value 0.545, respectively). Conclusions: This pilot study demonstrates the feasibility of testing for ctDNA following potentially curative resection in PB malignancies. Presence of ctDNA may be associated with increased relapse risk; further studies are required to increase sample size and assess clinical implications.

Molecular profiling of advanced pancreatic ductal adenocarcinoma (PDAC): Role of ctDNA.

425 Background: Molecular profiling of tumour samples and circulating tumour DNA (ctDNA) may inform treatment of advanced cancer; the role of ctDNA to predict progression-free-survival (PFS) and overall survival (OS) in advanced PDAC is not fully understood. Methods: Eligible patients: those diagnosed with advanced PDAC undergoing molecular profiling [tumour (Foundation Medicine CDx/Caris) or ctDNA (FoundationMedicine Liquid (72 cancer-related genes))]. Baseline patient characteristics and molecular profiling outcomes, including mutant allele frequency (MAF) for pathological alterations were extracted. The primary aim was to assess the impact of presence of ctDNA at time of systemic chemotherapy initiation on PFS and OS. Results: Total of 26 samples (ctDNA 18 samples and 8 tumour samples) from 25 patients diagnosed with advanced PDAC underwent molecular profiling. When the whole population was analysed, the rate of sample analysis failure seemed to be higher when tumour tissue was tested (37.5%) compared to ctDNA (5.56%); p-value 0.072. The overall rate of identification of pathological findings was 72.73%, with 18.18% of patients having targetable findings [EGFRmut (1 patient), KRAS G12C mut (1 patient), FGFR2 fusion (1 patient), RNF43 mut (1 patient)]; these findings impacted treatment management in one patient only (RNF43 mutation; Wnt inhibitor). Variants of unknown significance were identified in 63.64% of samples. Patients with ctDNA analysis at time of palliative chemotherapy initiation (16 samples; 15 patients) were analysed [6 female (40.00%), median age 69.57 years (range 51.61-81.49), metastatic disease (66.67%), 80% first-line (80%), 20% second-line]. Pathological mutations were identified in 9/15 (60.00%) of these patients (KRAS mutation identified in 6/9). After median follow-up of 8.33 months from sample acquisition, 80% and 53.33% of patients had progressed and died, respectively. Median estimated PFS and OS were 5.65 months (95% CI 1.59-8.17) and 7.80 months (95% CI 4.13-not reached). Presence (vs absence) of pathological alterations in ctDNA showed a trend towards shorter PFS (2.91 vs 6.51 months; HR 1.38 (95% CI 0.40-4.77)) and OS (6.12 vs 9.72 months; HR 2.03 (95% CI 0.60-6.82)). Conclusions: This pilot study demonstrates the feasibility of ctDNA analysis in patients with advanced PDAC prior to initiation of palliative therapy. The presence of pathological alterations in ctDNA may prognosticate for worse PFS and OS. Larger studies are required to confirm these findings.

Circulating tumour DNA as a biomarker in resectable and irresectable stage IV colorectal cancer; a systematic review and meta-analysis.

For patients with metastatic colorectal cancer, stratification for treatment (surgery or chemotherapy) is often based on crude clinicopathological characteristics like tumour size and number of lesions. Circulating tumour DNA (ctDNA) acts as a potential biomarker of disease trajectory and biology, allowing better stratification. This study aims to systematically review ctDNA in stage IV colorectal cancer to assess its potential role as a prospective biomarker to guide management decisions. A literature search was performed to identify studies where the measurement of ctDNA in stage IV colorectal cancer was correlated with a clinical outcome (radiological response, secondary resection rate, PFS, DFS or OS). Twenty-eight studies were included, reporting on 2823 patients. Circulating tumour DNA was detectable in between 80% and 90% of patients prior to treatment. Meta-analysis identified a strong correlation between detectable ctDNA after treatment (surgery or chemotherapy) and overall survival (HR 2.2, 95% CI 1.79-2.69, p<0.00001), as well as progression-free survival (HR 3.15, 95% CI 2.10-4.73, p<0.00001). ctDNA consistently offered an early marker of long-term prognosis in irresectable disease, with changes after one cycle of systemic therapy demonstrating prognostic value. In resectable disease treated with curative intent, detection of ctDNA offered a lead time over radiological recurrence of 10 months. Circulating tumour DNA is detectable in the majority of resectable and irresectable patients. The presence of ctDNA is clearly associated with shorter overall survival, with changes in ctDNA an early biomarker of adverse disease behaviour. Prospective trials are essential to test its clinical efficacy.

Peroxidase activity of hemoglobin and heme destruction in the presence of hydrogen peroxide and CT-DNA

The aim of this study was to investigate the peroxidase activity of Hb with different concentrations of hydrogen peroxide and compare it with hypochlorous acid effect on Hb. Hypochlorous acid at higher concentrations decomposed Hb and heme, releasing fee iron ion from the metal center. High concentrations of hydrogen peroxide switched the peroxidase activity of Hb towards the partial Hb and heme destruction. Heme alone was degraded showing that the Hb conformation and protein environment protects Hb from the distraction in the presence of highly increased hydrogen peroxide concentration that occurs as a result of oxidative stress. In the presence of CT-DNA acted inhibition of the peroxidase activity of Hb was observed signaling inhibited hydrogen peroxide consumption.

Association of clinical, instrumental and molecular genetic predictors with the risk of development and tumor progression of melanocytic intraocular neoplasms

Purpose : to analyze the frequency and possible associations of GNAQ and GNA11 oncogenes in peripheral blood ctDNA, as well as the relationship of the ABCB1/MDR1 rs1045642 polymorphic marker gene with the clinical and instrumental characteristics of nevi and initial choroidal melanoma. Material and methods . A prospective study of 81 previously untreated patients (84 eyes, mean age 57.8 ± 13.8 years) included general ophthalmological examinations and special instrumental diagnostics: ultrasound checkups, spectral optical coherent tomography (SOCT), OCT angiography. Depending on the nature of the pathology, the patients were divided into three groups: I — with benign choroidal nevus (23 eyes, 28 %; mean age 61.1 ± 13.6 years); II — with suspicious choroidal nevus (24 eyes, 29 %; mean age 54.8 ± 13.0 years); III — small choroidal melanoma (37 eyes, 43 %; mean age 56.2 ± 14.8 years). Genotyping was performed by melting curve analysis. Results . A significant association was revealed between the presence of ctDNA (GNAQ/GNA11 oncogenes) and the CC genotype of the ABCB1 gene with the risk of developing a small melanoma of the choroid and choroidal nevi. In patients with suspicious choroidal nevus, there is an unfavorable significance of mutations in the GNAQ/GNA11 genes (ctDNA). A relative unfavorable predictive significance of the presence of mutations in the GNAQ/GNA11 genes in peripheral blood ctDNA of patients with a benign choroidal nevus was suggested. No significant associations between GNAQ/GNA11 mutations, ABCB1 gene polymorphism and clinical/instrumental tumor features were found. Conclusion . The revealed features can be used for screening the patients with melanocytic intraocular tumors and for developing modern approaches to predicting the course of UM in the early preclinical period.

The mutational landscape of spinal chordomas and their sensitive detection using circulating tumor DNA.

Background Chordomas are the most common primary spinal column malignancy in the United States. The aim of this study was to determine whether chordomas may be detected by evaluating mutations in circulating tumor DNA (ctDNA).Methods Thirty-two patients with a biopsy-confirmed diagnosis of chordoma had blood drawn pre-operatively and/or at follow-up appointments. Mutations in the primary tumor were identified by whole exome sequencing and liquid biopsy by ddPCR and/or RACE-Seq was used to detect one or more of these mutations in plasma ctDNA at concurrent or later time points.Results At the time of initial blood draw, 87.1% of patients were ctDNA positive (P <.001). Follow-up blood draws in twenty of the patients suggest that ctDNA levels may reflect the clinical status of the disease. Patients with positive ctDNA levels were more likely to have greater mutant allele frequencies in their primary tumors (P = .004) and undergo radiotherapy (P = .02), and the presence of ctDNA may correlate with response to systemic chemotherapy and/or disease recurrence.Conclusions Detection of ctDNA mutations may allow for the detection and monitoring of disease progression for chordomas.

Circulating Methylated DNA to Monitor the Dynamics of RAS Mutation Clearance in Plasma from Metastatic Colorectal Cancer Patients.

Simple SummaryOngoing clinical trials are recently investigating the efficacy of second-line EGFR inhibitors in initially RAS mutant metastatic colorectal cancer patients who convert to RAS wild-type in plasma, as assessed by circulating tumor DNA (ctDNA) analysis. To this purpose, discriminating between patients with a real clearance of RAS mutations in plasma (real wild-type) from those who do not shed ctDNA is mandatory. The aim of the present study was to confirm the presence of ctDNA in patients with RAS mutation clearance in plasma at different time points, using a colon-cancer-specific five-gene methylation panel. The methylation test confirmed the presence of ctDNA in most RAS wild-type samples at the time of disease progression, thus confirming that the negative selection of RAS mutant clones during the clonal evolution of mutant RAS colorectal cancer is not an infrequent event.The clearance of RAS mutations in plasma circulating tumor DNA (ctDNA) from originally RAS-mutant metastatic colorectal cancer (mCRC) has been recently demonstrated. Clinical trials investigating whether RAS mutant mCRC who “convert” to wild-type in plasma might benefit from EGFR blockade are ongoing. Detection of tumor-specific DNA methylation alterations in ctDNA has been suggested as a specific tool to confirm the tumoral origin of cell-free DNA. We monitored RAS clearance in plasma from patients with RAS-mutant mCRC at baseline (pre-treatment) (T0); after 4 months of first-line therapy (T1); at the time of first (T2) and second (T3) progression. A five-gene methylation panel was used to confirm the presence of ctDNA in samples in which RAS mutation clearance was detected. At T1, ctDNA analysis revealed wild-type RAS status in 83% of samples, all not methylated, suggesting at this time point the lack of ctDNA shedding. At T2, ctDNA analysis revealed wild-type RAS status in 83% of samples, of which 62.5% were found methylated. At T3, 50% of wild-type RAS samples were found methylated. Non-methylated samples were found in patients with lung or brain metastases. This five-gene methylation test might be useful to confirm the presence of ctDNA in RAS wild-type plasma samples.

Meta-analysis of the studies dedicated to the predictive significance of circulating tumor DNA in pancreatic cancer

The method of liquid biopsy allows detection of circulating tumor DNA (ctDNA) in patient blood, but the clinical significance of this approach in pancreatic cancer is still unclear. In this regard, we have carried out a meta-analysis of the studies dedicated to the predictive significance of ctDNA in pancreatic cancer.&#x0D; Materials and methods.We carried out the search for the articles and abstracts in PubMed, ASCO and ESMO databases published before February 2020, containing data about the connection between ctDNA and the prognosis of pancreatic cancer. The exclusion criteria were the studies including 10 or less participating patients, absence of the data about the relative risk of mortality and/or progression, and the 95% confidence interval. The meta-analysis was carried out by using the Review Manager software (RevMan), Version 5.3.&#x0D; Results.There were no significant systematic errors associated with the publications. The presence of ctDNA in patient blood showed poor overall survival of patients (odds ratio OR 2.21, 95% confidence interval CI 1.35-3.33,p=0.001) regardless of the prevalence of the disease. In case of the resectable process, the detection of ctDNA in patient blood both before and after surgery was a factor of worse progression-free survival (OR 2.32, 95% CI 1.543.5,p0.001 and OR 3.06, 95% CI 1.635.76,р=0.0005 and overall survival (OR2.01, 95% CI 1.123.63,р=0,02 and OR 3.39, 95% CI 2.125.44,р0.00001, respectively).&#x0D; Conclusions.The detection of ctDNA in the bloodstream in pancreatic cancer patients is a factor of poor prognosis in both localized and advanced cancer. It is very important to make further prospective studies to develop the optimal protocol for detecting ctDNA in patient bloodstream.

Circulating Tumour DNA in Advanced Melanoma Patients Ceasing PD1 Inhibition in the Absence of Disease Progression.

Simple SummaryImmunotherapy is an effective treatment that harnesses the immune system to fight cancer. Drugs such as immune checkpoint inhibitors have demonstrated efficacy in the treatment of advanced melanoma. However, the optimal duration of treatment is not well established. The aim of our retrospective study was to analyse the outcomes of patients who have stopped immunotherapy treatment for advanced melanoma after durable disease control. Furthermore, we assessed circulating tumour DNA (ctDNA), which is shed from the tumour into the bloodstream, to determine its validity as a predictive biomarker of disease progression after treatment was stopped. We demonstrated that stopping treatment after durable disease control results in excellent short- to medium-term prognosis and ctDNA present at the time of stopping treatment is a strong predictor of disease recurrence.Immunotherapy is an important and established treatment option for patients with advanced melanoma. Initial anti-PD1 trials arbitrarily defined a two-year treatment duration, but a shorter treatment duration may be appropriate. In this study, we retrospectively assessed 70 patients who stopped anti-PD1 therapy in the absence of progressive disease (PD) to determine clinical outcomes. In our cohort, the median time on treatment was 11.8 months. Complete response was attained at time of anti-PD1 discontinuation in 61 (87%). After a median follow up of 34.2 months (range: 2–70.8) post discontinuation, 81% remained disease free. Using ddPCR, we determine the utility of circulating tumour DNA (ctDNA) to predict progressive disease after cessation (n = 38). There was a significant association between presence of ctDNA at cessation and disease progression (p = 0.012, Fisher’s exact test) and this conferred a negative and positive predictive value of 0.82 (95% CI: 0.645–0.930) and 0.80 (95% CI 0.284–0.995), respectively. Additionally, dichotomised treatment-free survival in patients with or without ctDNA at cessation was significantly longer in the latter group (p < 0.001, HR: 0.008, 95% CI: 0.001–0.079). Overall, our study confirms that durable disease control can be achieved with cessation of therapy in the absence of disease progression and undetectable ctDNA at cessation was associated with longer treatment-free survival.

Abstract 720: Analysis of circulating tumor DNA after neoadjuvant therapy to predict disease relapse in patients with stage II-III breast cancer

Abstract The therapeutic efficacy of neoadjuvant chemotherapy for patients with breast cancer is usually assessed by the reduction of tumor size and lymph nodes. The pathologic complete response (pCR) is considered as a marker related to good prognosis. However, patients who can achieve pCR is a minor population, and pCR may be not a robust marker predicting prognosis in patients with ER-positive breast cancer. Additional markers allowing the assessment of prognosis is needed. Circulating tumor DNA (ctDNA) has been proved as a marker to detect disease progression in metastatic breast cancer and predict relapse in patients after resection of breast cancer. Therefore, we would like to assess the value of ctDNA in breast cancer patients receiving neoadjuvant therapy. The ctDNA was collected before and after completion of the neoadjuvant chemotherapy (Tend). The presence of ctDNA was detected by ultra-deep targeted next-generation sequencing. We enrolled 82 patients and 62 patients were found to have ctDNA as the biomarkers. The presence of ctDNA included nonsynonymous variants and copy number variations in TP53, PIK3CA, HER2, CDH1, PTEN, S100A, CCND1 and c-MYC. Among the 62 patients, there were 24 patients with hormone receptor (+)/Her2(-), 20 with Her2(+) and 18 with triple-negative breast cancer (TNBC). Pathologic complete response (pCR) occurred in 9 patients, partial response in 32 patients and stable/progression in 21 patients, according to the RECIST 1.1 criteria. Univariate analysis showed tumor reduction&amp;gt;50% (p=0.032), number of metastatic lymph node (p=0.039), and status of ctDNA at Tend (p&amp;lt;0.001) were significantly associated with disease-free survival (DFS). The 5-year DFS of patients with undetectable ctDNA at Tend (74.0%, 95% confidence interval (CI) range 98%-50%) was significantly superior to those with detected ctDNA (16.8%, 95% CI range 36.1%-0%). Multivariate analysis revealed that status of ctDNA at Tend was the only one independent factor predicting relapse (hazard ratio 7.74, 95% CI 2.81-21.35). Two patients had pCR and were found to have alterations of ctDNA at Tend; one with TNBC had distant metastases at 6 months after mastectomy and the other with Her2 (+) breast cancer had brain metastasis at the end of adjuvant trastuzumab treatment. Therefore, we concluded that the presence of ctDNA at the end of the neoadjuvant chemotherapy was a poor prognostic factor predicting relapse in patients with stage II-III breast cancer. Citation Format: Po-Han Lin, Sung-Sheng Kuo, Chiun-Sheng Huang. Analysis of circulating tumor DNA after neoadjuvant therapy to predict disease relapse in patients with stage II-III breast cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 720.

Interactions of mono spermine porphyrin derivative with DNAs.

In this work, we have characterized the interactions of monospermine porphyrin derivative with calf thymus DNA (ct-DNA) and poly (dG-dC)2 in both B and Z conformation. By several spectroscopic techniques (UV-vis, electronic circular dichroism and resonance light scattering), the binding modes of monospermine porphyrin derivative with different DNA sequences have been elucidated. In the presence of ct-DNA, the porphyrin binds along the external double helix as well as in the presence of B conformation of poly (dG-dC)2 . Whilst when the Z form of the poly (dG-dC)2 is induced, a slight intercalation of the porphyrin between the basis has been detected.

Circulating tumor DNA as a marker of minimal residual disease following local treatment of metastases from colorectal cancer

Background Local treatment of liver and/or lung metastases from colorectal cancer (CRC) is increasingly used in daily practice and comprises resection, radiofrequency ablation (RFA) and stereotactic radiotherapy (SBRT). The need for prognostic markers for patients undergoing such treatment is currently unmet. We investigated post-treatment circulating tumor-specific DNA (ctDNA) analysis and address a possible prognostic value in a pilot study. Materials From July 2015 to September 2017, patients undergoing standard of care local treatment of liver and/or lung metastases were included in a prospective translational study. Blood samples were drawn 2 weeks after local treatment and during follow-up. CtDNA was detected by ddPCR and a mass spectrometry-based platform MassARRAY®. Results Post treatment blood samples were available for 35 patients including five with detectable ctDNA (KRAS mutation, n = 2; NRAS mutation, n = 2; BRAF mutation, n = 1) by ddPCR. 17 out of 35 patients (49%) developed recurrence within a median of 273 days (95%CI 95–NA) among patients positive for ctDNA, while the median time to recurrence was not reached for the group of patients negative for ctDNA (p = .03). Conclusion The presence of ctDNA following local treatment of metastatic CRC is associated with an increased risk of recurrence and a short time to failure.

Abstract 724: Application of oncoMonitor™ ctDNA tracking technology for monitoring of therapy and early detection of recurrence in metastatic colorectal cancer

Abstract Introduction: Liquid biopsy based on detection of circulating tumor DNA (circulating-tumor DNA, ctDNA) is finding new applications in clinical management of solid cancers. From the initial use as an alternative to classical tissue biopsy for investigation of molecuar profiles in prediction or resistance detection of targeted therapies the approach is now applied to monitoring of minimal residual disease (MRD). Patients with metastatic colorectal cancer undergoing liver resection are subject to standard post-operative surveillance based mainly on periodic CT or PET/CT imaging (complemented eventually by MR imaging). Some reports have previously demonstrated benefit from performing a ctDNA detection complimentary to standard imaging to improve follow-up efficiency. Methods: In this report we demonstrate use of our oncoMonitor™ ctDNA technology, based on detection of somatic mutations derived from tumor in patient plasma. Unlike various approaches targeting oncogenic hotspot mutants, this technology can trace virtually any somatic mutation found in any gene (e.g. a tumor supressor). The relatively simple approach is based on rapid scanning of 100 to 140bp target amplicons for presence of mutations revealed based on differential melting by high-resolution denaturing capillary electrophoresis (DCE). The detection sensitivity is 0.1% MAF. Patients: In our concordance study during which a total of 41 patients with metatatic colorectal cancer were followed for 3 years from metastasectomy. oncoMonitor™ tast was applied to evaluate ctDNA along with standard CT or PET/CT imaging and tumor markers (CEA/CA19-9). Results: A total of 23 recurrences were clinically confirmed during surveillance. All recurrences were detected by oncoMonitor™ (23/23, 100%), while at the same time only 17 instances were detected by CT or PET/CT imaging (17/23, 73.9%) and 16 by tumor markers CEA/CA19-9 (16/23, 69.6%). There were 2 patients after R0 resection with persistent ctDNA positivity who had recurrence within 6 months from surgery. Conclusions: oncoMonitor™ technology is suitable for quick and sensitive detection of ctDNA in plasma of patients with metatatic colorectal cancer after liver resection. Evaluation of ctDNA presence improves efficiency of post-operative surveillance. Supported by Czech Ministry of Health project no. 17-31909A. Citation Format: Marek Minarik, Barbora Belsanova, Renata Ptackova, Tereza Halkova, Filip Pazdirek, Jiri Pudil, Miroslav Levy, Jaromir Simsa, Jiri Hoch, Miroslav Ryska, Lubos Petruzelka, Lucie Benesova. Application of oncoMonitor™ ctDNA tracking technology for monitoring of therapy and early detection of recurrence in metastatic colorectal cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 724.

Circulating Tumor DNA in KRAS positive colorectal cancer patients as a prognostic factor - a systematic review and meta-analysis.

Liquid biopsy is a novel tool in oncology. It provides minimally invasive detection of tumor specific DNA. This review summarizes data on presence of circulating tumor DNA in serum or plasma of CRC patients as a potential negative prognostic factor. The systematic review was performed according to PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses). The search was performed using PubMed, Web of Science and Scopus. In total 18 articles with a total of 1779 patients met the inclusion criteria. Six out of 8 studies found that presence of ctDNA in plasma/serum was associated with inferior overall survival. All 6 studies found that high concentrations of ctDNA in plasma/serum was associated with inferior overall survival. Presence or high concentrations of KRAS mutation in plasma or serum were associated with inferior prognosis. Establishing cut-off concentrations is warranted for further clinical implementation of liquid biopsy.

Longitudinal monitoring of circulating tumour DNA improves prognostication and relapse detection in gastroesophageal adenocarcinoma

BackgroundGastroesophageal adenocarcinoma (GOA) has poor clinical outcomes and lacks reliable blood markers. Here we present circulating tumour DNA (ctDNA) as an emerging biomarker.MethodsForty patients (17 palliative and 23 curative) were followed by serial plasma monitoring. Primary tumour DNA was analysed by targeted next-generation sequencing to identify somatic single-nucleotide variants (SNVs), and Nanostring nCounter® to detect copy number alterations (CNAs). Patient-specific SNVs and CNA amplifications (CNAamp) were analysed in plasma using digital droplet PCR and quantitative PCR, respectively.ResultsThirty-five patients (13 palliative, 22 curative) had ≥1 SNVs and/or CNAamp detected in primary tumour DNA suitable for tracking in plasma. Eighteen of 35 patients (nine palliative, nine curative) had ≥1 ctDNA-positive plasma sample. Detection of postoperative ctDNA predicted short RFS (190 vs 934 days, HR = 3.7, p = 0.028) and subsequent relapse (PPV for relapse 0.83). High ctDNA levels (>60.5 copies/ml) at diagnosis of metastatic disease predicted poor OS (90 vs 372 days, HR = 11.7 p < 0.001).ConclusionSensitive ctDNA detection allows disease monitoring and prediction of short OS in metastatic patients. Presence of ctDNA postoperatively predicts relapse and defines a ‘molecular relapse’ before overt clinical disease. This lead time defines a potential therapeutic window for additional anticancer therapy.

Monitoring of Early Changes of Circulating Tumor DNA in the Plasma of Rectal Cancer Patients Receiving Neoadjuvant Concomitant Chemoradiotherapy: Evaluation for Prognosis and Prediction of Therapeutic Response.

Introduction: Patients with locally advanced rectal cancer (LARC) are undergoing neoadjuvant chemoradiotherapy (NCRT) prior to surgery. Although in some patients the NCRT is known to prevent local recurrence, it is also accompanied by side effects. Accordingly, there is an unmet need to identify predictive markers allowing to identify non-responders to avoid its adverse effects. We monitored circulating tumor DNA (ctDNA) as a potential liquid biopsy-based biomarker. We have investigated ctDNA changes plasma during the early days of NCRT and its relationship to the overall therapy outcome.Methods and Patients: The studied cohort included 36 LARC patients (stage II or III) undergoing NCRT with subsequent surgical treatment. We have detected somatic mutations in tissue biopsies taken during endoscopic examination prior to the therapy. CtDNA was extracted from patient plasma samples prior to therapy and at the end of the first week. In order to optimize the analytical costs of liquid-biopsy testing, we have utilized a two-level approach in which first a low-cost detection method of denaturing capillary electrophoresis was used followed by examination of initially negative samples by a high-sensitivity BEAMING assay. The ctDNA was related to clinical parameters including tumor regression grade (TRG) and TNM tumor staging.Results: We have detected a somatic mutation in 33 out of 36 patients (91.7%). Seven patients (7/33, 21.2%) had ctDNA present prior to therapy. The ctDNA positivity before treatment reduced post-operative disease-free survival and overall survival by an average of 1.47 and 1.41 years, respectively (p = 0.015, and p = 0.010). In all patients, ctDNA was strongly reduced or completely eliminated from plasma by the end of the first week of NCRT, with no correlation to any of the parameters analyzed.Conclusions: The baseline ctDNA presence represented a statistically significant negative prognostic biomarker for the overall patient survival. As ctDNA was reduced indiscriminately from circulation of all patients, dynamics during the first week of NCRT is not suited for predicting the outcome of LARC. However, the general effect of rapid ctDNA disappearance apparently occurring during the initial days of NCRT is noteworthy and should further be studied.

Tumor-related mutations in cell-free DNA in pre-operative plasma as a prognostic indicator of recurrence in endometrial cancer

ObjectivesCirculating tumor DNA (ctDNA) has potential as a basis for understanding the molecular features of a tumor non-invasively and for use as a diagnostic, prognostic, and disease-monitoring marker. The aim...