scholarly journals Efficient detection and post-surgical monitoring of colon cancer with a multi-marker DNA methylation liquid biopsy

2021 ◽  
Vol 118 (5) ◽  
pp. e2017421118
Author(s):  
Shengnan Jin ◽  
Dewen Zhu ◽  
Fanggui Shao ◽  
Shiliang Chen ◽  
Ying Guo ◽  
...  
Keyword(s):  
Early Stage ◽  
Cancer Recurrence ◽  
Cost Effective ◽  
Circulating Tumor Dna ◽  
Plasma Samples ◽  
Liquid Biopsies ◽  
Efficient Detection ◽  
Radiologic Imaging ◽  
Tumor Dna

Multiplex assays, involving the simultaneous use of multiple circulating tumor DNA (ctDNA) markers, can improve the performance of liquid biopsies so that they are highly predictive of cancer recurrence. We have developed a single-tube methylation-specific quantitative PCR assay (mqMSP) that uses 10 different methylation markers and is capable of quantitative analysis of plasma samples with as little as 0.05% tumor DNA. In a cohort of 179 plasma samples from colorectal cancer (CRC) patients, adenoma patients, and healthy controls, the sensitivity and specificity of the mqMSP assay were 84.9% and 83.3%, respectively. In a head-to-head comparative study, the mqMSP assay also performed better for detecting early-stage (stage I and II) and premalignant polyps than a published SEPT9 assay. In an independent longitudinal cohort of 182 plasma samples (preoperative, postoperative, and follow-up) from 82 CRC patients, the mqMSP assay detected ctDNA in 73 (89.0%) of the preoperative plasma samples. Postoperative detection of ctDNA (within 2 wk of surgery) identified 11 of the 20 recurrence patients and was associated with poorer recurrence-free survival (hazard ratio, 4.20; P = 0.0005). With subsequent longitudinal monitoring, 14 patients (70%) had detectable ctDNA before recurrence, with a median lead time of 8.0 mo earlier than seen with radiologic imaging. The mqMSP assay is cost-effective and easily implementable for routine clinical monitoring of CRC recurrence, which can lead to better patient management after surgery.

scholarly journals Detection of KRAS Mutations in Circulating Tumor DNA by Digital PCR in Early Stages of Pancreatic Cancer

2016 ◽  
Vol 62 (11) ◽  
pp. 1482-1491 ◽  
Author(s):  
Nora Brychta ◽  
Thomas Krahn ◽  
Oliver von Ahsen
Keyword(s):  
Pancreatic Cancer ◽  
Early Stage ◽  
Digital Pcr ◽  
Circulating Tumor Dna ◽  
Surgical Removal ◽  
Plasma Samples ◽  
Kras Mutations ◽  
Detection Rates ◽  
Early Stages ◽  
Tumor Dna

Abstract BACKGROUND Since surgical removal remains the only cure for pancreatic cancer, early detection is of utmost importance. Circulating biomarkers have potential as diagnostic tool for pancreatic cancer, which typically causes clinical symptoms only in advanced stage. Because of their high prevalence in pancreatic cancer, KRAS proto-oncogene, GTPase [KRAS (previous name: Kirsten rat sarcoma viral oncogene homolog)] mutations may be used to identify tumor-derived circulating plasma DNA. Here we tested the diagnostic sensitivity of chip based digital PCR for the detection of KRAS mutations in circulating tumor DNA (ctDNA) in early stage pancreatic cancer. METHODS We analyzed matched plasma (2 mL) and tumor samples from 50 patients with pancreatic cancer. Early stages (I and II) were predominant (41/50) in this cohort. DNA was extracted from tumor and plasma samples and tested for the common codon 12 mutations G12D, G12V, and G12C by chip-based digital PCR. RESULTS We identified KRAS mutations in 72% of the tumors. 44% of the tumors were positive for G12D, 20% for G12V, and 10% for G12C. One tumor was positive for G12D and G12V. Analysis of the mutations in matched plasma samples revealed detection rates of 36% for G12D, 50% for G12V, and 0% for G12C. The detection appeared to be correlated with total number of tumor cells in the primary tumor. No KRAS mutations were detected in 20 samples of healthy control plasma. CONCLUSIONS Our results support further evaluation of tumor specific mutations as early diagnostic biomarkers using plasma samples as liquid biopsy.

scholarly journals ctDNAtools: An R package to work with sequencing data of circulating tumor DNA

2020 ◽  
Author(s):  
Amjad Alkodsi ◽  
Leo Meriranta ◽  
Annika Pasanen ◽  
Sirpa Leppä
Keyword(s):  
Residual Disease ◽  
Fragment Size ◽  
R Package ◽  
Monte Carlo Sampling ◽  
Circulating Tumor Dna ◽  
Cancer Diagnostics ◽  
Sequencing Data ◽  
Liquid Biopsies ◽  
Tumor Dna

AbstractSummarySequencing of cell-free DNA (cfDNA) including circulating tumor DNA (ctDNA) in minimally-invasive liquid biopsies is rapidly maturing towards clinical utility for cancer diagnostics. However, the publicly available bioinformatics tools for the specialized analysis of ctDNA sequencing data are still scarce. Here, we present the ctDNAtools R package, which provides functionalities for testing minimal residual disease (MRD) and analyzing cfDNA fragmentation. MRD detection in ctDNAtools utilizes a Monte Carlo sampling approach to test ctDNA positivity through tracking a set of pre-detected reporter mutations in follow-up samples. Additionally, ctDNAtools includes various functionalities to study cfDNA fragment size histograms, profiles and fragment ends patterns.AvailabilityThe ctDNAtools package is freely available under MIT license at https://github.com/alkodsi/ctDNAtools.

scholarly journals Clinical utility of circulating tumor DNA as a response and follow-up marker in cancer therapy

2020 ◽  
Vol 39 (3) ◽  
pp. 999-1013 ◽  
Author(s):  
Pieter A. Boonstra ◽  
Thijs T. Wind ◽  
Michel van Kruchten ◽  
Ed Schuuring ◽  
Geke A. P. Hospers ◽  
...  
Keyword(s):  
Cancer Therapy ◽  
Treatment Response ◽  
Clinical Utility ◽  
Tumor Response ◽  
Circulating Tumor Dna ◽  
Tumor Burden ◽  
Liquid Biopsies ◽  
Secondary Resistance ◽  
Tumor Dna

Abstract Response evaluation for cancer treatment consists primarily of clinical and radiological assessments. In addition, a limited number of serum biomarkers that assess treatment response are available for a small subset of malignancies. Through recent technological innovations, new methods for measuring tumor burden and treatment response are becoming available. By utilization of highly sensitive techniques, tumor-specific mutations in circulating DNA can be detected and circulating tumor DNA (ctDNA) can be quantified. These so-called liquid biopsies provide both molecular information about the genomic composition of the tumor and opportunities to evaluate tumor response during therapy. Quantification of tumor-specific mutations in plasma correlates well with tumor burden. Moreover, with liquid biopsies, it is also possible to detect mutations causing secondary resistance during treatment. This review focuses on the clinical utility of ctDNA as a response and follow-up marker in patients with non-small cell lung cancer, melanoma, colorectal cancer, and breast cancer. Relevant studies were retrieved from a literature search using PubMed database. An overview of the available literature is provided and the relevance of ctDNA as a response marker in anti-cancer therapy for clinical practice is discussed. We conclude that the use of plasma-derived ctDNA is a promising tool for treatment decision-making based on predictive testing, detection of resistance mechanisms, and monitoring tumor response. Necessary steps for translation to daily practice and future perspectives are discussed.

Potential utility of methylation levels detected from circulating tumor DNA (ctDNA) in predicting molecular residual disease (MRD) in patients with resected non-small cell lung cancer (NSCLC).

2020 ◽  
Vol 38 (15_suppl) ◽  
pp. e15535-e15535
Author(s):  
Hong HU ◽  
Hang Li ◽  
Zelin Ma ◽  
Jiaqing Xiang ◽  
Yawei Zhang ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Prediction Model ◽  
Methylation Level ◽  
Early Stage ◽  
Circulating Tumor Dna ◽  
Post Surgery ◽  
Resected Nsclc ◽  
Resected Tumor ◽  
Tumor Dna

e15535 Background: To improve the prognosis of resected lung cancer patients, growing efforts are being invested in finding the most optimal approach to detect MRD and predict relapse. In our study, we aimed to investigate the utility of ctDNA methylation profiling in detecting MRD from patients with resected early-stage NSCLC. Methods: Surgically-resected tumor tissues were obtained from 65 patients diagnosed with resectable stage IA-III NSCLC with various histological subtypes. Matched blood samples were also collected before surgery (baseline) and during regular follow-up after 2-8 weeks of surgery. Comprehensive somatic mutation and methylation level profile from circulating tumor DNA (ctDNA) were performed using unique molecular identifier-based targeted sequencing and targeted bisulfite sequencing, respectively. A tumor-informed MRD prediction model was constructed based on methylation levels obtained from the patient’s resected tumor tissue to calculate the corresponding methylation signal intensity from the matched plasma sample of the patient at baseline or other time points during follow-up, which is reflected as MRD score. Results: Of the 28 patients with baseline ctDNA methylation data, 28.6% (8/28) of the patients had elevated ctDNA methylation levels at first post-operative follow-up (F1) as compared to baseline, indicating the possibility of MRD. Meanwhile, of the 20 patients (71.4%, 20/28) who had reduced ctDNA methylation levels at F1, elevation of ctDNA methylation level was detected from 3 and 1 patients at second (F2) and third (F3) follow-up, respectively. Based on the MRD prediction model, 17.9% (5/28) of the patients had higher MRD scores at F1. Of the 23 patients with lower MRD scores at F1, 5, 1 and 1 patients had an elevation in MRD scores at F2, F3, and F4, respectively, which indicates a possible MRD. Disease relapse was radiologically confirmed after 10-16 months post-surgery in three patients with concomitant elevation of ctDNA methylation level and MRD score during follow-up between 2-9 months prior to radiologic relapse. Conclusions: Our results demonstrate that post-operative ctDNA methylation levels could be used to detect MRD in patients with resected NSCLC. Moreover, ctDNA methylation-based prediction model of MRD could serve as a potential model to predict relapse in early-stage as well as disease progression in advanced-stage NSCLC.

scholarly journals Liquid Biopsies in Hepatocellular Carcinoma: Are We Winning?

2020 ◽  
Vol 9 (5) ◽  
pp. 1541 ◽  
Author(s):  
Tudor Mocan ◽  
André L. Simão ◽  
Rui E. Castro ◽  
Cecília M. P. Rodrigues ◽  
Artur Słomka ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Clinical Performance ◽  
Circulating Tumor Dna ◽  
Liquid Biopsies ◽  
Disease Biomarkers ◽  
Common Cancer ◽  
Tumor Dna ◽  
Tumor Proteins

Hepatocellular carcinoma (HCC) represents the sixth most common cancer worldwide and the third most common cause of cancer-related death. One of the major problems faced by researchers and clinicians in this area is the lack of reliable disease biomarkers, which would allow for an earlier diagnosis, follow-up or prediction of treatment response, among others. In this regard, the “HCC circulome”, defined as the pool of circulating molecules in the bloodstream derived from the primary tumor, represents an appealing target, the so called liquid biopsy. Such molecules encompass circulating tumor proteins, circulating tumor cells (CTCs), extracellular vesicles (EVs), tumor-educated platelets (TEPs), and circulating tumor nucleic acids, namely circulating tumor DNA (ctDNA) and circulating tumor RNA (ctRNA). In this article, we summarize recent findings highlighting the promising role of liquid biopsies as novel potential biomarkers in HCC, emphasizing on its clinical performance.

scholarly journals The Promise of Circulating Tumor DNA (ctDNA) in the Management of Early-Stage Colon Cancer: A Critical Review

Cancers ◽  
2020 ◽  
Vol 12 (10) ◽  
pp. 2808
Author(s):  
Sakti Chakrabarti ◽  
Hao Xie ◽  
Raul Urrutia ◽  
Amit Mahipal
Keyword(s):  
Colon Cancer ◽  
Adjuvant Therapy ◽  
Risk Stratification ◽  
Early Stage ◽  
Cancer Recurrence ◽  
Circulating Tumor Dna ◽  
Clinicopathologic Characteristics ◽  
Early Stage Colon Cancer ◽  
Risk Of Cancer ◽  
Tumor Dna

The current standard treatment for patients with early-stage colon cancer consists of surgical resection, followed by adjuvant therapy in a select group of patients deemed at risk of cancer recurrence. The decision to administer adjuvant therapy, intended to eradicate the clinically inapparent minimal residual disease (MRD) to achieve a cure, is guided by clinicopathologic characteristics of the tumor. However, the risk stratification based on clinicopathologic characteristics is imprecise and results in under or overtreatment in a substantial number of patients. Emerging research indicates that the circulating tumor DNA (ctDNA), a fraction of cell-free DNA (cfDNA) in the bloodstream that originates from the neoplastic cells and carry tumor-specific genomic alterations, is a promising surrogate marker of MRD. Several recent studies suggest that ctDNA-guided risk stratification for adjuvant therapy outperforms existing clinicopathologic prognostic indicators. Preliminary data also indicate that, aside from being a prognostic indicator, ctDNA can inform on the efficacy of adjuvant therapy, which is the underlying scientific rationale for several ongoing clinical trials evaluating ctDNA-guided therapy escalation or de-escalation. Furthermore, serial monitoring of ctDNA after completion of definitive therapy can potentially detect cancer recurrence much earlier than conventional surveillance methods that may provide a critical window of opportunity for additional curative-intent therapeutic interventions. This article presents a critical overview of published studies that evaluated the clinical utility of ctDNA in the management of patients with early-stage colon cancer, and discusses the potential of ctDNA to transform the adjuvant therapy strategies.

scholarly journals Rescue of Non-Informative Circulating Tumor DNA to Monitor the Mutational Landscape in NSCLC

Cancers ◽  
2020 ◽  
Vol 12 (7) ◽  
pp. 1917
Author(s):  
Stefanie Mayer ◽  
Gerlinde Schmidtke-Schrezenmeier ◽  
Christian Buske ◽  
Frank G. Rücker ◽  
Thomas F.E. Barth ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Cost Effective ◽  
Circulating Tumor Dna ◽  
Driver Mutations ◽  
Genetic Profile ◽  
Plasma Samples ◽  
Plasma Dna ◽  
Technical Improvement ◽  
Mutational Landscape ◽  
Tumor Dna

In non-small cell lung cancer (NSCLC) the usage of plasma-derived circulating tumor DNA (ctDNA) have come into focus to obtain a comprehensive genetic profile of a given lung cancer. Despite the usage of specific sampling tubes, archived plasma samples as well as inappropriately treated blood samples still cause a loss of information due to cell lysis and contamination with cellular DNA. Our aim was to establish a reliable protocol to rescue ctDNA from such non-informative samples to monitor the mutational landscape in NSCLC. As a proof-of-concept study we used archived plasma samples derived from whole blood EDTA samples of 51 patients suffering from NSCLC. Analysis of the isolated plasma DNA determined only a small fraction of ctDNA in a range of 90–250 bp. By applying a specific purification procedure, we were able to increase the informative ctDNA content and improve in a cohort of 42 patients the detection of driver mutations from 32% to 79% of the mutations found in tissue biopsies. Thus, we present here an easy to perform, time and cost effective procedure to rescue non-informative ctDNA samples, which is sufficient to detect oncogenic mutations in NGS approaches and is therefore a valuable technical improvement for laboratories handling liquid biopsy samples.

scholarly journals Circulating tumor DNA dynamics and recurrence risk in patients undergoing curative intent resection of colorectal cancer liver metastases: A prospective cohort study

PLoS Medicine ◽  
2021 ◽  
Vol 18 (5) ◽  
pp. e1003620
Author(s):  
Jeanne Tie ◽  
Yuxuan Wang ◽  
Joshua Cohen ◽  
Lu Li ◽  
Wei Hong ◽  
...  
Keyword(s):  
Adjuvant Chemotherapy ◽  
Neoadjuvant Chemotherapy ◽  
Circulating Tumor Dna ◽  
Prognostic Impact ◽  
Plasma Samples ◽  
End Of Treatment ◽  
Ctdna Analysis ◽  
Serial Samples ◽  
Tumor Dna

Background In patients with resectable colorectal liver metastases (CRLM), the role of pre- and postoperative systemic therapy continues to be debated. Previous studies have shown that circulating tumor DNA (ctDNA) analysis, as a marker of minimal residual disease, is a powerful prognostic factor in patients with nonmetastatic colorectal cancer (CRC). Serial analysis of ctDNA in patients with resectable CRLM could inform the optimal use of perioperative chemotherapy. Here, we performed a validation study to confirm the prognostic impact of postoperative ctDNA in resectable CRLM observed in a previous discovery study. Methods and findings We prospectively collected plasma samples from patients with resectable CRLM, including presurgical and postsurgical samples, serial samples during any pre- or postoperative chemotherapy, and serial samples in follow-up. Via targeted sequencing of 15 genes commonly mutated in CRC, we identified at least 1 somatic mutation in each patient’s tumor. We then designed a personalized assay to assess 1 mutation in plasma samples using the Safe-SeqS assay. A total of 380 plasma samples from 54 patients recruited from July 2011 to Dec 2014 were included in our analysis. Twenty-three (43%) patients received neoadjuvant chemotherapy, and 42 patients (78%) received adjuvant chemotherapy after surgery. Median follow-up was 51 months (interquartile range, 31 to 60 months). At least 1 somatic mutation was identified in all patients’ tumor tissue. ctDNA was detectable in 46/54 (85%) patients prior to any treatment and 12/49 (24%) patients after surgery. There was a median 40.93-fold (19.10 to 87.73, P < 0.001) decrease in ctDNA mutant allele fraction with neoadjuvant chemotherapy, but ctDNA clearance during neoadjuvant chemotherapy was not associated with a better recurrence-free survival (RFS). Patients with detectable postoperative ctDNA experienced a significantly lower RFS (HR 6.3; 95% CI 2.58 to 15.2; P < 0.001) and overall survival (HR 4.2; 95% CI 1.5 to 11.8; P < 0.001) compared to patients with undetectable ctDNA. For the 11 patients with detectable postoperative ctDNA who had serial ctDNA sampling during adjuvant chemotherapy, ctDNA clearance was observed in 3 patients, 2 of whom remained disease-free. All 8 patients with persistently detectable ctDNA after adjuvant chemotherapy have recurred. End-of-treatment (surgery +/− adjuvant chemotherapy) ctDNA detection was associated with a 5-year RFS of 0% compared to 75.6% for patients with an undetectable end-of-treatment ctDNA (HR 14.9; 95% CI 4.94 to 44.7; P < 0.001). Key limitations of the study include the small sample size and the potential for false-positive findings with multiple hypothesis testing. Conclusions We confirmed the prognostic impact of postsurgery and posttreatment ctDNA in patients with resected CRLM. The potential utility of serial ctDNA analysis during adjuvant chemotherapy as an early marker of treatment efficacy was also demonstrated. Further studies are required to define how to optimally integrate ctDNA analyses into decision-making regarding the use and timing of adjuvant therapy for resectable CRLM. Trial registration ACTRN12612000345886.

scholarly journals BC-Monitor: Towards a Routinely Accessible Circulating Tumor DNA-Based Tool for Real-Time Monitoring Breast Cancer Progression and Treatment Effectiveness

Cancers ◽  
2021 ◽  
Vol 13 (14) ◽  
pp. 3489
Author(s):  
Katalin Priskin ◽  
Sára Pólya ◽  
Lajos Pintér ◽  
Gábor Jaksa ◽  
Bernadett Csányi ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Real Time ◽  
Cancer Progression ◽  
Palliative Therapy ◽  
Circulating Tumor Dna ◽  
Plasma Samples ◽  
Breast Cancer Patients ◽  
Plasma Dna ◽  
Tumor Dna

Circulating tumor DNA (ctDNA) is increasingly employed in the screening, follow-up, and monitoring of the continuously evolving tumor; however, most ctDNA assays validated for clinical use cannot maintain the right balance between sensitivity, coverage, sample requirements, time, and cost. Here, we report our BC-monitor, a simple, well-balanced ctDNA diagnostic approach using a gene panel significant in breast cancer and an optimized multiplex PCR-based NGS protocol capable of identifying allele variant frequencies below 1% in cell-free plasma DNA. We monitored a cohort of 45 breast cancer patients prospectively enrolled into our study receiving neoadjuvant chemotherapy or endocrine therapy or palliative therapy for metastatic diseases. Their tumor mutation status was examined in the archived tumor samples and plasma samples collected before and continuously during therapy. Traceable mutations of the used 38-plex NGS assay were found in approximately two-thirds of the patients. Importantly, we detected new pathogenic variants in follow-up plasma samples that were not detected in the primary tumor and baseline plasma samples. We proved that the BC-monitor can pre-indicate disease progression four–six months earlier than conventional methods. Our study highlights the need for well-designed ctDNA monitoring during treatment and follow-up, integrated into a real-time treatment assessment, which could provide information on the active tumor DNA released into the blood.

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