Liquid Biopsy from Bile-Circulating Tumor DNA in Patients with Biliary Tract Cancer

Although liquid biopsy of blood is useful for cancer diagnosis and prediction of prognosis, diagnostic and prognostic value of ctDNA in bile fluid for BTCs are not clear yet. To determine whether liquid biopsy for circulating tumor DNA (ctDNA) can replace tissue biopsy when assessing somatic mutations in biliary tract cancers (BTCs). Bile samples were obtained from 42 patients with BTC. Matched formalin-fixed paraffin-embedded (FFPE) samples were obtained from 20 of these patients and matched plasma samples from 16 of them. Droplet digital PCR (ddPCR) was used for detection KRAS somatic mutation. KRAS mutations were identified in the bile ctDNA of 20 of 42 (48%) patients. Patients with mutant KRAS showed significantly worse survival than those with wild-type KRAS (2-year survival rates: 0% vs. 55.5%, respectively; p = 0.018). There was 80.0% mutational concordance between the paired bile ctDNA and FFPE samples, and 42.9% between the plasma and FFPE samples. On transcriptomic sequencing of one set of paired bile and FFPE samples, expression level of KRAS-associated signaling oncogenes in the bile and tissue samples showed a strong positive correlation (r = 0.991, p < 0.001). Liquid biopsy of bile reliably detect mutational variants within the bile ctDNA of BTC patients. These results suggest that bile is an effective biopsy fluid for ctDNA analysis.

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

Clinical Chemistry ◽

10.1373/clinchem.2016.257469 ◽

2016 ◽

Vol 62 (11) ◽

pp. 1482-1491 ◽

Cited By ~ 55

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.

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Quantitative Analysis and Monitoring of EZH2 Mutations Using Liquid Biopsy in Follicular Lymphoma

Genes ◽

10.3390/genes11070785 ◽

2020 ◽

Vol 11 (7) ◽

pp. 785

Author(s):

Ákos Nagy ◽

Bence Bátai ◽

Alexandra Balogh ◽

Sarolta Illés ◽

Gábor Mikala ◽

...

Keyword(s):

Follicular Lymphoma ◽

Mutation Analysis ◽

Liquid Biopsy ◽

Digital Pcr ◽

Circulating Tumor Dna ◽

Allele Frequencies ◽

Variant Allele ◽

Monitoring Tool ◽

Positron Emission ◽

Tumor Dna

Recent advances in molecular technologies enable sensitive and quantitative assessment of circulating tumor DNA, offering a noninvasive disease monitoring tool for patients with malignant disorders. Here, we demonstrated on four follicular lymphoma cases that circulating tumor DNA based EZH2 mutation analysis performed by a highly sensitive droplet digital PCR method may be a valuable treatment monitoring approach in EZH2 mutant follicular lymphoma. EZH2 variant allele frequencies changed in parallel with the volume of metabolically active tumor sites observed on 18F-fluorodeoxyglucose positron emission tomography combined with computer tomography (PET-CT) scans. Variant allele frequencies of EZH2 mutations decreased or were eliminated rapidly upon successful treatment, with treatment failure being associated with elevated EZH2 variant allele frequencies. We also demonstrated spatial heterogeneity in a patient with two different EZH2 mutations in distinct anatomical sites, with both mutations simultaneously detected in the liquid biopsy specimen. In summary, circulating tumor DNA based EZH2 mutation analysis offers a rapid, real-time, radiation-free monitoring tool for sensitive detection of EZH2 mutations deriving from different anatomical sites in follicular lymphoma patients receiving immunochemotherapy.

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Liquid biopsy for patients with IBD-associated neoplasia

BMC Cancer ◽

10.1186/s12885-020-07699-z ◽

2020 ◽

Vol 20 (1) ◽

Author(s):

Hideaki Kinugasa ◽

Sakiko Hiraoka ◽

Kazuhiro Nouso ◽

Shumpei Yamamoto ◽

Mami Hirai ◽

...

Keyword(s):

Liquid Biopsy ◽

Target Genes ◽

Tumor Tissue ◽

Digital Pcr ◽

Circulating Tumor Dna ◽

Tissue Samples ◽

Non Invasive ◽

Tumor Tissues ◽

Inflammatory Bowel

Abstract Background It is often difficult to diagnose inflammatory bowel disease (IBD)-associated neoplasia endoscopically due to background inflammation. In addition, due to the absence of sensitive tumor biomarkers, countermeasures against IBD-associated neoplasia are crucial. The purpose of this study is to develop a new diagnostic method through the application of liquid biopsy. Methods Ten patients with IBD-associated cancers and high-grade dysplasia (HGD) with preserved tumor tissue and blood were included. Tumor and non-tumor tissues were analyzed for 48 cancer-related genes using next-generation sequencing. Simultaneously, circulating tumor DNA (ctDNA) was analyzed for mutations in the target genes using digital PCR. Results Out of 10 patients, seven had IBD-related cancer and three had IBD-related HGD. Two patients had carcinoma in situ; moreover, three had stageII and two had stage III. To avoid false positives, the mutation rate cutoff was set at 5% based on the control results; seven of 10 (70%) tumor tissue samples were mutation-positive. Mutation frequencies for each gene were as follows: TP53 (20.9%; R136H), TP53 (25.0%; C110W), TP53 (8.5%; H140Q), TP53 (31.1%; R150W), TP53 (12.8%; R141H), KRAS (40.0%; G12V), and PIK3CA (34.1%; R 88Q). The same mutations were detected in the blood of these seven patients. However, no mutations were detected in the blood of the remaining three patients with no tumor tissue mutations. The concordance rate between tumor tissue DNA and blood ctDNA was 100%. Conclusion Blood liquid biopsy has the potential to be a new method for non-invasive diagnosis of IBD-associated neoplasia.

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Measuring KRAS Mutations in Circulating Tumor DNA by Droplet Digital PCR and Next-Generation Sequencing

Translational Oncology ◽

10.1016/j.tranon.2018.07.013 ◽

2018 ◽

Vol 11 (5) ◽

pp. 1220-1224 ◽

Cited By ~ 29

Author(s):

Christina Demuth ◽

Karen-Lise Garm Spindler ◽

Julia S. Johansen ◽

Niels Pallisgaard ◽

Dorte Nielsen ◽

...

Keyword(s):

Next Generation Sequencing ◽

Digital Pcr ◽

Circulating Tumor Dna ◽

Droplet Digital Pcr ◽

Next Generation ◽

Kras Mutations ◽

Tumor Dna ◽

Generation Sequencing

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Blood-based genomic profiling of circulating tumor DNA from patients with advanced biliary tract cancer.

Journal of Clinical Oncology ◽

10.1200/jco.2019.37.15_suppl.1576 ◽

2019 ◽

Vol 37 (15_suppl) ◽

pp. 1576-1576

Author(s):

Junying Wang ◽

Jia Song ◽

Jing Zhao ◽

Yuzi Zhang ◽

Shangli Cai ◽

...

Keyword(s):

Allele Frequency ◽

Biliary Tract ◽

Biliary Tract Cancer ◽

Tumor Heterogeneity ◽

Circulating Tumor Dna ◽

Pi3k Pathway ◽

Chinese Patients ◽

Tissue Samples ◽

Tract Cancer ◽

Tumor Dna

1576 Background: Biliary tract cancer (BTC) is a highly lethal malignancy as diagnosis occurring at late stages and marginally sensitive to chemotherapy. Increasing evidence indicates targeted therapeutics may provide new hope for improving clinical response in BTC, hence better comprehending the genomic profile is particularly important. However, tissue of BTC is highly wide tumor heterogeneity and often inadequate for molecular characterization, a proper method is urgently needed. Circulating tumor DNA (ctDNA) is an emerging technology for detecting actionable alterations, and may be regarded as a reliable tool to reveal genomic signature. Methods: Next-generation sequencing (NGS) targeted 150 cancer-related genes was used to detect blood-based ctDNA from 154 Chinese patients with BTC. The mean sequencing depth was more than 3000×. Somatic genomic alternations (GA) including single nucleotide variation (SNV), copy number variation (CNV) and fusion were analyzed and compared with an internal tissue genomic database (545 Chinese patients with BTC) tested by NGS and TCGA database (n = 227) tested by the whole exome sequencing (WES). Allele frequency (AF) represented the percentage of mutant allele reads relative to total allele reads (mutant plus wild type). Maximum somatic allele frequency (MSAF) was defined as the maximum AF (0.1% < AF < 35%) of all the somatic alterations identified per sample. Results: Among ctDNA database, at least one GA was found in 95% (147/154) of samples (a median of 4 GA per patient). The median MSAF across all cases was 6.47% (range, 0%-34.8%). Pathologic type (P < 0.001) and sex (P < 0.001) were significantly related with MSAF, respectively. Frequencies of SNV in commonly mutated genes from ctDNA were similar to those observed among tissue samples, like TP53 (35.1% vs 40.4%) and KRAS (20.1% vs 22.6%), however, a little lower in TCGA database (TP53 24.2%; KRAS 10.1%). Besides, the consistency of CNV detected from ctDNA and tissue was relatively poor, and tumor heterogeneity might be in charge of this phenomenon. Among the highly frequent mutations (AF > 5%) in ctDNA, 45% of genes was considered as druggable targets, such as EGFR/RAS/RAF pathway and AKT/mTOR/PI3K pathway. Conclusions: These findings demonstrated that ctDNA tested by NGS was feasible in revealing genomic profiles and identifying potential therapeutic targets. Noninvasive ctDNA could be used as a complementary approach to tissue testing in patients with metastatic BTC.

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Epigenetic Analysis of Circulating Tumor DNA in Localized and Metastatic Prostate Cancer: Evaluation of Clinical Biomarker Potential

Cells ◽

10.3390/cells9061362 ◽

2020 ◽

Vol 9 (6) ◽

pp. 1362

Author(s):

Marianne Trier Bjerre ◽

Maibritt Nørgaard ◽

Ole Halfdan Larsen ◽

Sarah Østrup Jensen ◽

Siri H. Strand ◽

...

Keyword(s):

Prostate Cancer ◽

De Novo ◽

Digital Pcr ◽

Circulating Tumor Dna ◽

Healthy Controls ◽

Tissue Samples ◽

Castration Resistant ◽

Clinical Biomarker ◽

Epigenetic Analysis ◽

Tumor Dna

Novel and minimally-invasive prostate cancer (PCa)-specific biomarkers are needed to improve diagnosis and risk stratification. Here, we investigated the biomarker potential in localized and de novo metastatic PCa (mPCa) of methylated circulating tumor DNA (ctDNA) in plasma. Using the Marmal-aid database and in-house datasets, we identified three top candidates specifically hypermethylated in PCa tissue: DOCK2, HAPLN3, and FBXO30 (specificity/sensitivity: 80%–100%/75–94%). These candidates were further analyzed in plasma samples from 36 healthy controls, 61 benign prostatic hyperplasia (BPH), 102 localized PCa, and 65 de novo mPCa patients using methylation-specific droplet digital PCR. Methylated ctDNA for DOCK2/HAPLN3/FBXO30 was generally not detected in healthy controls, BPH patients, nor in patients with localized PCa despite a positive signal in 98%–100% of matched radical prostatectomy tissue samples. However, ctDNA methylation of DOCK2, HAPLN3, and/or FBXO30 was detected in 61.5% (40/65) of de novo mPCa patients and markedly increased in high- compared to low-volume mPCa (89.3% (25/28) vs. 32.1% (10/31), p < 0.001). Moreover, detection of methylated ctDNA was associated with significantly shorter time to progression to metastatic castration resistant PCa, independent of tumor-volume. These results indicate that methylated ctDNA (DOCK2/HAPLN3/FBXO30) may be potentially useful for identification of hormone-naïve mPCa patients who could benefit from intensified treatment.

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Genomic Profiling of Blood-Derived Circulating Tumor DNA from Patients with Advanced Biliary Tract Cancer

Pathology & Oncology Research ◽

10.3389/pore.2021.1609879 ◽

2021 ◽

Vol 27 ◽

Author(s):

Chen Chen ◽

Tao Wang ◽

Mengmei Yang ◽

Jia Song ◽

Mengli Huang ◽

...

Keyword(s):

Biliary Tract ◽

Biliary Tract Cancer ◽

Circulating Tumor Dna ◽

Chinese Patients ◽

Ras Signaling ◽

Genomic Alterations ◽

Tissue Samples ◽

Genomic Changes ◽

Tract Cancer ◽

Tumor Dna

Background: Biliary tract cancer is a highly lethal malignancy with poor clinical outcome. Accumulating evidence indicates targeted therapeutics may provide new hope for improving treatment response in BTC, hence better understanding the genomic profile is particularly important. Since tumor tissue may not be available for some patients, a complementary method is urgently needed. Circulating tumor DNA (ctDNA) provides a noninvasive means for detecting genomic alterations, and has been regarded as a promising tool to guide clinical therapies.Methods: Next-generation sequencing of 150 cancer-related genes was used to detect gene alterations in blood-derived ctDNA from 154 Chinese patients with BTC. Genomic alterations were analyzed and compared with an internal tissue genomic database and TCGA database.Results: 94.8% patients had at least one change detected in their ctDNA. The median maximum somatic allele frequency was 6.47% (ranging 0.1–34.8%). TP53 and KRAS were the most often mutated genes. The frequencies of single nucleotide variation in commonly mutated genes in ctDNA were similar to those detected in tissue samples, TP53 (35.1 vs. 40.4%) and KRAS (20.1 vs. 22.6%). Pathway analysis revealed that mutated genes were mapped to several key pathways including PI3K-Akt, p53, ErbB and Ras signaling pathway. In addition, patients harboring LRP1B, TP53, and ErbB family mutations presented significantly higher tumor mutation burden.Conclusions: These findings demonstrated that ctDNA testing by NGS was feasible in revealing genomic changes and could be a viable alternative to tissue biopsy in patients with metastatic BTC.

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Greater than two coexisting mutations in KRAS and NRAS identified in the circulating tumor DNA fraction of liquid biopsies by NGS and confirmed with ddPCR.

Journal of Clinical Oncology ◽

10.1200/jco.2020.38.15_suppl.e15563 ◽

2020 ◽

Vol 38 (15_suppl) ◽

pp. e15563-e15563

Author(s):

Hala Boulos ◽

Robert Tell ◽

Nike Beaubier ◽

Richard Blidner

Keyword(s):

Liquid Biopsy ◽

Detection Threshold ◽

Treatment Resistance ◽

Digital Pcr ◽

Circulating Tumor Dna ◽

Precision Oncology ◽

Single Nucleotide Variants ◽

Liquid Biopsies ◽

Low Frequencies ◽

Tumor Dna

e15563 Background: Liquid biopsies are increasingly utilized as a non-invasive tool in precision oncology to assess tumor mutational profiles in order to select targeted therapies, detect treatment resistance, and monitor disease progression in cancer patients. Additionally, liquid biopsies may provide a more comprehensive representation of tumor heterogeneity than standard tissue biopsies. However, limitations such as scarcity of circulating tumor DNA (ctDNA) and/or variants at low frequencies can be technically challenging to detect by next-generation sequencing (NGS) assays. Here, we use NGS to detect greater than two KRAS/NRAS mutations coexisting in single samples at low variant allele frequencies (VAFs). Methods: The Tempus xF liquid biopsy NGS assay is designed to detect actionable oncologic targets spanning 105 genes in plasma. The assay was validated to reliably detect single-nucleotide variants at 0.25% VAF, indels and copy number variants at 0.5% VAF, and fusions at 1% VAF with 96.2%-100% specificity and 97.4%-100% sensitivity. Pre-designed digital PCR assays were modified to measure 10ng of cell-free DNA (cfDNA) on a droplet-digital PCR (ddPCR) platform. Results: Overall, we report 100% positive predictive value and high correlation between ddPCR results and xF VAF, as well as in individual variants, such as KRAS G12D. Unexpectedly, we detected more than two coexisting KRAS/NRAS mutations at a low VAF in the plasma samples. To orthogonally confirm these results, ddPCR was deployed to independently measure the presence of each cfDNA variant with a sensitivity of 0.09% VAF. Subsequent ddPCR analysis of all targeted variants were concordant with NGS results. Conclusions: The occurrence of multiple KRAS and NRAS mutations in a single sample is quite uncommon and may be falsely interpreted as an NGS artifact. However, verification of this phenomenon by ddPCR confirmed the validity of the NGS liquid biopsy approach. These results highlight the capability of the Tempus xF assay to detect low-frequency variants, including those that fall below the validated detection threshold, which is essential for the diagnosis of early disease.

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The detection of specific hypermethylated WIF1 and NPY genes in circulating DNA by crystal digital PCR™ is a powerful new tool for colorectal cancer diagnosis and screening

BMC Cancer ◽

10.1186/s12885-021-08816-2 ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Alexis Overs ◽

Mylène Flammang ◽

Eric Hervouet ◽

Laurent Bermont ◽

Jean-Luc Pretet ◽

...

Keyword(s):

Liquid Biopsy ◽

Digital Pcr ◽

Circulating Tumor Dna ◽

Point Of View ◽

University Hospital ◽

Liquid Biopsies ◽

Tissue Samples ◽

Mutation Status ◽

Tumor Tissues ◽

Tumor Stages

Abstract Background In oncology, liquid biopsy is of major relevance from theranostic point of view. The searching for mutations in circulating tumor DNA (ctDNA) in case of colorectal cancers (CRCs) allows the optimization of patient care. In this context, independent of mutation status biomarkers are required for its detection to confirm the presence of ctDNA in liquid biopsies. Indeed, the hypermethylation of NPY and WIF1 genes appear to be an ideal biomarker for the specific detection of ctDNA in CRCs. The objective of this work is to develop the research of hypermethylation of NPY and WIF1 by Crystal Digital PCR™ for the detection of ctDNA in CRCs. Methods Detection of hypermethylated NPY and WIF1 was developed on Cristal digital PCR™. Biological validation was performed from a local cohort of 22 liquid biopsies and 23 tissue samples from patients with CRC. These patients were treated at the University Hospital of Besancon (France). Results The local cohort study confirmed that NPY and WIF1 were significantly hypermethylated in tumor tissues compared to adjacent non-tumor tissues (WIF1 p < 0.001; NPY p < 0.001; non-parametric Wilcoxon paired-series test). Histological characteristics, tumor stages or mutation status were not correlated to the methylation profiles. On the other hand, hypermethylation of NPY or WIF1 in liquid biopsy had a 95.5% [95%CI 77–100%] sensitivity and 100% [95%CI 69–100%] specificity. Conclusion Using Crystal digital PCR™, this study shows that hypermethylation of NPY and WIF1 are constant specific biomarkers of CRCs regardless of a potential role in carcinogenesis.

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An optimized ultra-deep massively parallel sequencing with unique molecular identifier tagging for detection and quantification of circulating tumor DNA from lung cancer patients.

Journal of Global Oncology ◽

10.1200/jgo.2019.5.suppl.55 ◽

2019 ◽

Vol 5 (suppl) ◽

pp. 55-55

Author(s):

Hong-Anh Thi Pham ◽

Le Son Tran ◽

Uyen Vu Tran ◽

Thanh-Truong Tran ◽

Hoai-Nghia Nguyen ◽

...

Keyword(s):

Liquid Biopsy ◽

Massively Parallel Sequencing ◽

Circulating Tumor Dna ◽

Massively Parallel ◽

Tissue Samples ◽

Platform Comparison ◽

Cross Platform ◽

Unique Molecular Identifier ◽

Tumor Dna ◽

Detection And Quantification

55 Background: The identification and quantification of actionable mutations are of critical importance for effective genotype-directed therapies, prognosis and drug response monitoring in patients with non-small-cell lung cancer (NSCLC). Although tumor tissue biopsy remains the gold standard for diagnosis of NSCLC, the analysis of plasma circulating tumor DNA (ctDNA), known as liquid biopsy, has recently emerged as an alternative and noninvasive approach for exploring tumor genetic constitution. In this study, we developed a mutation detection approach for liquid biopsy using ultra-deep massively parallel sequencing (MPS) with unique molecular identifier (UID) tagging and evaluated its performance for the identification and quantification of tumor-derived mutations from plasma of patients with advanced NSCLC. Methods: Tissue biopsy and plasma samples were collected from a total of 58 patients diagnosed with NSCLC in Vietnam. Genetic alterations in four driver genes including EGFR, KRAS, NRAS and BRAF were identified by using ultra-deep MPS combined with UID tagging. Subsequently, the concordance rate of mutation testing between matched plasma and tissue samples was assessed. Additionally, a commercially available ddPCR (Bio-rad) assay was used to conduct a cross-platform comparison with ultra-deep MPS for the detection and quantification of the three most common actionable EGFR mutations (del19, L858R and T790M). Results: Compared to the mutations detected in paired tissue samples, the plasma based ultra-deep MPS achieved high concordance rate of 87.5%. Cross-platform comparison with droplet digital PCR demonstrated comparable detection performance (91.4% concordance, Cohen's kappa coefficient of 0.85 with 95% CI = 0.72 – 0.97) and great reliability in quantification of mutation allele frequency (Intraclass correlation coefficient of 0.96 with 95% CI = 0.90 – 0.98). Conclusions: Our results highlight the potential application of liquid biopsy using ultra-deep MPS as a routine assay in clinical practice for both detection and quantification of actionable mutation landscape in NSCLC patients.

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