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.

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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High-Accuracy Determination of Microsatellite Instability Compatible with Liquid Biopsies

Clinical Chemistry ◽

10.1093/clinchem/hvaa013 ◽

2020 ◽

Vol 66 (4) ◽

pp. 606-613 ◽

Cited By ~ 3

Author(s):

Amanda Bortolini Silveira ◽

François-Clément Bidard ◽

Amélie Kasperek ◽

Samia Melaabi ◽

Marie-Laure Tanguy ◽

...

Keyword(s):

Microsatellite Instability ◽

Large Scale ◽

Digital Pcr ◽

Circulating Tumor Dna ◽

Diagnostic Tools ◽

Tumor Biomarker ◽

Analytical Sensitivity ◽

Liquid Biopsies ◽

Tumor Dna ◽

Large Scale Screening

Abstract Background Microsatellite instability (MSI) has recently emerged as a predictive pan-tumor biomarker of immunotherapy efficacy, stimulating the development of diagnostic tools compatible with large-scale screening of patients. In this context, noninvasive detection of MSI from circulating tumor DNA stands as a promising diagnostic and posttreatment monitoring tool. Methods We developed drop-off droplet-digital PCR (ddPCR) assays targeting BAT-26, activin A receptor type 2A (ACVR2A), and defensin beta 105A/B (DEFB105A/B) microsatellite markers. Performances of the assays were measured on reconstitution experiments of various mutant allelic fractions, on 185 tumor samples with known MSI status, and on 72 blood samples collected from 42 patients with advanced colorectal or endometrial cancers before and/or during therapy. Results The 3 ddPCR assays reached analytical sensitivity <0.1% variant allelic frequency and could reliably detect and quantify MSI in both tumor and body fluid samples. High concordance between MSI status determination by the three-marker ddPCR test and the reference pentaplex method were observed (100% for colorectal tumors and 93% for other tumor types). Moreover, the 3 assays showed correlations with r ≥ 0.99 with other circulating tumor DNA markers and their dynamic during treatment correlated well with clinical response. Conclusions This innovative approach for MSI detection provides a noninvasive, cost-effective, and fast diagnostic tool, well suited for large-scale screening of patients that may benefit from immunotherapy agents, as well as for monitoring treatment responses.

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Rational Development of Liquid Biopsy Analysis in Renal Cell Carcinoma

Cancers ◽

10.3390/cancers13225825 ◽

2021 ◽

Vol 13 (22) ◽

pp. 5825

Author(s):

Kate I. Glennon ◽

Mahafarin Maralani ◽

Narges Abdian ◽

Antoine Paccard ◽

Laura Montermini ◽

...

Keyword(s):

Renal Cell Carcinoma ◽

Cell Carcinoma ◽

Liquid Biopsy ◽

Renal Cell ◽

Somatic Mutations ◽

Circulating Tumor Dna ◽

Precision Oncology ◽

Liquid Biopsies ◽

Disease Evolution ◽

Ctdna Analysis

Renal cell carcinoma (RCC) is known for its variable clinical behavior and outcome, including heterogeneity in developing relapse or metastasis. Recent data highlighted the potential of somatic mutations as promising biomarkers for risk stratification in RCC. Likewise, the analysis of circulating tumor DNA (ctDNA) for such informative somatic mutations (liquid biopsy) is considered an important advance for precision oncology in RCC, allowing to monitor molecular disease evolution in real time. However, our knowledge about the utility of ctDNA analysis in RCC is limited, in part due to the lack of RCC-appropriate assays for ctDNA analysis. Here, by interrogating different blood compartments in xenograft models, we identified plasma cell-free (cf) DNA and extracellular vesicles (ev) DNA enriched for RCC-associated ctDNA. Additionally, we developed sensitive targeted sequencing and bioinformatics workflows capable of detecting somatic mutations in RCC-relevant genes with allele frequencies ≥ 0.5%. Applying this assay to patient-matched tumor and liquid biopsies, we captured tumor mutations in cf- and ev-DNA fractions isolated from the blood, highlighting the potentials of both fractions for ctDNA analysis. Overall, our study presents an RCC-appropriate sequencing assay and workflow for ctDNA analysis and provides a proof of principle as to the feasibility of detecting tumor-specific mutations in liquid biopsy in RCC patients.

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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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Difference in appearance of KRAS mutated circulating tumor DNA in patients with metastatic colorectal cancer during treatments.

Journal of Clinical Oncology ◽

10.1200/jco.2017.35.15_suppl.e23025 ◽

2017 ◽

Vol 35 (15_suppl) ◽

pp. e23025-e23025

Author(s):

Yuji Takayama ◽

Koichi Suzuki ◽

Kosuke Ichida ◽

Taro Fukui ◽

Fumiaki Watanabe ◽

...

Keyword(s):

Colorectal Cancer ◽

Metastatic Colorectal Cancer ◽

Kras Mutation ◽

Treatment Resistance ◽

Digital Pcr ◽

Circulating Tumor Dna ◽

Oil Droplets ◽

Tumor Tissues ◽

The Difference ◽

Tumor Dna

e23025 Background: Emergence of KRAS mutation in blood is observed in colorectal cancer patients who undergo chemotherapy, but its clinical significance is not well known. In this study, we focused on the difference in appearance of KRAS mutated circulating tumor DNA (MctDNA) and elucidated its association with treatments. Methods: Four hundred and fifty-one plasma samples were collected prospectively from 85 patients (pts) who underwent chemotherapy due to metastatic colorectal cancer in 2014 - 2016. Seven types of KRAS mutation in MctDNA were detected by droplet digital PCR creating oil droplets. To exclude false positive detection, mutation was validated. MctDNA amplified in oil droplets was selectively sorted by On-chip sorting system and mutation was determined by Sanger sequencing. Results: KRAS assessment in tumor tissues showed 29 pts with KRAS mutation (MT), 56 pts without KRAS mutation (WT). Among 29 pts with MT, KRAS assessment in plasma displayed 23 pts with MctDNA and 6 pts without MctDNA. The type of mutation in MctDNA was consistent with that detected in tumor tissues, indicating mutual exclusivity in KRAS mutation was confirmed. In 56 pts with WT, 28 pts showed MctDNA during treatments. Difference in appearance of MctDNA was recognized in several treatments. Gradual increase in detection of MctDNA was observed with anti-EGFR antibody, resulted in treatment resistance. Transient spike elevation was frequently seen in TAS-102, which associated with drug response. No specific appearance was recognized during treatments with other drugs including anti-VEGF antibody. MctDNA in oil droplets were successfully sorted even if a few droplets were targeted, and mutation was confirmed. Conclusions: Difference in appearance of MctDNA may associate with treatment response in patients with metastatic colorectal cancer during treatments. [Table: see text]

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

Cancers ◽

10.3390/cancers13184581 ◽

2021 ◽

Vol 13 (18) ◽

pp. 4581

Author(s):

Jin-Yi Han ◽

Keun Soo Ahn ◽

Tae-Seok Kim ◽

Yong Hoon Kim ◽

Kwang Bum Cho ◽

...

Keyword(s):

Biliary Tract ◽

Liquid Biopsy ◽

Survival Rates ◽

Digital Pcr ◽

Circulating Tumor Dna ◽

Strong Positive Correlation ◽

Kras Mutations ◽

Tissue Samples ◽

Ffpe Samples ◽

Tumor Dna

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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Plasma Circulating Tumor DNA Levels for the Monitoring of Melanoma Patients: Landscape of Available Technologies and Clinical Applications

BioMed Research International ◽

10.1155/2017/5986129 ◽

2017 ◽

Vol 2017 ◽

pp. 1-8 ◽

Cited By ~ 23

Author(s):

Benoit Busser ◽

Julien Lupo ◽

Lucie Sancey ◽

Stéphane Mouret ◽

Patrice Faure ◽

...

Keyword(s):

Acquired Resistance ◽

Digital Pcr ◽

Circulating Tumor Dna ◽

Clinical Applications ◽

Molecular Heterogeneity ◽

Liquid Biopsies ◽

Melanoma Patients ◽

Ctdna Analysis ◽

Next Generation Sequencing Ngs ◽

Tumor Dna

Melanoma is a cutaneous cancer with an increasing worldwide prevalence and high mortality due to unresectable or metastatic stages. Mutations inBRAF,NRAS, orKITare present in more than 60% of melanoma cases, but a useful blood-based biomarker for the clinical monitoring of melanoma patients is still lacking. Thus, the analysis of circulating tumor cells (CTCs) and/or cell-free circulating tumor DNA (ctDNA) analysis from blood (liquid biopsies) appears to be a promising noninvasive, repeatable, and systemic sampling tool for detecting and monitoring melanoma. Here, we review the molecular biology-based strategies used for ctDNA quantification in melanoma patients, as well as their main clinical applications. Droplet digital PCR (ddPCR) and next generation sequencing (NGS) technologies appear to be two versatile and complementary strategies to study rare variant mutations for the detection and monitoring of melanoma progression. Among the different clinical uses of ctDNA, we highlight the assessment of molecular heterogeneity and the identification of genetic determinants for targeted therapy as well as the analysis of acquired resistance. Importantly, ctDNA quantification might also be a novel biomarker with a prognostic value for melanoma patients.

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The Role of the Liquid Biopsy in Decision-Making for Patients with Non-Small Cell Lung Cancer

Journal of Clinical Medicine ◽

10.3390/jcm9113674 ◽

2020 ◽

Vol 9 (11) ◽

pp. 3674

Author(s):

D. Akhoundova ◽

J. Mosquera Martinez ◽

L. E. Musmann ◽

C. Britschgi ◽

C. Rütsche ◽

...

Keyword(s):

Lung Cancer ◽

Clinical Practice ◽

Cancer Patients ◽

Liquid Biopsy ◽

Clinical Management ◽

Circulating Tumor Dna ◽

Precision Oncology ◽

Liquid Biopsies ◽

Lung Cancer Patients ◽

Molecular Alterations

Liquid biopsy is a rapidly emerging tool of precision oncology enabling minimally invasive molecular diagnostics and longitudinal monitoring of treatment response. For the clinical management of advanced stage lung cancer patients, detection and quantification of circulating tumor DNA (ctDNA) is now widely adopted into clinical practice. Still, interpretation of results and validation of ctDNA-based treatment decisions remain challenging. We report here our experience implementing liquid biopsies into the clinical management of lung cancer. We discuss advantages and limitations of distinct ctDNA assay techniques and highlight our approach to the analysis of recurrent molecular alterations found in lung cancer. Moreover, we report three exemplary clinical cases illustrating the complexity of interpreting liquid biopsy results in clinical practice. These cases underscore the potential and current limitations of liquid biopsy, focusing on the difficulty of interpreting discordant findings. In our view, despite all current limitations, the analysis of ctDNA in lung cancer patients is an essential and highly versatile complementary diagnostic tool for the clinical management of lung cancer patients in the era of precision oncology.

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Extracellular Vesicle-Derived DNA vs. CfDNA as a Biomarker for the Detection of Colon Cancer

Genes ◽

10.3390/genes12081171 ◽

2021 ◽

Vol 12 (8) ◽

pp. 1171

Author(s):

Kavita Thakur ◽

Manu Smriti Singh ◽

Sara Feldstein-Davydova ◽

Victoria Hannes ◽

Dov Hershkovitz ◽

...

Keyword(s):

Colon Carcinoma ◽

Liquid Biopsy ◽

Tumor Tissue ◽

Mutation Detection ◽

Extracellular Vesicle ◽

Circulating Tumor Dna ◽

P Value ◽

Liquid Biopsies ◽

Targeted Ngs ◽

Tumor Dna

Liquid biopsy has emerged as a promising non-invasive way to diagnose tumor and monitor its progression. Different types of liquid biopsies have different advantages and limitations. In the present research, we compared the use of two types of liquid biopsy, extracellular vesicle-derived DNA (EV-DNA) and cell-free DNA (cfDNA) for identifying tumor mutations in patients with colon carcinoma. Method: DNA was extracted from the tumor tissue of 33 patients diagnosed with colon carcinoma. Targeted NGS panel, based on the hotspots panel, was used to identify tumor mutations. Pre-surgery serum and plasma were taken from the patients in which mutation was found in the tumor tissue. Extracellular vesicles were isolated from the serum followed by the extraction of EV-DNA. CfDNA was extracted from the plasma. The mutations found in the tumor were used to detect the circulating tumor DNA using ultra-deep sequencing. We compared the sensitivity of mutation detection and allele frequency obtained in EV-DNA and cfDNA. Results: The sensitivity of mutation detection in EV-DNA and cfDNA was 61.90% and 66.67%, respectively. We obtained almost identical sensitivity of mutation detection in EV-DNA and cfDNA in each of the four stages of colon carcinoma. The total DNA concentration and number mutant copies were higher in cfDNA vs. EV-DNA (p value = 0.002 and 0.003, respectively). Conclusion: Both cfDNA and EV-DNA can serve as tumor biomarkers. The use of EV-DNA did not lead to improved sensitivity or better detection of tumor DNA in the circulation.

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Circulating tumor DNA (ctDNA) kinetics to predict survival in patients (pts) with unresectable or metastatic melanoma treated with dabrafenib (D) or D + trametinib (T).

Journal of Clinical Oncology ◽

10.1200/jco.2019.37.15_suppl.9510 ◽

2019 ◽

Vol 37 (15_suppl) ◽

pp. 9510-9510

Author(s):

Mahrukh M Syeda ◽

JENNIFER M WIGGINS ◽

Broderick Corless ◽

Georgina V. Long ◽

Keith Flaherty ◽

...

Keyword(s):

Metastatic Melanoma ◽

Detection Threshold ◽

Digital Pcr ◽

Mek Inhibitor ◽

Circulating Tumor Dna ◽

Braf V600e ◽

Phase 3 ◽

Paired Samples ◽

Tumor Dna ◽

Clinical Trial Information

9510 Background: There are no validated blood-based biomarkers to monitor efficacy in pts with advanced melanoma. Lactate dehydrogenase (LDH) is an established prognostic factor; however, it is not normally used to inform treatment decisions. ctDNA at baseline (BL) is associated with a poor prognosis in pts treated with BRAF inhibitors, but no studies have examined the association between serial changes in ctDNA and survival after BRAF and/or MEK inhibitor therapy. Methods: We measured BRAF V600E/K ctDNA at BL and wk 4 in plasma samples from a pooled population of pts with unresectable or metastatic melanoma treated with D or D+T in the phase 3 COMBI-d trial (NCT01584648). We used mutation-specific droplet digital PCR assays; ctDNA results were categorized as positive/negative (pos/neg) using an analytically validated detection threshold of 0.25 copies/mL. Progression-free (PFS) and overall survival (OS) were analyzed in all pts and by BL LDH level (> or < upper limit of normal). Results: BL ctDNA was detectable in 320/345 pts (92.7%) and was not associated with survival. Nearly all pts had a considerable drop in ctDNA after 4 wks of therapy; 201 pts had paired samples (BL and wk 4) and detectable ctDNA at BL. In 80/201 pts (40%) whose ctDNA changed from pos at BL to neg at wk 4, PFS and OS were prolonged vs in 121/201 (60%) whose ctDNA remained pos (median PFS, 12.9 [95% CI, 9.2-20.2] mo vs 7.1 [5.5-8.9] mo; HR, 0.55 [0.39-0.76]; P = .0003; median OS, 28.2 [20.5-48.8] mo vs 14.6 [11.8-18.7] mo; HR, 0.56 [0.40-0.79]; P = .0007). Undetectable ctDNA at wk 4 was associated with prolonged PFS and OS, especially in pts with high BL LDH (Table). Conclusions: Particularly in pts with high LDH, on-treatment ctDNA monitoring may be helpful for early identification of pts likely to benefit from D or D+T. All ctDNA Samples at Wk 4. Clinical trial information: NCT01584648. [Table: see text]

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Translational Utility of Liquid Biopsies in Thyroid Cancer Management

Cancers ◽

10.3390/cancers13143443 ◽

2021 ◽

Vol 13 (14) ◽

pp. 3443

Author(s):

Ayanthi A. Wijewardene ◽

Marthe Chehade ◽

Matti L. Gild ◽

Roderick J. Clifton-Bligh ◽

Martyn Bullock

Keyword(s):

Thyroid Cancer ◽

Cancer Patients ◽

Liquid Biopsy ◽

Circulating Tumor Dna ◽

Liquid Biopsies ◽

Future Directions ◽

Cancer Management ◽

Diagnostic Role ◽

Tumor Dna

Liquid biopsies are a novel technique to assess for either circulating tumor cells (CTC) or circulating tumor DNA (ctDNA and microRNA (miRNA)) in peripheral blood samples of cancer patients. The diagnostic role of liquid biopsy in oncology has expanded in recent years, particularly in lung, colorectal and breast cancer. In thyroid cancer, the role of liquid biopsy in either diagnosis or prognosis is beginning to translate from the lab to the clinic. In this review, we describe the evolution of liquid biopsies in detecting CTC, ctDNA and miRNA in thyroid cancer patients, together with its limitations and future directions in clinical practice.

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