Circulating tumor DNA tracking through driver mutations as a liquid biopsy-based biomarker for uveal melanoma

Abstract Background Uveal melanoma (UM) is the most common intraocular tumor in adults. Despite good primary tumor control, up to 50% of patients develop metastasis, which is lethal. UM often presents asymptomatically and is usually diagnosed by clinical examination and imaging, making it one of the few cancer types diagnosed without a biopsy. Hence, alternative diagnostic tools are needed. Circulating tumor DNA (ctDNA) has shown potential as a liquid biopsy target for cancer screening and monitoring. The aim of this study was to evaluate the feasibility and clinical utility of ctDNA detection in UM using specific UM gene mutations. Methods We used the highly sensitive digital droplet PCR (ddPCR) assay to quantify UM driver mutations (GNAQ, GNA11, PLCβ4 and CYSTLR2) in cell-free DNA (cfDNA). cfDNA was analyzed in six well established human UM cell lines with known mutational status. cfDNA was analyzed in the blood and aqueous humor of an UM rabbit model and in the blood of patients. Rabbits were inoculated with human UM cells into the suprachoroidal space, and mutated ctDNA was quantified from longitudinal peripheral blood and aqueous humor draws. Blood clinical specimens were obtained from primary UM patients (n = 14), patients presenting with choroidal nevi (n = 16) and healthy individuals (n = 15). Results The in vitro model validated the specificity and accuracy of ddPCR to detect mutated cfDNA from UM cell supernatant. In the rabbit model, plasma and aqueous humor levels of ctDNA correlated with tumor growth. Notably, the detection of ctDNA preceded clinical detection of the intraocular tumor. In human specimens, while we did not detect any trace of ctDNA in healthy controls, we detected ctDNA in all UM patients. We observed that UM patients had significantly higher levels of ctDNA than patients with nevi, with a strong correlation between ctDNA levels and malignancy. Noteworthy, in patients with nevi, the levels of ctDNA highly correlated with the presence of clinical risk factors. Conclusions We report, for the first time, compelling evidence from in vitro assays, and in vivo animal model and clinical specimens for the potential of mutated ctDNA as a biomarker of UM progression. These findings pave the way towards the implementation of a liquid biopsy to detect and monitor UM tumors.

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Plasma circulating tumor DNA-based genetic profiling of lung cancer patients in Vietnam using ultra-deep massive parallel sequencing with unique identifier tagging.

Journal of Global Oncology ◽

10.1200/jgo.2019.5.suppl.58 ◽

2019 ◽

Vol 5 (suppl) ◽

pp. 58-58

Author(s):

Huy Phuoc Do ◽

Thao Thanh Tran Nguyen ◽

Uyen Vu Tran ◽

Thanh-Truong Tran ◽

Anh-Thu Huynh Dang ◽

...

Keyword(s):

Lung Cancer ◽

Liquid Biopsy ◽

Circulating Tumor Dna ◽

Massive Parallel Sequencing ◽

Driver Mutations ◽

Unique Identifier ◽

Parallel Sequencing ◽

Cancer Driver ◽

Lung Cancer Patients ◽

Tumor Dna

58 Background: Lung cancer is by far the leading cause of cancer death worldwide, with non-small cell lung cancer (NSCLC) accounting for the majority of cases. Genotype-directed therapy becomes a promising method for cancer treatment beside surgery and chemo-radiotherapy. Liquid biopsy using massive parallel sequencing has emerged as a non-invasive alternative procedure in profiling cancer driver mutations. In this study, we report the spectrum of clinically actionable mutations in plasma circulating tumor DNA of 299 non-small cell lung cancer patients using ultra-deep massive parallel sequencing with unique identifier tagging. Methods: Plasma circulating tumor DNA was extracted, ligated with unique identifier (Swift Bioscience), enriched of the target coding regions of EGFR, KRAS, NRAS, BRAF and the breakpoints of ALK, ROS1 (IDT) and sequenced using NextSeq 550 (Illumina) at mean coverage depth of 20,000X. Results: Out of 299 patients tested, 128 (42,8%) carried driver mutations. Genetic alterations were identified in EGFR (79 samples, 26,4%), KRAS (30 samples, 10%), ALK (7 samples, 2,34%), ROS1 (6 samples, 2%), BRAF (3 samples, 1%). There was no sample with NRAS mutation. In 79 EGFR-cases, there were 23 carry two pathogenic variants. 28 mutation types of EGFR were found including 19 indels and 9 missense variants L858R and T790M were the major ones. One case was found with concomitant EGFR and BRAF. Our study showed the spectrum and frequency of the cancer driver mutations detected in liquid biopsy was correlated to those detected in tissue biopsy samples. Conclusions: For the first time the spectrum of mutation types in liquid biopsy of Vietnamese NSCLC patients were investigated and showed the correlation with those detected in tissue biopsy samples.

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Faculty Opinions recommendation of Clinical applications of circulating tumor cells and circulating tumor DNA as liquid biopsy.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.726213668.793535268 ◽

2017 ◽

Author(s):

Wafik El-Deiry

Keyword(s):

Tumor Cells ◽

Circulating Tumor Cells ◽

Liquid Biopsy ◽

Circulating Tumor Dna ◽

Clinical Applications ◽

Tumor Dna

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Mutated circulating tumor DNA as a liquid biopsy in lung cancer detection and treatment

Molecular Oncology ◽

10.1002/1878-0261.12983 ◽

2021 ◽

Author(s):

Martyna Filipska ◽

Rafael Rosell

Keyword(s):

Lung Cancer ◽

Cancer Detection ◽

Liquid Biopsy ◽

Circulating Tumor Dna ◽

Lung Cancer Detection ◽

Tumor Dna

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Diving into the Pleural Fluid: Liquid Biopsy for Metastatic Malignant Pleural Effusions

Cancers ◽

10.3390/cancers13112798 ◽

2021 ◽

Vol 13 (11) ◽

pp. 2798

Author(s):

Maria Alba Sorolla ◽

Anabel Sorolla ◽

Eva Parisi ◽

Antonieta Salud ◽

José M. Porcel

Keyword(s):

Pleural Fluid ◽

Liquid Biopsy ◽

Cell Types ◽

Circulating Tumor Dna ◽

Therapeutic Interventions ◽

Driver Mutations ◽

Pleural Effusions ◽

Malignant Pleural Effusions ◽

Micro Rnas ◽

Low Sensitivity

Liquid biopsy is emerging as a promising non-invasive diagnostic tool for malignant pleural effusions (MPE) due to the low sensitivity of conventional pleural fluid (PF) cytological examination and the difficulty to obtain tissue biopsies, which are invasive and require procedural skills. Currently, liquid biopsy is increasingly being used for the detection of driver mutations in circulating tumor DNA (ctDNA) from plasma specimens to guide therapeutic interventions. Notably, malignant PF are richer than plasma in tumor-derived products with potential clinical usefulness, such as ctDNA, micro RNAs (miRNAs) and long non-coding RNAs (lncRNAs), and circulating tumor cells (CTC). Tumor-educated cell types, such as platelets and macrophages, have also been added to this diagnostic armamentarium. Herein, we will present an overview of the role of the preceding biomarkers, collectively known as liquid biopsy, in PF samples, as well as the main technical approaches used for their detection and quantitation, including a proper sample processing. Technical limitations of current platforms and future perspectives in the field will also be addressed. Using PF as liquid biopsy shows promise for use in current practice to facilitate the diagnosis and management of metastatic MPE.

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Molecular disease monitoring using circulating tumor DNA in myelodysplastic syndromes

Blood ◽

10.1182/blood-2016-09-740308 ◽

2017 ◽

Vol 129 (12) ◽

pp. 1685-1690 ◽

Cited By ~ 29

Author(s):

Paul Yeh ◽

Michael Dickinson ◽

Sarah Ftouni ◽

Tane Hunter ◽

Devbarna Sinha ◽

...

Keyword(s):

Treatment Failure ◽

Myelodysplastic Syndromes ◽

Circulating Tumor Dna ◽

Driver Mutations ◽

Disease Monitoring ◽

Key Points ◽

Tumor Dna ◽

Predict Treatment Failure

Key PointsCirculating tumor DNA can monitor disease and predict treatment failure by tracking driver mutations and karyotypic abnormalities in MDS.

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Liquid biopsy in NSCLC: a new challenge in radiation therapy

Exploration of Targeted Anti-tumor Therapy ◽

10.37349/etat.2021.00038 ◽

2021 ◽

Author(s):

Annarita Perillo ◽

Mohamed Vincenzo Agbaje Olufemi ◽

Jacopo De Robbio ◽

Rossella Margherita Mancuso ◽

Anna Roscigno ◽

...

Keyword(s):

Lung Cancer ◽

Liquid Biopsy ◽

Residual Disease ◽

Biological Fluids ◽

Circulating Tumor Dna ◽

Minimum Residual ◽

Nsclc Patients ◽

Choice Of Therapy ◽

Tumor Dna

Lung cancer is the most common cancer and the leading cause of cancer mortality worldwide. To date, tissue biopsy has been the gold standard for the diagnosis and the identification of specific molecular mutations, to guide choice of therapy. However, this procedure has several limitations. Liquid biopsy could represent a solution to the intrinsic limits of traditional biopsy. It can detect cancer markers such as circulating tumor DNA or RNA (ctDNA, ctRNA), and circulating tumor cells, in plasma, serum or other biological fluids. This procedure is minimally invasive, reproducible and can be used repeatedly. The main clinical applications of liquid biopsy in non-small cell lung cancer (NSCLC) patients are the early diagnosis, stratification of the risk of relapse, identification of mutations to guide application of targeted therapy and the evaluation of the minimum residual disease. In this review, the current role of liquid biopsy and associated markers in the management of NSCLC patients was analyzed, with emphasis on ctDNA and CTCs, and radiotherapy.

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Value of Liquid Biopsy Monitoring of Circulating Tumor DNA in HPV-Associated Oropharyngeal Cancer

Practica oto-rhino-laryngologica Suppl ◽

10.5631/jibirinsuppl.156.1 ◽

2021 ◽

Vol 156 (0) ◽

pp. 1-7

Author(s):

Atsushi Imai ◽

Kiyoshi Misawa ◽

Satoshi Yamada ◽

Jun Okamura ◽

Daiki Mochizuki ◽

...

Keyword(s):

Oropharyngeal Cancer ◽

Liquid Biopsy ◽

Circulating Tumor Dna ◽

Tumor Dna

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Phase II study of anti-EGFR rechallenge therapy with panitumumab driven by circulating tumor DNA molecular selection in metastatic colorectal cancer: The CHRONOS trial.

Journal of Clinical Oncology ◽

10.1200/jco.2021.39.15_suppl.3506 ◽

2021 ◽

Vol 39 (15_suppl) ◽

pp. 3506-3506

Author(s):

Andrea Sartore-Bianchi ◽

Filippo Pietrantonio ◽

Sara Lonardi ◽

Benedetta Mussolin ◽

Francesco Rua ◽

...

Keyword(s):

Colorectal Cancer ◽

Clinical Trial ◽

Metastatic Colorectal Cancer ◽

Phase Ii ◽

Primary Endpoint ◽

Liquid Biopsy ◽

Objective Response ◽

Circulating Tumor Dna ◽

Type I ◽

Tumor Dna

3506 Background: Despite advances in molecular segmentation of metastatic colorectal cancer (mCRC), beyond RAS status therapeutic actionability remains confined to the limited subgroups of ERBB2 amplified, BRAF mutated and MSI-H patients. Optimization of available treatments is therefore warranted. Rechallenge with anti-EGFR monoclonal antibodies is often empirically used with some benefit as late-line therapy. We previously found that mutant RAS and EGFR ectodomain clones, which emerge in blood during EGFR blockade, decline upon antibody withdrawal leading to regain drug sensitivity. Based on this rationale, we designed CHRONOS, a multicenter phase II trial of anti-EGFR therapy rechallenge guided by monitoring of the mutational status of RAS, BRAF and EGFR in circulating tumor DNA (ctDNA). To our knowledge, this is the first interventional clinical trial of liquid biopsy for driving anti-EGFR rechallenge therapy in mCRC. Methods: Eligible patients were PS ECOG 0-2 RAS/BRAF WT mCRC having first achieved an objective response and then progression in any treatment line with an anti-EGFR antibody containing regimen, displaying RAS, BRAF and EGFR ectodomain WT status in ctDNA at molecular screening after progression to the last anti-EGFR-free regimen. Clonal evolution in ctDNA was analyzed by ddPCR and next generation sequencing. Panitumumab 6 mg/kg was administered IV every two weeks until progression. The primary endpoint was objective response rate (ORR) by RECIST version 1.1 with independent central review. 27 total patients and 6 responses were required to declare the study positive (power = 85%, type I error = 0.05). Results: Between Aug 19, 2019 and Nov 6, 2020 52 patients were screened by liquid biopsy and 36 (69%) were negative in ctDNA for RAS/BRAF/EGFR mutations. Of these, 27 patients were enrolled in 4 centers. Median age was 64 years (range: 42-80). PS ECOG was 0/50%, 1/46%, 2/4%. Previous anti-EGFR was administered in 1st line in 63%, 2nd in 15% and > 2nd in 22%. Median number of previous treatments was 3. The primary endpoint was met, with 8/27 partial responses (PR) observed (2 unconfirmed) (ORR = 30%, 95% CI: 12-47%). Stable disease (SD) was obtained in 11/27 (40%, 95% CI: 24-59%), lasting > 4 months in 8/11. Disease control rate (PR plus SD > 4 months) was therefore obtained in 16/27 (59%, 95% CI: 41-78%). Median progression-free survival was 16 weeks. Median duration of response was 17 weeks (1 ongoing). Maximal grade toxicity was G3, limited to dermatological and occurring in 19% of patients. ctDNA dynamics were studied in all patients. Conclusions: Liquid biopsy-driven rechallenge with anti-EGFR antibodies leads to further objective responses in one third of patients. Genotyping tumor DNA in the blood to direct therapy can be effectively incorporated in the management of advanced CRCs. Clinical trial information: 2016-002597-12.

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