Genetic Characterization of Cancer of Unknown Primary Using Liquid Biopsy Approaches

Cancers of unknown primary (CUPs) comprise a heterogeneous group of rare metastatic tumors whose primary site cannot be identified after extensive clinical–pathological investigations. CUP patients are generally treated with empirical chemotherapy and have dismal prognosis. As recently reported, CUP genome presents potentially druggable alterations for which targeted therapies could be proposed. The paucity of tumor tissue, as well as the difficult DNA testing and the lack of dedicated panels for target gene sequencing are further relevant limitations. Here, we propose that circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA) could be used to identify actionable mutations in CUP patients. Blood was longitudinally collected from two CUP patients. CTCs were isolated with CELLSEARCH® and DEPArrayTM NxT and Parsortix systems, immunophenotypically characterized and used for single-cell genomic characterization with Ampli1TM kits. Circulating cell-free DNA (ccfDNA), purified from plasma at different time points, was tested for tumor mutations with a CUP-dedicated, 92-gene custom panel using SureSelect Target Enrichment technology. In parallel, FFPE tumor tissue was analyzed with three different assays: FoundationOne CDx assay, DEPArray LibPrep and OncoSeek Panel, and the SureSelect custom panel. These approaches identified the same mutations, when the gene was covered by the panel, with the exception of an insertion in APC gene. which was detected by OncoSeek and SureSelect panels but not FoundationOne. FGFR2 and CCNE1 gene amplifications were detected in single CTCs, tumor tissue, and ccfDNAs in one patient. A somatic variant in ARID1A gene (p.R1276∗) was detected in the tumor tissue and ccfDNAs. The alterations were validated by Droplet Digital PCR in all ccfDNA samples collected during tumor evolution. CTCs from a second patient presented a pattern of recurrent amplifications in ASPM and SEPT9 genes and loss of FANCC. The 92-gene custom panel identified 16 non-synonymous somatic alterations in ccfDNA, including a deletion (I1485Rfs∗19) and a somatic mutation (p. A1487V) in ARID1A gene and a point mutation in FGFR2 gene (p.G384R). Our results support the feasibility of non-invasive liquid biopsy testing in CUP cases, either using ctDNA or CTCs, to identify CUP genetic alterations with broad NGS panels covering the most frequently mutated genes.

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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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Liquid Biopsy Detects Relapse Five Months Earlier than Regular Clinical Follow-Up and Guides Targeted Treatment in Breast Cancer

Case Reports in Oncological Medicine ◽

10.1155/2019/6545298 ◽

2019 ◽

Vol 2019 ◽

pp. 1-4

Author(s):

Fiona Tsui-Fen Cheng ◽

Nina Lapke ◽

Chin-Chu Wu ◽

Yen-Jung Lu ◽

Shu-Jen Chen ◽

...

Keyword(s):

Breast Cancer ◽

Early Detection ◽

Liquid Biopsy ◽

Digital Pcr ◽

Circulating Tumor Dna ◽

Genetic Alterations ◽

Targeted Treatment ◽

Breast Cancer Patients ◽

Ctdna Analysis

Genetic alterations in circulating tumor DNA (ctDNA) are an emerging biomarker for the early detection of relapse and have the potential to guide targeted treatment. ctDNA analysis is often performed by droplet digital PCR; however, next-generation sequencing (NGS) allows multigene testing without having to access a tumor sample to identify target alterations. Here, we report the case of a stage III hormone receptor-positive breast cancer patient who remained symptomless after receiving surgery and adjuvant chemotherapy. Liquid biopsy analysis by NGS revealed the presence of a ctDNA PIK3CA N345K mutation five months before the detection of relapse with multiple liver metastases by regular clinical follow-up. To date, clinical implications of the PIK3CA N345K variant remain insufficiently investigated; however, everolimus treatment resulted in the shrinkage of tumor lesions and decreased the levels of tumor markers. Four months after treatment initiation, a second ctDNA analysis suggested a relapse, and the patient clinically progressed after five months of everolimus therapy. This case report demonstrates the value of ctDNA analysis by NGS for the early detection of relapse in breast cancer patients. The study further indicates its usefulness for the choice of targeted treatments, suggesting that the variant PIK3CA N345K might be associated with everolimus sensitivity.

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Liquid biopsy in peritoneal fluid and plasma as a prognostic factor in advanced colorectal and appendiceal tumors after complete cytoreduction and hyperthermic intraperitoneal chemotherapy

Therapeutic Advances in Medical Oncology ◽

10.1177/1758835920981351 ◽

2020 ◽

Vol 12 ◽

pp. 175883592098135

Author(s):

Irene López-Rojo ◽

Susana Olmedillas-López ◽

Pedro Villarejo Campos ◽

Víctor Domínguez Prieto ◽

Javier Barambio Buendía ◽

...

Keyword(s):

Prognostic Factor ◽

Intraperitoneal Chemotherapy ◽

Liquid Biopsy ◽

Peritoneal Fluid ◽

Hyperthermic Intraperitoneal Chemotherapy ◽

Digital Pcr ◽

Circulating Tumor Dna ◽

Before And After ◽

Colorectal Origin ◽

Systemic Relapse

Background: Positive cytology has been identified as an independent negative prognostic factor in patients with peritoneal metastases (PM) of colorectal origin. Liquid biopsy in plasma may detect increasing levels of circulating tumor DNA (ctDNA) and could help predict systemic relapse in patients with colorectal cancer, but little is known about the role of liquid biopsy in peritoneal fluid. The aim of this study was to evaluate the prognostic value of peritoneal fluid and plasma liquid biopsy in patients undergoing complete cytoreductive surgery and hyperthermic intraperitoneal chemotherapy (CC-HIPEC). Methods: A longitudinal prospective study was designed in patients with KRAS-mutated colorectal or appendiceal primary tumor, including PM of colorectal origin, pseudomyxoma peritonei and patients at high risk of developing PM (selected for second-look surgery). Eleven patients were recruited according to inclusion and exclusion criteria. ctDNA from plasma and peritoneal fluid before and after HIPEC was studied by droplet digital PCR looking for KRAS mutation. A close follow-up was scheduled (mean of 28.5 months) to monitor for systemic and peritoneal recurrences. Results: All patients with positive plasma postHIPEC had systemic relapse and four patients died as a result, while those with negative plasma postHIPEC did not relapse. Patients with negative peritoneal ctDNA after CC-HIPEC did not present peritoneal relapse. Of six patients with positive peritoneal ctDNA postHIPEC, two presented peritoneal recurrence and four systemic relapses. Conclusions: Treatment with CC-HIPEC does not always neutralize ctDNA in peritoneal fluid, and its persistence after treatment may predict adverse outcome. Despite being a proof of concept, an adequate correlation between liquid biopsy in plasma and peritoneal fluid with both systemic and peritoneal relapse has been observed.

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Multiple Hotspot Mutations Scanning by Single Droplet Digital PCR

Clinical Chemistry ◽

10.1373/clinchem.2017.272518 ◽

2018 ◽

Vol 64 (2) ◽

pp. 317-328 ◽

Cited By ~ 21

Author(s):

Charles Decraene ◽

Amanda B Silveira ◽

François-Clément Bidard ◽

Audrey Vallée ◽

Marc Michel ◽

...

Keyword(s):

Liquid Biopsy ◽

Limit Of Detection ◽

Clinical Importance ◽

Digital Pcr ◽

Circulating Tumor Dna ◽

Droplet Digital Pcr ◽

Egfr Mutations ◽

Detection Accuracy ◽

Exon 2 ◽

Exon 19

Abstract BACKGROUND Progress in the liquid biopsy field, combined with the development of droplet digital PCR (ddPCR), has enabled noninvasive monitoring of mutations with high detection accuracy. However, current assays detect a restricted number of mutations per reaction. ddPCR is a recognized method for detecting alterations previously characterized in tumor tissues, but its use as a discovery tool when the mutation is unknown a priori remains limited. METHODS We established 2 ddPCR assays detecting all genomic alterations within KRAS exon 2 and EGFR exon 19 mutation hotspots, which are of clinical importance in colorectal and lung cancer, with use of a unique pair of TaqMan® oligoprobes. The KRAS assay scanned for the 7 most common mutations in codons 12/13 but also all other mutations found in that region. The EGFR assay screened for all in-frame deletions of exon 19, which are frequent EGFR-activating events. RESULTS The KRAS and EGFR assays were highly specific and both reached a limit of detection of <0.1% in mutant allele frequency. We further validated their performance on multiple plasma and formalin-fixed and paraffin-embedded tumor samples harboring a panel of different KRAS or EGFR mutations. CONCLUSIONS This method presents the advantage of detecting a higher number of mutations with single-reaction ddPCRs while consuming a minimum of patient sample. This is particularly useful in the context of liquid biopsy because the amount of circulating tumor DNA is often low. This method should be useful as a discovery tool when the tumor tissue is unavailable or to monitor disease during therapy.

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OS1.4 Liquid biopsy of the CSF in a series of GBM patients: preliminary results

Neuro-Oncology ◽

10.1093/neuonc/noz126.017 ◽

2019 ◽

Vol 21 (Supplement_3) ◽

pp. iii6-iii6

Author(s):

R Rudà ◽

F Bruno ◽

F De Bacco ◽

F Orzan ◽

P Cassoni ◽

...

Keyword(s):

Liquid Biopsy ◽

Point Mutations ◽

Digital Pcr ◽

Genetic Alterations ◽

Copy Number Variations ◽

Molecular Profile ◽

Genetic Profile ◽

Radiological Features ◽

Meningeal Involvement ◽

Idh Mutations

Abstract BACKGROUND Liquid biopsy (LB) by cerebrospinal fluid (CSF) can be useful to identify circulating tumour DNA (ctDNA), thus offering information about the heterogeneity of the neoplastic genome. The aim of our study is to assess the effectiveness of LB of the CSF in detecting ctDNA which mirrors the genetic profile of the tumoural tissue, and to investigate the clinical and radiological aspects influencing the availability of ctDNA. MATERIAL AND METHODS Tumoral tissue and CSF samples of 13 GBM patients undergoing surgery was collected. CSF was withdrawn from the very proximity of the tumoural surface before the excision. DNA extracted from tissue samples was analysed by qPCR to identify typical genetic alterations such as copy number variations (EGFR, PDGFRA, CDK4, MDM2, CDKN2A), and point mutations (TP53, PTEN, IDH, NRAS, PI3K1, pTERT). CtDNA extracted from CSF was analysed by droplet digital PCR to assess the presence of the alterations found in the matching tissue. Both contrast-enhanced (CE) and FLAIR volumes of the lesions were measured in the pre-surgical MRI. Linear and logarithmic regressions were employed for the statistical analysis. RESULTS From June 2016 to February 2017 we prospectively collected 13 GBM patients. Median age was 73 years. All lesions showed CE at the MRI; other radiological findings included necrosis (84.6%), oedema (76.9%), cortical, ventricular or meningeal involvement (76.9%, 30.8%, and 15.4%). Median volumes of CE and FLAIR lesions were 28.6 and 25.5 cm3, with a median FLAIR/CE ratio of 72.9. Surgery was subtotal (<95%) in all patients. All GBM tissues were tested for the following alterations: EGFR, PDGFRA, CDK4, MDM2, CDKN2A; 76.9% were tested for TP53, PTEN, and IDH mutations; 38.5% for NRAS and pTERT mutations; 30.8% for PI3KR1 mutation. MGMT methylation was assessed in 12 cases (92.3%) and found in 7 (58.3%). Median CSF volume, ctDNA quantity and concentration were 0.45 mL, 59.64 ng, and 0.42 ng/μL. Processable DNA was found in 11 CSF specimens (84.6%), in 8 of which (61.5%) it carried the same alteration expressed by the tumoural cells of the matched tissue, while in 3 cases (23.1%) it seemed to have a different genetic profile; finally, in 2 cases it was not possible to detect any circulating DNA in the CSF. Preliminary data on 13 patients suggest that the ctDNA concentration in the CSF could be related to the FLAIR/CE ratio as measured in the MRI before surgery (p = 0.02). Other correlations between the molecular and the radiological features are still being exploring. CONCLUSION Our study confirms that LB of CSF can detect ctDNA carrying the same molecular profile harboured in the tumour. Therefore, it seems to be an accurate method to identify markers useful for the diagnosis and the monitoring of the disease. Additionally, our ongoing study is trying to demonstrate a potential correlation between radiological features of the tumour and availability of ctDNA in CSF.

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Liquid biopsy leads to a paradigm shift in cancer treatment.

Journal of Clinical Oncology ◽

10.1200/jco.2016.34.4_suppl.602 ◽

2016 ◽

Vol 34 (4_suppl) ◽

pp. 602-602

Author(s):

Koichi Suzuki ◽

Yuji Takayama ◽

Kosuke Ichida ◽

Taro Fukui ◽

Nao Kakizawa ◽

...

Keyword(s):

Liquid Biopsy ◽

Multiple Testing ◽

Poor Response ◽

Digital Pcr ◽

Circulating Tumor Dna ◽

Initial Assessment ◽

Tumor Tissues ◽

Time Changes ◽

Colorectal Cancer Patients ◽

Monitoring Treatment

602 Background: Liquid biopsy represents an ideal non-invasive tool allowing multiple testing over time, monitoring real time changes occurring within the tumor and evaluation of therapeutic response. Here we show monitoring of KRAS mutation in detection of circulating tumor DNA (ctDNA) during treatments for metastatic colorectal cancer patients (pts). Methods: 238 plasma samples were collected from 57 pts who underwent chemotherapy. KRAS mutant ctDNA (MctDNA) was determined by digital PCR as a tool of Liquid biopsy, detecting rare mutant clones in blood. Results: KRAS assessment in tumor tissues showed 19 pts with mutation (M) and 38 without mutation (W). Among 19 pts with M, 12 pts displayed MctDNA at the initial assessment and 7 pts showed no MctDNA. Then, one (M*) of 7 pts exhibited MctDNA during treatments. While 38 pts with W showed no MctDNA before chemotherapy except one pt (M**), 4 pts exhibited MctDNA after treatments with different regimens. MctDNA was detectable in the blood of these 4 pts prior to radiographic detection of disease progression. Regardless of KRAS status in tumor tissues, poor response was seen in pts with MctDNA. Conclusions: Liquid biopsy provides us a circulating biomarker for monitoring treatment response and choice of optimal regimens. [Table: see text]

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Circulating tumor DNA molecular profiling in rare cancer patients from the MASTER KEY Project.

Journal of Clinical Oncology ◽

10.1200/jco.2019.37.15_suppl.e14532 ◽

2019 ◽

Vol 37 (15_suppl) ◽

pp. e14532-e14532

Author(s):

Hitomi Sumiyoshi Okuma ◽

Kan Yonemori ◽

Takuji Seo ◽

Emi Noguchi ◽

Keiko Wakakuwa ◽

...

Keyword(s):

Cancer Patients ◽

Salivary Gland Tumor ◽

Circulating Tumor Dna ◽

Genetic Alterations ◽

Unknown Primary ◽

Genomic Alteration ◽

Cancer Type ◽

Rare Cancer ◽

Registry Study ◽

Tumor Dna

e14532 Background: In April 2017, MASTER KEY Project, composed of a prospective registry study part and a multiple clinical trials part, was established to promote treatment development for rare cancers, which is lacking standard or investigational therapeutic options. Circulating tumor DNA (ctDNA) analysis by next-generation sequencing (NGS) has provided new insight into personalized medicine in a more accessible, non-invasive manner; however, most reports are focused on common cancers. We report genetic alterations detected by ctDNA NGS analysis in rare solid cancers. Methods: Prospectively consented patients (pts), also enrolled in MASTER KEY registry study, had ctDNA NGS testing at a CLIA-certified lab (Guardant360) for point mutations, indels, copy number amplifications, fusions, and microsatellite instability. Alterations were assessed for incidence according to cancer type, functional impact, therapeutic implications, and comparison with tissue NGS. Main inclusion criteria: 1) age ≥16, 2) histological diagnosis of rare cancer (annual incidence less than 6 cases per 100,000 population), cancer of unknown primary (CUP), or rare tissue subtypes of common cancers, 3) active metastatic / unresectable cancer, 4) a written consent. Results: From Nov. 2018 to Jan 2019, 50 pts had Guardant360 testing. Diseases included: soft tissue sarcoma (28), ovarian carc. (7), CUP (4), salivary gland tumor (3), thyroid carc. (2), GIST (1), adrenal cortical carc. (1), rare subtype of GI tract (1), malignant mesothelioma (1), nephroblastoma (1), NET (1), NUT carc. (1). All Japanese, male/female = 14/36, median age 61, ECOG performance status 0/1/2/3 = 30/19/0/1. 76% of pts (38/50) had ≥1 alteration detected, with median number of 2 alterations (range: 0-9), with VAF(0.1-60.3%); 87%(33/38) of those pts had a variant most likely pathogenic; 61% (20/33) of those pts had a variant potential for clinical action. Mean TAT was 10.3 days. 19 pts had tissue NGS testing and in 3 pts, alterations were detected by ctDNA NGS but not by tissue NGS. Updated results of 100 patients will be presented at the conference. Conclusions: Most rare cancer patients with advanced disease had a detectable genomic alteration by Guardant360. Clinical trial information: UMIN000034394.

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ESR1 Gene Mutation in Hormone Receptor-Positive HER2-Negative Metastatic Breast Cancer Patients: Concordance Between Tumor Tissue and Circulating Tumor DNA Analysis

Frontiers in Oncology ◽

10.3389/fonc.2021.625636 ◽

2021 ◽

Vol 11 ◽

Author(s):

Loredana Urso ◽

Grazia Vernaci ◽

Jessica Carlet ◽

Marcello Lo Mele ◽

Matteo Fassan ◽

...

Keyword(s):

Breast Cancer ◽

Metastatic Breast Cancer ◽

Hormone Receptor ◽

Liquid Biopsy ◽

Tumor Tissue ◽

Metastatic Breast ◽

Metastatic Tumor ◽

Circulating Tumor Dna ◽

Hormone Receptor Positive ◽

Tumor Dna

Endocrine therapy represents the cornerstone of treatment in hormone receptor-positive (HR+), HER2-negative metastatic breast cancer (mBC). The natural course of this disease is marked by endocrine resistance, mainly due to Estrogen Receptor 1 (ESR1) acquired mutations. The aim of this study is to evaluate the concordance between ESR1 status in metastatic tumor specimens and matched circulating tumor DNA (ctDNA). Forty-three patients with HR+, HER2-negative mBC underwent both a metastatic tumor biopsy and a liquid biopsy at the time of disease progression. DNA extracted from formalin fixed paraffin embedded (FFPE) tumor specimens and ctDNA from matched plasma were analyzed by droplet digital (dd)PCR for the main ESR1 mutations (Y537S, Y537C, Y537N, D538G, E380Q). We observed a total mutation rate of 21%. We found six mutations on tissue biopsy: Y537S (1), D538G (2), Y537N (1), E380Q (2). Three patients with no mutations in tumor tissue had mutations detected in ctDNA. The total concordance rate between ESR1 status on tumor tissue and plasma was 91%. Our results confirm the potential role of liquid biopsy as a non-invasive alternative to tissue biopsy for ESR1 mutation assessment in mBC patients.

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The prospect of using liquid biopsy in diagnosis and treatment strategy in patients with carcinomas of unknown primary

Advances in molecular oncology ◽

10.17650/2313-805x-2020-7-4-10-19 ◽

2021 ◽

Vol 7 (4) ◽

pp. 10-19

Author(s):

I. B. Kononenko ◽

M. G. Filippova ◽

A. V. Snegovoy ◽

S. L. Gutorov

Keyword(s):

Tumor Cells ◽

Liquid Biopsy ◽

Tissue Specificity ◽

Circulating Tumor Dna ◽

Patient Treatment ◽

Unknown Primary ◽

Molecular Characteristics ◽

Scientific Databases ◽

Circulating Free Dna ◽

Personalized Approach

Background. The search and use of new molecular prognostic and predictive markers of efficacy detected by liquid biopsy are aimed at understanding the biology of Carcinomas of Unknown Primary (CUP) and improving results of patient treatment. The review is devoted to advances in scientific and clinical research on this issue.Materials and methods. In order to assess the current state of the problem, a search and analysis of relevant data of the scientific databases PubMed, Medline, RISC was carried out.Results. The scientific rationale for the use of liquid biopsy in clinical practice to improve the treatment of cancer patients with CUP is presented. The results of the use of modern approaches to the analysis of fluid biopsy samples in CUP are presented. In particular, the features of using circulating free DNA, circulating tumor DNA, circulating tumor cells in the analysis are considered. The modern possibilities of determining tissue specificity using liquid biopsy in CUP are discussed. The prospects for the development of liquid biopsy for improving diagnostics, determining the prognosis of the disease, and choosing a strategy for treating CUP and the treatment monitoring have been determined.Conclusion. A review of the literature confirms that modern methods of molecular profiling of tumor cells obtained both as a result of liquid biopsy and tissue biopsies will make a significant contribution to the determination of tissue specificity, molecular characteristics of CUP for a personalized approach in order to improve strategies and treatment outcomes for patients with CUP.

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Translational Application of Circulating DNA in Oncology: Review of the Last Decades Achievements

Cells ◽

10.3390/cells8101251 ◽

2019 ◽

Vol 8 (10) ◽

pp. 1251 ◽

Cited By ~ 21

Author(s):

Tuaeva ◽

Falzone ◽

Porozov ◽

Nosyrev ◽

Trukhan ◽

...

Keyword(s):

Early Diagnosis ◽

Liquid Biopsy ◽

Prognostic Significance ◽

Circulating Tumor Dna ◽

Molecular Techniques ◽

Unknown Primary ◽

Diagnostic Strategies ◽

Early Diagnosis Of Cancer ◽

Mutational Status ◽

Ctdna Analysis

In recent years, the introduction of new molecular techniques in experimental and clinical settings has allowed researchers and clinicians to propose circulating-tumor DNA (ctDNA) analysis and liquid biopsy as novel promising strategies for the early diagnosis of cancer and for the definition of patients’ prognosis. It was widely demonstrated that through the non-invasive analysis of ctDNA, it is possible to identify and characterize the mutational status of tumors while avoiding invasive diagnostic strategies. Although a number of studies on ctDNA in patients’ samples significantly contributed to the improvement of oncology practice, some investigations generated conflicting data about the diagnostic and prognostic significance of ctDNA. Hence, to highlight the relevant achievements obtained so far in this field, a clearer description of the current methodologies used, as well as the obtained results, are strongly needed. On these bases, this review discusses the most relevant studies on ctDNA analysis in cancer, as well as the future directions and applications of liquid biopsy. In particular, special attention was paid to the early diagnosis of primary cancer, to the diagnosis of tumors with an unknown primary location, and finally to the prognosis of cancer patients. Furthermore, the current limitations of ctDNA-based approaches and possible strategies to overcome these limitations are presented.

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