• Adverse effects
• Early detection of cancer
• Fanconi anemia
• Genetic predisposition to disease
• Head and neck neoplasms
• Screening

• Humans
• Fanconi Anemia/complications
• Fanconi Anemia/genetics
• Fanconi Anemia/diagnosis
• Early Detection of Cancer/methods
• Squamous Cell Carcinoma of Head and Neck/diagnosis
• Squamous Cell Carcinoma of Head and Neck/pathology
• Squamous Cell Carcinoma of Head and Neck/etiology
• Squamous Cell Carcinoma of Head and Neck/genetics
• Head and Neck Neoplasms/diagnosis
• Head and Neck Neoplasms/pathology
• Head and Neck Neoplasms/etiology
• Head and Neck Neoplasms/genetics
• Mouth Neoplasms/diagnosis
• Mouth Neoplasms/pathology
• Mouth Neoplasms/etiology
• Mouth Neoplasms/genetics
• High-Throughput Nucleotide Sequencing

• Cell-free DNA
• Head and neck squamous cell carcinoma
• Liquid biopsy
• Non-invasive biomarker
• Personalized medicine

• NGS
• WGS
• ctCNA
• ddPCR
• digital droplet PCR
• next-generation sequencing
• soft tissue sarcoma
• whole-genome sequencing

• Biomarkers
• Head and neck squamous cell carcinoma
• Liquid biopsy
• Next-generation sequencing
• Tumor heterogeneity
• circulating tumor DNA

• Adult
• Aged
• Female
• Humans
• Male
• Middle Aged
• Biomarkers, Tumor/blood
• Biomarkers, Tumor/genetics
• Circulating Tumor DNA/blood
• Circulating Tumor DNA/genetics
• Genetic Heterogeneity
• Head and Neck Neoplasms/genetics
• Head and Neck Neoplasms/blood
• Head and Neck Neoplasms/pathology
• Head and Neck Neoplasms/surgery
• High-Throughput Nucleotide Sequencing/methods
• Mutation
• Neoplasm Recurrence, Local/genetics
• Neoplasm Recurrence, Local/blood
• Neoplasm, Residual
• Prospective Studies
• Squamous Cell Carcinoma of Head and Neck/genetics
• Squamous Cell Carcinoma of Head and Neck/blood
• Squamous Cell Carcinoma of Head and Neck/pathology
• Squamous Cell Carcinoma of Head and Neck/surgery

• HPV16 detection
• ddPCR
• digital PCR assay
• direct detection
• liquid biopsies
• rapid and sensitive assay

• Humans
• Liquid Biopsy/methods
• Human papillomavirus 16/genetics
• Human papillomavirus 16/isolation & purification
• Sensitivity and Specificity
• DNA, Viral/genetics
• DNA, Viral/isolation & purification
• Papillomavirus Infections/diagnosis
• Papillomavirus Infections/virology
• Polymerase Chain Reaction/methods
• Head and Neck Neoplasms/virology
• Head and Neck Neoplasms/diagnosis
• Cell-Free Nucleic Acids/genetics

• This study was supported by grants from the National Institutes of Health (CA096512, CA284554, CA278812 and CA124332 to S.-J. Gao), UPMC Hillman Cancer Center Startup Fund to S.-J. Gao, and in part by award P30CA047904.

• Circulating tumor cells
• cancer
• clinical oncology.
• immunotherapy
• inhibitor therapy
• theranosis

• Humans
• Neoplastic Cells, Circulating/pathology
• Neoplastic Cells, Circulating/metabolism
• Neoplastic Cells, Circulating/drug effects
• Neoplasms/diagnosis
• Neoplasms/therapy
• Neoplasms/drug therapy
• Neoplasms/pathology
• Theranostic Nanomedicine
• Immunotherapy
• Precision Medicine
• Biomarkers, Tumor

• Cancer
• Circulating tumor DNA
• Electrochemical biosensor
• Point of care diagnostics

• Biomarkers
• Cell-free nucleic acids
• Circulating tumor DNA
• Squamous cell carcinoma of head and neck

• DNA methylation
• cell free DNA
• droplet digital PCR
• head and neck squamous cell carcinoma
• liquid biopsy

• Humans
• Squamous Cell Carcinoma of Head and Neck/genetics
• DNA Methylation
• Genes, Homeobox
• Carcinoma, Squamous Cell/pathology
• Homeodomain Proteins/genetics
• Homeodomain Proteins/metabolism
• Polymerase Chain Reaction
• Cytoskeletal Proteins/genetics
• Cell-Free Nucleic Acids/genetics
• Head and Neck Neoplasms/genetics
• Biomarkers, Tumor/metabolism

• biosensor
• cancer screening
• giant magnetoresistance
• multiplexed assay
• point-of-care

• NGS
• cfDNA
• liquid biopsy

• Biomarkers
• Circulating tumour DNA
• DNA alterations
• Head and neck cancer
• Liquid biopsy
• Precision medicine
• Tumour DNA

• Humans
• Squamous Cell Carcinoma of Head and Neck/diagnosis
• Squamous Cell Carcinoma of Head and Neck/genetics
• Circulating Tumor DNA/genetics
• Biomarkers, Tumor/genetics
• Head and Neck Neoplasms/diagnosis
• Head and Neck Neoplasms/genetics
• Prognosis

• APP2002576 National Health and Medical Research Council
• APP2012560 National Health and Medical Research Council
• Royal Brisbane and Women's Hospital Foundation
• Garnett Passe and Rodney Williams Memorial Foundation

• biosensing
• head and neck cancers
• high throughput screening
• microfluidics
• personalized medicine
• tumor-on-chip

• cell-free DNA
• distant metastasis
• liquid biopsy
• mutation burden
• oral cancer
• whole-exome sequencing

• 108-2314-B-195 -002 -MY2 National Science and Technology Council (NSTC), Taiwan
• 110-2314-B-195 -015 -MY3 National Science and Technology Council (NSTC), Taiwan
• MMH-E-105-12 and MMH-E-108-12 Mackay Memorial Hospital

• cell-free DNA (cfDNA)
• head and neck squamous cell carcinoma (HNSCC)
• liquid biopsy
• monitoring
• prognostication
• resistance

• ctDNA
• gastric cancer
• liquid biopsy

Head and neck squamous cell carcinomas (HNSCCs) represent the most common epithelial tumors that arise from mucosa of the oral cavity, pharynx, and larynx. The development of HNSCCs is usually associated with tobacco use, alcohol consumption, and human papillomavirus (HPV) infection. Most HNSCCs are diagnosed in advanced states, leading to a worse clinical outcome. Screening tests based on potential biomarkers associated with HNSCCs could improve this scenario. Liquid biopsy has emerged as a promising area of cancer investigation, offering a minimally invasive approach to track circulating biomarkers in body fluids that could potentially contribute to the diagnosis, predict prognosis, and monitor response to treatment. This review will discuss translational studies describing the clinical applications of liquid biopsy in HPV-negative and HPV-positive HNSCCs focused on circulating nucleic acids [cell-free DNA (cfDNA) and cell-free RNA (cfRNA)], circulating tumor cells (CTCs), and extracellular vesicles (EVs), which can be found in plasma, serum, and saliva.

Digital PCR is the most advanced PCR technology. However, due to the high price of the digital PCR analysis instrument, this powerful nucleic acid detection technology is still difficult to be popularized in the general biochemistry laboratory. Moreover, one of the biggest disadvantages of commercial digital PCR systems is the poor versatility of reagents: each instrument can only be used for a few customized kits. Herein, we built a low-cost digital PCR system. The system only relies on low-cost traditional flat-panel PCR equipment to provide temperature conditions for commercial dPCR chips, and the self-made fluorescence detection system is designed and optically optimized to meet a wide range of reagent requirements. More importantly, our system not only has a low cost (<8000 US dollars) but also has a much higher universality for nucleic acid detection reagents than the traditional commercial digital PCR system. In this study, several samples were tested. The genes used in the experiment were plasmids containing UPE-1a fragment, TP53 reference DNA, hepatitis B virus DNA, leukemia sample, SARS-COV-2 DNA, and SARS-COV-2 RNA. Under the condition that DNA can be amplified normally, the function of the dPCR system can be realized with simpler and low-price equipment. Some DNA cannot be detected by using the commercial dPCR system because of the special formula when it is configured as the reaction solution, but these DNA fluorescence signals can be clearly detected by our system, and the concentration can be calculated. Our system is more applicable than the commercial dPCR system to form a new dPCR system that is smaller and more widely applicable than commercially available machinery.

• Exosomes
• NGS
• circulating biomarkers
• ctDNA
• ddPCR
• epigenetics
• liquid biopsy
• miRNAs
• oral cancer
• qPCR

The major cause of treatment failure after allogeneic stem cell transplantation (allo-HSCT) is due to relapse of the underlying disease. Novel methods and strategies are needed to detect early relapse after allo-HSCT. The present study reports the clinical utility of monitoring measurable residual disease (MRD) and mixed chimerism (MC) by droplet-digital PCR in circulating cell-free DNA (cfDNA) in 62 patients with myeloid malignancies undergoing allo-HSCT. MC in circulating cfDNA at an optimal threshold of 18% discriminated patients with hematological relapse from patients in complete remission after allo-HSCT. Most of the mutations identified using a targeted next-generation sequencing (NGS) panel were detected in cfDNA at relapse and were suitable for the monitoring of MRD. In several cases, mutations were detected earlier in cfDNA than in peripheral blood mononuclear cells. In conclusion, longitudinal analysis of cfDNA for MRD and MC can be used as a complementary tool for early detection of relapse in patients after allo-HSCT and could be used to guide clinical interventions.

Relapse of the underlying disease is a frequent complication after allogeneic hematopoietic stem cell transplantation (allo-HSCT). In this study, we describe the clinical utility of measurable residual disease (MRD) and mixed chimerism (MC) assessment in circulating cell-free DNA (cfDNA) analysis to detect earlier relapse in patients with hematological malignancies after allo-HSCT. A total of 326 plasma and peripheral blood mononuclear cell (PBMCs) samples obtained from 62 patients with myeloid malignancies were analyzed by droplet-digital PCR (median follow-up: 827 days). Comparison of MC in patients at relapse and in complete remission identified an optimal discriminating threshold of 18% of recipient-derived cfDNA. After performing a targeted next-generation sequencing (NGS) panel, 136 mutations in 58 patients were detected. In a total of 119 paired samples, the putative mutations were detected in both cfDNA and PBMCs in 73 samples (61.3%). In 45 samples (37.8%) they were detected only in cfDNA, and in only one patient (0.9%) were they detected solely in DNA from PBMCs. Hence, in 6 out of 23 patients (26%) with relapse after allo-HSCT, MRD positivity was detected earlier in cfDNA (mean 397 days) than in DNA derived from PBMCs (mean 451 days). In summary, monitoring of MRD and MC in cfDNA might be useful for earlier relapse detection in patients with myeloid malignancies after allo-HSCT.

• circulating tumor DNA
• exosomes
• gene methylation
• head and neck cancer
• head and neck squamous cell carcinoma
• liquid biopsy

• K12 CA138464 NCI NIH HHS
• K12CA138464 National Institute of Health

Platinum-based agents are one of the most widely used chemotherapy drugs for various types of cancer. However, one of the main challenges in the application of platinum drugs is resistance, which is currently being widely investigated. Epigenetic DNA methylation-based biomarkers are promising to aid in the selection of patients, helping to foresee their platinum therapy response in advance. These biomarkers enable minimally invasive patient sample collection, short analysis, and good sensitivity. Hence, improved methodologies for the detection and quantification of DNA methylation biomarkers will facilitate their use in the choice of an optimal treatment strategy.

Platinum-based chemotherapy is routinely used for the treatment of several cancers. Despite all the advances made in cancer research regarding this therapy and its mechanisms of action, tumor resistance remains a major concern, limiting its effectiveness. DNA methylation-based biomarkers may assist in the selection of patients that may benefit (or not) from this type of treatment and provide new targets to circumvent platinum chemoresistance, namely, through demethylating agents. We performed a systematic search of studies on biomarkers that might be predictive of platinum-based chemotherapy resistance, including in vitro and in vivo pre-clinical models and clinical studies using patient samples. DNA methylation biomarkers predictive of response to platinum remain mostly unexplored but seem promising in assisting clinicians in the generation of more personalized follow-up and treatment strategies. Improved methodologies for their detection and quantification, including non-invasively in liquid biopsies, are additional attractive features that can bring these biomarkers into clinical practice, fostering precision medicine.

• DNA methylation
• biomarker
• cancer
• epigenetics
• platinum-based chemotherapy

Head and neck cancer is the sixth most common type of solid tumor and harbors a poor prognosis since most patients are diagnosed at an advanced stage. The study of different tumor components in the blood, saliva or other body fluids is called liquid biopsy. The introduction of novel diagnostic tools such as liquid biopsy could aid in achieving earlier diagnoses and more accurate disease monitoring during treatment. In this manuscript, the reader will find an in-depth review of the current evidence and a future perspective on the role of liquid biopsy in head and neck cancer.

Head and neck cancer (HNC) is currently the sixth most common solid malignancy, accounting for a 50% five-year mortality rate. In the past decade, substantial improvements in understanding its molecular biology have allowed for a growing development of new biomarkers. Among these, the field of liquid biopsy has seen a sustained growth in HNC, demonstrating the feasibility to detect different liquid biomarkers such as circulating tumor DNA (ctDNA), circulating tumor cells (CTC), extracellular vesicles and microRNAs. Liquid biopsy has been studied in HPV-negative squamous cell carcinoma of the head and neck (SCCHN) but also in other subentities such as HPV-related SCCHN, EBV-positive nasopharyngeal cancer and oncogene-driven salivary gland cancers. However, future studies should be internally and externally validated, and ideally, clinical trials should incorporate the use of liquid biomarkers as endpoints in order to prospectively demonstrate their role in HNC. A thorough review of the current evidence on liquid biopsy in HNC as well as its prospects will be conducted.

• CTC
• EBV
• Epstein-Barr virus
• HPV
• circulating tumor DNA
• circulating tumor cells
• ctDNA
• extracellular vesicles
• head and neck cancer
• human papillomavirus
• liquid biopsy
• microRNAs

Recurrences remain an important problem in laryngeal squamous cell carcinoma. Little has been described about histological characteristics of the primary laryngeal tumor that may be associated with recurrences. Identifying risk factors for recurrences might help in adapting treatment or follow-up. Using real-life population-based data, we aimed to identify histological features of the primary tumor associated with recurrences and overall survival.

Demographic, clinical and treatment information on all first primary invasive laryngeal tumors diagnosed in 2010–2014 (N = 3705) were extracted from the population-based nationwide Netherlands cancer registry (NCR) and linked to PALGA, the nationwide Dutch pathology registry, to obtain data on histological factors and recurrences. For a random 1502 patients histological information i.e., keratinization, perineural invasion (PNI+), vascular invasion (VI+), growth pattern, degree of differentiation, extracapsular spread (ECS+), cartilage- and bone invasion and extralaryngeal extension, was manually extracted from narrative pathology reports and analyzed for locoregional recurrence and overall survival using cox regression analysis.

In total, 299 patients developed a locoregional recurrence and 555 patients died. Keratinization (HR = 0.96 (95%CI: 0.68–1.34) p = 0.79), two or three adverse characteristics (PNI+, VI+, non-cohesive growth) (HR = 1.38 (95% CI: 0.63–3.01) p = 0.42), and ECS+ (HR = 1.38 (95% CI: 0.48–4.02) p = 0.55) were not associated to recurrence. For death, also no significant association was found.

In this population-based real-life dataset on laryngeal carcinoma in the Netherlands, histological factors were not associated with locoregional recurrences or overall survival, but future studies should investigate the role of these features in treatment decisions.

The online version contains supplementary material available at 10.1186/s12885-022-09533-0.

• Histology
• Laryngeal neoplasms
• Pathology
• Recurrence
• Squamous cell carcinoma
• Survival

Advanced cutaneous squamous cell carcinoma (cSCC) encompasses unresectable and metastatic disease. Although immune checkpoint inhibition has been approved for this entity recently, a considerable proportion of cases is associated with significant morbidity and mortality. Clinical, histopathological, and radiological criteria are used for current diagnostics, classification, and therapeutic decision-making. The identification of complex molecular biomarkers to accurately stratify patients is a not yet accomplished requirement to further shift current diagnostics and care to a personalized precision medicine. This article highlights new insights into the mutational profile of cSCC, summarizes current diagnostic and therapeutic standards, and discusses emerging diagnostic approaches with emphasis on liquid biopsy and tumor tissue-based analyses.

• head and neck squamous cell carcinoma
• liquid biopsy
• minimal residual disease
• relapse
• surveillance

• Biomarkers, Tumor/genetics
• Early Detection of Cancer
• Head and Neck Neoplasms/diagnosis
• Head and Neck Neoplasms/genetics
• Humans
• Liquid Biopsy
• Neoplasm Recurrence, Local/diagnosis
• Neoplasm Recurrence, Local/genetics
• Sensitivity and Specificity
• Squamous Cell Carcinoma of Head and Neck/diagnosis
• Squamous Cell Carcinoma of Head and Neck/genetics
• Survival Analysis
• Time-to-Treatment

Pancreatic ductal adenocarcinoma (PDAC) patients eligible for curative surgery undergo unpredictable disease relapse. Even patients with a good pathological response after neoadjuvant treatment (NAT) remain susceptible to recurrent PDAC. Molecular analysis of R0 margins may identify patients with a worse prognosis. The molecular status of mutant KRAS (exon 2, codon 12/13) was analysed retrospectively by digital droplet PCR in tumour areas, venous and resection margins of resected tumours, either undergoing up-front surgery (UFS) or after NAT with a good pathological response. Expectedly, tumour tissues or remnants from patients who underwent NAT presented lower KRAS mutant allele frequencies (MAF) than patients eligible for UFS. Similarly, ypT1 tumour MAFs were greater than the ypT0 tumour remnant MAFs in the NAT group. Mutant KRAS status in margins did not distinguish NAT subgroups. It was not predictive of shorter recurrence-free or overall survival within or between groups. KRAS-double negativity in both venous and resection margins did not identify patients with a better prognosis, regardless of the groups. The cohorts ‘sizes were small due to limited numbers of patients meeting the inclusion criteria, but KRAS-positivity or MAFs in resection and venous margins did not carry prognostic value. Comparison of margins from good versus bad responders receiving NAT may provide better clinical value.

• Cancer biomarkers
• Cancer diagnosis
• Circulating tumor DNA (ctDNA)

• Cancer
• Diagnostics
• Drug screening
• High throughput
• Microfluidics
• Nanofluidics

Circulating cell-free DNA (cfDNA) in the plasma of cancer patients contains cell-free tumour DNA (ctDNA) derived from tumour cells and it has been widely recognized as a non-invasive source of tumour DNA for diagnosis and prognosis of cancer. Molecular profiling of ctDNA is often performed using targeted sequencing or low-coverage whole genome sequencing (WGS) to identify tumour specific somatic mutations or somatic copy number aberrations (sCNAs). However, these approaches cannot efficiently detect all tumour-derived genomic changes in ctDNA.

We performed WGS analysis of cfDNA from 4 breast cancer patients and 2 patients with benign tumours. We sequenced matched germline DNA for all 6 patients and tumour samples from the breast cancer patients. All samples were sequenced on Illumina HiSeqXTen sequencing platform and achieved approximately 30x, 60x and 100x coverage on germline, tumour and plasma DNA samples, respectively.

The mutational burden of the plasma samples (1.44 somatic mutations/Mb of genome) was higher than the matched tumour samples. However, 90% of high confidence somatic cfDNA variants were not detected in matched tumour samples and were found to comprise two background plasma mutational signatures. In contrast, cfDNA from the di-nucleosome fraction (300 bp–350 bp) had much higher proportion (30%) of variants shared with tumour. Despite high coverage sequencing we were unable to detect sCNAs in plasma samples.

Deep sequencing analysis of plasma samples revealed higher fraction of unique somatic mutations in plasma samples, which were not detected in matched tumour samples. Sequencing of di-nucleosome bound cfDNA fragments may increase recovery of tumour mutations from plasma.

The online version contains supplementary material available at 10.1186/s12885-021-09160-1.

Levels of circulating tumor DNA (ctDNA) in liquid biopsies may serve as a sensitive biomarker for real-time, minimally-invasive tumor diagnostics and monitoring. However, detecting ctDNA is challenging, as much fewer than 5% of the cell-free DNA in the blood typically originates from the tumor. To detect lowly abundant ctDNA molecules based on somatic variants, extremely sensitive sequencing methods are required. Here, we describe a new technique, CyclomicsSeq, which is based on Oxford Nanopore sequencing of concatenated copies of a single DNA molecule. Consensus calling of the DNA copies increased the base-calling accuracy ~60×, enabling accurate detection of TP53 mutations at frequencies down to 0.02%. We demonstrate that a TP53-specific CyclomicsSeq assay can be successfully used to monitor tumor burden during treatment for head-and-neck cancer patients. CyclomicsSeq can be applied to any genomic locus and offers an accurate diagnostic liquid biopsy approach that can be implemented in clinical workflows.

• circulating tumour DNA (ctDNA)
• diagnostic biomarker
• head and neck cancer squamous cell carcinoma (HNSCC)
• liquid biopsy

• Biomolecules
• Optical imaging
• Sensor

Non-invasive prenatal diagnosis (NIPD) of isolated cell-free DNA from maternal plasma has been applied to detect monogenic diseases in the fetus. Droplet digital PCR (ddPCR) is a sensitive and quantitative technique for NIPD. In the present study, the development and evaluation of ddPCR-based assays for common α and β-thalassemia variants amongst the Asian population was described; specifically, Southeast Asian (SEA) deletion, HbE, and 41/42 (-CTTT). SEA is caused by deletion of a 20 kb region surrounding the α-globin gene, whilst HbE and 41/42 (-CTTT) are caused by point mutations on the β-globin gene. Cell-free DNA samples from 46 singleton pregnant women who were carriers of these mutations were isolated and quantified using ddPCR with specially designed probes for each target allele. Allelic copy number calculation and likelihood ratio tests were used to classify fetal genotypes. Classification performances were evaluated against ground truth fetal genotypes obtained from conventional amniocentesis. Copy number variation analysis of SEA deletion accurately classified fetal genotypes in 20 out of 22 cases with an area under the receiver operating characteristic curve of 0.98 for detecting Hb Bart's hydrops fetalis. For HbE cases, 10 out of 16 samples were correctly classified, and three were inconclusive. For 41/42 (-CTTT) cases, 2 out of 8 were correctly classified, and four were inconclusive. The correct genotype was not rejected in any inconclusive case and may be resolved with additional ddPCR experiments. These results indicate that ddPCR-based analysis of maternal plasma can become an accurate and effective NIPD for SEA deletion α-(0) thalassemia. Although the performance of ddPCR on HbE and 41/42 (-CTTT) mutations were not sufficient for clinical application, these results may serve as a foundation for future works in this field.

• cell-free fetal DNA
• droplet digital PCR
• non-invasive prenatal test
• α-thalassemia
• β-thalassemia

TP53 mutation is a driver mutation of oral carcinogenesis. This study investigated cancerous and cell-free DNA (cfDNA) in patients with oral squamous cell carcinoma (OSCC) to detect the target hotspot somatic mutation of TP53.

TP53 target hotspot mutations were determined in surgically resected primary tumor samples from 107 OSCC patients.

Cancerous and cfDNA samples were examined for mutations through droplet digital polymerase chain reaction (ddPCR) by using mutation-specific assays. The ddPCR results were evaluated alongside clinicopathological data.

In total, 23 cases had target TP53 mutations in varying degrees. We found that OSCC had relatively low cfDNA shedding, and mutations were at low allele frequencies. Of these 23 cases, 13 had target TP53 mutations in their corresponding cfDNA. Target somatic mutations in cancerous DNA and cfDNA are related to cervical lymph node metastasis. The cfDNA concentration is related to primary tumor size, lymph node metastasis, and OSCC stage.

Our results show that the detection of TP53 target somatic mutations in OSCC patients by using ddPCR is technically feasible. Low levels of cfDNA may produce different results between cancerous tissue and cfDNA analyses. Future research on cfDNA may quantify diagnostic biomarkers in the surveillance of OSCC patients.

• cfDNA
• ddPCR
• oral cancer
• somatic mutation
• tp53

• Australian Research Council
• Japan Society for the Promotion of Science

• circulating tumor DNA
• circulating tumor cells
• exosomes
• head and neck cancer
• head and neck squamous cell carcinoma
• liquid biopsy

Human papillomaviruses (HPVs) are associated with invasive malignancies, including almost 100% of cervical cancers (CECs), and 35–70% of oropharyngeal cancers (OPCs). HPV infection leads to clinical implications in related tumors by determining better prognosis and predicting treatment response, especially in OPC. Currently, specific and minimally invasive tests allow for detecting HPV-related cancer at an early phase, informing more appropriately therapeutical decisions, and allowing for timely disease monitoring. A blood-based biomarker detectable in liquid biopsy represents an ideal candidate, and the use of circulating HPV DNA (ct-DNA) itself could offer the highest specificity for such a scope. Circulating HPV DNA is detectable in the greatest part of patients affected by HPV-related cancers, and studies have demonstrated its potential usefulness for CEC and OPC clinical management. Unfortunately, when using conventional polymerase chain reaction (PCR), the detection rate of serum HPV DNA is low. Innovative techniques such as droplet-based digital PCR and next generation sequencing are becoming increasingly available for the purpose of boosting HPV ct-DNA detection rate. We herein review and critically discuss the most recent and representative literature, concerning the role of HPV ctDNA in OPC and CEC in the light of new technologies that could improve the potential of this biomarker in fulfilling many of the unmet needs in the clinical management of OPC and CEC patients.

• cervical cancer
• circulating HPV DNA
• liquid biopsy
• oropharyngeal cancer

• Biomarkers
• Cell-free DNA (cfDNA)
• Circulating tumour DNA (ctDNA)
• Liquid biopsy
• Precision medicine

Circulating tumor DNA (ctDNA) has become an attractive biomarker in human oncology, and its use may be informative in canine cancer. Thus, we used droplet digital PCR or PCR for antigen receptor rearrangement, to explore tumor-specific point mutations, copy number alterations, and chromosomal rearrangements in the plasma of cancer-affected dogs. We detected ctDNA in 21/23 (91.3%) of histiocytic sarcoma (HS), 2/8 (25%) of oral melanoma, and 12/13 (92.3%) of lymphoma cases. The utility of ctDNA in diagnosing HS was explored in 133 dogs, including 49 with HS, and the screening of recurrent PTPN11 mutations in plasma had a specificity of 98.8% and a sensitivity between 42.8 and 77% according to the clinical presentation of HS. Sensitivity was greater in visceral forms and especially related to pulmonary location. Follow-up of four dogs by targeting lymphoma-specific antigen receptor rearrangement in plasma showed that minimal residual disease detection was concordant with clinical evaluation and treatment response. Thus, our study shows that ctDNA is detectable in the plasma of cancer-affected dogs and is a promising biomarker for diagnosis and clinical follow-up. ctDNA detection appears to be useful in comparative oncology research due to growing interest in the study of natural canine tumors and exploration of new therapies.