ESMO recommendations on the use of circulating tumour DNA assays for patients with cancer: a report from the ESMO Precision Medicine Working Group.

Circulating tumour DNA (ctDNA) assays conducted on plasma are rapidly developing a strong evidence base for use in patients with cancer. The European Society for Medical Oncology convened an expert working group to review the analytical and clinical validity and utility of ctDNA assays. For patients with advanced cancer, validated and adequately sensitive ctDNA assays have utility in identifying actionable mutations to direct targeted therapy, and may be used in routine clinical practice, provided the limitations of the assays are taken into account. Tissue-based testing remains the preferred test for many cancer patients, due to limitations of ctDNA assays detecting fusion events and copy number changes, although ctDNA assays may be routinely used when faster results will be clinically important, or when tissue biopsies are not possible or inappropriate. Reflex tumour testing should be considered following a non-informative ctDNA result, due to false-negative results with ctDNA testing. In patients treated for early-stage cancers, detection of molecular residual disease or molecular relapse, has high evidence of clinical validity in anticipating future relapse in many cancers. Molecular residual disease/molecular relapse detection cannot be recommended in routine clinical practice, as currently there is no evidence for clinical utility in directing treatment. Additional potential applications of ctDNA assays, under research development and not recommended for routine practice, include identifying patients not responding to therapy with early dynamic changes in ctDNA levels, monitoring therapy for the development of resistance mutations before clinical progression, and in screening asymptomatic people for cancer. Recommendations for reporting of results, future development of ctDNA assays and future clinical research are made.

Delay-induced resonance suppresses damping-induced unpredictability.

Combined effects of the damping and forcing in the underdamped time-delayed Duffing oscillator are considered in this paper. We analyse the generation of a certain damping-induced unpredictability due to the gradual suppression of interwell oscillations. We find the minimal amount of the forcing amplitude and the right forcing frequency to revert the effect of the dissipation, so that the interwell oscillations can be restored, for different time delay values. This is achieved by using the delay-induced resonance, in which the time delay replaces one of the two periodic forcings present in the vibrational resonance. A discussion in terms of the time delay of the critical values of the forcing for which the delay-induced resonance can tame the dissipation effect is finally carried out. This article is part of the theme issue 'Vibrational and stochastic resonance in driven nonlinear systems (part 1)'.

Oral cancer awareness in North-Western Spain: a population-based study.

BACKGROUND: An early diagnosis depends greatly on patient awareness. Thus, the aim of this study was to investigate general awareness of oral cancer and knowledge about its risk factors, signs and symptoms. MATERIAL AND METHODS: Cross-sectional population-based survey of randomly selected respondents conducted from March 1, 2015 to 30 June 2016. RESULTS: A total of 5,727 people entered the survey (response rate: 53%). When asked what cancers participants had heard about, 20.3% mentioned oral cancer. Regarding risk factors, tobacco was mentioned by 55.3% of the sample (n=3,169), followed by alcohol (12.5%; n=708), poor oral hygiene (10.8%; n=618), diet (6.5%; n=377), and genetics (4.5%; n=248). CONCLUSIONS: General population has low awareness of oral cancer with poor knowledge of risk factors and main alarm signs. In addition, individuals in the risk group scored lower values in the main variables analysed; even those highly educated showed insufficient awareness and knowledge of oral cancer. In these circumstances, there is clear need for educational interventions tailored to the target audience and aimed at increasing knowledge and awareness of oral cancer to promote primary prevention of oral cancer and minimising the time interval of patients with symptomatic oral cancer in their path to treatment.

[Changes in the Objective Structured Clinical Examination (OSCE) of University Schools of Medicine during COVID-19. Experience with a computer-based case simulation OSCE (CCS-OSCE)]. / Cambios en el examen clínico objetivo y estructurado (ECOE) de las facultades de Medicina durante la COVID-19. Experiencia de una ECOE de casos-clínicos computarizados simulados (ECOE-CCS) conjunta.

BACKGROUND AND OBJECTIVES: The COVID-19 pandemic has forced universities to move the completion of university studies online. Spain's National Conference of Medical School Deans coordinates an objective, structured clinical competency assessment called the Objective Structured Clinical Examination (OSCE), which consists of 20 face-to-face test sections for students in their sixth year of study. As a result of the pandemic, a computer-based case simulation OSCE (CCS-OSCE) has been designed. The objective of this article is to describe the creation, administration, and development of the test. MATERIALS AND METHODS: This work is a descriptive study of the CCS-OSCE from its planning stages in April 2020 to its administration in June 2020. RESULTS: The CCS-OSCE evaluated the competences of anamnesis, exploration, clinical judgment, ethical aspects, interprofessional relations, prevention, and health promotion. No technical or communication skills were evaluated. The CCS-OSCE consisted of ten test sections, each of which had a 12-minutes time limit and ranged from six to 21 questions (mean: 1.1 minutes/question). The CCS-OSCE used the virtual campus platform of each of the 16 participating medical schools, which had a total of 2,829 students in their sixth year of study. It was jointly held on two dates in June 2020. CONCLUSIONS: The CCS-OSCE made it possible to bring together the various medical schools and carry out interdisciplinary work. The CCS-OSCE conducted may be similar to Step 3 of the United States Medical Licensing Examination.

Clinical development of therapies targeting TGFß: current knowledge and future perspectives.

Transforming growth factor beta (TGFß) is a pleiotropic cytokine that plays a key role in both physiologic and pathologic conditions, including cancer. Importantly, TGFß can exhibit both tumor-suppressive and oncogenic functions. In normal epithelial cells TGFß acts as an antiproliferative and differentiating factor, whereas in advanced tumors TGFß can act as an oncogenic factor by creating an immune-suppressive tumor microenvironment, and inducing cancer cell proliferation, angiogenesis, invasion, tumor progression, and metastatic spread. A wealth of preclinical findings have demonstrated that targeting TGFß is a promising means of exerting antitumor activity. Based on this rationale, several classes of TGFß inhibitors have been developed and tested in clinical trials, namely, monoclonal, neutralizing, and bifunctional antibodies; antisense oligonucleotides; TGFß-related vaccines; and receptor kinase inhibitors. It is now >15 years since the first clinical trial testing an anti-TGFß agent was engaged. Despite the promising preclinical studies, translation of the basic understanding of the TGFß oncogenic response into the clinical setting has been slow and challenging. Here, we review the conclusions and status of all the completed and ongoing clinical trials that test compounds that inhibit the TGFß pathway, and discuss the challenges that have arisen during their clinical development. With none of the TGFß inhibitors evaluated in clinical trials approved for cancer therapy, clinical development for TGFß blockade therapy is primarily oriented toward TGFß inhibitor combinations. Immune checkpoint inhibitors are considered candidates, albeit with efficacy anticipated to be restricted to specific populations. In this context, we describe current efforts in the search for biomarkers for selecting the appropriate cancer patients who are likely to benefit from anti-TGFß therapies. The knowledge accumulated during the last 15 years of clinical research in the context of the TGFß pathway is crucial to design better, innovative, and more successful trials.

People would rather see a physician than a dentist when experiencing a long-standing oral ulceration. A population-based study in Spain.

BACKGROUND: Primary care physicians have been reported to be the first choice for patients with oral ulcerations. This study investigates the health-seeking behaviour of lay public in Galicia (North-western Spain) if experiencing a long-standing oral ulceration. MATERIAL AND METHODS: Cross-sectional population-based survey of randomly selected respondents conducted from March 1, 2015 to 30 June 2016. RESULTS: A total of 5,727 pedestrians entered the study (response rate: 53%), mostly in the 45-64 age group (30.2%; n=1,728), 47.7% of them (n=2,729) were males. Most participants (42.1%; n=2,411) reported to visit their dentist once a year and had secondary or compulsory education as their highest educational achievement (28.18%, n=1,614; 28%, n=1,600 respectively). When questioned what they would do if they had a wound/ulceration lasting longer than 3 weeks, most participants answered they would go to see their primary care physician (62.8%; n=3,597) and less than one quarter of the sample (23.8%; n=1,371) would seek consultation with their dentist. CONCLUSIONS: General Galician population would seek professional consultation about a long-standing oral ulceration, relying mostly on primary care physicians. Those neglecting these lesions are elderly, less-schooled people and unaware of oral cancer.

Cerebrospinal fluid cell-free tumour DNA as a liquid biopsy for primary brain tumours and central nervous system metastases.

Challenges in obtaining tissue specimens from patients with brain tumours limit the diagnosis and molecular characterisation and impair the development of better therapeutic approaches. The analysis of cell-free tumour DNA in plasma (considered a liquid biopsy) has facilitated the characterisation of extra-cranial tumours. However, cell-free tumour DNA in plasma is limited in quantity and may not reliably capture the landscape of genomic alterations of brain tumours. Here, we review recent work assessing the relevance of cell-free tumour DNA from cerebrospinal fluid in the characterisation of brain cancer. We focus on the advances in the use of the cerebrospinal fluid as a source of cell-free tumour DNA to facilitate diagnosis, reveal actionable genomic alterations, monitor responses to therapy, and capture tumour heterogeneity in patients with primary brain tumours and brain and leptomeningeal metastases. Profiling cerebrospinal fluid cell-free tumour DNA provides the opportunity to precisely acquire and monitor genomic information in real time and guide precision therapies.

How liquid biopsies can change clinical practice in oncology.

Cell-free DNA fragments are shed into the bloodstream by tumor cells. The analysis of circulating tumor DNA (ctDNA), commonly known as liquid biopsy, can be exploited for a variety of clinical applications. ctDNA is being used to genotype solid cancers non-invasively, to track tumor dynamics and to detect the emergence of drug resistance. In a few settings, liquid biopsies have already entered clinical practice. For example, ctDNA is used to guide treatment in a subset of lung cancers. In this review, we discuss how recent improvements in the sensitivity and accuracy of ctDNA analyses have led to unprecedented advances in this research field. We further consider what is required for the routine deployment of liquid biopsies in the clinical diagnostic space. We pinpoint technical hurdles that liquid biopsies have yet to overcome, including preanalytical and analytical challenges. We foresee how liquid biopsies will transform clinical practice: by complementing (or replacing) imaging to monitor treatment response and by detecting minimal residual disease after surgery with curative intent.

The European Society for Medical Oncology (ESMO) Precision Medicine Glossary.

Background: Precision medicine is rapidly evolving within the field of oncology and has brought many new concepts and terminologies that are often poorly defined when first introduced, which may subsequently lead to miscommunication within the oncology community. The European Society for Medical Oncology (ESMO) recognises these challenges and is committed to support the adoption of precision medicine in oncology. To add clarity to the language used by oncologists and basic scientists within the context of precision medicine, the ESMO Translational Research and Personalised Medicine Working Group has developed a standardised glossary of relevant terms. Materials and methods: Relevant terms for inclusion in the glossary were identified via an ESMO member survey conducted in Autumn 2016, and by the ESMO Translational Research and Personalised Medicine Working Group members. Each term was defined by experts in the field, discussed and, if necessary, modified by the Working Group before reaching consensus approval. A literature search was carried out to determine which of the terms, 'precision medicine' and 'personalised medicine', is most appropriate to describe this field. Results: A total of 43 terms are included in the glossary, grouped into five main themes-(i) mechanisms of decision, (ii) characteristics of molecular alterations, (iii) tumour characteristics, (iv) clinical trials and statistics and (v) new research tools. The glossary classes 'precision medicine' or 'personalised medicine' as technically interchangeable but the term 'precision medicine' is favoured as it more accurately reflects the highly precise nature of new technologies that permit base pair resolution dissection of cancer genomes and is less likely to be misinterpreted. Conclusions: The ESMO Precision Medicine Glossary provides a resource to facilitate consistent communication in this field by clarifying and raising awareness of the language employed in cancer research and oncology practice. The glossary will be a dynamic entity, undergoing expansion and refinement over the coming years.

Early evolutionary divergence between papillary and anaplastic thyroid cancers.

Background: Papillary thyroid cancer (PTC) is the most common thyroid carcinoma and exhibits an almost uniformly good prognosis, while anaplastic thyroid cancer (ATC) is less frequent and is one of the most aggressive cancers usually resistant to conventional treatment. Current hypothesis posits that ATC derives from PTC through the progressive acquisition of a discrete number of genomic alterations and implies that the mutational landscape of ATC resembles that of PTC. However, the clinical behaviour of ATC and PTC is radically different. We decided to address the disconnection between the clinical behaviour of ATC and PTC and the proposed model of the progressive development of ATC from PTC. Patients and methods: We carried out exome sequencing of DNA from 14 ATC specimens including three cases of concomitant ATC and PTC as well as their corresponding normal DNA from 14 patients. The sequencing results were validated using droplet digital PCR. We carried out immunohistochemistry and immunofluorescence studies of the concomitant ATC and PTC cases. In addition, we integrated our sequencing results with the existing TCGA data. Results: Most of the somatic mutations identified in the ATC component differed from the ones in PTC in the cases of concomitant ATC and PTC. The trunks of the phylogenetic trees representing the somatic mutations were short with long branches. In one case of concomitant PTC and ATC specimens, we observed an infiltration of PTC cells within the ATC component. Moreover, we integrated our results with data obtained from TCGA and observed that the most frequent mutations found in ATC presented high cancer cell fraction values and were significantly different from the PTC ones. Conclusion: ATC diverge from PTC early in tumour development and both tumour types evolve independently. Our work allows the understanding of the relationship between ATC and PTC facilitating the clinical management of these malignancies.

Shorter specialist time intervals are associated with advanced stage on symptomatic oral cancer.

OBJECTIVES: As longer times from the first symptom to diagnosis and treatment of oral cancers have been linked to poorer outcomes, this study investigates the contribution of the specialist to this time (STI). SUBJECTS AND METHODS: A series of 228 oral/oropharyngeal squamous cell carcinoma patients were retrospectively studied to determine the STI and its related factors. RESULTS: Patients were mostly males (n = 170; 74.5%), (50.7% stages I-II), mean age = 61.4 ± 12.5 years. The STI median was 6 days (X±SD:6.8 ± 5.6 days). Time first symptom to diagnosis was 64 days (X±SD:91.0 ± 84.6 days). Univariate regression unveiled a significant association between STI and TNM stage, which was confirmed by multivariate regression. CONCLUSIONS: Specialist time interval is a short time interval in oral cancer diagnosis, imposing a limited time burden in the context of the whole interval until diagnosis. However, there seems to be room for improvement and a possible target for future interventions to shorten STI particularly for patients at early stages after their disease has been disclosed.

A Dramatic Case of Odynophagia.

We report the case of a 77-year-old male with a history of aortic stenosis and interstitial lung disease, who debuted 3 years ago with an outbreak of necrotic and very painful canker sores. The severity of the lesions and their refractory response to treatment led to several hospital admissions and multiple consultations to different specialists (ENT, rheumatology, dermatology, ophthalmology, cardiology, and internal medicine). During this time, the patient received central parenteral nutrition with an episode of catheter-related septicemia, and he came to require psychiatric assistance for autolytic ideation. Numerous diagnostic tests were performed with inconclusive results, including biopsy of the lesion (histological study, immunohistochemistry for CD68 + , CD4 + , CD8 + , CD20 + , MCT +, and cytomegalovirus, PAS, Grocott-Gomori and Zielh-Neelsen staining, and in situ hybridization for Epstein Barr virus). Numerous treatments were unsuccessfully tested until thalidomide was administered, thus completely remitting lesions but leaving retractable scarring sequelae. Since then, the patient has had two recurrences, coinciding with the reduction of thalidomide dosages, which were controlled by increasing the dose of the immunomodulator. Recurrent necrotizing major aphthous stomatitis (Sutton's disease) is a clinical variant of recurrent aphthous stomatitis that may have a dramatic course. Unfortunately, the lack of etiopathogenetic uniformity precludes any specific treatment. In severe cases, immunomodulators, including thalidomide, may represent a valid therapeutic option.

The length of patient and primary care time interval in the pathways to treatment in symptomatic oral cancer. A quantitative systematic review.

OBJECTIVES: To examine the relative length of the patient and primary care intervals in symptomatic oral cancer. DESIGN: Quantitative systematic review. SEARCH STRATEGY: Oral cancer OR oral squamous cell carcinoma OR oropharyngeal cancer AND time interval OR diagnostic delay. SETTING: Primary and secondary care. PARTICIPANTS: Oral and oropharyngeal cancer patients. MAIN OUTCOME MEASURES: We computed five measures (patient, primary care, diagnosis, total diagnosis and total treatment intervals). Most studies did not provide any dispersion measure. We then used the sample size of each study to compute a weighted average of the mean intervals. When the median was provided, we assumed normality of the distribution of the means and used the median as a proxy of the mean. RESULTS: A total of 1089 articles were identified, and 22 met the inclusion criteria, reporting on 2710 patients from Europe, USA, India, Australia, Japan, Argentina and Iran. The weighted average of patient interval was 80.3 days. Primary care interval was five times shorter: 15.8 days. The diagnostic interval was appreciably shorter (47.9 days) when compared with the patient interval during symptomatic period. CONCLUSIONS: Patient interval represents the major component of waiting times since the detection of the first signs/symptoms to the definitive diagnosis of oral cancer. Thus, strategies focused on high-risk patients should be prioritised. Interventions aimed at optimising the health systems should be implemented by monitoring and facilitating diagnostic and treatment pathways of patients with oral cancer.

An antisense oligonucleotide targeting TGF-ß2 inhibits lung metastasis and induces CD86 expression in tumor-associated macrophages.

BACKGROUND: The transforming growth factor (TGF)-ß pathway is a well-described inducer of immunosuppression and can act as an oncogenic factor in advanced tumors. Several preclinical and clinical studies show that the TGF-ß pathway can be considered a promising molecular target for cancer therapy. The human genome has three TGF-ß isoforms and not much is known about the oncogenic response to each of the isoforms. Here, we studied the antitumor response to ISTH0047, a recently developed locked nucleic acid-modified antisense oligonucleotide targeting TGF-ß2. MATERIALS AND METHODS: We have studied the anticancer response to ISTH0047 using gymnotic delivery in tumor cell cultures and in in vivo preclinical orthotopic mouse models for primary tumors (breast and kidney tumors) and lung metastasis. RESULTS: We observed that ISTH0047 is able to significantly reduce TGF-ß2 mRNA and protein levels without altering the levels of TGF-ß1 and TGF-ß3. ISTH0047 prevented lung metastasis in syngeneic orthotopic renal cell carcinoma (RENCA) and breast cancer (4T1) tumor models. In addition, using an orthotopic xenograft model of a lung cancer cell line (CRL5807) that mainly expresses TGF-ß2, we observed that ISTH0047 had an important effect on the lung microenvironment inhibiting the growth of lung lesions. ISTH0047 treatment re-educated macrophages in the lung parenchyma to express the tumor-suppressive factor, CD86. CONCLUSION: Overall, our data point to TGF-ß2 as a therapeutic target and ISTH0047 as a novel anticancer drug to prevent lung metastasis by impacting on the tumor niche, in part, through the induction of CD86 in tumor-associated macrophages.

Surviving at high elevations: an inter- and intra-specific analysis in a mountain bird community.

Elevation represents an important selection agent on self-maintenance traits and correlated life histories in birds, but no study has analysed whether life-history variation along this environmental cline is consistent among and within species. In a sympatric community of passerines, we analysed how the average adult survival of 25 open-habitat species varied with their elevational distribution and how adult survival varied with elevation at the intra-specific level. For such purpose, we estimated intra-specific variation in adult survival in two mountainous species, the Water pipit (Anthus spinoletta) and the Northern wheatear (Oenanthe oenanthe) in NW Spain, by means of capture-recapture analyses. At the inter-specific level, high-elevation species showed higher survival values than low elevation ones, likely because a greater allocation to self-maintenance permits species to persist in alpine environments. At the intra-specific level, the magnitude of survival variation was lower by far. Nevertheless, Water pipit survival slightly decreased at high elevations, while the proportion of transient birds increased. In contrast, no such relationships were found in the Northern wheatear. Intra-specific analyses suggest that living at high elevation may be costly, such as for the Water pipit in our case study. Therefore, it seems that a species can persist with viable populations in uplands, where extrinsic mortality is high, by increasing the investment in self-maintenance and prospecting behaviours.

Assessment of time intervals in the pathway to oral cancer diagnosis in north-westerm Spain. Relative contribution of patient interval.

BACKGROUND: Despite continuous advances in diagnosis and therapy, oral cancers are mostly diagnosed at advanced stages with minor survival improvements in the last two decades. Both phenomena have been attributed to delays in the diagnosis. This study aims at quantifying the time elapsed until definitive diagnosis in these patients and the patient interval's contribution. MATERIAL AND METHODS: A hospital-based, ambispective, observational study was undertaken on incident cases with a pathological diagnosis of oral squamous cell carcinoma recruited during 2015 at the Oral and Maxillofacial Surgery services of CHUAC (A Coruña) and POVISA (Vigo) hospitals. RESULTS: 74 consecutive oral cancer patients (59.5% males; median age: 65.0 years (IQ:57-74)) were studied. Most cases (52.7%; n=39) were at advanced stages (TNM III-IV) at diagnosis. The period since first sign/symptom until the patient seeks health care was the longest interval in the pathway to diagnosis and treatment (median: 31.5 days; IQR= 7.0 - 61.0) and represents >60% of the interval since symptom onset until referral to specialised care (pre-referral interval). The average interval assigned to the patient resulted to be relatively larger than the time elapsed since the patient is seen at primary care until a definitive diagnosis is reached (diagnostic interval). Median of the referral interval for primary care professionals: 6.5 days (IQR= 0.0 - 49.2) and accounts for 35% (19% - 51%) of the diagnostic interval. CONCLUSIONS: The patient interval is the main component of the pathway to treatment since the detection of a bodily change until the definitive diagnosis. Therefore, strategies focused on risk groups to shorten this interval should be implemented in order to ease an early diagnosis of symptomatic oral cancer.

Distance of the alveolar antral artery from the alveolar crest. Related factors and surgical considerations in sinus floor elevation.

BACKGROUND: In a variable proportion of maxillary sinuses alveolar antral artery is located close to the residual ridge, increasing the chances for haemorrhagic complications during sinus floor elevation procedures. MATERIAL AND METHODS: Retrospective observational study of CBCT explorations performed for implant-treatment planning. The upper first molar area was selected for this study. The relative uncertainty (standard deviation of the measurement divided by its mean and expressed as a percentage from 0% to 100%) was chosen for determining the observational errors. For modeling the chances of AAA detection, the generalized additive models (GAM) approach was chosen. RESULTS: A total of 240 maxillary sinuses were studied (46.25% males) whose median median age was 58 years old (IQR: 52-66). Univariate models showed that the chances for an AAA-alvelar crest distance ≤15mm increase in wider sinuses with lower, subsinusally edentulous crests. When distance is considered as a continuous variable, the best mutivariate model showed an explained deviance of 67% and included AAA diameter, distance AAA-sinus floor, sinus width, and shape, height and width of the residual ridge. Thinner AAAs are found closer to the crest (within the ≤15mm safe distance). CONCLUSIONS: Bearing in mind the inclusion criteria and the limitations of this investigation, it is concluded that there is a high proportion of maxillary sinuses where AAA describes a course close to the alveolar crest (≤15mm), which was classically considered a safe distance for SFE. This position is related to the presence of atrophic crests (depressed ridge form) and wide maxillary sinuses where the distance of the vessel to the floor of the sinus is small. This information may permit a better surgical planning of SFE procedures.

Capturing intra-tumor genetic heterogeneity by de novo mutation profiling of circulating cell-free tumor DNA: a proof-of-principle.

BACKGROUND: Plasma-derived cell-free tumor DNA (ctDNA) constitutes a potential surrogate for tumor DNA obtained from tissue biopsies. We posit that massively parallel sequencing (MPS) analysis of ctDNA may help define the repertoire of mutations in breast cancer and monitor tumor somatic alterations during the course of targeted therapy. PATIENT AND METHODS: A 66-year-old patient presented with synchronous estrogen receptor-positive/HER2-negative, highly proliferative, grade 2, mixed invasive ductal-lobular carcinoma with bone and liver metastases at diagnosis. DNA extracted from archival tumor material, plasma and peripheral blood leukocytes was subjected to targeted MPS using a platform comprising 300 cancer genes known to harbor actionable mutations. Multiple plasma samples were collected during the fourth line of treatment with an AKT inhibitor. RESULTS: Average read depths of 287x were obtained from the archival primary tumor, 139x from the liver metastasis and between 200x and 900x from ctDNA samples. Sixteen somatic non-synonymous mutations were detected in the liver metastasis, of which 9 (CDKN2A, AKT1, TP53, JAK3, TSC1, NF1, CDH1, MML3 and CTNNB1) were also detected in >5% of the alleles found in the primary tumor sample. Not all mutations identified in the metastasis were reliably identified in the primary tumor (e.g. FLT4). Analysis of ctDNA, nevertheless, captured all mutations present in the primary tumor and/or liver metastasis. In the longitudinal monitoring of the patient, the mutant allele fractions identified in ctDNA samples varied over time and mirrored the pharmacodynamic response to the targeted therapy as assessed by positron emission tomography-computed tomography. CONCLUSIONS: This proof-of-principle study is one of the first to demonstrate that high-depth targeted MPS of plasma-derived ctDNA constitutes a potential tool for de novo mutation identification and monitoring of somatic genetic alterations during the course of targeted therapy, and may be employed to overcome the challenges posed by intra-tumor genetic heterogeneity. REGISTERED CLINICAL TRIAL: www.clinicaltrials.gov, NCT01090960.