Background/Objectives: Although the overall survival prognosis of patients in advanced stages of pancreatic ductal adenocarcinoma (PDAC) is poor, typically ranging from days to months from diagnosis, there are rare cases of patients remaining in therapy for longer periods of time. Early estimations of survival prognosis would allow rational decisions on complex therapy interventions, including radical surgery and robust systemic therapy regimens. Understandably, there is great interest in finding prognostic markers that can be used for patient stratification. We determined the role of various KRAS mutations in the prognosis of PDAC patients using biopsy samples and circulating tumor DNA. Methods: A total of 118 patients with PDAC, clinically confirmed by endoscopic ultrasound-guided fine-needle aspiration biopsy (EUS-FNB), were included in the study. DNA was extracted from cytological slides following a standard cytology evaluation to ensure adequacy (viability and quantity) and to mark the tumor cell fraction. Circulating tumor DNA (ctDNA) was extracted from plasma samples of 45 patients in stage IV of the disease. KRAS mutations in exons 12 and 13 were detected by denaturing capillary electrophoresis (DCE), revealing a minute presence of mutation-specific heteroduplexes. Kaplan-Meier survival curves were calculated for individual KRAS mutation types. Results:KRAS mutations were detected in 90% of tissue (106/118) and 44% of plasma (20/45) samples. All mutations were localized at exon 2, codon 12, with G12D (GGT > GAT) being the most frequent at 44% (47/106) and 65% (13/20), followed by other types including G12V (GGT > GTT) at 31% (33/106) and 10% (2/20), G12R (GGT > CGT) at 17% (18/106) and 10% (2/20), G12C (GGT/TGT) at 5% (5/106) and 0% (0/20) and G12S (GGT/AGT) at 1% (1/106) and 5% (1/20) in tissue and plasma samples, respectively. Two patients had two mutations simultaneously (G12V + G12S and G12D + G12S) in both types of samples (2%, 2/106 and 10%, 2/20 in tissue and plasma samples, respectively). The median survival of patients with the G12D mutation in tissues was less than half that of other patients (median survival 101 days, 95% CI: 80-600 vs. 228 days, 95% CI: 184-602), with a statistically significant overall difference in survival (p = 0.0080, log-rank test), and furthermore it was less than that of all combined patients with other mutation types (101 days, 95% CI: 80-600 vs. 210 days, 95% CI: 161-602, p = 0.0166). For plasma samples, the survival of patients with this mutation was six times shorter than that of patients without the G12D mutation (27 days, 95% CI: 8-334 vs. 161 days, 95% CI: 107-536, p = 0.0200). In contrast, patients with detected KRAS G12R in the tissue survived nearly twice as long as other patients in the aggregate (286 days, 95% CI: 70-602 vs. 162 days, 95% CI: 122-600, p = 0.0374) or patients with other KRAS mutations (286 days, 95% CI: 70-602 vs. 137 days, 95% CI: 107-600, p = 0.0257). Conclusions: Differentiation of specific KRAS mutations in EUS-FNB and ctDNA (above all, the crucial G12D and G12R) is feasible in routine management of PDAC patients and imperative for assessment of prognosis.

• Klíčová slova
• EUS-FNB, KRAS, ctDNA, liquid biopsy, mutation type, pancreatic cancer, prognosis,
• MeSH
• biopsie tenkou jehlou pod endosonografickou kontrolou * MeSH
• cirkulující nádorová DNA genetika   krev   MeSH
• dospělí MeSH
• duktální karcinom slinivky břišní * genetika   patologie   krev   MeSH
• lidé středního věku MeSH
• lidé MeSH
• mutace * MeSH
• nádorové biomarkery genetika   MeSH
• nádory slinivky břišní * genetika   patologie   mortalita   MeSH
• prognóza MeSH
• protoonkogenní proteiny p21(ras) * genetika   MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• tekutá biopsie metody   MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• cirkulující nádorová DNA MeSH
• KRAS protein, human MeSH Prohlížeč
• nádorové biomarkery MeSH
• protoonkogenní proteiny p21(ras) * MeSH

BACKGROUND: Melanoma is the most aggressive skin cancer with ability to recur also after early-stage tumor surgery. The aim was to identify early-stage melanoma patients at high risk of recurrence using liquid biopsy, estimating of mutated BRAF ctDNA and the level of tumor marker S100B in plasma. METHODS: Eighty patients were enrolled in the study. BRAF V600E mutation was determined in FFPE tissue and plasma samples using ultrasensitive ddPCR with pre-amplification. The level of S100B was determined in plasma by immunoassay chemiluminescent method. RESULTS: The best prediction of melanoma recurrence after surgery was observed in patients with combined high level of S100B (S100Bhigh) and ctDNA BRAFV600E (BRAFmut) in preoperative (57.1% vs. 12.5%, p = 0.025) as well as postoperative blood samples (83.3% vs. 14.3%, resp., p = 0.001) in comparison with low S100B and BRAF wild-type. Similarly, patients with preoperative and postoperative S100Bhigh and BRAFmut experienced worse prognosis (DFI p = 0.05, OS p = 0.131 and DFI p = 0.001, OS = 0.001, resp.). CONCLUSION: We observed the benefit of the estimation of combination of S100B and ctDNA BRAFmut in peripheral blood for identification of patients at high risk of recurrence and unfavorable prognosis. SIGNIFICANCE: There is still no general consensus on molecular markers for deciding the appropriateness of adjuvant treatment of early-stage melanoma. We have shown for the first time that the combined determination of the ctDNA BRAFmut oncogene (liquid biopsy) and the high level of tumor marker S100B in pre- and postoperative plasma samples can identify patients with the worst prognosis and the highest risk of tumor recurrence. Therefore, modern adjuvant therapy would be appropriate for these patients with resectable melanoma, regardless of disease stage.

• Klíčová slova
• BRAF V600E, S100B, ctDNA, ddPCR, melanoma,
• MeSH
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• lokální recidiva nádoru * genetika   krev   MeSH
• melanom * genetika   krev   patologie   diagnóza   chirurgie   MeSH
• mutace * MeSH
• nádorové biomarkery * krev   genetika   MeSH
• nádory kůže * krev   genetika   patologie   diagnóza   chirurgie   MeSH
• prediktivní hodnota testů MeSH
• prognóza MeSH
• protoonkogenní proteiny B-Raf * genetika   krev   MeSH
• S-100 kalcium vázající protein G, podjednotka beta * krev   genetika   MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• staging nádorů MeSH
• tekutá biopsie metody   MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• BRAF protein, human MeSH Prohlížeč
• nádorové biomarkery * MeSH
• protoonkogenní proteiny B-Raf * MeSH
• S-100 kalcium vázající protein G, podjednotka beta * MeSH
• S100B protein, human MeSH Prohlížeč

BACKGROUND: Prostate cancer is the second leading cause of male cancer-related deaths in Western countries, which is predominantly attributed to the metastatic castration-resistant stage of the disease (CRPC). There is an urgent need for better prognostic and predictive biomarkers, particularly for androgen receptor targeted agents and taxanes. METHODS: We have searched the PubMed database for original articles and meta-analyses providing information on blood-based markers for castration-resistant prostate cancer monitoring, risk group stratification and prediction of therapy response. RESULTS: The molecular markers are discussed along with the standard clinical parameters, such as prostate specific antigen, lactate dehydrogenase or C-reactive protein. Androgen receptor (AR) alterations are commonly associated with progression to CRPC. These include amplification of AR and its enhancer, point mutations and splice variants. Among DNA methylations, a novel 5-hydroxymethylcytosine activation marker of TOP2A and EZH2 has been identified for the aggressive disease. miR-375 is currently the most promising candidate among non-coding RNAs and sphingolipid analysis has recently emerged as a novel approach. CONCLUSIONS: The promising biomarkers have the potential to improve the care of metastatic prostate cancer patients, however, they need further validation for routine implementation.

• Klíčová slova
• biomarker, castration-resistant prostate cancer, liquid biopsy, progression monitoring, therapy response,
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH

BACKGROUND: Despite advances in early detection and therapies, cancer is still one of the most common causes of death worldwide. Since each tumor is unique, there is a need to implement personalized care and develop robust tools for monitoring treatment response to assess drug efficacy and prevent disease relapse. MAIN BODY: Recent developments in liquid biopsies have enabled real-time noninvasive monitoring of tumor burden through the detection of molecules shed by tumors in the blood. These molecules include circulating tumor nucleic acids (ctNAs), comprising cell-free DNA or RNA molecules passively and/or actively released from tumor cells. Often highlighted for their diagnostic, predictive, and prognostic potential, these biomarkers possess valuable information about tumor characteristics and evolution. While circulating tumor DNA (ctDNA) has been in the spotlight for the last decade, less is known about circulating tumor RNA (ctRNA). There are unanswered questions about why some tumors shed high amounts of ctNAs while others have undetectable levels. Also, there are gaps in our understanding of associations between tumor evolution and ctNA characteristics and shedding kinetics. In this review, we summarize current knowledge about ctNA biology and release mechanisms and put this information into the context of tumor evolution and clinical utility. CONCLUSIONS: A deeper understanding of the biology of ctDNA and ctRNA may inform the use of liquid biopsies in personalized medicine to improve cancer patient outcomes.

• Klíčová slova
• Biomarkers, Cell-free DNA, Circulating tumor DNA, Circulating tumor RNA, Clinical application, Liquid biopsy, Precision oncology, Shedding mechanisms,
• MeSH
• biologie MeSH
• klinická relevance MeSH
• lidé MeSH
• nádorové biomarkery genetika   MeSH
• nádorové cirkulující buňky * MeSH
• nádory * patologie   MeSH
• RNA MeSH
• volné cirkulující nukleové kyseliny * genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Research Support, N.I.H., Extramural MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH
• Názvy látek
• nádorové biomarkery MeSH
• RNA MeSH
• volné cirkulující nukleové kyseliny * MeSH

Head and neck cancer (HNC) remains one of the leading causes of mortality worldwide due to tumor diagnosis at a late stage, loco-regional aggression, and distant metastases. A standardized diagnostic procedure for HNC is a tissue biopsy that cannot faithfully portray the in-depth tumor dynamics. Therefore, there is an urgent need to develop simple, accurate, and non-invasive methods for cancer detection and follow-up. A saliva-based liquid biopsy allows convenient, non-invasive, and painless collection of high volumes of this biofluid, with the possibility of repetitive sampling, all enabling real-time monitoring of the disease. No approved clinical test for HNC has yet been established. However, epigenetic changes in saliva circulating cell-free DNA (cfDNA) have the potential for a wide range of clinical applications. Therefore, the aim of this review is to present an overview of cfDNA-based methylation patterns in saliva for early detection of HNC, with particular attention to circulating tumor DNA (ctDNA). Due to advancements in isolation and detection technologies, as well as next- and third-generation sequencing, recent data suggest that salivary biomarkers may be successfully applied for early detection of HNC in the future, but large prospective clinical trials are still warranted.

• Klíčová slova
• DNA methylation, biomarker, cell-free DNA (cfDNA), circulating tumor DNA (ctDNA), early cancer detection, head and neck cancer, liquid biopsy, saliva,
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH

BACKGROUND/AIM: Circulating tumour DNA (ctDNA) represents an emerging biomarker in non-small cell lung cancer (NSCLC). We focused on the combination of ctDNA and positron emission tomography/computed tomography (PET/CT) in the follow-up monitoring of advanced-stage NSCLC patients treated with chemotherapy. PATIENTS AND METHODS: Eighty-four patients were enrolled in this study. 18F-fluorodeoxyglucose PET/CT and ctDNA assessments were performed at baseline and after two cycles of chemotherapy (follow-up). RESULTS: There was a correlation of ctDNA with metabolic tumour volume (MTV), total lesion glycolysis (TLG), and iodine concentration (IC) at baseline (p=0.001, p=0.001, p=0.003) and at follow-up (p=0.006, p=0.002, p=0.001). The objective response was associated with follow-up ctDNA (p<0.001) and the change of all PET/CT parameters. ROC analyses showed that the combination of follow-up ctDNA with changes in SUVmax is very promising for the estimation of objective response and progression-free survival. CONCLUSION: The combination of ctDNA assessment with PET/CT is a promising approach for the follow-up monitoring of therapy response and prognosis estimation of advanced-stage NSCLC patients.

• Klíčová slova
• Circulating tumour DNA, PET/CT, liquid biopsy, non-small cell lung cancer, therapy response,
• MeSH
• cirkulující nádorová DNA * genetika   MeSH
• fluorodeoxyglukosa F18 metabolismus   terapeutické užití   MeSH
• glykolýza MeSH
• lidé MeSH
• nádory plic * diagnostické zobrazování   farmakoterapie   genetika   MeSH
• nemalobuněčný karcinom plic * diagnostické zobrazování   farmakoterapie   genetika   MeSH
• PET/CT metody   MeSH
• prognóza MeSH
• prospektivní studie MeSH
• radiofarmaka terapeutické užití   MeSH
• retrospektivní studie MeSH
• tumor burden MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• cirkulující nádorová DNA * MeSH
• fluorodeoxyglukosa F18 MeSH
• radiofarmaka MeSH

BACKGROUND: Implementation of adjuvant therapies in non-metastatic melanoma improved treatment outcomes in some patients; however, adjuvant therapy can be associated with significant cost and risk of toxicity. Therefore, there is an unmet need to better identify patients at high risk of recurrence. PATIENTS AND METHODS: We carried out an ultrasensitive droplet digital PCR (ddPCR)-based detection of BRAFV600E-mutated circulating tumor DNA (ctDNA) from blood samples prospectively collected before surgery, 1 hour after surgery, and then serially during follow-up. RESULTS: In 80 patients (stages ≤III), BRAFV600E mutations were detected in 47.2% of tissue, in 37.7% of ctDNA samples collected before surgery, and in 25.9% of ctDNA samples collected 1 hour after surgery. Patients with detected ctDNA in blood collected 1 hour after surgery compared to patients without detected ctDNA had higher likelihood of melanoma recurrence (P < 0.001) and shorter median disease-free survival (P = 0.001) and overall survival (P = 0.003). CONCLUSIONS: Ultrasensitive ddPCR can detect ctDNA in pre- and post-surgical blood samples from patients with resectable melanoma. Detection of ctDNA in post-surgical samples is associated with inferior treatment outcomes.

• Klíčová slova
• circulating tumor DNA, liquid biopsy, melanoma,
• MeSH
• cirkulující nádorová DNA * genetika   MeSH
• lidé MeSH
• melanom * genetika   MeSH
• mutace * MeSH
• polymerázová řetězová reakce MeSH
• protoonkogenní proteiny B-Raf * genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• BRAF protein, human MeSH Prohlížeč
• cirkulující nádorová DNA * MeSH
• protoonkogenní proteiny B-Raf * MeSH

(1) Background: Head and Neck Squamous Cell Carcinoma (HNSCC) is one of the most common malignancies globally. An early diagnosis of this disease is crucial, and the detection of gene mutations in circulating tumour DNA (ctDNA) through a liquid biopsy is a promising non-invasive diagnostic method. This review aims to provide an overview of ctDNA mutations in HNSCC patients and discuss the potential use of this tool in diagnosis and prognosis. (2) Methods: A systematic search for articles published in the English language between January 2000 and April 2021 in the Medline and Scopus databases was conducted. (3) Results: A total of 10 studies published in nine publications were selected and analysed. Altogether, 390 samples were obtained from HNSCC patients, and 79 control samples were evaluated. The most often explored gene mutation in ctDNA was TP53. (4) Conclusions: The examination of a larger group of gene mutations and the use of a combination of multiple detection methods contribute to a higher detection rate of mutated ctDNA. More studies are necessary to verify these conclusions and to translate them into clinical practice.

• Klíčová slova
• circulating tumour DNA (ctDNA), diagnostic biomarker, head and neck cancer squamous cell carcinoma (HNSCC), liquid biopsy,
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH

Circulating tumor DNA (ctDNA) sequencing is being rapidly adopted in precision oncology, but the accuracy, sensitivity and reproducibility of ctDNA assays is poorly understood. Here we report the findings of a multi-site, cross-platform evaluation of the analytical performance of five industry-leading ctDNA assays. We evaluated each stage of the ctDNA sequencing workflow with simulations, synthetic DNA spike-in experiments and proficiency testing on standardized, cell-line-derived reference samples. Above 0.5% variant allele frequency, ctDNA mutations were detected with high sensitivity, precision and reproducibility by all five assays, whereas, below this limit, detection became unreliable and varied widely between assays, especially when input material was limited. Missed mutations (false negatives) were more common than erroneous candidates (false positives), indicating that the reliable sampling of rare ctDNA fragments is the key challenge for ctDNA assays. This comprehensive evaluation of the analytical performance of ctDNA assays serves to inform best practice guidelines and provides a resource for precision oncology.

• MeSH
• cirkulující nádorová DNA genetika   MeSH
• individualizovaná medicína * MeSH
• lékařská onkologie * MeSH
• lidé MeSH
• limita detekce MeSH
• nádory genetika   MeSH
• reprodukovatelnost výsledků MeSH
• sekvenční analýza DNA normy   MeSH
• směrnice pro lékařskou praxi jako téma MeSH
• vysoce účinné nukleotidové sekvenování metody   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Research Support, N.I.H., Intramural MeSH
• validační studie MeSH
• Názvy látek
• cirkulující nádorová DNA MeSH

BACKGROUND: The use of liquid biopsy is of potential high importance for children with high grade (HGG) and diffuse midline gliomas (DMG), particularly where surgical procedures are limited, and invasive biopsy sampling not without risk. To date, however, the evidence that detection of cell-free DNA (cfDNA) or circulating tumor DNA (ctDNA) could provide useful information for these patients has been limited, or contradictory. METHODS: We optimized droplet digital PCR (ddPCR) assays for the detection of common somatic mutations observed in pediatric HGG/DMG, and applied them to liquid biopsies from plasma, serum, cerebrospinal fluid (CSF), and cystic fluid collected from 32 patients. RESULTS: Although detectable in all biomaterial types, ctDNA presented at significantly higher levels in CSF compared to plasma and/or serum. When applied to a cohort of 127 plasma specimens from 41 patients collected from 2011 to 2018 as part of a randomized clinical trial in pediatric non-brainstem HGG/DMG, ctDNA profiling by ddPCR was of limited use due to the small volumes (mean = 0.49 mL) available. In anecdotal cases where sufficient material was available, cfDNA concentration correlated with disease progression in two examples each of poor response in H3F3A_K27M-mutant DMG, and longer survival times in hemispheric BRAF_V600E-mutant cases. CONCLUSION: Tumor-specific DNA alterations are more readily detected in CSF than plasma. Although we demonstrate the potential of the approach to assessing tumor burden, our results highlight the necessity for adequate sample collection and approach to improve detection if plasma samples are to be used.

• Klíčová slova
• CSF, DIPG, HGG, cfDNA, ctDNA, plasma,
• Publikační typ
• časopisecké články MeSH