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Genomic analysis, performed on tumoral tissue DNA and on circulating tumor DNA (ctDNA) from blood, is the cornerstone of precision cancer medicine. Herein, we characterized the clinical prognostic implications of the concordance of alterations in major cancer genes between tissue- and blood-derived DNA in a pan-cancer cohort. The molecular profiles of both liquid (Guardant Health) and tissue (Foundation Medicine) biopsies from 433 patients were analyzed. Mutations and amplifications of cancer genes scored by these two tests were assessed. In 184 (42.5%) patients, there was at least one mutual gene alteration. The mean number of mutual gene-level alterations in the samples was 0.67 per patient (range: 0-5). A higher mutual gene-level alteration number correlated with shorter overall survival (OS). As confirmed in multivariable analysis, patients with ≥2 mutual gene-level alterations in blood and tissue had a hazard ratio (HR) of death of 1.49 (95% confidence interval [CI]=1-2.2; P=0.047), whereas patients with ≥3 mutual gene-level alterations had an HR of death 2.38 (95% CI=1.47-3.87; P=0.0005). Together, our results show that gene-level concordance between tissue DNA and ctDNA analysis is prevalent and is an independent factor predicting significantly shorter patient survival., (© 2023 The Authors. Molecular Oncology published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Introduction: Nucleic acid sequencing technologies have advanced significantly in recent years, thereby allowing for the development of liquid biopsies as new means to detect cancer biomarkers and cancer heterogenicity. Most of the assays available, clinically, focus on cell free DNA (cfDNA), however, cell free RNA (cfRNA) is also present. cfRNA has the potential to complement and improve cancer detection especially in cancers like lung cancer, which are usually only diagnosed at late stages and therefore have poor long-term survival outcomes., Methods: Remnant EDTA plasma was collected from lung cancer patients and non-cancer individuals at the University of Maryland Medical Center. RNA was extracted and processed for next generation sequencing with a tagmentation-based library preparation approach., Results: cfRNA was successfully extracted and sequenced from 52 EDTA-treated plasma samples with volumes as low as 1.5 mL. This quantity was sufficient to prepare libraries with the length of libraries averaging from 264 bp to 381 bp and resulted in over 2.2 to 3.6 million total sequence reads respectively. Sequential dilution of cfRNA samples from healthy individuals indicated that the starting cfRNA concentration influenced the detection of differentially expressed genes., Conclusions: This proof-of-concept study provides a framework for screening cfRNA for identifying biomarkers for early detection of lung cancer (and other cancers), using minimal amounts of samples (1.5 mL) from standard EDTA 3-mL collection tubes routinely used for patient care. Further studies in large populations are required to establish limit of detection and other parameters including precision, accuracy, sensitivity, and specificity, to standardize this method., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Amol Carl Shetty and Kristin Mullins disclose discounted reagents from Illumina, Inc. Sanford A Stass discloses grants from NIH/NCI, and reduction in cost of reagents from Illumina. Chamindi Seneviratne discloses discounted reagents from Illumina, Inc, and grants from NIH/NIAAA (K23AA020899) for the collection of control samples identified as “Set 2”, and 5R01AA026291. Feng Jiang discloses grants FDA-5U01FD005946-06 and NCI-UH2CA229132., (Copyright © 2023 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.)

Precision medicine has transformed the way urothelial carcinoma is managed. However, current practices are limited by the availability of tissue samples for genomic profiling and the spatial and temporal molecular heterogeneity observed in many studies. Among rapidly advancing genomic sequencing technologies, non-invasive liquid biopsy has emerged as a promising diagnostic tool to reproduce tumour genomics, and has shown potential to be integrated in several aspects of clinical care. In urothelial carcinoma, liquid biopsies such as plasma circulating tumour DNA (ctDNA) and urinary tumour DNA (utDNA) have been investigated as a surrogates for tumour biopsies and might bridge many shortfalls currently faced by clinicians. Both ctDNA and utDNA seem really promising in urothelial carcinoma diagnosis, staging and prognosis, response to therapy monitoring, detection of minimal residual disease and surveillance. The use of liquid biopsies in patients with urothelial carcinoma could further advance precision medicine in this population, facilitating personalized patient monitoring through non-invasive assays., (© 2023. Springer Nature Limited.)

Competing Interests: Conflicts of interest The authors declare no conflicts of interest relating to this manuscript, as per the seminar article structure. The manuscript is an extended summary of an online seminar and therefore reflects presentations of already published work and details of already established clinical trials.

Introduction: Lung cancer is a leading cause of death in patients with cancer. Early diagnosis is crucial to improve the prognosis of patients with lung cancer. Plasma circulating cell-free DNA (cfDNA) contains comprehensive genetic and epigenetic information from tissues throughout the body, suggesting that early detection of lung cancer can be done non-invasively, conveniently, and cost-effectively using high-sensitivity techniques such as sequencing., Areas Covered: In this review, we summarize the latest technological innovations, coupled with next-generation sequencing (NGS), regarding genomic alterations, methylation, and fragmentomic features of cfDNA for the early detection of lung cancer, as well as their clinical advances. Additionally, we discuss the suitability of study designs for diagnostic accuracy evaluation for different target populations and clinical questions., Expert Opinion: Currently, cfDNA-based early screening and diagnosis of lung cancer faces many challenges, such as unsatisfactory performance, lack of quality control standards, and poor repeatability. However, the progress of several large prospective studies employing epigenetic features has shown promising predictive performance, which has inspired cfDNA sequencing for future clinical applications. Furthermore, the development of multi-omics markers for lung cancer, including genome-wide methylation and fragmentomics, is expected to play an increasingly important role in the future.

Introduction: Advances in genomics have facilitated the application of cell-free DNA (cfDNA) and circulating tumor DNA (ctDNA) in phase II and phase III clinical trials. The various mutations of cfDNA/ctDNA have been correlated with clinical features. Advances in next-generation sequencing (NGS) and digital droplet PCR have paved the way for identifying cfDNA/ctDNA mutations., Areas Covered: Herein, the biology of ctDNA and its function in clinical application in metastasis, which may lead to improved clinical management of metastatic cancer patients, are comprehensively reviewed., Expert Opinion: Metastatic cancer ctDNA shows the greatest frequency of mutations in TP53, HER-2, KRAS, and EGFR genes (alteration frequency of > 50%). Therefore, identifying key mutations frequently present in metastatic cancers can help identify patients with pre-malignant tumors before cancer progression. Studying ctDNA can help determine the prognosis and select appropriate treatments for affected patients. Nevertheless, the obstacles to detecting and analyzing ctDNA should be addressed before translation into routine practice. Also, more clinical trials should be conducted to study the significance of ctDNA in commonly diagnosed malignancies. Given the recent advances in personalized anti-neoplastic treatments, further studies are needed to detect a panel of ctDNA and patient-specific ctDNA for various cancers.

Esophageal cancer (EC) has one of the highest mortality rates among cancers, making it imperative that therapies are optimized and dynamically adapted to individuals. In this regard, liquid biopsy is an increasingly important method for residual disease monitoring. However, conflicting detection rates (14% versus 60%) and varying cell-free circulating tumor DNA (ctDNA) levels (0.07% versus 0.5%) have been observed in previous studies. Here, we aim to resolve this discrepancy. For 19 EC patients, a complete set of cell-free DNA (cfDNA), formalin-fixed paraffin-embedded tumor tissue (TT) DNA and leukocyte DNA was sequenced (139 libraries). cfDNA was examined in biological duplicates and/or longitudinally, and TT DNA was examined in technical duplicates. In baseline cfDNA, mutations were detected in 12 out of 19 patients (63%); the median ctDNA level was 0.4%. Longitudinal ctDNA changes were consistent with clinical presentation. Considerable mutational diversity was observed in TT, with fewer mutations in cfDNA. The most recurrently mutated genes in TT were TP53 , SMAD4 , TSHZ3 , and SETBP1 , with SETBP1 being reported for the first time. ctDNA in blood can be used for therapy monitoring of EC patients. However, a combination of solid and liquid samples should be used to help guide individualized EC therapy.

Purpose: GI cancers commonly spread to the peritoneal cavity, particularly from primary adenocarcinomas of the stomach and appendix. Peritoneal metastases are difficult to visualize on cross-sectional imaging and cause substantial morbidity and mortality. The purpose of this study was to determine whether serial highly sensitive tumor-informed circulating tumor DNA (ctDNA) measurements could longitudinally track changes in disease burden and inform clinical care., Methods: This was a retrospective case series of patients with gastric or appendiceal adenocarcinoma and isolated peritoneal disease that was radiographically occult. Patients underwent quantitative tumor-informed ctDNA testing (Signatera) as part of routine clinical care. No interventions were prespecified based on ctDNA results., Results: Of 13 patients studied, the median age was 65 (range, 45-75) years, with 7 (54%) women, 5 (38%) patients with gastric, and 8 (62%) patients with appendiceal adenocarcinoma. Eight (62%) patients had detectable ctDNA at baseline measurement, with median value 0.13 MTM/mL (range, 0.06-11.68), and assay was technically unsuccessful in two cases with appendiceal cancer because of limited tumor tissue. Five (100%) patients with gastric cancer and 3 (50%) patients with appendiceal cancer had detectable ctDNA at baseline. Although baseline levels of ctDNA were low, longitudinal assessment tracked with changes in disease burden among patients undergoing chemotherapy for metastatic disease. In two patients undergoing surveillance after definitive surgical management of gastric adenocarcinoma, detection of ctDNA prompted diagnosis of isolated peritoneal disease., Conclusion: Quantitative tumor-informed serial ctDNA testing aids clinical management of patients with isolated peritoneal disease. Low levels of baseline ctDNA suggest a role for highly sensitive ctDNA approaches over panel-based testing. Further exploration of this approach should be considered in patients with isolated peritoneal malignant disease.

Saliva is an emerging source of disease biomarkers, particularly for cancers of the head and neck. Although analysis of cell-free DNA (cfDNA) in saliva holds promise as a liquid biopsy for cancer detection, currently there are no standardized methodologies for the collection and isolation of saliva for the purposes of studying DNA. Here, we evaluated various saliva collection receptacles and DNA purification techniques, comparing DNA quantity, fragment size, source, and stability. Then, using our optimized techniques, we tested the ability to detect human papillomavirus (HPV) DNA- a bona fide cancer biomarker in a subset of head and neck cancers- from patient saliva samples. For saliva collection, we found that the Oragene OG-600 receptacle yielded the highest concentration of total salivary DNA as well as short fragments <300 bp corresponding to mononucleosomal cell-free DNA. Moreover, these short fragments were stabilized beyond 48 hours after collection in contrast to other saliva collection receptacles. For DNA purification from saliva, the QIAamp Circulating Nucleic Acid kit yielded the highest concentration of mononucleosome-sized DNA fragments. Freeze-thaw of saliva samples did not affect DNA yield or fragment size distribution. Salivary DNA isolated from the OG-600 receptacle was found to be composed of both single and double-stranded DNA, including mitochondrial and microbial sources. While levels of nuclear DNA were consistent over time, levels of mitochondrial and microbial DNA were more variable and increased 48 hours after collection. Finally, we found that HPV DNA was stable in OG-600 receptacles, was reliably detected within the saliva of patients with HPV-positive head and neck cancer, and was abundant among mononucleosome-sized cell-free DNA fragments. Our studies have defined optimal techniques for isolating DNA from saliva that will contribute to future applications in liquid biopsy-based cancer detection., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: SVB is inventor on patents related to cell-free DNA mutation and methylation analysis technologies that have been licensed to Roche Molecular Diagnostics and Adela, respectively. SVB is a cofounder of, has ownership in, and serves in a leadership role at Adela. This does not alter our adherence to PLOS ONE policies on sharing data and materials., (Copyright: © 2023 Brooks et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Circulating tumor DNA (ctDNA) has emerged as a promising biomarker of minimal residual disease (MRD) in solid tumors. There is increasing evidence to suggest that the detection of ctDNA following curative‑intent treatments has high potential in anticipating future relapse in various solid tumors. Multiple liquid biopsy technical approaches and commercial platforms, including tumor‑informed and tumor‑agnostic ctDNA assays, have been developed for ctDNA‑based MRD detection in solid tumors. Accurate ctDNA‑based MRD analysis remains a critical technical challenge due to the very low concentration of ctDNA in peripheral blood samples, particularly in cancer patients following a curative‑intent surgery or treatment. The present review summarizes the current key technical approaches that can be used to analyze ctDNA in the surveillance of MRD in solid tumors and provides a brief update on current commercial assays or platforms available for ctDNA‑based MRD detection. The available evidence to date supporting ctDNA as a biomarker for detection of MRD in various types of solid tumors is also reviewed. In addition, technical and biological variables and considerations in pre‑analytical and analytical steps associated with ctDNA‑based MRD detection are discussed.

Cholangiocarcinoma is a malignant epithelial tumor arising from bile ducts that is frequently fatal. Diagnosis is difficult due to tumor location in the biliary tract. Earlier diagnosis requires less invasive methods of identifying effective biomarkers for cholangiocarcinoma. The present study investigated the genomic profiles of cell-free DNA (cfDNA) and DNA from corresponding primary cholangiocarcinomas using a targeted sequencing panel. Somatic mutations in primary tumor DNA and circulating tumor DNA (ctDNA) were compared and clinical applications of ctDNA validated in patients with cholangiocarcinoma. A comparison of primary tumor DNA and ctDNA identified somatic mutations in patients with early cholangiocarcinomas that showed clinical feasibility for early screening. The predictive value of single-nucleotide variants (SNVs) of preoperative plasma cfDNA positive for somatic mutations of the primary tumor was 42%. The sensitivity and specificity of postoperative plasma SNVs in detecting clinical recurrence were 44% and 45%, respectively. Targetable fibroblast growth factor receptor 2 (FGFR2) and Kirsten rat sarcoma virus (KRAS) mutations were detected in 5% of ctDNA samples from patients with cholangiocarcinoma. These findings showed that genomic profiling of cfDNA was useful in clinical evaluation, although ctDNA had limited ability to detect mutations in cholangiocarcinoma patients. Serial monitoring of ctDNA is important clinically and in assessing real-time molecular aberrations in cholangiocarcinoma patients.

Background: Circulating tumor DNA (ctDNA) sequencing is a promising approach for tailoring therapy in patients with cancer. We report hereby the results from a prospective study where we investigated the impact of comprehensive molecular profiling of ctDNA in patients with advanced solid tumors., Patients and Methods: Genomic analysis was performed using the FoundationOne Liquid CDx Assay [324 genes, tumor mutational burden (TMB), microsatellite instability status]. Each individual genomic report was reviewed and discussed weekly by a multidisciplinary tumor board (MTB). Actionable targets were classified by ESMO Scale for Clinical Actionability of Molecular Targets (ESCAT) tier leading to molecular-based treatment suggestions wherever it was possible., Results: Between December 2020 and November 2021, 1772 patients with metastatic solid tumors underwent molecular profiling. Median time to assay results was 12 days. Results were contributive for 1658 patients (94%). At least one actionable target was detected in 1059 patients (64%) with a total of 1825 actionable alterations including alteration of the DNA damage repair response pathway (n = 336, 18%), high TMB (>16 mutations/Mb; n = 243, 13%), PIK3CA mutations (n = 150, 8%), ERBB family pathway alterations (n = 127, 7%), PTEN alterations (n = 95, 5%), FGFR alterations (n = 67, 4%) and MET activations (n = 13, 0.7%). The MTB recommended a matched therapy for 597 patients (56%) with a total of 819 therapeutic orientations: clinical trials (n = 639, 78%), off-label/compassionate use (n = 81, 10%), approved drug (n = 51, 6%), and early access program (n = 48, 6%). In total, 122 patients (21%) were treated. Among the assessable patients (n = 107), 4 (4%) had complete response, 35 (33%) had partial response, 27 (25%) had stable disease, and 41 (38%) a progressive disease as best response. The median progression-free survival and median overall survival were 4.7 months (95% confidence interval 2.7-6.7 months) and 8.3 months (95% confidence interval 4.7-11.9 months) respectively., Conclusions: ctDNA sequencing with a large panel is an efficient approach to match patients with advanced cancer with targeted therapies., (Copyright © 2023 European Society for Medical Oncology. Published by Elsevier Ltd. All rights reserved.)

Purpose: To evaluate early circulating tumor DNA (ctDNA) kinetics using a tumor-naïve assay and correlate it with clinical outcomes in early phase immunotherapy (IO) trials., Methods: Plasma samples were analyzed using a 425-gene next-generation sequencing panel at baseline and before cycle 2 (3-4 weeks) in patients with advanced solid tumors treated with investigational IO agents. Variant allele frequency (VAF) for mutations in each gene, mean VAF (mVAF) from all mutations, and change in mVAF between both time points were calculated. Hyperprogression (HyperPD) was measured using Matos and Caramella criteria., Results: A total of 162 plasma samples were collected from 81 patients with 27 different tumor types. Patients were treated in 37 different IO phase I/II trials, 72% of which involved a PD-1/PD-L1 inhibitor. ctDNA was detected in 122 plasma samples (75.3%). A decrease in mVAF from baseline to precycle 2 was observed in 24 patients (37.5%) and was associated with longer progression-free survival (hazard ratio [HR], 0.43; 95% CI, 0.24 to 0.77; P < .01) and overall survival (HR, 0.54; 95% CI, 0.3 to 0.96; P = .03) compared with an increase. These differences were more marked if there was a >50% decrease in mVAF for both progression-free survival (HR, 0.29; 95% CI, 0.13 to 0.62; P < .001) and overall survival (HR, 0.23; 95% CI, 0.09 to 0.6; P = .001). No differences in mVAF changes were observed between the HyperPD and progressive disease patients., Conclusion: A decrease in ctDNA within 4 weeks of treatment was associated with treatment outcomes in patients in early phase IO trials. Tumor-naïve ctDNA assays may be useful for identifying early treatment benefits in phase I/II IO trials.

Objectives: To evaluate DNA ploidy and S-phase fraction (SPF) in non-Lynch colonic adenocarcinoma, ulcerative colitis (UC), Crohn disease (CD) which are known as risk factors, and colitis. We correlated ploidy and SPF with tumor grading, staging and BRAF expression., Methods: All studied adenocarcinomas have intact mismatch repair genes as proved by immunohistochemistry. All were assessed for ploidy by automated image-based DNA cytometry and histograms were drawn. Immunostaining by anti-BRAF V600E was performed. Diagnostic laparoscopy (DL) was done as a preliminary step for staging GI cancers., Results: there is significant difference in DNA ploidy between groups; 77.5% and 17.5% of aneuploid cases are adenocarcinoma and UC. Groups are compared in terms of 2C, 4C, above 4C DNA content and SPF and significant difference is principally found between adenocarcinoma group and others. In adenocarcinomas, DNA ploidy is significantly correlated with tumor staging and grading. Regarding BRAF expression, there is significant difference between groups; all adenocarcinomas, 83.33% of UC were positive, while all cases of colitis, bilharzial colitis, CD were negative. There is significant relation between BRAF and SPF among all diploid cases including adenocarcinoma, and among non-neoplastic diploid cases. There is direct significant relation between BRAF intensity and adenocarcinoma staging. There is no significant difference between BRAF and ploidy among UC cases, although 75% of aneuploid UC are positive. DL helps in GI cancer staging. Routine laparoscopy before laparotomy, especially in cancers which have equivocal operability helps to avoid unnecessary laparotomies., Conclusion: Based on significant difference in ploidy between adenocarcinoma and UC and between SPF and ploidy, assessment of ploidy by DNA cytometry for UC and other colitis could therefore predict impending malignant transformation before development of colonic dysplasia. Also measuring SPF in adenocarcinoma helps to select patients who could greatly benefit from chemotherapy. DL has vital role in staging GI cancers.

Sampling circulating tumor DNA (ctDNA) using liquid biopsies offers clinically important benefits for monitoring cancer progression. A single ctDNA sample represents a mixture of shed tumor DNA from all known and unknown lesions within a patient. Although shedding levels have been suggested to hold the key to identifying targetable lesions and uncovering treatment resistance mechanisms, the amount of DNA shed by any one specific lesion is still not well characterized. We designed the Lesion Shedding Model (LSM) to order lesions from the strongest to the poorest shedding for a given patient. By characterizing the lesion-specific ctDNA shedding levels, we can better understand the mechanisms of shedding and more accurately interpret ctDNA assays to improve their clinical impact. We verified the accuracy of the LSM under controlled conditions using a simulation approach as well as testing the model on three cancer patients. The LSM obtained an accurate partial order of the lesions according to their assigned shedding levels in simulations and its accuracy in identifying the top shedding lesion was not significantly impacted by number of lesions. Applying LSM to three cancer patients, we found that indeed there were lesions that consistently shed more than others into the patients' blood. In two of the patients, the top shedding lesion was one of the only clinically progressing lesions at the time of biopsy suggesting a connection between high ctDNA shedding and clinical progression. The LSM provides a much needed framework with which to understand ctDNA shedding and to accelerate discovery of ctDNA biomarkers. The LSM source code has been available in the IBM BioMedSciAI Github (https://github.com/BiomedSciAI/Geno4SD)., (© The Author(s) 2023. Published by Oxford University Press.)

DNA-methylation alterations are common in cancer and display unique characteristics that make them ideal markers for tumor quantification and classification. Here we present MIMESIS, a computational framework exploiting minimal DNA-methylation signatures composed by a few dozen informative DNA-methylation sites to quantify and classify tumor signals in tissue and cell-free DNA samples. Extensive analyses of multiple independent and heterogenous datasets including >7200 samples demonstrate the capability of MIMESIS to provide precise estimations of tumor content and to enable accurate classification of tumor type and molecular subtype. To assess our framework for clinical applications, we designed a MIMESIS-informed assay incorporating the minimal signatures for breast cancer. Using both artificial samples and clinical serial cell-free DNA samples from patients with metastatic breast cancer, we show that our approach provides accurate estimations of tumor content, sensitive detection of tumor signal and the ability to capture clinically relevant molecular subtype in patients' circulation. This study provides evidence that our extremely parsimonious approach can be used to develop cost-effective and highly scalable DNA-methylation assays that could support and facilitate the implementation of precision oncology in clinical practice., (© The Author(s) 2023. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Background: Anal squamous cell carcinoma (SCCA) is an uncommon malignancy with a rising incidence that has a high cure rate in its early stages. There is an unmet need for a reliable method to monitor response to treatment and assist in surveillance. Circulating tumor DNA (ctDNA) testing has shown great promise in other solid tumors for monitoring disease progression and detecting relapse in real time. This study aimed to determine the feasibility and use of personalized and tumor-informed ctDNA testing in SCCA., Patients and Methods: We analyzed real-world data from 251 patients (817 plasma samples) with stages I-IV SCCA, collected between 11/5/19 and 5/31/22. The tumor genomic landscape and feasibility of ctDNA testing was examined for all patients. The prognostic value of longitudinal ctDNA testing was assessed in patients with clinical follow-up (N = 37)., Results: Whole-exome sequencing analysis revealed PIK3CA as the most commonly mutated gene, and no associations between mutations and stage. Anytime ctDNA positivity and higher ctDNA levels (MTM/mL) were associated with metastatic disease (P = .004). For 37 patients with clinical follow-up, median follow-up time was 21.0 months (range: 4.1-67.3) post-diagnosis. For patients with stages I-III disease, anytime ctDNA-positivity after definitive treatment was associated with reduced DFS (HR: 28.0; P = .005)., Conclusions: Our study demonstrates the feasibility of personalized and tumor-informed ctDNA testing as an adjunctive tool in patients with SCCA as well as potential use for detection of molecular/minuteimal residual disease, and relapse during surveillance. Prospective studies are needed to better evaluate the use of ctDNA testing in this indication., (© The Author(s) 2022. Published by Oxford University Press.)

Objective: To explore the clinical diagnostic value of DNA image cytometry (DNA-ICM) ploidy analysis in malignant pleural effusion cancer screening, this study analyzed the effect of exfoliated cell smears (ECSs), cell blocks (CBs), and immunochemistry., Method: A total of 830 cases of pleural effusion were considered for the DNA-ICM ploidy analysis. The ECSs were centrifuged, the CBs were formed, and the DNA-ICM ploidy analysis was carried out in the diagnosis of malignant pleural effusion. Immunochemistry and biopsy was applied to differentiate between benign and malignant pleural effusion and to determine the source of the latter. The sensitivity and specificity differences between the three methods alone and in combination were compared., Results: The sensitivity of the DNA-ICM, ECS, and CB methods was 96.28%, 94.93%, and 95.95%, respectively, and the specificity of each method was 86.52%, 87.08%, and 86.14%, respectively. The sensitivity and specificity of the combined diagnosis method were 99.32% and 75.09%, respectively. Among the 22 cases diagnosed as positive in the DNA-ICM ploidy analysis but negative in the ECS and CB analyses, four cases were diagnosed as positive by comprehensive clinical diagnosis., Conclusion: The sensitivity and specificity of DNA-ICM ploidy analysis are high; the positive detection rate of pleural fluid cytology is effectively increased, and the missed detection rate of cell pathologies is effectively reduced. The combination of the three methods significantly improves the specificity and sensitivity of the diagnosis of malignant pleural effusion, and immunochemistry with CBs can be used to accurately analyze the primary tumor site., (© 2022 Wiley Periodicals LLC.)

Purpose: Circulating tumor DNA (ctDNA) has the potential to guide therapy selection and monitor treatment response in patients with metastatic cancer. However, germline and clonal hematopoiesis-associated alterations can confound identification of tumor-specific mutations in cell-free DNA (cfDNA), often requiring additional sequencing of tumor tissue. The current study assessed whether ctDNA-based treatment response monitoring could be performed in a tumor tissue-independent manner by combining ultra-deep targeted sequencing analyses of cfDNA with patient-matched white blood cell (WBC)-derived DNA., Experimental Design: In total, 183 cfDNA and 49 WBC samples, along with 28 tissue samples, from 52 patients with metastatic colorectal cancer participating in the prospective phase III CAIRO5 clinical trial were analyzed using an ultra-deep targeted sequencing liquid biopsy assay., Results: The combined cfDNA and WBC analysis prevented false-positives due to germline or hematopoietic variants in 40% of patients. Patient-matched tumor tissue sequencing did not provide additional information. Longitudinal analyses of ctDNA were more predictive of overall survival than standard-of-care radiological response evaluation. ctDNA mutations related to primary or acquired resistance to panitumumab were identified in 42% of patients., Conclusions: Accurate calling of ctDNA mutations for treatment response monitoring is feasible in a tumor tissue-independent manner by combined cfDNA and patient-matched WBC genomic DNA analysis. This tissue biopsy-independent approach simplifies sample logistics and facilitates the application of liquid biopsy ctDNA testing for evaluation of emerging therapy resistance, opening new avenues for early adaptation of treatment regimens., (©2022 The Authors; Published by the American Association for Cancer Research.)

Precision medicine has driven a major change in the treatment of many forms of cancer. The discovery that each patient is different and each tumor mass has its own characteristics has shifted the focus of basic and clinical research to the singular individual. Liquid biopsy (LB), in this sense, presents new scenarios in personalized medicine through the study of molecules, factors, and tumor biomarkers in blood such as circulating tumor cells (CTCs), circulating tumor DNA (ctDNA), exosomes and circulating tumor microRNAs (ct-miRNAs). Moreover, its easy application and complete absence of contraindications for the patient make this method applicable in a great many fields. Melanoma, given its highly heterogeneous characteristics, is a cancer form that could significantly benefit from the information linked to liquid biopsy, especially in the treatment management. In this review, we will focus our attention on the latest applications of liquid biopsy in metastatic melanoma and possible developments in the clinical setting.

Compared to stage I-III gastric cancer (GC), the level of cell-free DNA (cfDNA) was significantly higher in stage IV GC. The mutation patterns of different metastatic patterns between cfDNA and tumor DNA in stage IV GC have not yet been reported. We used next-generation sequencing (NGS) to analyze cfDNA and tumor DNA in 56 stage IV GC patients. Tumor DNA and cfDNA were analyzed using a 29-gene NGS panel. In tumor samples, the most commonly mutated gene was TP53 (64%), followed by ARID1A (62%), KMT2C (60%) and KMT2D (58%). In cfDNA samples, the most commonly mutated genes were FAT4 (19%) and MACF1 (19%), followed by KMT2D (18%), ARID1A (14%) and LRP1B (14%). The concordance of mutation patterns in these 29 genes was 42.0% between cfDNA and tumor DNA. A specificity of 100% was found when using the mutation status of cfDNA to predict mutations in tumor samples. The sensitivity of the mutation status of cfDNA to predict mutation in tumor samples was highest in FAT4 (88.9%), followed by MACF1 (80%), CDH1 (75%) and PLB1 (75%). For cfDNA with PLB1 mutations, patients were more likely to develop distant lymphatic metastasis than peritoneal metastasis. Patients with multiple-site metastases had significantly more mutated spots than patients with single-site metastasis. Due to the high sensitivity and specificity of some genes in the prediction of mutation in tumor samples, monitoring the mutation pattern of cfDNA may be useful in the stage IV GC treatment.

Background: Sputum cell-free DNA (cfDNA) has been confirmed to be a valued surrogate sample for detection of EGFR mutations in patients with lung adenocarcinoma (LAC). Whether it is suitable for detection of mutations of multiple driver genes has not been reported., Methods: A total of 83 patients with LAC were enrolled and their sputum and paired tumor samples were collected. A next-generation sequencing (NGS)-based 10-gene panel was used to test sputum supernatant-derived cfDNA and paired tumor DNA. The sputum sediments were used for cytological evaluation., Results: The total positive rates of hotspot mutations of the 10 driver genes in sputum cfDNA and matched tissue samples were 65.1% and 77.1%, respectively. The overall detection sensitivity of variants in sputum cfDNA was 81.3% (95% confidence interval [CI], 69.2%, 89.5%) and the specificity was 100% (95% CI, 79.1%, 100%). The sensitivities of testing sputum cfDNA from patients with stage IIIB-IV was 87.0% (95% CI, 74.5%, 94.1%); the sensitivities of testing sputum cfDNA from patients with malignant sputum was 92.3% (95% CI, 78.0%, 98.0%); and the sensitivity of testing sputum cfDNA from patients with malignant sputum in stage IIIB-IV were 94.1% (95% CI, 78.9%, 99.0%)., Conclusions: This study demonstrated that sputum cfDNA were successfully used for the detection of multiple driver genes by NGS. Sputum cfDNA could be a valuable surrogate clinical sample for all-in-one test of mutations to guide target therapies, especially for patients with advanced LAC and malignant sputum., (© 2022 American Cancer Society.)

Colorectal cancer (CRC) is a very common gastrointestinal tumor, ranking second in the global cause of cancer death. Because of the invasive nature of biopsy and cannot reflect the heterogeneity of tumor or monitor the dynamic progress of tumor, it is necessary to induce a novel noninvasive method to improve the current treatment strategies of colorectal cancer. Among all the components of liquid biopsy, circulating tumor DNA (ctDNA) may have the best future. CtDNA maintains the same genomic characteristics as those in matched tumor tissues, so it allows quantitative evaluation and analysis of mutation load in body fluid. Furthermore, because the half-life of ctDNA is from 16 min to several hours in circulation, the circulating ctDNA can be measured repeatedly within a certain period to monitor the response of CRC to treatment, the occurrence of drug resistance, and the diagnosis of recurrence., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Early detection is critical for minimizing mortality from cancer. Plasma cell-free DNA (cfDNA) contains the signatures of tumor DNA, allowing us to quantify the signature and diagnose early-stage tumors. Here, we report a novel tumor fragment quantification method, TOF (Tumor Originated Fragment) for the diagnosis of lung cancer by quantifying and analyzing both the plasma cfDNA methylation patterns and fragmentomic signatures. TOF utilizes the amount of ctDNA predicted from the methylation density information of each cfDNA read mapped on 6243 lung-tumor-specific CpG markers. The 6243 tumor-specific markers were derived from lung tumor tissues by comparing them with corresponding normal tissues and healthy blood from public methylation data. TOF also utilizes two cfDNA fragmentomic signatures: 1) the short fragment ratio, and 2) the 5' end-motif profile. We used 298 plasma samples to analyze cfDNA signatures using enzymatic methyl-sequencing data from 201 lung cancer patients and 97 healthy controls. The TOF score showed 0.98 of the area under the curve in correctly classifying lung cancer from normal samples. The TOF score resolution was high enough to clearly differentiate even the early-stage non-small cell lung cancer patients from the healthy controls. The same was true for small cell lung cancer patients., Competing Interests: Declaration of competing interest Jong Bhak is the CEO and a major shareholder of Clinomics Inc., Korea. Other authors declare no competing interests., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

There is no useful biomarker to evaluate treatment response and early relapse in head and neck squamous cell carcinoma (HNSCC). Circulating tumor DNA (ctDNA) is a promising biomarker for detecting minimal residual diseases and monitoring treatment effect. We investigated whether individualized ctDNA analysis could help monitor treatment response and relapse in HNSCC. Mutation analysis of tumor and peripheral blood mononuclear cell (PBMC) DNAs of 26 patients with HNSCC was performed using a custom squamous cell carcinoma (SCC) panel. The identified individualized mutated genes were defined as ctDNA candidates. We investigated whether frequent ctDNA monitoring via digital PCR (dPCR) is clinically valid for HNSCC patients. TP53 was the most frequently mutated gene and was detected in 14 of 24 cases (58.2%), wherein two cases were excluded owing to the absence of tumor-specific mutations in the SCC panel. Six cases were excluded because of undesignable and unusable primer-probes for dPCR. Longitudinal ctDNA was monitored in a total of 18 cases. In seven cases, ctDNA tested positive again or did not test negative, and all seven cases relapsed after initial curative treatment. In 11 cases, after initial curative treatment, ctDNA remained negative and patients were alive without recurrence. Patients who remained negative for ctDNA during follow-up after initial curative treatment (n = 11) had a significantly better prognosis than those who reverted to ctDNA positivity (n = 7; p < 0.0001; log-rank test). Individualized ctDNA monitoring using SCC panel and dPCR might be a novel and promising biomarker for HNSCC., (© 2022 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.)

Comprehensive genomic profiling is increasingly used to facilitate precision oncology based on molecular stratification. In addition to conventional tissue comprehensive genomic profiling, comprehensive genomic profiling of circulating tumor DNA has become widely utilized in cancer care owing on its advantages, including less invasiveness, rapid turnaround time, and capturing heterogeneity. However, circulating tumor DNA comprehensive genomic profiling has some limitations, mainly false negatives due to low levels of plasma circulating tumor deoxyribonucleic acid and false positives caused by clonal hematopoiesis. Nevertheless, no guidelines and recommendations fully address these issues. Here, an expert panel committee involving representatives from 12 Designated Core Hospitals for Cancer Genomic Medicine in Japan was organized to develop expert consensus recommendations for the use of circulating tumor deoxyribonucleic acid-based comprehensive genomic profiling. The aim was to generate guidelines for clinicians and allied healthcare professionals on the optimal use of the circulating tumor DNA assays in advanced solid tumors and to aid the design of future clinical trials that utilize and develop circulating tumor DNA assays to refine precision oncology. Fourteen clinical questions regarding circulating tumor deoxyribonucleic acid comprehensive genomic profiling including the timing of testing and considerations for interpreting results were established by searching and curating associated literatures, and corresponding recommendations were prepared based on the literature for each clinical question. Final consensus recommendations were developed by voting to determine the level of each recommendation by the Committee members., (© 2022 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

Somatic mutations of genes involved in NF-κB, PI3K/AKT, NOTCH, and JAK/STAT signaling pathways play an important role in the pathogenesis of Hodgkin lymphoma (HL). HL tumor cells form only about 5% of the tumor mass; however, it was shown that HL tumor-derived DNA could be detected in the bloodstream. This circulating tumor DNA (ctDNA) reflects the genetic profile of HL tumor cells and can be used for qualitative and quantitative analysis of tumor-specific somatic DNA mutations within the concept of liquid biopsy. Overall, the most frequently mutated gene in HL is STAT6; however, the exact spectrum of mutations differs between individual HL histological subtypes. Importantly, reduction of ctDNA plasma levels after initial treatment is highly correlated with prognosis. Therefore, ctDNA shows great promise as a novel tool for non-invasive tumor genome analysis for biomarker driven therapy as well as for superior minimal residual disease monitoring and treatment resistance detection. Here, we summarize the recent advancements of ctDNA analysis in HL with focus on ctDNA detection methodologies, genetic profiling of HL and its clonal evolution, and the emerging prognostic value of ctDNA., (© 2022. The Author(s).)

Cancer is still ranked as a leading cause of death according to estimates from the World Health Organization (WHO) and the strong link between tumor viruses and human cancers have been proved for almost six decades. Cell-free DNA (cfDNA) has drawn enormous attention for its dynamic, instant, and noninvasive advantages as one popular type of cancer biomarker. cfDNAs are mainly released from apoptotic cells and exosomes released from cancer cells, including those infected with viruses. Although cfDNAs are present at low concentrations in peripheral blood, they can reflect tumor load with high sensitivity. Considering the relevance of the tumor viruses to the associated cancers, cfDNAs derived from viruses may serve as good biomarkers for the early screening, diagnosis, and treatment monitoring. In this review, we summarize the methods and newly developed analytic techniques for the detection of cfDNAs from different body fluids, and discuss the implications of cfDNAs derived from different tumor viruses in the detection and treatment monitoring of virus-associated cancers. A better understanding of cfDNAs derived from tumor viruses may help formulate novel antitumoral strategies to decrease the burden of cancers that attributed to viruses., (© 2022 Wiley Periodicals LLC.)

Purpose: Circulating tumor DNA (ctDNA) has good clinical guiding value for metastatic breast cancer (MBC) patients. This study aimed to apply a novel genetic analysis approach for therapeutic prediction based on ctDNA alterations., Method: This nonrandomized, multicenter study recruited 223 MBC patients (NCT05079074). Plasma samples were collected for target-capture deep sequencing of ctDNA at baseline, after the 2nd cycle of treatment, and when progressive disease (PD) was evaluated. Samples were categorized into four levels according to the number of ctDNA alterations: level 1 (no alterations), level 2 (1-2 alterations), level 3 (3-4 alterations) and level 4 (≥5 alterations). According to ctDNA alteration level and variant allele frequency (VAF), a novel ctDNA-level Response Evaluation Criterion in Solid Tumors (ctle-RECIST) was established to assess treatment response and predict progression-free survival (PFS)., Results: The median PFS in level 1 (6.63 months) patients was significantly longer than that in level 2-4 patients (level 2: 5.70 months; level 3-4: 4.90 months, p < 0.05). After 2 cycles of treatment, based on ctle-RECIST, the median PFS of level-based disease control rate (lev-DCR) patients was significantly longer than that of level-based PD (lev-PD) patients [HR 2.42 (1.52-3.85), p < 0.001]. In addition, we found that ctDNA level assessment could be a good supplement to radiologic assessment. The median PFS in the dual-DCR group tended to be longer than that in the single-DCR group [HR 1.41 (0.93-2.13), p = 0.107]., Conclusion: The ctDNA alteration level and ctle-RECIST could be novel biomarkers of prognosis and could complement radiologic assessment in MBC., Competing Interests: Declaration of competing interest The authors have no relevant financial or non-financial interests to disclose., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Ploidy analysis is the fundamental method of measuring DNA content. For decades, the principal way of conducting ploidy analysis was through flow cytometry. A flow cytometer is a specialized tool for analyzing cells in a solution. This is convenient in laboratory environments, but prohibits measurement reproducibility and the complete detachment of sample preparation from data acquisition and analysis, which seems to have become paramount with the constant decrease in the number of pathologists per capita all over the globe. As more open computer-aided systems emerge in medicine, the demand for overcoming these shortcomings, and opening access to even more (and more flexible) options, has also emerged. Image-based analysis systems can provide an alternative to these types of workloads, placing the abovementioned problems in a different light. Flow cytometry data can be used as a reference for calibrating an image-based system. This article aims to show an approach to constructing an image-based solution for ploidy analysis, take measurements for a basic comparison of the data produced by the two methods, and produce a workflow with the ultimate goal of calibrating the image-based system.

Background and Aims: Circulating tumor DNA (ctDNA) has been recognized as a reliable source to reflect the molecular and genetic landscape of corresponding tumors in recent years. In this study, we tested the application of a cancer genomic panel sequencing on the cerebrospinal fluid (CSF)-derived ctDNA for the less invasive detection and diagnosis of glioma., Materials and Methods: CtDNA was extracted from 26 CSF samples and subject to a cancer genomic panel sequencing of 520 genes to analyze the mutation profiles and tumor mutation burden (TMB), which were compared with their corresponding tumor DNA samples. Associations between mutations or TMB and clinical characteristics were also evaluated., Results: A high detection rate of ctDNA (24/26, 92.3%) was observed in CSF. CtDNA mutations had high concordance rates with tumor DNA, especially in non-copy number variations and in glioblastoma. CSF ctDNA TMB also exhibited a strong correlation with tumor DNA TMB (R 2  = 0.879, P < 0.001), particularly in glioblastoma (R 2  = 0.992, P < 0.001). Age was significantly associated with CSF ctDNA TMB in glioma patients., Conclusion: We established a less invasive but effective molecular diagnostic approach using a cancer genomic panel sequencing system targeting CSF ctDNA for glioma, especially in glioblastoma., (Copyright © 2022. Published by Elsevier B.V.)

Background: Circulating tumour DNA (ctDNA) has been spotlighted as an attractive biomarker because of its easy accessibility and real-time representation of tumour genetic profile. However, the clinical utility of longitudinal ctDNA monitoring has not been clearly defined., Methods: Serial blood samples were obtained from metastatic colorectal cancer patients undergoing first-line chemotherapy. ctDNA was sequenced using a targeted next-generation sequencing platform which included 106 genes. Changes in ctDNA profile and treatment outcome were comprehensively analysed., Results: A total of 272 samples from 62 patients were analysed. In all, 90.3% of patients had detectable ctDNA mutation before treatment. ctDNA clearance after chemotherapy was associated with longer progression-free survival which was independent of radiological response (adjusted hazard ratio 0.22, 95% confidence interval 0.10-0.46). Longitudinal monitoring was able to detect ctDNA progression which preceded radiological progressive disease (PD) in 58.1% (median 3.3 months). Diverse resistant mutations (34.9%) and gene amplification (7.0%) at the time of PD were discovered. For 16.3% of the PD patients, the newly identified mutations could be potential candidates of targeted therapy or clinical trial., Conclusion: ctDNA profile provided a more accurate landscape of tumour and dynamic changes compared to radiological evaluation. Longitudinal ctDNA monitoring may improve personalised treatment decision-making., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Genomic profiling of bronchoalveolar lavage (BAL) samples may be useful for tumor profiling and diagnosis in the clinic. Here, we compared tumor-derived mutations detected in BAL samples from subjects with non-small cell lung cancer (NSCLC) to those detected in matched plasma samples. Cancer Personalized Profiling by Deep Sequencing (CAPP-Seq) was used to genotype DNA purified from BAL, plasma, and tumor samples from patients with NSCLC. The characteristics of cell-free DNA (cfDNA) isolated from BAL fluid were first characterized to optimize the technical approach. Somatic mutations identified in tumor were then compared with those identified in BAL and plasma, and the potential of BAL cfDNA analysis to distinguish lung cancer patients from risk-matched controls was explored. In total, 200 biofluid and tumor samples from 38 cases and 21 controls undergoing BAL for lung cancer evaluation were profiled. More tumor variants were identified in BAL cfDNA than plasma cfDNA in all stages (P < 0.001) and in stage I to II disease only. Four of 21 controls harbored low levels of cancer-associated driver mutations in BAL cfDNA [mean variant allele frequency (VAF) = 0.5%], suggesting the presence of somatic mutations in nonmalignant airway cells. Finally, using a Random Forest model with leave-one-out cross-validation, an exploratory BAL genomic classifier identified lung cancer with 69% sensitivity and 100% specificity in this cohort and detected more cancers than BAL cytology. Detecting tumor-derived mutations by targeted sequencing of BAL cfDNA is technically feasible and appears to be more sensitive than plasma profiling. Further studies are required to define optimal diagnostic applications and clinical utility., Significance: Hybrid-capture, targeted deep sequencing of lung cancer mutational burden in cell-free BAL fluid identifies more tumor-derived mutations with increased allele frequencies compared with plasma cell-free DNA. See related commentary by Rolfo et al., p. 2826., (©2022 The Authors; Published by the American Association for Cancer Research.)

Background: Observation of anticancer therapy effect by monitoring of minimal residual disease (MRD) is becoming an important tool in management of non-small cell lung cancer (NSCLC). The approach is based on periodic detection and quantification of tumor-specific somatic DNA mutation in circulating tumor DNA (ctDNA) extracted from patient plasma. For such repetitive testing, complex liquid-biopsy techniques relying on ultra-deep NGS sequencing are impractical. There are other, cost-effective, methods for ctDNA analysis, typically based on quantitative PCR or digital PCR, which are applicable for detecting specific individual mutations in hotspots. While such methods are routinely used in NSCLC therapy prediction, however, extension to cover broader spectrum of mutations (e.g., in tumor suppressor genes) is required for universal longitudinal MRD monitoring. Methods: For a set of tissue samples from 81 NSCLC patients we have applied a denaturing capillary electrophoresis (DCE) for initial detection of somatic mutations within 8 predesigned PCR amplicons covering oncogenes and tumor suppressor genes. Mutation-negative samples were then subjected to a large panel NGS sequencing. For each patient mutation found in tissue was then traced over time in ctDNA by DCE. Results: In total we have detected a somatic mutation in tissue of 63 patients. For those we have then prospectively analyzed ctDNA from collected plasma samples over a period of up to 2 years. The dynamics of ctDNA during the initial chemotherapy therapy cycles as well as in the long-term follow-up matched the clinically observed response. Conclusion: Detection and quantification of tumor-specific mutations in ctDNA represents a viable complement to MRD monitoring during therapy of NSCLC patients. The presented approach relying on initial tissue mutation detection by DCE combined with NGS and a subsequent ctDNA mutation testing by DCE only represents a cost-effective approach for its routine implementation., Competing Interests: MM is employed by Elphogene company. Elphogene is currently providing oncoMonitor liquid biopsy/ctDNA test, which is in part utilizing the DCE mutation detection technology presented in this work. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Benesova, Ptackova, Halkova, Semyakina, Svaton, Fiala, Pesek and Minarik.)

Dying tumor cells shed DNA fragments into the circulation that are known as circulating tumor DNA (ctDNA). Liquid biopsy tests aim to detect cancer using known markers, including genetic alterations and epigenetic profiles of ctDNA. Despite various advantages, the major limitation remains the low fraction of tumor-originating DNA fragments in a high background of normal blood-cell originating fragments in the cell-free DNA (cfDNA) pool in plasma. Deep targeted sequencing of cfDNA allows for enrichment of fragments in known cancer marker-associated regions of the genome, thus increasing the chances of detecting the low fraction variant harboring fragments. Most targeted sequencing panels are designed to include known recurrent mutations or methylation markers of cancer. Here, we propose the integration of cancer-specific chromatin accessibility states into panel designs for liquid biopsy. Using machine learning approaches, we first identify accessible and inaccessible chromatin regions specific to each major human cancer type. We then introduce a score that quantifies local chromatin accessibility in tumor relative to blood cells and show that this metric can be useful for prioritizing marker regions with higher chances of being detected in cfDNA for inclusion in future panel designs., (© 2022. The Author(s).)

Early recurrence detection of head and neck squamous cell carcinoma (HNSCC) is important for improving prognosis. Recently, circulating tumor DNA (ctDNA) has been reported to be useful in early detection or treatment response determination in various carcinomas. This study aimed to identify the utility of ctDNA for predicting recurrent metastasis in patients with HNSCC. We collected pre-treatment tissues (malignant and normal tissues) and multiple plasma samples before and after treatment for 20 cases of HNSCC treated with radical therapy. ctDNA was detected in pre-treatment plasma in 10 cases; however, there were no significant associations with tumor recurrence and staging. During follow-up, ctDNA was detected in 5 of the 7 plasma samples of recurrent cases but not in the 13 recurrence-free cases. Moreover, there was a significant difference in post-treatment relapse-free survival time between the groups with and without detected ctDNA (20.6 ± 7.7 vs. 9.6 ± 9.1 months, respectively; log-rank test, p < 0.01). Moreover, for two of the five cases with ctDNA detected after treatment, ctDNA detection was a more sensitive predictor of recurrence than imaging studies. ctDNA detection during treatment follow-up was useful in patients with HNSCC for predicting the response to treatment and recurrent metastasis., (© 2022. The Author(s).)

Breast cancer is a spatially and temporally dynamic disease in which differently evolving genetic clones are responsible for progression and clinical outcome. We review tumor heterogeneity and clonal evolution from studies comparing primary tumors and metastasis and discuss plasma circulating tumor DNA as a powerful real-time approach for monitoring the clonal landscape of breast cancer during treatment and recurrence. We found only a few early studies exploring clonal evolution and heterogeneity through analysis of multiregional tissue biopsies of different progression steps in comparison with circulating tumor DNA (ctDNA) from blood plasma. The model of linear progression seemed to be more often reported than the model of parallel progression. The results show complex routes to metastasis, however, and plasma most often reflected metastasis more than primary tumor. The described patterns of evolution and the polyclonal nature of breast cancer have clinical consequences and should be considered during patient diagnosis and treatment selection. Current studies focusing on the relevance of clonal evolution in the clinical setting illustrate the role of liquid biopsy as a noninvasive biomarker for monitoring clonal progression and response to treatment. In the clinical setting, circulating tumor DNA may be an ideal support for tumor biopsies to characterize the genetic landscape of the metastatic disease and to improve longitudinal monitoring of disease dynamics and treatment effectiveness through detection of residual tumor after resection, relapse, or metastasis within a particular patient., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Uveal melanoma (UM) is the most common primary intraocular malignancy affecting adults. Despite successful local treatment of the primary tumour, metastatic disease develops in up to 50% of patients. Metastatic UM carries a particularly poor prognosis, with no effective therapeutic option available to date. Genetic studies of UM have demonstrated that cytogenetic features, including gene expression, somatic copy number alterations and specific gene mutations can allow more accurate assessment of metastatic risk. Pre-emptive therapies to avert metastasis are being tested in clinical trials in patients with high-risk UM. However, current prognostic methods require an intraocular tumour biopsy, which is a highly invasive procedure carrying a risk of vision-threatening complications and is limited by sampling variability. Recently, a new diagnostic concept known as "liquid biopsy" has emerged, heralding a substantial potential for minimally invasive genetic characterisation of tumours. Here, we examine the current evidence supporting the potential of blood circulating tumour cells (CTCs), circulating tumour DNA (ctDNA), microRNA (miRNA) and exosomes as biomarkers for UM. In particular, we discuss the potential of these biomarkers to aid clinical decision making throughout the management of UM patients., (© 2022. The Author(s).)

RET fusion has emerged as a targetable driver in non-small-cell lung cancer. A comparative analysis on RET fusions at DNA [DNA sequencing (DNA-seq)] and RNA [RNA sequencing (RNA-seq)] levels was performed in this study. Archived tumor samples from 54 non-small-cell lung cancer patients with DNA-level noncanonical RET fusions were selected for RNA-seq. RNA-seq identified RET fusion transcripts in 41 of 44 samples passing quality control. In the subset of cases harboring RET 3'-end fusions and predicted to produce in-frame proteins (group A; n = 33), RNA-seq identified the same 3'-end fusions in 32 (96.9%). A total of 26 of 32 also had a reciprocal RET 5'-end fusion detected by DNA-seq that was not transcribed. In the subset with DNA-level out-of-frame RET fusions (group B; n = 9), RNA-seq identified in-frame RET fusion transcripts in 8 cases (88.9%). In the subset only identified with a RET 5'-end fusion by DNA-seq (group C; n = 2), RNA-seq detected the corresponding 3'-end fusion in one case. The discordant DNA- and RNA-level fusions observed in group B may be mediated by complex genomic rearrangement events and transcriptional or post-transcriptional processes. In conclusion, DNA-seq demonstrates a high concordance of 96.9% on detecting in-frame RET fusion, but shows a low concordance on detecting out-of-frame RET fusion and RET 5'-end fusion compared with RNA-seq., (Copyright © 2022 Association for Molecular Pathology and American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Background: The most widely used noninvasive screening tests for colorectal cancer are fecal occult blood tests. Stool DNA test was developed in recent years. However, direct comparative analyses of these tests within the same population are still sparse., Methods: A total of 2,842 participants who visited outpatient clinics or cancer screening centers were enrolled. Stool DNA test-I (KRAS, BMP3, NDRG4, and hemoglobin immunochemical tests), stool DNA test-II (SDC2 and SFRP2 tests), and fecal immunochemical test (FIT) alone were performed and colonoscopy was used as the gold standard among 2,240 participants. Forty-two and 302 participants had colorectal cancer and advanced adenomas (AA), respectively., Results: The sensitivity for colorectal cancer of stool DNA test-I, -II, and FIT was 90.5%, 92.9%, and 81.0%, respectively. The sensitivity for advanced neoplasm (AN; colorectal cancer plus AA) of stool DNA test-I, -II, and FIT was 34.9%, 42.2%, and 25.9%, respectively. The specificity of stool DNA test-I, -II, and FIT was 91.4%, 93.3%, and 96.8%, respectively, among those with negative results on colonoscopy. When the specificity of FIT was adjusted to match that of stool DNA tests by changing the threshold, no significant difference was seen in the sensitivities among the three tests for detecting colorectal cancer. For AN, the sensitivity of FIT was higher than DNA test-I and similar to DNA test-II under the same specificities., Conclusions: There was no significant advantage of the two stool DNA tests compared with FIT in detecting colorectal cancer or AN in this study., Impact: Our findings do not support extensive use of stool DNA tests instead of FIT., (©2021 American Association for Cancer Research.)

Evaluation of a cancer cell fraction is important for accurate molecular analysis, and pathological analysis is the gold standard for evaluation. Despite the potential convenience, no established molecular markers for evaluation are available. In this study, we aimed to identify ovarian cancer cell fraction markers using DNA methylation highly specific to ovarian cancer cells. Using genome-wide DNA methylation data, we screened candidate marker genes methylated in 30 ovarian cancer FFPE samples and 12 high-grade serous ovarian cancer cell lines, and unmethylated in two female leucocytes and two normal fallopian epithelial cell samples. Methylation levels of two genes, SIM1, and ZNF154, showed high correlation with pathological cancer cell fractions among the 30 ovarian cancer FFPE samples (R = 0.61 for SIM1, 0.71 for ZNF154). For cost-effective analysis of FFPE samples, pyrosequencing primers were designed, and successfully established for SIM1 and ZNF154. Correlation between a pathological cancer cell fraction and methylation levels obtained by pyrosequencing was confirmed to be high (R = 0.53 for SIM1, 0.64 for ZNF154). Finally, an independent validation cohort of 29 ovarian cancer FFPE samples was analyzed. ZNF154 methylation showed a high correlation with the pathological cancer cell fraction (R = 0.77, P < 0.0001). Therefore, the ZNF154 methylation level was considered to be useful for the estimation of ovarian cancer cell fraction, and is expected to help accurate molecular analysis., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Background: Circulating tumor DNA (ctDNA) has become one of the crucial components for cancer detection with the increase of precision medicine practice. ctDNA has great potential as a blood-based biomarker for the detection and treatment of cancer in its early stages. The purpose of this article was to discuss ctDNA and how it can be utilized to detect cancer. The benefits and drawbacks of this cancer detection technology, as well as the field's future possibilities in various cancer management scenarios, are discussed. MAIN TEXT: ctDNA has clinical applications in disease diagnosis and monitoring. It can be used to identify mutations of interest and genetic heterogeneity. Another use of ctDNA is to monitor the effects of therapy by detecting mutation-driven resistance. Different technologies are being used for the detection of ctDNA. Next-generation sequencing, digital PCR, real-time PCR, and mass spectrometry are used. Using dPCR makes it possible to partition and analyze individual target sequences from a complex mixture. Mass-spectrometry technology enables accurate detection and quantification of ctDNA mutations at low frequency. Surface-enhanced Raman spectroscopy (SERS) and UltraSEEK are two systems based on this technology. There is no unified standard for detecting ctDNA as it exists in a low concentration in blood. As there is no defined approach, false positives occur in several methods due to inadequate sensitivities. Techniques used in ctDNA are costly and there is a limitation in clinical settings., Short Conclusion: A detailed investigation is urgently needed to increase the test's accuracy and sensitivity. To find a standard marker for all forms of cancer DNA, more study is needed. Low concentrations of ctDNA in a sample require improved technology to provide the precision that low concentrations of ctDNA in a sample afford., (© 2022. The Author(s).)