Detection of cell-free tumor DNA in cerebrospinal fluid as a diagnostic biomarker for leptomeningeal melanoma metastasis: A case series.

Leptomeningeal melanoma metastases (LMM) are associated with poor survival. Diagnosis is based on clinical presentation, brain MRI and cerebrospinal fluid (CSF) analysis. Inconclusive findings at initial presentation can delay treatment. In this single-center case series, detection of BRAFV600- and NRASQ61-mutant cell-free tumor DNA (cfDNA) in CSF was evaluated as a complementary diagnostic biomarker. In 12 patients with clinical suspicion of LMM, a retrospective analysis of MRI, CSF cytology and cfDNA analysis on 1 mL of CSF using the Idylla® platform was carried out. Nine patients displayed MRI abnormalities suggesting LMM. CSF analysis identified malignant cells in three patients (including one without MRI abnormalities). BRAFV600- or NRASQ61-mutant cfDNA was detected in CSF of nine patients (eight with and one without MRI abnormalities; all patients with positive CSF cytology). Subsequent follow-up confirmed LMM in all patients with positive and in one patient with a negative CSF cfDNA analysis (sensitivity 81.8%; specificity 100%). Our findings suggest that analyzing BRAFV600- and NRASQ61-mutant cfDNA in CSF using the Idylla® platform holds promise as a sensitive and specific complementary diagnostic biomarker for LMM, particularly in case of inconsistency between imaging and CSF cytology. The 110-min analysis can facilitate urgent treatment decisions.

Error-Corrected Deep Targeted Sequencing of Circulating Cell-Free DNA from Colorectal Cancer Patients for Sensitive Detection of Circulating Tumor DNA.

Circulating tumor DNA (ctDNA) is a promising biomarker, reflecting the presence of tumor cells. Sequencing-based detection of ctDNA at low tumor fractions is challenging due to the crude error rate of sequencing. To mitigate this challenge, we developed ultra-deep mutation-integrated sequencing (UMIseq), a fixed-panel deep targeted sequencing approach, which is universally applicable to all colorectal cancer (CRC) patients. UMIseq features UMI-mediated error correction, the exclusion of mutations related to clonal hematopoiesis, a panel of normal samples for error modeling, and signal integration from single-nucleotide variations, insertions, deletions, and phased mutations. UMIseq was trained and independently validated on pre-operative (pre-OP) plasma from CRC patients (n = 364) and healthy individuals (n = 61). UMIseq displayed an area under the curve surpassing 0.95 for allele frequencies (AFs) down to 0.05%. In the training cohort, the pre-OP detection rate reached 80% at 95% specificity, while it was 70% in the validation cohort. UMIseq enabled the detection of AFs down to 0.004%. To assess the potential for detection of residual disease, 26 post-operative plasma samples from stage III CRC patients were analyzed. From this we found that the detection of ctDNA was associated with recurrence. In conclusion, UMIseq demonstrated robust performance with high sensitivity and specificity, enabling the detection of ctDNA at low allele frequencies.

Liquid Biopsy in H3K27M Diffuse Midline Glioma.

Diffuse midline glioma (DMG) with H3K27M mutation is an aggressive and difficult to treat pediatric brain tumor. Recurrent gain of function mutations in H3.3 (H3.3A) and H3.1 (H3C2) at the 27th lysine to methionine (H3K27M) are seen in over 2/3 of DMGs, and are associated with a worse prognosis. Due to the anatomical location of DMG, traditional biopsy carries risk for neurologic injury as it requires penetration of vital midline structures. Further, radiographic (MRI) monitoring of DMG often shows non-specific changes, which makes therapeutic monitoring difficult. This indicates a critical need for more minimally invasive methods, such as liquid biopsy, to understand, diagnose, and monitor H3K27M DMG. Here we review the use of all modalities to date to detect biomarkers of H3K27M in CSF, blood, and urine, and compare their effectiveness in detection, diagnosis, and monitoring treatment response. We provide specific detail of recent efforts to monitor CSF and plasma H3K27M cell-free DNA in patients undergoing therapy with the imipridone ONC201. Lastly, we discuss the future of therapeutic monitoring of H3K27M-DMG, including biomarkers such as mitochondrial DNA, mutant and modified histones, and novel sequencing-based approaches for improved detection methods.

Tracking tumor evolution during the first-line treatment in brain glioma via serial profiling of cell-free tumor DNA from tumor in situ fluid.

Objective: Tumor in situ fluid (TISF) refers to the fluid within surgical cavities of glioma. Several studies preliminarily proved the value of cell-free tumor DNA (cf-tDNA) from TISF in the dynamic characterization of the glioma genome. Here, we assessed the potential utility of TISF cf-tDNA in broad aspects of tumor evolution under therapeutic pressure. Methods: This study was conducted under an Institutional Review Board-approved protocol at Henan Provincial People's Hospital (China). Cf-tDNA samples were sequenced with a designed 68-gene panel. A total of 205 cf-tDNA samples from 107 patients were studied. The clinical relevance of serial cf-tDNA profiling during the postoperative course was analyzed. Results: At least one tumor mutations were detected in 179/205 (87.3%) TISF cf-tDNA samples. Serial cf-tDNA was complementary to molecular residual disease and to initial tumors. Serial cf-tDNA revealed the selection of pre-existing mismatch repair-deficient cells by temozolomide as a resistant mechanism. Cf-tDNA parameters during treatment were predictive of recurrence, and serial cf-tDNA monitoring had diagnostic value for early recurrence. A total of 223 potentially actionable genomic alterations were assessed in cf-tDNA samples, wherein 78% were not found in any tumor tissue. Conclusions: In conclusion, serial TISF cf-tDNA profiling is valuable in tracking the tumor evolution of glioma during treatment and may be a feasible non-invasive option for monitoring glioma in future prospective studies and clinical practice.

Evaluation of commercial kits for isolation and bisulfite conversion of circulating cell-free tumor DNA from blood.

BACKGROUND: DNA methylation biomarkers in circulating cell-free DNA (cfDNA) have great clinical potential for cancer management. Most methods for DNA methylation analysis require bisulfite conversion, causing DNA degradation and loss. This is particularly challenging for cfDNA, which is naturally fragmented and normally present in low amounts. The aim of the present study was to identify an optimal combination of cfDNA isolation and bisulfite conversion kits for downstream analysis of DNA methylation biomarkers in plasma. RESULTS: Of the five tested bisulfite conversion kits (EpiJET Bisulfite Conversion Kit, EpiTect Plus DNA Bisulfite Kit (EpiTect), EZ DNA Methylation-Direct Kit, Imprint DNA Modification Kit (Imprint) and Premium Bisulfite Kit), the highest and lowest DNA yield and recovery were achieved using the EpiTect kit and the Imprint kit, respectively, with more than double the amount of DNA for the EpiTect kit. Of the three tested cfDNA isolation kits (Maxwell RSC ccfDNA Plasma Kit, QIAamp Circulating Nucleic Acid Kit (CNA) and QIAamp MinElute ccfDNA Mini Kit), the CNA kit yielded around twice as much cfDNA compared to the two others kits, although with more high molecular weight DNA present. When comparing various combinations of cfDNA isolation kits and bisulfite conversion kits, the CNA kit and the EpiTect kit were identified as the best-performing combination, resulting in the highest yield of bisulfite converted cfDNA from normal plasma, as measured by droplet digital PCR (ddPCR). As a proof of principle, this kit combination was used to process plasma samples from 13 colorectal cancer patients for subsequent ddPCR methylation analysis of BCAT1 and IKZF1. Methylation of BCAT1 and/or IKZF1 was identified in 6/10 (60%) stage IV patients and 1/3 (33%) stage III patients. CONCLUSIONS: Based on a thorough evaluation of five bisulfite conversion kits and three cfDNA isolation kits, both individually and in combination, the CNA kit and the EpiTect kit were identified as the best-performing kit combination, with highest DNA yield and recovery across a range of DNA input amounts. The combination was successfully used for detection of clinically relevant DNA methylation biomarkers in plasma from cancer patients.

Clinical utility of a plasma-based comprehensive genomic profiling test in patients with non-small cell lung cancer in Korea.

OBJECTIVES: Plasma-based comprehensive circulating cell-free DNA (cfDNA) next generation sequencing (NGS) has shown utility in advanced non-small cell lung cancer (aNSCLC). The aim of this study was to determine the feasibility of cfDNA-based NGS to identify actionable gene alterations in patients with aNSCLC. PATIENTS AND METHODS: This single-center non-interventional retrospective study evaluated Korean patients with biopsy-confirmed stage III/IV non-squamous aNSCLC. Tissue biopsy samples were collected at baseline, and/or at progression and analysed with Standard of Care (SOC) testing; cfDNA was analyzed by NGS in some patients concurrently. RESULTS: aNSCLC patients with cfDNA test results (n = 405) were categorized into three groups: treatment naïve (n = 182), progressive aNSCLC after chemotherapy and/or immunotherapy (n = 157), and progressive aNSCLC after tyrosine kinase inhibitors (TKIs) (n = 66). Clinically informative driver mutations were identified for 63.5% of patients which were classified as OncoKB Tiers 1 (44.2%), 2 (3.4%), tier 3 (18.9%), and 4 (33.5%). Concordance between cfDNA NGS and tissue SOC methods for concurrently collected tissue samples (n = 221) with common EGFR mutations or ALK/ROS1 fusions was 96.9%. cfDNA analysis identified tumor genomic alterations in 13 patients that were unidentified with tissue testing, enabling initiation of targeted treatment. CONCLUSIONS: In clinical practice, results of cfDNA NGS are highly concordant with those of tissue SOC testing in aNSCLC patients. Plasma analysis identified actionable alterations that were missed or not evaluated by tissue testing, enabling the initiation of targeted therapy. Results from this study add to the body of evidence in the support routine use of cfDNA NGS for patients with aNSCLC.

Peritoneal Cell-Free Tumor DNA is a Biomarker of Locoregional and Peritoneal Recurrence in Resected Pancreatic Ductal Adenocarcinomas.

BACKGROUND: Recurrence after curative-intent pancreatectomy for pancreatic ductal adenocarcinomas (PDAC) is quite frequent with locoregional and peritoneal recurrence in about one-third of cases. We hypothesize that peritoneal cell-free tumor DNA (ptDNA) present in the intraoperative peritoneal lavage (PL) fluid may be used as a predictive biomarker of locoregional and peritoneal recurrence. PATIENTS AND METHODS: Under institutional review board (IRB)-approved protocol, pre- and postresection PL fluids were collected from PDAC patients undergoing curative-intent pancreatectomy. PL fluids from PDAC patients with pathologically proven peritoneal metastasis were also collected as positive controls. Cell-free DNA was extracted from PL fluids. Droplet digital PCR (ddPCR) was performed using ddPCR KRAS G12/G13 screening kit. Recurrence-free survival (RFS) based on KRAS-mutant ptDNA level was determined using Kaplan-Meier methods. RESULTS: KRAS-mutant ptDNA was detected in PL fluids from all PDAC patients. KRAS-mutant ptDNA was detected in 11/21 (52%) preresection and 15/18 (83%) postresection PL fluid samples. With a median follow-up of 23.6 months, 12 patients developed recurrence (8 locoregional/peritoneal recurrence, 9 pulmonary/hepatic recurrence); 5/8 (63%) and 6/6 (100%) patients with mutant allele frequency (MAF) of > 0.10% in pre- and postresection PL fluids, respectively, developed recurrence. Using a cutoff value of 0.10% MAF, the presence of KRAS-mutant ptDNA in postresection PL fluid predicted a significantly shortened time to locoregional and peritoneal recurrence (median RFS of 8.9 months versus not reached, P = 0.003). CONCLUSIONS: This study suggests that ptDNA in postresection PL fluids may be a useful biomarker to predict locoregional and peritoneal recurrence in resected PDAC patients.

Plasma cell-free tumor DNA, PIK3CA and TP53 mutations predicted inferior endocrine-based treatment outcome in endocrine receptor-positive metastatic breast cancer.

PURPOSE: How to factor both tumor burden and oncogenic genomic mutations as variables to predict the outcome of endocrine-based therapy (ET) in ER-positive/HER2-negative metastatic breast cancer patients (MBC) remains to be explored. METHOD: Blood samples prospectively collected from 163 ER-positive/HER2-negative female MBC patients, before ET, were used for cell-free tumor DNA (cfDNA) analysis. cfDNA was subjected to next-generation sequencing (NGS) to interrogate oncogenic PIK3CA hotspot and TP53 DNA-binding domain (DBD) mutations, including single nucleotide variants (SNVs) or small insertions and deletions (InDels). The variant calling threshold was set at 0.5%. Progression-free survival (PFS) was measured from the start of the ET treatment to the time of disease progression of the same treatment regimen. RESULTS: Overall, the median PFS was 8.3 months (95% CI 5.7-11.1 months). The median cfDNA was 38.5 ng (range 4.4-1935 ng). The proportion of patients with PIK3CA and TP53 alterations were 25.1 and 15.3%, respectively. Patients with high total cfDNA (HR 1.74, p = 0.003), PIK3CA mutation (HR 1.74, p = 0.007), and TP53 mutation (HR 1.64, p = 0.047) in liquid biopsy conferred worse outcome after ET. Even for patients with low tumor burden, the detrimental effect of PIK3CA or TP53 mutation remained significant (p < 0.001). For patients with either PIK3CA (p < 0.001) or TP53 mutation (p = 0.004), there was significant positive correlation between allele frequency (AF) and total cfDNA. CONCLUSION: After adjustment of cfDNA level, PIK3CA and TP53 mutations observed in liquid biopsy exerted detrimental effects on the outcome of ET-based regimens. The AF of PIK3CA or TP53 may be a surrogate marker for PFS.

Liquid biopsy at the frontier in renal cell carcinoma: recent analysis of techniques and clinical application.

Renal cell carcinoma (RCC) is a major pathological type of kidney cancer and is one of the most common malignancies worldwide. The unremarkable symptoms of early stages, proneness to postoperative metastasis or recurrence, and low sensitivity to radiotherapy and chemotherapy pose a challenge for the diagnosis and treatment of RCC. Liquid biopsy is an emerging test that measures patient biomarkers, including circulating tumor cells, cell-free DNA/cell-free tumor DNA, cell-free RNA, exosomes, and tumor-derived metabolites and proteins. Owing to its non-invasiveness, liquid biopsy enables continuous and real-time collection of patient information for diagnosis, prognostic assessment, treatment monitoring, and response evaluation. Therefore, the selection of appropriate biomarkers for liquid biopsy is crucial for identifying high-risk patients, developing personalized therapeutic plans, and practicing precision medicine. In recent years, owing to the rapid development and iteration of extraction and analysis technologies, liquid biopsy has emerged as a low cost, high efficiency, and high accuracy clinical detection method. Here, we comprehensively review liquid biopsy components and their clinical applications over the past 5 years. Additionally, we discuss its limitations and predict its future prospects.

An Overview of Circulating Cell-Free Nucleic Acids in Diagnosis and Prognosis of Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer due to its molecular heterogeneity and poor clinical outcomes. Analysis of circulating cell-free tumor nucleic acids (ctNAs) can improve our understanding of TNBC and provide efficient and non-invasive clinical biomarkers that may be representative of tumor heterogeneity. In this review, we summarize the potential of ctNAs to aid TNBC diagnosis and prognosis. For example, tumor fraction of circulating cell-free DNA (TFx) may be useful for molecular prognosis of TNBC: high TFx levels after neoadjuvant chemotherapy have been associated with shorter progression-free survival and relapse-free survival. Mutations and copy number variations of TP53 and PIK3CA/AKT genes in plasma may be important markers of TNBC onset, progression, metastasis, and for clinical follow-up. In contrast, the expression profile of circulating cell-free tumor non-coding RNAs (ctncRNAs) can be predictive of molecular subtypes of breast cancer and thus aid in the identification of TBNC. Finally, dysregulation of some circulating cell-free tumor miRNAs (miR17, miR19a, miR19b, miR25, miR93, miR105, miR199a) may have a predictive value for chemotherapy resistance. In conclusion, a growing number of efforts are highlighting the potential of ctNAs for future clinical applications in the diagnosis, prognosis, and follow-up of TNBC.

Hepatitis B Virus DNA Integration Drives Carcinogenesis and Provides a New Biomarker for HBV-related HCC.

Hepatitis B virus (HBV) DNA integration is an incidental event in the virus replication cycle and occurs in less than 1% of infected hepatocytes during viral infection. However, HBV DNA is present in the genome of approximately 90% of HBV-related HCCs and is the most common somatic mutation. Whole genome sequencing of liver tissues from chronic hepatitis B patients showed integration occurring at random positions in human chromosomes; however, in the genomes of HBV-related HCC patients, there are integration hotspots. Both the enrichment of the HBV-integration proportion in HCC and the emergence of integration hotspots suggested a strong positive selection of HBV-integrated hepatocytes to progress to HCC. The activation of HBV integration hotspot genes, such as telomerase (TERT) or histone methyltransferase (MLL4/KMT2B), resembles insertional mutagenesis by oncogenic animal retroviruses. These candidate oncogenic genes might shed new light on HBV-related HCC biology and become targets for new cancer therapies. Finally, the HBV integrations in individual HCC contain unique sequences at the junctions, such as virus-host chimera DNA (vh-DNA) presumably being a signature molecule for individual HCC. HBV integration may thus provide a new cell-free tumor DNA biomarker to monitor residual HCC after curative therapies or to track the development of de novo HCC.

The current status of cell-free human papillomavirus DNA as a biomarker in cervical cancer and other HPV-associated tumors: A review.

Tumor cells release fragments of their DNA into the circulation, so called cell-free tumor DNA (ctDNA), allowing for analysis of tumor DNA in a simple blood test, that is, liquid biopsy. Cervical cancer is one of the most common malignancies among women worldwide and high-risk human papillomavirus (HR-HPV) is the cause of the majority of cases. HR-HPV integrates into the host genome and is often present in multiple copies per cell and should thus also be released as ctDNA. Such ctHPV DNA is therefore a possible biomarker in cervical cancer. In this review, we first give a background on ctDNA in general and then a comprehensive review of studies on ctHPV DNA in cervical cancer and pre-malignant lesions that may develop in cervical cancer. Furthermore, studies on ctHPV DNA in other HPV related malignancies (eg, head-and-neck and anogenital cancers) are briefly reviewed. We conclude that detection of ctHPV DNA in plasma from patients with cervical cancer is feasible, although optimized protocols and ultra-sensitive techniques are required for sufficient sensitivity. Results from retrospective studies in both cervical cancer and other HPV-related malignancies suggests that ctHPV DNA is a promising prognostic biomarker, for example, for detecting relapses early. This paves the way for larger, preferably prospective studies investigating the clinical value of ctHPV DNA as a biomarker in cervical cancer. However, there are conflicting results whether ctHPV DNA can be found in blood from patients with pre-malignant lesions and further studies are needed to fully elucidate this question.

Prognosticators of osimertinib treatment outcomes in patients with EGFR-mutant non-small cell lung cancer and leptomeningeal metastasis.

PURPOSE: Leptomeningeal metastasis (LM) is a serious complication of non-small cell lung cancer (NSCLC), particularly in patients with EGFR mutations. In this study, we investigated the survival outcomes of patients with EGFR-mutant NSCLC who have developed LM and explored the factors associated with their survival. METHODS: From April 2018 to November 2021, patients with EGFR-mutant NSCLC who underwent cerebrospinal fluid (CSF) sampling under the clinical suspicion of LM were enrolled. The patients' clinicodemographic characteristics, treatment history including whole-brain radiation therapy (WBRT), overall survival (OS), and intracranial progression-free survival (icPFS) were measured. EGFR mutations in cell-free tumor DNA (ctDNA) of CSF, including T790M mutation, were analyzed. RESULTS: We enrolled 62 patients with NSCLC. The median time form diagnosis to LM was 23.1 months and 16 (25.8%) patients had history of prior third-generation EGFR-TKI use. EGFR mutation in CSF ctDNA was detected in 53 patients (85.5%); of them, 10 (16.1%) had T790M mutation. The patients' icPFS and OS after osimertinib were 6.43 and 9.37 months, respectively, and were comparable among patients with different sensitive EGFR mutations, indicating that EGFR mutation status did not affect osimertinib efficacy. Patients who received WBRT after LM had numerically higher icPFS and OS compared to those without. Multivariate analysis revealed that lack of prior exposure to third-generation EGFR-TKI was associated with better OS. CONCLUSIONS: Osimertinib is effective in patients with EGFR-mutant NSCLC who developed LM and prior third-generation EGFR-TKI use was associated with poor survival in these patients. The role of WBRT warrants further investigation.

Circulating tumor cells and cell-free tumor DNA analyses in urothelial cancer using the LiquidBiopsy platform.

Background: Emerging data suggested that liquid biopsy such as detection of circulating tumor cells (CTCs) and cell-free tumor DNA analysis augments the management of patients with urothelial cancer (UC). We presented our pilot experience of liquid biopsy using the Ion Torrent platform to detect CTCs and genomic alterations in UC. Materials and methods: Blood or urine samples from 16 patients were subjected to CTC and plasma/urine cell-free tumor DNA isolation for next generation sequencing (NGS) using the Ion S5 system to detect mutations among 50 oncogenes on the Ion AmpliSeq Cancer Hotspot Panel. Results: The Ion Torrent platform detected a higher number of CTCs than those in previous studies using the CellSearchTM system. Overall, mutations were detected in 13/16 (81.3%) patients with a median number of 18 (range 12-25). NGS isolated 17 hotspot mutations from 11 genes and 41 novel genomic alterations from 24 genes, some of which are supposed to be clinically actionable. Conclusions: The Ion Torrent platform efficiently detected CTCs compared with previous reports. NGS with the present system also allowed for detection of gene alterations which are likely to be therapeutic targets and provided an attractive tool to guide personalized therapy for patients with advanced UC.

Clinical implementation of plasma cell-free circulating tumor DNA quantification by digital droplet PCR for the monitoring of Ewing sarcoma in children and adolescents.

Background: Treatment stratification and response assessment in pediatric sarcomas has relied on imaging studies and surgical/histopathological evidence of vital tumor cells. Such studies and evidence collection processes often involve radiation and/or general anesthesia in children. Cell-free circulating tumor DNA (ctDNA) detection in blood plasma is one available method of so-called liquid biopsies that has been shown to correlate qualitatively and quantitatively with the existence of vital tumor cells in the body. Our clinical observational study focused on the utility and feasibility of ctDNA detection in pediatric Ewing sarcoma (EWS) as a marker of minimal residual disease (MRD). Patients and methods: We performed whole genome sequencing (WGS) to identify the exact breakpoints in tumors known to carry the EWS-FLI1 fusion gene. Patient-specific fusion breakpoints were tracked in peripheral blood plasma using digital droplet PCR (ddPCR) before, during, and after therapy in six children and young adults with EWS. Presence and levels of fusion breakpoints were correlated with clinical disease courses. Results: We show that the detection of ctDNA in the peripheral blood of EWS patients (i) is feasible in the clinical routine and (ii) allows for the longitudinal real-time monitoring of MRD activity in children and young adults. Although changing ctDNA levels correlated well with clinical outcome within patients, between patients, a high variability was observed (inter-individually). Conclusion: ctDNA detection by ddPCR is a highly sensitive, specific, feasible, and highly accurate method that can be applied in EWS for follow-up assessments as an additional surrogate parameter for clinical MRD monitoring and, potentially, also for treatment stratification in the near future.

Circulating cell-free tumor human papillomavirus DNA is a promising biomarker in cervical cancer.

OBJECTIVES: Tumor cells release fragments of their DNA into the circulation, so called cell-free tumor DNA (ctDNA) or liquid biopsy. Here, we analyze if cell-free human papillomavirus DNA (ctHPV DNA) is detectable before, during and after treatment, in patients with cervical cancer or pre-malignant lesions that may develop into cervical cancer, and whether ctHPV DNA levels were correlated to patient or tumor characteristics and outcome. Furthermore, total cell-free DNA load is studied using cfAlbumin DNA as a surrogate marker. METHODS: 18 patients with locally advanced CC (LACC), 15 patients with early stage CC (ESCC) and 21 patients with pre-malignant lesions, all with verified HPV16, 18 or 45-positive lesions, were included. Pre- during- and post-treatment plasma were tested for HPV16, 18 & 45 and total cfDNA load using droplet digital PCR. RESULTS: ctHPV DNA was found in 94.4% and 26.7% of pre-treatment plasma of patients with LACC and ESCC respectively, while all samples from patients with pre-malignant lesions were negative. Higher levels of ctHPV DNA were correlated to higher FIGO2018 stage. Patients with LACC and persistent ctHPV DNA at end-of-treatment had significantly worse progression-free survival (PFS) than patients who had cleared the ctHPV DNA (p = 0.007). Patients with total ctDNA-levels above median in pre-treatment plasma had a worse PFS (p = 0.026), compared to patients with total ctDNA-levels below median. CONCLUSION: ctHPV DNA is a promising prognostic biomarker in locally advanced cervical cancer that should be studied further for clinical use.

Circulating tumor DNA for diagnosis, prognosis and treatment of gastrointestinal malignancies.

Minimally invasive detection of circulating tumor DNA (ctDNA) in peripheral blood or other body fluids of patients with gastrointestinal malignancies via liquid biopsy has emerged as a promising biomarker. This is urgently needed, as conventional imaging and plasma protein-derived biomarkers lack sensitivity and specificity in prognosis, early detection of relapse or treatment monitoring. This review summarizes the potential role of liquid biopsy in diagnosis, prognosis and treatment monitoring of gastrointestinal malignancies, including upper gastrointestinal, liver, bile duct, pancreatic and colorectal cancer. CtDNA can now be part of the clinical routine as a promising, highly sensitive and specific biomarker with a broad range of applicability. Liquid-biopsy based postoperative relapse prediction could lead to improved survival by intensification of adjuvant treatment in patients identified to be at risk of early recurrence. Moreover, ctDNA allows monitoring of antineoplastic treatment success, with identification of potentially developed resistance or therapeutic targets during the course of treatment. It may also assist in early change of chemotherapy in metastatic gastrointestinal malignancies prior to imaging findings of relapse. Nevertheless, clinical utility is dependent on the tumor's entity and burden.

The Influence of Proteins on Fate and Biological Role of Circulating DNA.

Circulating DNA has already proven itself as a valuable tool in translational medicine. However, one of the overlooked areas of circulating DNA research is its association with different proteins, despite considerable evidence that this association might impact DNA's fate in circulation and its biological role. In this review, we attempt to shed light on current ideas about circulating DNA origins and forms of circulation, known biological effects, and the clinical potential of circulating tumor deoxyribonucleoprotein complexes.

Combined Analysis of Disseminated Tumor Cells (DTCs) and Circulating Tumor DNA (ctDNA) in a Patient Suffering from Triple Negative Breast Cancer Revealed Elevated Risk.

BACKGROUND: Disseminated tumor cells (DTCs) in bone marrow aspirates of patients with primary breast cancer may serve as independent prognostic markers associated with impaired survival. Due to limited therapy options and high risk of recurrence particularly, women diagnosed with the aggressive triple negative breast cancer (TNBC) require personalized treatment choices. Genetic profiling of circulating cell-free tumor DNA (ctDNA) might help to find individual treatment options and to monitor disease course. METHODS: Here we report the case of a 66-year-old patient with TNBC. She received neoadjuvant chemotherapy (NACT) that had to be interrupted due to intolerance. Surgical resection of the residual tumor resulted in pathologic complete response (pCR), though. RESULTS: Bone marrow aspiration during surgery revealed an unusual high number of DTCs and thus elevated risk for recurrence. Analysis of pre-surgical blood and urine samples revealed the presence of plasma-derived and urinary ctDNA after NACT and indicated poor prognosis. Subsequent targeted sequencing showed that pathogenic variants occurred in urinary and plasma-derived ctDNA emphasizing the potential of liquid biopsy usage for early detection of relapse. Despite the detection of residual molecular disease after NACT, the presented patient reached pCR and could benefit from standard treatment until present. CONCLUSIONS: In this case, liquid biopsy based biomarkers did not necessarily correlate to clinical outcome. Further, ctDNA analysis did not reveal approved therapeutic options to target the identified pathogenic variants. Adjuvant bisphosphonate treatment was applied based on the positive DTC status and may improve the patients' prognosis. Further investigations are required to identify TNBC patients at risk for recurrence.

Cell-Free Tumor DNA (cf-tDNA) Liquid Biopsy: Current Methods and Use in Brain Tumor Immunotherapy.

Gliomas are tumors derived from mutations in glial brain cells. Gliomas cause significant morbidity and mortality and development of precision diagnostics and novel targeted immunotherapies are critically important. Radiographic imaging is the most common technique to diagnose and track response to treatment, but is an imperfect tool. Imaging does not provide molecular information, which is becoming critically important for identifying targeted immunotherapies and monitoring tumor evolution. Furthermore, immunotherapy induced inflammation can masquerade as tumor progression in images (pseudoprogression) and confound clinical decision making. More recently, circulating cell free tumor DNA (cf-tDNA) has been investigated as a promising biomarker for minimally invasive glioma diagnosis and disease monitoring. cf-tDNA is shed by gliomas into surrounding biofluids (e.g. cerebrospinal fluid and plasma) and, if precisely quantified, might provide a quantitative measure of tumor burden to help resolve pseudoprogression. cf-tDNA can also identify tumor genetic mutations to help guide targeted therapies. However, due to low concentrations of cf-tDNA, recovery and analysis remains challenging. Plasma cf-tDNA typically represents <1% of total cf-DNA due to the blood-brain barrier, limiting their usefulness in practice and motivating the development and use of highly sensitive and specific detection methods. This mini review summarizes the current and future trends of various approaches for cf-tDNA detection and analysis, including new methods that promise more rapid, lower-cost, and accessible diagnostics. We also review the most recent clinical case studies for longitudinal disease monitoring and highlight focus areas, such as novel accurate detection methodologies, as critical research priorities to enable translation to clinic.