Hypermethylation of DNA Methylation Markers in Non-Cirrhotic Hepatocellular Carcinoma.

Aberrant DNA methylation changes have been reported to be associated with carcinogenesis in cirrhotic HCC, but DNA methylation patterns for these non-cirrhotic HCC cases were not examined. Therefore, we sought to investigate DNA methylation changes on non-cirrhotic HCC using reported promising DNA methylation markers (DMMs), including HOXA1, CLEC11A, AK055957, and TSPYL5, on 146 liver tissues using quantitative methylation-specific PCR and methylated DNA sequencing. We observed a high frequency of aberrant methylation changes in the four DMMs through both techniques in non-cirrhotic HCC compared to cirrhosis, hepatitis, and benign lesions (p < 0.05), suggesting that hypermethylation of these DMMs is specific to non-cirrhotic HCC development. Also, the combination of the four DMMs exhibited 78% sensitivity at 80% specificity with an AUC of 0.85 in discriminating non-cirrhotic HCC from hepatitis and benign lesions. In addition, HOXA1 showed a higher aberrant methylation percentage in non-cirrhotic HCC compared to cirrhotic HCC (43.3% versus 13.3%, p = 0.039), which was confirmed using multivariate linear regression (p < 0.05). In summary, we identified aberrant hypermethylation changes in HOXA1, CLEC11A, AK055957, and TSPYL5 in non-cirrhotic HCC tissues compared to cirrhosis, hepatitis, and benign lesions, providing information that could be used as potentially detectable biomarkers for these unusual HCC cases in clinical practice.

Liquid Biopsies for Colorectal Cancer and Advanced Adenoma Screening and Surveillance: What to Measure?

Colorectal cancer (CRC) colonoscopic surveillance is effective but burdensome. Circulating tumor DNA (ctDNA) analysis has emerged as a promising, minimally invasive tool for disease detection and management. Here, we assessed which ctDNA assay might be most suitable for a ctDNA-based CRC screening/surveillance blood test. In this prospective, proof-of-concept study, patients with colonoscopies for Lynch surveillance or the National Colorectal Cancer screening program were included between 7 July 2019 and 3 June 2022. Blood was drawn, and if advanced neoplasia (adenoma with villous component, high-grade dysplasia, ≥10 mm, or CRC) was detected, it was analyzed for chromosomal copy number variations, single nucleotide variants, and genome-wide methylation (MeD-seq). Outcomes were compared with corresponding patients' tissues and the MeD-seq results of healthy blood donors. Two Lynch carriers and eight screening program patients were included: five with CRC and five with advanced adenomas. cfDNA showed copy number variations and single nucleotide variants in one patient with CRC and liver metastases. Eight patients analyzed with MeD-seq showed clustering of Lynch-associated and sporadic microsatellite instable lesions separate from microsatellite stable lesions, as did healthy blood donors. In conclusion, whereas copy number changes and single nucleotide variants were only detected in one patient, cfDNA methylation profiles could discriminate all microsatellite instable advanced neoplasia, rendering this tool particularly promising for LS surveillance. Larger studies are warranted to validate these findings.

mFast-SeqS-based aneuploidy score in circulating cell-free DNA is a prognostic biomarker in prostate cancer.

Multiple prognostic biomarkers, including circulating tumour cell (CTC) counts, exist in metastatic castration-resistant prostate cancer (mCRPC) patients, but none of them have been implemented into daily clinical care. The modified fast aneuploidy screening test-sequencing system (mFast-SeqS), which yields a genome-wide aneuploidy score, is able to reflect the fraction of cell-free tumour DNA (ctDNA) within cell-free DNA (cfDNA) and may be a promising biomarker in mCRPC. In this study, we investigated the prognostic value of dichotomized aneuploidy scores (< 5 vs. ≥ 5) as well as CTC counts (< 5 vs. ≥ 5) in 131 mCRPC patients prior to treatment with cabazitaxel. We validated our findings in an independent cohort of 50 similarly treated mCRPC patients. We observed that, similar to the dichotomized CTC count [HR: 2.92; 95% confidence interval (CI);1.84-4.62], dichotomized aneuploidy scores (HR: 3.24; CI: 2.12-4.94) significantly correlated with overall survival in mCRPC patients. We conclude that a dichotomized aneuploidy score from cfDNA is a prognostic marker for survival in mCRPC patients within our discovery cohort and in an independent mCRPC validation cohort. Therefore, this easy and robust minimally-invasive assay can be readily implemented as a prognostic marker in mCRPC. A dichotomized aneuploidy score might also be used as a stratification factor in clinical studies to account for tumour load.

ESR1 Methylation Measured in Cell-Free DNA to Evaluate Endocrine Resistance in Metastatic Breast Cancer Patients.

ESR1 methylation was proposed as mechanism for endocrine resistance in metastatic breast cancer patients. To evaluate its potential as a minimally invasive biomarker, we investigated the feasibility of measuring ESR1 methylation in cell-free DNA (cfDNA) and its association with endocrine resistance. First, we provided evidence that demethylation in vitro restores ER expression. Subsequently, we found that ESR1 methylation in cfDNA was not enriched in endocrine-resistant versus endocrine-sensitive patients. Interestingly, we found a correlation between ESR1 methylation and age. Publicly available data confirm an age-related increase in ESR1 methylation in leukocytes, confounding the determination of the ESR1 methylation status of tumors using cfDNA.

Validity and utility of HER2/ERBB2 copy number variation assessed in liquid biopsies from breast cancer patients: A systematic review.

Addition of HER2-targeted treatment in breast cancer is mainly determined by the HER2-receptor status of primary breast cancer tissue, since longitudinal sampling is burdensome and often not feasible. However, tumor heterogeneity and receptor conversion occur and may lead to improper diagnose as disease progresses. It is however possible that patients with their applied receptor status misjudged may benefit from HER2-targeted therapy. Liquid biopsies provide a useful source of information for receptor status and determine prognosis or evaluate therapy effectiveness. In this review, we give a comprehensive overview of current analytical and clinical validity and clinical utility of liquid biopsies for HER2 assessment. We systematically searched publications in Cochrane, Embase, PubMed and Google Scholar databases until April 2021 reporting on HER2-positivity in liquid biopsies and its concordance with tumor tissue, or on prognostic and/or predictive value of HER2-positivity in liquid biopsies. Applying our selection criteria, we identified 57 studies; 43 studies on circulating tumor cells, 13 studies on circulating tumor DNA and 1 study on extracellular vesicles. Most studies showed an association with outcome or treatment response, but used methods differed greatly, lacked a gold standard and evaluated patient groups differently hampering interpretation of results. We conclude that well-designed studies to determine assay validity and clinical validity and utility of HER2 assessment through liquid biopsies are currently lacking.

Genome-wide aneuploidy detected by mFast-SeqS in circulating cell-free DNA is associated with poor response to pembrolizumab in patients with advanced urothelial cancer.

Second-line treatment with immune checkpoint inhibition in patients with metastatic urothelial cancer (mUC) has a low success rate (~ 20%). Circulating tumour-derived DNA (ctDNA) levels may guide patient stratification, provided that an affordable and robust assay is available. Here, we investigate whether the modified fast aneuploidy screening test-sequencing system (mFast-SeqS) may provide such an assay. To this end, mFast-SeqS was performed on cell-free DNA (cfDNA) from 74 patients with mUC prior to treatment with pembrolizumab. Results were associated with corresponding tissue-based profiles, plasma-based variant allele frequencies (VAFs) and clinical response. We found that plasma-derived mFast-SeqS-based aneuploidy scores significantly correlated with those observed in the corresponding tumour tissue as well as with the ctDNA level in the plasma. In multivariate logistic regression analysis, a high aneuploidy score was independently associated with lack of clinical benefit from treatment with pembrolizumab. In conclusion, mFast-SeqS provides a patient-friendly, high-throughput and affordable method to estimate ctDNA level. Following independent validation, this test could be used to stratify mUC patients for response prior to the initiation of treatment with pembrolizumab.

A pipeline for copy number profiling of single circulating tumour cells to assess intrapatient tumour heterogeneity.

Intrapatient tumour heterogeneity is likely a major determinant of clinical outcome in cancer patients. To assess heterogeneity in a minimally invasive manner, methods to perform single circulating tumour cell (CTC) genomics at high resolution are necessary. However, due to the rarity of CTCs, development of such methods is challenging. Here, we developed a modular single CTC analysis pipeline to assess intrapatient heterogeneity by copy number (CN) profiling. To optimize this pipeline, spike-in experiments using MCF-7 breast cancer cells were performed. The VyCAP puncher system was used to isolate single cells. The quality of whole genome amplification (WGA) products generated by REPLI-g and Ampli1™ methods, as well as the results from the Illumina Truseq and the Ampli1™ LowPass library preparation techniques, was compared. Moreover, a bioinformatic pipeline was designed to generate CN profiles from single CTCs. The optimal combination of Ampli1™ WGA and Illumina Truseq library preparation was successfully validated on patient-derived CTCs. In conclusion, we developed a novel modular pipeline to isolate single CTCs and subsequently generate detailed patient-derived CN profiles that allow assessment of intrapatient heterogeneity in future studies.

High-throughput and affordable genome-wide methylation profiling of circulating cell-free DNA by methylated DNA sequencing (MeD-seq) of LpnPI digested fragments.

BACKGROUND: DNA methylation detection in liquid biopsies provides a highly promising and much needed means for real-time monitoring of disease load in advanced cancer patient care. Compared to the often-used somatic mutations, tissue- and cancer-type specific epigenetic marks affect a larger part of the cancer genome and generally have a high penetrance throughout the tumour. Here, we describe the successful application of the recently described MeD-seq assay for genome-wide DNA methylation profiling on cell-free DNA (cfDNA). The compatibility of the MeD-seq assay with different types of blood collection tubes, cfDNA input amounts, cfDNA isolation methods, and vacuum concentration of samples was evaluated using plasma from both metastatic cancer patients and healthy blood donors (HBDs). To investigate the potential value of cfDNA methylation profiling for tumour load monitoring, we profiled paired samples from 8 patients with resectable colorectal liver metastases (CRLM) before and after surgery. RESULTS: The MeD-seq assay worked on plasma-derived cfDNA from both EDTA and CellSave blood collection tubes when at least 10 ng of cfDNA was used. From the 3 evaluated cfDNA isolation methods, both the manual QIAamp Circulating Nucleic Acid Kit (Qiagen) and the semi-automated Maxwell® RSC ccfDNA Plasma Kit (Promega) were compatible with MeD-seq analysis, whereas the QiaSymphony DSP Circulating DNA Kit (Qiagen) yielded significantly fewer reads when compared to the QIAamp kit (p < 0.001). Vacuum concentration of samples before MeD-seq analysis was possible with samples in AVE buffer (QIAamp) or water, but yielded inconsistent results for samples in EDTA-containing Maxwell buffer. Principal component analysis showed that pre-surgical samples from CRLM patients were very distinct from HBDs, whereas post-surgical samples were more similar. Several described methylation markers for colorectal cancer monitoring in liquid biopsies showed differential methylation between pre-surgical CRLM samples and HBDs in our data, supporting the validity of our approach. Results for MSC, ITGA4, GRIA4, and EYA4 were validated by quantitative methylation specific PCR. CONCLUSIONS: The MeD-seq assay provides a promising new method for cfDNA methylation profiling. Potential future applications of the assay include marker discovery specifically for liquid biopsy analysis as well as direct use as a disease load monitoring tool in advanced cancer patients.

A Systematic Review of the Use of Circulating Cell-Free DNA Dynamics to Monitor Response to Treatment in Metastatic Breast Cancer Patients.

Monitoring treatment response in metastatic breast cancer currently consists mainly of radiological and clinical assessments. These methods have high inter-observer variation, suboptimal sensitivity to determine response to treatment and give little insight into the biological characteristics of the tumor. Assessing circulating tumor DNA (ctDNA) over time could be employed to address these limitations. Several ways to quantify and characterize ctDNA exist, based on somatic mutations, copy number variations, methylation, and global circulating cell-free DNA (cfDNA) fragment sizes and concentrations. These methods are being explored and technically validated, but to date none of these methods are applied clinically. We systematically reviewed the literature on the use of quantitative ctDNA measurements over time to monitor response to systemic therapy in patients with metastatic breast cancer. Cochrane, Embase, PubMed and Google Scholar databases were searched to find studies focusing on the use of cfDNA to longitudinally monitor treatment response in advanced breast cancer patients until October 2020. This resulted in a total of 33 studies which met the inclusion criteria. These studies were heterogeneous in (pre-)processing procedures, applied techniques and design. An association between ctDNA and treatment response was found in most of the included studies, independent of the applied assay. To implement ctDNA-based response monitoring into daily clinical practice for metastatic breast cancer patients, sample (pre-) processing procedures need to be standardized and large prospectively collected sample cohorts with well annotated clinical follow-up are required to establish its clinical validity.

Detection of Aneuploidy in Cerebrospinal Fluid from Patients with Breast Cancer Can Improve Diagnosis of Leptomeningeal Metastases.

PURPOSE: Detection of leptomeningeal metastasis is hampered by limited sensitivities of currently used techniques: MRI and cytology of cerebrospinal fluid (CSF). Detection of cell-free tumor DNA in CSF has been proposed as a tumor-specific candidate to detect leptomeningeal metastasis at an earlier stage. The aim of this study was to investigate mutation and aneuploidy status in CSF-derived cell-free DNA (cfDNA) of patients with breast cancer with a clinical suspicion of leptomeningeal metastasis. EXPERIMENTAL DESIGN: cfDNA was isolated from stored remnant CSF and analyzed by targeted next-generation sequencing (NGS; n = 30) and the modified fast aneuploidy screening test-sequencing system (mFAST-SeqS; n = 121). The latter method employs selective amplification of long interspaced nuclear elements sequences that are present throughout the genome and allow for fast and cheap detection of aneuploidy. We compared these results with the gold standard to diagnose leptomeningeal metastasis: cytology. RESULTS: Leptomeningeal metastasis was cytology proven in 13 of 121 patients. Low DNA yields resulted in insufficient molecular coverage of NGS for the majority of samples (success rate, 8/30). The mFAST-SeqS method, successful in 112 of 121 (93%) samples, detected genome-wide aneuploidy in 24 patients. Ten of these patients had cytology-proven leptomeningeal metastasis; 8 additional patients were either concurrently diagnosed with central nervous system metastases by radiological means or developed these soon after the lumbar puncture. The remaining six cases were suspected of leptomeningeal metastasis, but could not be confirmed by cytology or imaging. Aneuploidy was associated with development of leptomeningeal metastasis and significantly worse overall survival. CONCLUSIONS: Aneuploidy in CSF-derived cfDNA may provide a promising biomarker to improve timely detection of leptomeningeal metastasis.