Investigating the Z-scan technique for quantifying circulating cell-free DNA (ccfDNA) extracted from blood plasma as a potential biomarker for various cancers.

The Z-scan technique is a nonlinear optical method that has found applications in characterizing various materials, particularly those exhibiting nonlinear optical response (NLOR). This study applies the continuous wave (CW) Z-scan technique to examine the NLOR in terms of the nonlinear optical phase shifts(ΔΦ0) exhibited by the ccfDNA extracted from blood plasma samples collected from a group constituting 30 cancer-diagnosed patients and another group constituting 30 non-diagnosed individuals. The cancer group exhibited significantly higherΔΦ0versus incident power slopes compared to the non-cancer group (0.34 versus 0.12) providing a clear distinction between the two groups. The receiver operating characteristic (ROC) curve analysis of the results indicates a clear separation between cancer and non-cancer groups, along with a 94% accuracy rate of the data. The Z-scan results are corroborated by spectrophotometric analysis, revealing a consistent trend in the concentration values of ccfDNA samples extracted from both cancerous and non-cancerous samples, measuring 3.24 and 1.41 respectively. Additionally, more sensitive fluorometric analyses of the respective samples demonstrate significantly higher concentrations of ccfDNA in the cancer group, further affirming the correlation with the Z-scan results. The study suggests that the Z-scan technique holds promise as an effective method for cancer detection, potentially contributing to improved oncology diagnosis and prognosis in the future.

Mitochondrial Fraction of Circulating Cell-Free DNA as an Indicator of Human Pathology.

Circulating cell-free DNA (ccfDNA) of mitochondrial origin (ccf-mtDNA) consists of a minor fraction of total ccfDNA in blood or in other biological fluids. Aberrant levels of ccf-mtDNA have been observed in many pathologies. Here, we introduce a simple and effective standardized Taqman probe-based dual-qPCR assay for the simultaneous detection and relative quantification of nuclear and mitochondrial fragments of ccfDNA. Three pathologies of major burden, one malignancy (Breast Cancer, BrCa), one inflammatory (Osteoarthritis, OA) and one metabolic (Type 2 Diabetes, T2D), were studied. Higher levels of ccf-mtDNA were detected both in BrCa and T2D in relation to health, but not in OA. In BrCa, hormonal receptor status was associated with ccf-mtDNA levels. Machine learning analysis of ccf-mtDNA datasets was used to build biosignatures of clinical relevance. (A) a three-feature biosignature discriminating between health and BrCa (AUC: 0.887) and a five-feature biosignature for predicting the overall survival of BrCa patients (Concordance Index: 0.756). (B) a five-feature biosignature stratifying among T2D, prediabetes and health (AUC: 0.772); a five-feature biosignature discriminating between T2D and health (AUC: 0.797); and a four-feature biosignature identifying prediabetes from health (AUC: 0.795). (C) a biosignature including total plasma ccfDNA with very high performance in discriminating OA from health (AUC: 0.934). Aberrant ccf-mtDNA levels could have diagnostic/prognostic potential in BrCa and Diabetes, while the developed multiparameter biosignatures can add value to their clinical management.

Circulating cell-free DNA-based biomarkers for prognostication and disease monitoring in adrenocortical carcinoma.

OBJECTIVE: Adrenocortical carcinoma (ACC) is a rare aggressive cancer with heterogeneous behaviour. Disease surveillance relies on frequent imaging, which comes with significant radiation exposure. The aim of the study was to investigate the role of circulating cell-free DNA (ccfDNA)-related biomarkers (BMs) for prognostication and monitoring of ACC. DESIGN AND METHODS: We investigated 34 patients with ACC and 23 healthy subjects (HSs) as controls. Circulating cell-free DNA was extracted by commercial kits and ccfDNA concentrations were quantified by fluorimeter (BM1). Targeted sequencing was performed using a customized panel of 27 ACC-specific genes. Leucocyte DNA was used to discriminate somatic variants (BM2), while tumour DNA was sequenced in 22/34 cases for comparison. Serial ccfDNA samples were collected during follow-up in 19 ACC patients (median period 9 months) and analysed in relationship with standard radiological imaging. RESULTS: Circulating cell-free DNA concentrations were higher in ACC than HS (mean ± SD, 1.15 ± 1.56 vs 0.05 ± 0.05 ng/µL, P < .0001), 96% of them being above the cut-off of 0.146 ng/µL (mean HS + 2 SD, positive BM1). At ccfDNA sequencing, 47% of ACC showed at least 1 somatic mutation (positive BM2). A combined ccfDNA-BM score was strongly associated with both progression-free and overall survival (hazard ratio [HR] = 2.63; 95% CI, 1.13-6.13; P = .010, and HR = 5.98; 95% CI, 2.29-15.6; P = .0001, respectively). During disease monitoring, positive BM2 showed the best specificity (100%) and sensitivity (67%) to detect ACC recurrence or progress compared with BM1. CONCLUSION: ccfDNA-related BMs are frequently detected in ACC patients and represent a promising, minimally invasive tool to predict clinical outcome and complement surveillance imaging. Our findings will be validated in a larger cohort of ACCs with long-term follow-up.

Quantification of Circulating Cell-Free DNA in Idiopathic Parkinson's Disease Patients.

Parkinson's disease (PD) is one of the most common neurodegenerative disorders globally and leads to an excessive loss of dopaminergic neurons in the substantia nigra of the brain. Circulating cell-free DNA (ccf-DNA) are double-stranded DNA fragments of different sizes and origins that are released into the serum and cerebrospinal fluid (CSF) due to cell death (i.e., necrosis and apoptosis) or are actively released by viable cells via exocytosis and NETosis. Using droplet digital polymerase chain reaction (ddPCR), we comprehensively analyzed and distinguished circulating cell-free mitochondrial DNA (ccf mtDNA) and circulating cell-free nuclear DNA (ccfDNA) in the serum and CSF of PD and control patients. The quantitative analysis of serum ccf-DNA in PD patients demonstrated a significant increase in ccf mtDNA and ccfDNA compared to that in healthy control patients and a significantly higher copy of ccf mtDNA when compared to ccfDNA. Next, the serum ccf mtDNA levels significantly increased in male PD patients compared to those in healthy male controls. Furthermore, CSF ccf mtDNA in PD patients increased significantly compared to ccfDNA, and ccf mtDNA decreased in PD patients more than it did in healthy controls. These decreases were not statistically significant but were in agreement with previous data. Interestingly, ccf mtDNA increased in healthy control patients in both serum and CSF as compared to ccfDNA. The small sample size of serum and CSF were the main limitations of this study. To the best of our knowledge, this is the first comprehensive study on serum and CSF of PD patients using ddPCR to indicate the distribution of the copy number of ccf mtDNA as well as ccfDNA. If validated, we suggest that ccf mtDNA has greater potential than ccfDNA to lead the development of novel treatments for PD patients.

A Novel, Highly Sensitive Nucleic Acid Amplification Test Assay for the Diagnosis of Loiasis and its Use for Detection of Circulating Cell-Free DNA.

Mass drug administration programs targeting filarial infections depend on diagnostic tools that are sensitive and specific. The coendemicity of Loa loa with other filarial species often hampers the control programs. LL2634 was identified as the most promising target among several highly repeated targets, with sensitivity between 500 ag and 1 fg of genomic DNA. Using DNA from infected individuals, LL2643 quantitative polymerase chain reaction (qPCR) was positive in all individuals. LL2643 was detected in plasma-derived circulating cell-free DNA (ccfDNA) from 48 of 53 microfilariae-positive patients. Detection of ccfDNA in urine was possible, but it occurred rarely among those tested. Importantly, LL2643 ccfDNA became undetectable within 1 month following diethylcarbamazine (DEC) treatment and remained negative for at least a year. LL2643 offers a more sensitive and specific target for detection of L. loa infection and would be easily configurable to a point-of-contact assay. Clinical Trials Registration. NCT00001230 and NCT00090662.

Epigenetic liquid biopsies for minimal residual disease, what's around the corner?

Liquid biopsy assays for minimal residual disease (MRD) are used to monitor and inform oncological treatment and predict the risk of relapse in cancer patients. To-date, most MRD assay development has focused on targeting somatic mutations. However, epigenetic changes are more frequent and universal than genetic alterations in cancer and circulating tumor DNA (ctDNA) retains much of these changes. Here, we review the epigenetic signals that can be used to detect MRD, including DNA methylation alterations and fragmentation patterns that differentiate ctDNA from noncancerous circulating cell-free DNA (ccfDNA). We then summarize the current state of MRD monitoring; highlight the advantages of epigenetics over genetics-based approaches; and discuss the emerging paradigm of assaying both genetic and epigenetic targets to monitor treatment response, detect disease recurrence, and inform adjuvant therapy.

Liquid Biopsy for Advanced Cancer: An Amplicon-Based Massively Parallel Sequencing Panel Approach to Precision Oncology.

Although discovered in the 1940s (Mandel and Metais, C R Seances Soc Biol Fil 142:241-243, 1948), cell-free DNA has only recently become a tool practical for use in clinical settings. The challenges associated with detection of circulating tumor DNA (ctDNA) in patient plasma are many and exist in the pre-analytical, analytical, and post-analytical periods. Initiation of a ctDNA program in a small academic clinical laboratory setting can be challenging. Thus, cost-effective, fast methods should be leveraged to promote a self-supporting system. Any assay should be based on clinical utility and have the potential to adapt in order to maintain relevance in a rapidly developing genomic landscape. Herein is described one of many approaches to ctDNA mutation testing - a massively parallel sequencing (MPS) method that is widely applicable and relatively easy to perform. Sensitivity and specificity are enhanced by unique molecular identification tagging and deep sequencing.

Improvements in Quality Control and Library Preparation for Targeted Sequencing Allowed Detection of Potentially Pathogenic Alterations in Circulating Cell-Free DNA Derived from Plasma of Brain Tumor Patients.

Malignant gliomas are the most frequent primary brain tumors in adults. They are genetically heterogenous and invariably recur due to incomplete surgery and therapy resistance. Circulating tumor DNA (ctDNA) is a component of circulating cell-free DNA (ccfDNA) and represents genetic material that originates from the primary tumor or metastasis. Brain tumors are frequently located in the eloquent brain regions, which makes biopsy difficult or impossible due to severe postoperative complications. The analysis of ccfDNA from a patient's blood presents a plausible and noninvasive alternative. In this study, freshly frozen tumors and corresponding blood samples were collected from 84 brain tumor patients and analyzed by targeted next-generation sequencing (NGS). The cohort included 80 glioma patients, 2 metastatic cancer patients, and 2 primary CNS lymphoma (PCNSL) patients. We compared the pattern of genetic alterations in the tumor DNA (tDNA) with that of ccfDNA. The implemented technical improvements in quality control and library preparation allowed for the detection of ctDNA in 8 out of 84 patients, including 5 out of 80 glioma patients. In 32 out of 84 patients, we found potentially pathogenic genetic alterations in ccfDNA that were not detectable in tDNA. While sequencing ccfDNA from plasma has a low efficacy as a diagnostic tool for glioma patients, we concluded that further improvements in sample processing and library preparation can make liquid biopsy a valuable diagnostic tool for glioma patients.

Early Detection of Relapse by ctDNA Sequencing in a Patient with Metastatic Thymic Tumor and MEN1 Mosaicism.

CONTEXT: Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant disease caused by inactivating mutations in the MEN1 gene. In the literature, few cases of MEN1 have been reported because of mosaic MEN1 mutations. OBJECTIVE: We performed an extensive molecular characterization in several lesions and blood samples, including plasmatic circulating cell-free DNA (ccfDNA) in an exceptional case of a patient with MEN1 mosaicism causing primary hyperparathyroidism, multiple pancreatic neuroendocrine tumors (NETs), and a metastatic thymic NET. METHODS: Blood, ccfDNA and multiple tissue analysis were performed by next-generation sequencing. RESULTS: MEN1 mosaicism was confirmed by multiple tissue analysis. Somatic analysis of the largest pancreatic NET revealed the same MEN1 second-hit mutation as found in the thymic lesion, demonstrating its metastatic origin from the thymic lesion. Moreover, in ccfDNA we found the mosaic MEN1 mutation but also the somatic second-hit mutation found in the thymic primary tumor, revealing the presence of circulating tumor DNA (ctDNA). After surgical removal of the pancreatic metastasis, the mutated fraction of both mutations decreased, before increasing again several weeks before a new clinical relapse, suggesting that thymic ctDNA may be used as an early tumor biomarker. CONCLUSION: This exceptional MEN1 case highlighted (1) the importance of looking for MEN1 mosaicism, (2) that MEN1 mosaicism can cause very aggressive disease, and (3) the interest in analyzing ccfDNA for confirming MEN1 mosaicism but also as a potential tumor biomarker for NET.

Methylated ccfDNA from plasma biomarkers of Alzheimer's disease using targeted bisulfite sequencing.

Aim: Noninvasive biomarkers such as methylated ccfDNA from plasma could help to support the diagnosis of Alzheimer's disease (AD). Methods: A targeted sequencing protocol was developed to identify candidate biomarkers of AD in methylated ccfDNA extracted from plasma. Results: The authors identified differentially methylated CpGs, regions of which were the same as those identified in previous AD studies. Specifically, a differentially methylated CpG of the LHX2 gene previously identified in a plasma study of AD was replicated in the study. The MBP and DUSP22 regions have been identified in other brain studies of AD and in the authors' study. Conclusion: Although these biomarkers must be validated in other cohorts, methylated ccfDNA could be a relevant noninvasive biomarker in AD.

Currently, the diagnosis of Alzheimer's disease (AD) is based on symptoms and medical imaging, and definitive clinical diagnosis is only possible postmortem. The identification of noninvasive biomarkers such as methylated ccfDNA is crucial for the diagnosis, prognosis and monitoring of AD. However, the analysis of ccfDNA from plasma is a challenge because it is highly fragmented and present in low amounts and originates from various tissues. The authors developed a targeted sequencing protocol using genes previously reported in AD literature (brain, blood and plasma) to identify potential noninvasive biomarkers in plasma. The authors identified positions identical to those in the literature as well as potential novel sites located in the promoter, exon and intron regions of these genes. Although these results must be validated in a large cohort, methylated ccfDNA could be a useful noninvasive biomarker for AD.

Evaluation of circulating cell-free nucleic acids in health workers occupationally exposed to ionizing radiation.

Radiology workers might constantly be exposed to low-dose ionizing radiation due to their profession. Low doses of radiation in a short exposure time have the potential to alter the genome, which might potentially lead to diseases. The main objective of this study was to determine whether the amount of cell-free nucleic acids in plasma samples of radiation-exposed workers was different from the general public, in other words, non-exposed individuals. In this context, we investigated the association between radiation exposure and cell-free nucleic acids concentration by using radiation exposure parameters. The study consisted of 40 radiology workers and 40 individuals who were not exposed to ionizing radiation. The plasma concentrations of cell-free DNA, RNA, and miRNA were measured fluorometrically. We found that the ccfRNA concentration of the radiation-exposed group was significantly different from that of the non-exposed group (p = 0.0001). However, there are no differences between both groups in terms of ccfDNA and ccfmiRNA concentration. The concentration of ccfDNA is significantly correlated with working time in the fluoroscopy field (p < 0.05). We found that the concentration of ccfmiRNA was significantly correlated with working time in plain radiography (p < 0.01) and computed tomography (p < 0.05) and with total working time (p < 0.01). Similarly, the concentrations of ccfRNA were significantly correlated with working time in computed tomography (p < 0.01) and with the total working time (p < 0.05) of the workers. We found that imaging number in computed tomography significantly altered the level of ccfRNA (p = 0.006) and that working time in the computed tomography field significantly affected the ccfRNA concentration (p = 0.03, R2 = 0.36 for model). Finally, we determined that total working time was significantly associated with total ccfRNA concentration (p < 0.05, R2 = 0.25 for model). In conclusion, total RNA measured in radiation-exposed workers has the potential to predict the radiation exposure risk. Furthermore, total working time and working time in the tomography field significantly alter the level of free nucleic acids.

Comparison of methods for isolation and quantification of circulating cell-free DNA from patients with endometriosis.

RESEARCH QUESTION: Which is the optimal extraction method for isolating and quantifying circulating cell-free DNA (ccfDNA) from patients with endometriosis? Endometriosis is a common benign disease, associated with pain, infertility and reduced quality of life. Endometriosis is also a known risk factor for various cancers. Robust biomarkers for early detection and prediction of prognosis, however, are lacking. CcfDNA is an easy to obtain biomarker associated with prognosis of cancer patients and enables non-invasive analysis of somatic mutations. Recently, elevated levels of ccfDNA were detected in patients with endometriosis. DESIGN: Two different ccfDNA extraction methods were compared: Maxwell RSC ccfDNA plasma kit (Maxwell) and QiAamp minElute ccfDNA mini kit (QIAamp). The ccfDNA and circulating mitochondrial DNA (mtDNA) quantities from 34 patients diagnosed with endometriosis were analysed. Fluorometric measurement and quantitative reverse transcription polymerase chain reaction (qRT-PCR) of short and long ALU and mtDNA fragments were used to quantiy ccfDNA. RESULTS: The yield of ccfDNA isolated with the Maxwell method was significantly higher compared with the QIAamp method (P < 0.0001). Integrity of ccfDNA was significantly higher in the QIAamp isolate (P < 0.0001). Recovered mtDNA was not significantly different between both extraction methods used. CONCLUSIONS: The choice of extraction method can significantly influence the ccfDNA output and integrity. Both methods, however, enabled isolation of sufficient ccfDNA for further downstream applications. With this approach, isolation of ccfDNA could enable the non-invasive detection and analysis of somatic mutation within endometriosis tissue.

Eps15 Homology Domain-Containing Protein 3 Hypermethylation as a Prognostic and Predictive Marker for Colorectal Cancer.

Colorectal cancer (CRC) arises from chromosomal instability, resulting from aberrant hypermethylation in tumor suppressor genes. This study identified hypermethylated genes in CRC and investigated how they affect clinical outcomes. Methylation levels of specific genes were analyzed from The Cancer Genome Atlas dataset and 20 breast cancer, 16 esophageal cancer, 33 lung cancer, 15 uterine cancer, 504 CRC, and 9 colon polyp tissues and 102 CRC plasma samples from a Taiwanese cohort. In the Asian cohort, Eps15 homology domain-containing protein 3 (EHD3) had twofold higher methylation in 44.4% of patients with colonic polyps, 37.3% of plasma from CRC patients, and 72.6% of CRC tissues, which was connected to vascular invasion and high microsatellite instability. Furthermore, EHD3 hypermethylation was detected in other gastrointestinal cancers. In the Asian CRC cohort, low EHD3 mRNA expression was found in 45.1% of patients and was connected to lymph node metastasis. Multivariate Cox proportional-hazards survival analysis revealed that hypermethylation in women and low mRNA expression were associated with overall survival. In the Western CRC cohort, EHD3 hypermethylation was also connected to overall survival and lower chemotherapy and antimetabolite response rates. In conclusion, EHD3 hypermethylation contributes to the development of CRC in both Asian and Western populations.

Validation of a New Protocol to Collect and Isolate Plasma from Pregnant Women for Noninvasive Prenatal Testing (NIPT).

BACKGROUND: Most laboratories use specialized tubes (e.g., Streck) to recover circulating cell-free DNA (ccfDNA) for noninvasive prenatal testing (NIPT). We validated a low cost, simple procedure for collecting NIPT samples in remote laboratories that avoids highspeed centrifugation. EDTA gel blood sampling tube allows simple separation of plasma from blood cells. Decanted plasma is filtered to remove cell debris. The procedure can be performed within a few minutes after the blood centrifugation step, and ccfDNA-grade plasma can be frozen for transportation. METHODS: We recruited 51 pregnant women and collected blood in one EDTA-gel Greiner tube and two Streck tubes. All tubes were centrifuged at 1600 g x 10 min within 6 h of sample collection. Plasma from EDTA tubes was poured into a syringe cylinder and filtered through a 0.45 µm Millipore filter. Plasma from Streck tubes was recovered with a pipette and one was filtered as above while the second was centrifuged at 16 000 g. The ccfDNA was isolated and NGS sequencing libraries were prepared and sequenced on an Illumina system. Fetal fractions were estimated using SeqFF. This study had a power of 79% to detect a decrease of 1% in fetal fractions with the new method. RESULTS: We did not observe any significant difference between the three procedures for the fetal fraction nor for the quality or quantity of libraries produced. CONCLUSION: EDTA-gel tubes with filtration provide high quality plasma for ccfDNA analysis and can be sent frozen to the NIPT laboratory. This is economical and it frees the laboratory of time-consuming steps.

SMAD3 Hypomethylation as a Biomarker for Early Prediction of Colorectal Cancer.

The incidence and mortality rates of colorectal cancer (CRC) have been high in recent years. Prevention and early detection are crucial for decreasing the death rate. Therefore, this study aims to characterize the alteration patterns of mothers against decapentaplegic homolog 3 (SMAD3) in patients with CRC and its applications in early detection by using a genome-wide methylation array to identify an aberrant hypomethylation site in the intron position of the SMAD3 gene. Quantitative methylation-specific polymerase chain reaction showed that hypomethylated SMAD3 occurred in 91.4% (501/548) of Taiwanese CRC tissues and 66.6% of benign tubular adenoma polyps. In addition, SMAD3 hypomethylation was observed in 94.7% of patients with CRC from The Cancer Genome Atlas dataset. A decrease in circulating cell-free methylation SMAD3 was detected in 70% of CRC patients but in only 20% of healthy individuals. SMAD3 mRNA expression was low in 42.9% of Taiwanese CRC tumor tissues but high in 29.4% of tumors compared with paired adjacent normal tissues. Hypomethylated SMAD3 was found in cancers of the digestive system, such as liver cancer, gastric cancer, and colorectal cancer, but not in breast cancer, endometrial cancer, and lung cancer. In conclusion, SMAD3 hypomethylation is a potential diagnostic marker for CRC in Western and Asian populations.

Investigating the relationship between ccfDNA concentration, its integrity, and some individual factors in an Iranian population.

INTRODUCTION: Circulating cell-free DNA (ccfDNA) increases in some pathologic conditions like cancer. We aimed to investigate the correlation between some individual factors and the ccfDNA level in peripheral blood of Iranian in relation to prostate cancer. MATERIAL AND METHOD: 30 patients with prostate cancer (PCa), 40 with benign prostate hyperplasia (BPH), and 30 controls were studied. Personal information, ccfDNA concentration, and the integrity index were assessed for the correlation between the disease and different factors. The results were statistically analyzed using SPSS software. RESULTS: In PCa group, no association was found between total ccfDNA, BMI, BPH background, non-cancerous diseases, medications, PCa length, and job (p-value > 0.05). But, total ccfDNA had statistical associations with weight, family history of cancer, and location (p-value < 0.05). No association was between the integrity of ccfDNA, weight, the background of BPH, and family history of cancer. But, the integrity of ccfDNA was significantly associated with BMI and PCa length (p-value < 0.05).In BPH group, no association between total ccfDNA or the integrity of ccfDNA and the assessed factors was obtained (p-value > 0.05). In the normal group, neither statistical association was found between total ccfDNA, weight, BMI, and job, nor between the integrity of ccfDNA, weight, BMI, non-cancerous disease, drug, job, and location (p-value > 0.05). But, a statistical association was found between the integrity of ccfDNA and family history of cancer in the recent group (Based on 95% CI and P-value less than 0.05). CONCLUSION: ccfDNA and its integrity as possible prostate cancer biomarkers under the influence of individuals' physiological status are prone to the pathologic changes toward the disease. Further simultaneous study of the target groups could clarify this matter.

Comparison of Circulating Cell-Free DNA Extraction Methods for Downstream Analysis in Cancer Patients.

Circulating cell-free DNA (ccfDNA) may contain DNA originating from the tumor in plasma of cancer patients (ctDNA) and enables noninvasive cancer diagnosis, treatment predictive testing, and response monitoring. A recent multicenter evaluation of workflows by the CANCER-ID consortium using artificial spiked-in plasma showed significant differences and consequently the importance of carefully selecting ccfDNA extraction methods. Here, the quantity and integrity of extracted ccfDNA from the plasma of cancer patients were assessed. Twenty-one cancer patient-derived cell-free plasma samples were selected to compare the Qiagen CNA, Maxwell RSC ccfDNA plasma, and Zymo manual quick ccfDNA kit. High-volume citrate plasma samples collected by diagnostic leukapheresis from six cancer patients were used to compare the Qiagen CNA (2 mL) and QIAamp MinElute ccfDNA kit (8 mL). This study revealed similar integrity and similar levels of amplified short-sized fragments and tumor-specific mutants comparing the CNA and RSC kits. However, the CNA kit consistently showed the highest yield of ccfDNA and short-sized fragments, while the RSC and ME kits showed higher variant allelic frequencies (VAFs). Our study pinpoints the importance of standardizing preanalytical conditions as well as consensus on defining the input of ccfDNA to accurately detect ctDNA and be able to compare results in a clinical routine practice, within and between clinical studies.

Hypermethylation and decreased expression of TMEM240 are potential early-onset biomarkers for colorectal cancer detection, poor prognosis, and early recurrence prediction.

BACKGROUND: Gene silencing by aberrant DNA methylation of promoter regions remains the most dominant phenomenon occurring during tumorigenesis. Improving the early diagnosis, prognosis, and recurrence prediction of colorectal cancer using noninvasive aberrant DNA methylation biomarkers has encouraging potential. The aim of this study is to characterize the DNA methylation of the promoter region of TMEM240, as well as gene expression and its effect on cell biological functions and its applications in early detection and outcome prediction. RESULTS: Highly methylated CpG sites were identified in the TMEM240 gene by Illumina methylation 450K arrays in 26 Taiwanese patient paired samples and 38 paired samples from The Cancer Genome Atlas (TCGA) colorectal cancer dataset. Transient transfection and knockdown of TMEM240 were performed to demonstrate the role of TMEM240 in colorectal cancer cells. The data showed that TMEM240 could lead to G1 cell cycle arrest, repress cancer cell proliferation, and inhibit cancer cell migration. The quantitative methylation-specific real-time polymerase chain reaction (PCR) results revealed that 87.8% (480 of 547) of the colorectal cancer tumors had hypermethylated TMEM240, and this was also found in benign tubular adenomas (55.6%). Circulating cell-free methylated TMEM240 was detected in 13 of 25 (52.0%) Taiwanese colorectal cancer patients but in fewer (28.6%) healthy controls. In 72.0% (85/118) of tissue samples, TMEM240 mRNA expression was lower in Taiwanese CRC tumor tissues than in normal colorectal tissues according to real-time reverse transcription PCR results, and this was also found in benign tubular adenomas (44.4%). The TMEM240 protein was analyzed in South Korean and Chinese CRC patient samples using immunohistochemistry. The results exhibited low protein expression in 91.7% (100/109) of tumors and 75.0% (24/32) of metastatic tumors but exhibited high expression in 75.0% (6/8) of normal colon tissues. Multivariate Cox proportional hazards regression analysis found that mRNA expression of TMEM240 was significantly associated with overall, cancer-specific, and recurrence-free survival (p = 0.012, 0.007, and 0.022, respectively). CONCLUSIONS: Alterations in TMEM240 are commonly found in Western and Asian populations and can potentially be used for early prediction and as poor prognosis and early-recurrence biomarkers in colorectal cancer.

Targeted deep sequencing revealed variants in cell-free DNA of hormone receptor-positive metastatic breast cancer patients.

Cell-free DNA (cfDNA) is described to mirror intratumoral heterogeneity and gives insight about clonal evolution for improved therapeutic decisions. We sequenced cfDNA of a hormone receptor-positive, HER2-negative metastatic breast cancer (MBC) cohort with a high coverage to examine the prevalence and relevance of any detected variant. cfDNA of 44 MBC patients was isolated, followed by library construction using a customized targeted DNA panel with integrated unique molecular indices analyzing AKT1, AR, BRCA1, BRCA2, EGFR, ERCC4, ERBB2, ERBB3, ESR1, FGFR1, KRAS, MUC16, PIK3CA, PIK3R1, PTEN, PTGFR, and TGFB1. CfDNA was sequenced on the NextSeq® 550 platform (Illumina) and variants were analyzed with Ingenuity Variant Analysis (QIAGEN). We evaluated cfDNA variants in 40 of the 44 hormone receptor-positive and HER2-negative patients with a high mean coverage of 22,000×, resulting in MUC16, BRCA2, ERBB3, and AR variant calling in > 90% of the patients. 47% of all AR variants were pathogenic and at least one pathogenic or likely pathogenic variant was detected in each patient. A specific BRCA1 variant and > 3.5 pathogenic variants significantly associated with a reduced survival after diagnosis of metastasis. Longitudinal monitoring revealed an increase of pathogenic and likely pathogenic PIK3CA and ESR1 variant allele frequency under everolimus and exemestane, 8 months before proof of therapy failure by visual staging in one exemplary case. The identification of new variants with high prevalence, prognostic value, and dynamics under treatment by deep sequencing of cfDNA might empower sensitive monitoring and personalized therapeutic decisions.

Evaluating NISCH and CDH1 Promoter Hypermethylation in Nonsmokers, Cancer Free Smokers and Lung Cancer Patients: A Case Control Study.

Lung cancer has very high mortality due to late stage diagnosis not amenable to curative resection. Cancer specific methylation patterns of tumor suppressor genes may precede precursor lesions of lung cancer. Our aim was to evaluate the promoter hypermethylation of tumor suppressor gene NISCH and CDH1 in cfDNA from plasma of lung cancer patients and its possible correlation with smoking status and various clinicopathological parameters. Forty histopathologically confirmed lung cancer cases, thirty smoker and thirty nonsmoker controls were enrolled. Plasma cfDNA was extracted and subjected to bisulfite treatment followed by MS-PCR. Serum nischarin levels were estimated by ELISA. The frequency of promoter hypermethylation of NISCH and CDH1 was significantly higher in lung cancer patients as compared to lifelong non-smoker controls (p < 0.05). It did not vary with smoking status among cancer cases. No significant association was found with staging or histological grading. NISCH methylation was found to be significantly higher among smoker controls. Pack years and packs per day were significantly higher in the methylated group. Serum nischarin levels showed no significant association with NISCH methylation or clinicopathological variables. NISCH is highly methylated in both high risk smoker controls as well as cancerousnon-smokers and may mark the convergence of varied etiologies of lung cancer. Hence NISCH and CDH1 are highly methylated in plasma cfDNA of lung cancer patients.