False-Positive Liquid Biopsy Assays Secondary to Overlapping Aberrant Methylation from Non-Cancer Disease States
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PROVIDER: S-EPMC10697744 | biostudies-literature | 2023 Jan
REPOSITORIES: biostudies-literature
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Project description:Lung cancer is the leading cause of cancer-related deaths worldwide. Recent implementation of low-dose computed tomography (LDCT) screening is predicted to lead to diagnosis of lung cancer at an earlier stage, with survival benefit. However, there is still a pressing need for biomarkers that will identify individuals eligible for screening, as well as improve the diagnostic accuracy of LDCT. In addition, biomarkers for prognostic stratification of patients with early stage disease, and those that can be used as surrogates to monitor tumor evolution, will greatly improve clinical management. Molecular alterations found in the DNA of tumor cells, such as mutations, translocations and methylation, are reflected in DNA that is released from the tumor into the bloodstream. Thus, in recent years, circulating tumor DNA (ctDNA) has gained increasing attention as a noninvasive alternative to tissue biopsies and potential surrogate for the entire tumor genome. Activating gene mutations found in ctDNA have been proven effective in predicting response to targeted therapy. Analysis of ctDNA is also a valuable tool for longitudinal follow-up of cancer patients that does not require serial biopsies and may anticipate the acquisition of resistance. DNA methylation has also emerged as a promising marker for early detection, prognosis and real-time follow-up of tumor dynamics that is independent of the genomic composition of the primary tumor. This review summarizes the various investigational applications of methylated ctDNA in lung cancer reported to date. It also provides a brief overview of the technologies for analysis of DNA methylation in liquid biopsies, and the challenges that befall the implementation of methylated ctDNA into routine clinical practice.
| S-EPMC5099516 | biostudies-other
Project description:Circulating tumor DNA (ctDNA) analysis is a promising non-invasive technique for active surveillance after chemoradiotherapy for locally advanced resectable esophageal carcinoma. In other malignancies false-positive results in ctDNA analysis have been reported due to clonal hematopoiesis. In this case, we present a 66-year-old male who had adenocarcinoma of the gastroesophageal junction for which he received neoadjuvant chemoradiotherapy and underwent a transhiatal esophagectomy. Postoperatively our patient received follow-up with ctDNA analysis using next generation sequencing (NGS) and droplet digital PCR (ddPCR). This case report illustrates a number of the current challenges in ctDNA diagnostics in esophageal carcinoma. Firstly, the TP53 c.524G>A; p.R175H mutation that was found in preoperative tumor biopsies became detectable in ctDNA only after distant metastases had already been confirmed by clinical symptoms and standard imaging- and biopsy techniques. Secondly our patient repeatedly had false-positive outcomes of ctDNA analysis. Genomic analysis of white blood cells revealed that the origin of these discordant mutations lies in clonal hematopoiesis. Failure to detect TP53 c.524G>A; p.R175H in cell-free DNA (cfDNA) is most likely due to the amount of ctDNA in the cfDNA fraction being below the limit of detection for NGS and ddPCR analyses. Clinicians should be aware of the possibility of finding mutations originating from clonal hematopoiesis when using ctDNA analysis during active surveillance for esophageal carcinoma. We recommend correlation of mutations in cfDNA with mutations in tumor biopsies.
| S-EPMC8421960 | biostudies-literature
Project description:Galactomannan (GM) is a polysaccharide cell wall component released by Aspergillus spp., and an immunoenzymatic GM assay is used for the diagnosis of invasive pulmonary aspergillosis. We evaluated the cause of strong positivity for GM in patients with no typical signs of aspergillosis. Repeat assays were performed using different instruments and reagent lots, but there were no differences in results among the assays. Patients with strongly positive GM results were investigated. Medication histories revealed that 14 of 23 patients had been administered total parenteral nutrition solution from one manufacturer and 4 patients had been administered dextrose solution from a different manufacturer before being tested. The results of GM assays conducted on samples of dextrose solution and the glucose fraction of the total parenteral nutrition solution were strongly positive, confirming the causes of the false-positive reactions. We hypothesize that a trace amount of GM was introduced into the glucose-containing solutions because glucoamylase, which is necessary for the saccharification step of glucose synthesis, was derived from Aspergillus niger. To enhance patient care and prevent unnecessary antifungal prescriptions, healthcare providers and manufacturers of healthcare products need to be aware of the possibility of false-positive reactions for GM.
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Project description:Methylation of lysine and arginine residues is known to play a key role in regulating histone structure and function. However, methylation of other amino acid residues in histones has not been previously described. Using exhaustive nano-HPLC/MS/MS and blind protein sequence database searches, we tentatively assigned methylation to serine 28 of histone H3 from calf thymus. The assignment was in agreement with our stringent manual verification rules, coelution in HPLC/MS/MS with its corresponding synthetic peptide, the dynamic nature of such methylation in distinct cell lines, and isotopic labeling. However, careful inspection of the MS/MS and MS/MS/MS spectra of a series of synthetic peptides confirmed that methylation actually occurs on K27 rather than on S28. The misassignment was caused by the fact that the (y(9) + 14) of the putative S28-methylated peptide and (b(9) + 18) ions of the K27 methylated peptide share the same m/z value (m/z 801). This MS/MS peak was used as the major evidence to assign methylation to S28 (consecutive y(8) and (y(9) + 14) ions). MS/MS/MS analysis revealed the false positive nature of serine methylation: the ambiguous ion at m/z 801 is indeed (b(9) + 18), an ion resulting from an in vitro reaction in the gas phase during collisionally activated dissociation (CAD). When lysine (K27) was acetylated, the degree of such in vitro reactions was greatly reduced, and such reactions were completely eliminated when the C-terminus was blocked by carboxylic group derivatization. Moreover, such side-chain assisted C-terminal rearrangement was found to be charge dependent. In aggregate, these results suggest that extra caution should be taken in interpretation of post-translational modification (PTM) data and that MS/MS as well as MS/MS/MS of synthetic peptides are needed for verifying the identity of peptides bearing a novel PTM.
| S-EPMC2801770 | biostudies-literature
Project description:BackgroundLiquid biopsy enables the non-invasive analysis of genetic tumor variants in circulating free DNA (cfDNA) in plasma. Accurate analytical validation of liquid biopsy NGS assays is required to detect variants with low variant allele frequencies (VAFs).MethodsSix types of commercial cfDNA reference materials and 42 patient samples were analyzed using a duplex-sequencing-based liquid biopsy NGS assay.ResultsWe comprehensively evaluated the similarity of commercial cfDNA reference materials to native cfDNA. We observed significant differences between the reference materials in terms of wet-lab and sequencing quality as well as background noise. No reference material resembled native cfDNA in all performance metrics investigated. Based on our results, we established guidelines for the selection of appropriate reference materials for the different steps in performance evaluation. The use of inappropriate materials and cutoffs could eventually lead to a lower sensitivity for variant detection.ConclusionCareful consideration of commercial reference materials is required for performance evaluation of liquid biopsy NGS assays. While the similarity to native cfDNA aids in the development of experimental protocols, reference materials with well-defined variants are preferable for determining sensitivity and precision, which are essential for accurate clinical interpretation.
| S-EPMC10605119 | biostudies-literature
Project description:Retinoblastoma (RB) is a cancer that forms in the developing retina of babies and toddlers. The goal of therapy is to cure the tumor, save the eye and maximize vision. However, it is difficult to predict which eyes are likely to respond to therapy. Predictive molecular biomarkers are needed to guide prognosis and optimize treatment decisions. Direct tumor biopsy is not an option for this cancer; however, the aqueous humor (AH) is an alternate source of tumor-derived cell-free DNA (cfDNA). Here we show that DNA methylation profiling of the AH is a valid method to identify the methylation status of RB tumors. We identify 294 genes directly regulated by methylation that are implicated in p53 tumor suppressor (RB1, p53, p21, and p16) and oncogenic (E2F) pathways. Finally, we use AH to characterize molecular subtypes that can potentially be used to predict the likelihood of treatment success for retinoblastoma patients.
| S-EPMC9492718 | biostudies-literature
Project description:Here, we present a next-generation sequencing (NGS) methylation-based blood test called methylation DETEction of Circulating Tumour DNA (mDETECT) designed for the optimal detection and monitoring of metastatic triple-negative breast cancer (TNBC). Based on a highly multiplexed targeted sequencing approach, this assay incorporates features that offer superior performance and included 53 amplicons from 47 regions. Analysis of a previously characterised cohort of women with metastatic TNBC with limited quantities of plasma (<2 ml) produced an AUC of 0.92 for detection of a tumour with a sensitivity of 76% for a specificity of 100%. mDETECTTNBC was quantitative and showed superior performance to an NGS TP53 mutation-based test carried out on the same patients and to the conventional CA15-3 biomarker. mDETECT also functioned well in serum samples from metastatic TNBC patients where it produced an AUC of 0.97 for detection of a tumour with a sensitivity of 93% for a specificity of 100%. An assay for BRCA1 promoter methylation was also incorporated into the mDETECT assay and functioned well but its clinical significance is currently unclear. Clonal Hematopoiesis of Indeterminate Potential was investigated as a source of background in control subjects but was not seen to be significant, though a link to adiposity may be relevant. The mDETECTTNBC assay is a liquid biopsy able to quantitatively detect all TNBC cancers and has the potential to improve the management of patients with this disease.
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Project description:BackgroundErrors have seldom been evaluated in computer-aided detection on brain metastases. This study aimed to analyze false negatives (FNs) and false positives (FPs) generated by a brain metastasis detection system (BMDS) and by readers.MethodsA deep learning-based BMDS was developed and prospectively validated in a multicenter, multireader study. Ad hoc secondary analysis was restricted to the prospective participants (148 with 1,066 brain metastases and 152 normal controls). Three trainees and 3 experienced radiologists read the MRI images without and with the BMDS. The number of FNs and FPs per patient, jackknife alternative free-response receiver operating characteristic figure of merit (FOM), and lesion features associated with FNs were analyzed for the BMDS and readers using binary logistic regression.ResultsThe FNs, FPs, and the FOM of the stand-alone BMDS were 0.49, 0.38, and 0.97, respectively. Compared with independent reading, BMDS-assisted reading generated 79% fewer FNs (1.98 vs 0.42, P < .001); 41% more FPs (0.17 vs 0.24, P < .001) but 125% more FPs for trainees (P < .001); and higher FOM (0.87 vs 0.98, P < .001). Lesions with small size, greater number, irregular shape, lower signal intensity, and located on nonbrain surface were associated with FNs for readers. Small, irregular, and necrotic lesions were more frequently found in FNs for BMDS. The FPs mainly resulted from small blood vessels for the BMDS and the readers.ConclusionsDespite the improvement in detection performance, attention should be paid to FPs and small lesions with lower enhancement for radiologists, especially for less-experienced radiologists.
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Project description:Staphylococcus aureus can be distinguished from similar coagulase-positive staphylococci by its absence of ?-galactosidase activity. This is commonly tested using o-nitrophenyl-?-D-galactopyranoside (ONPG) as the substrate. Unexpectedly, 111 and 58 of 123 isolates displayed apparent ?-galactosidase activity in the ONPG assay and on the Vitek 2 system, respectively. Compositional analysis showed that the yellow coloration of the positive ONPG assay resulted from production of 2-aminophenoxazin-3-one. Alternative ?-galactosidase substrates like X-Gal (5-bromo-4-chloro-3-indolyl-?-D-galactopyranoside) should be used for testing staphylococci.
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Project description:Cervical cancer is the fourth most common cancer in women, which is associated in >95% with a high-risk human papillomavirus (HPV) infection. Methylation of specific genes has been closely associated with the progress of cervical high-grade dysplastic lesions to invasive carcinomas. Therefore, DNA methylation has been proposed as a triage for women infected with high-risk HPV. Methylation analyses of cervical cancer tissue have shown that cell adhesion molecule 1 (CADM1) and myelin and lymphocyte protein (MAL) methylation are present in over 90% of all cervical high-grade neoplasias and invasive cervical cancers. Here, we established a liquid biopsy-based assay to detect MAL and CADM1 methylation in cell free (cf)DNA of cervical cancer. Methylation of the target gene was validated on bisulfite converted smear-DNA from cervical dysplasia patients and afterward applied to cfDNA using quantitative real-time PCR. In 52 smears, a combined analysis of CADM1 and/or MAL (CADM1/MAL) showed methylation in 86.5% of the cases. In cfDNA samples of 24 cervical cancer patients, CADM1/MAL methylation was detected in 83.3% of the cases. CADM1/MAL methylation was detected already in 81.8% of stage I-II patients showing the high sensitivity of this liquid biopsy assay. In combination with a specificity of 95.5% towards healthy donors (HD) and an area under the curve (AUC) of 0.872 in the receiver operating characteristic (ROC) analysis, CADM1/MAL cfDNA methylation detection might represent a novel and promising liquid biopsy marker in cervical cancer.
| S-EPMC9406083 | biostudies-literature