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Increased Growth Differentiation Factor 15 Is Associated with Unfavorable Clinical Outcomes of Acute Ischemic Stroke

期刊: CLINICAL CHEMISTRY, 2019; 65 (4)

BACKGROUND: Growth differentiation factor 15 (GDF-15), a stress-responsive biomarker, is known to be independently associated with mortality and cardiovascular events in different disease settings, but data on the prognostic value of GDF-15 after stroke are limited. METHODS: Baseline serum GDF-15 was measured in 3066 acute ischemic stroke patients from the China Antihypertensive Trial in Acute Ischemic Stroke (CATIS). The primary outcome was a composite of death and major disability within 3 months. Secondary outcomes included death, major disability, vascular events, and stroke recurrence. The associations between GDF-15 and clinical outcomes after stroke were assessed by multivariate logistic regression or Cox proportional hazards models. RESULTS: At 3 months' follow-up, 676 (22.05%), 86 (2.80%), 81 (2.64%), and 51 (1.66%) patients had experienced major disability, death, vascular events, or stroke recurrence, respectively. After adjusting for age, sex, current smoking, alcohol consumption, and baseline National Institutes of Health Stroke Scale score, the odds ratio/hazard ratio (95% CI) of 1 SD higher of base-10 log-transformed GDF-15 was 1.26 (1.15-1.39) for primary outcome, 1.13 (1.02-1.25) for major disability, 1.79 (1.48-2.16) for death, and 1.26 (1.00-1.58) for vascular events. The addition of GDF-15 to established risk factors improved risk prediction of the composite outcome of death and major disability (c-statistic, net reclassification index, and integrated discrimination improvement, all P < 0.05). CONCLUSIONS: High GDF-15 concentrations are independently associated with adverse clinical outcomes of acute ischemic stroke, suggesting that baseline serum GDF-15 could provide additional information to identify ischemic stroke patients at high risk of poor prognosis. (C) 2018 American Association for Clinical Chemistry

IF:6.89

Novel Assay for Quantitative Analysis of DNA Methylation at Single-Base Resolution

期刊: CLINICAL CHEMISTRY, 2019; 65 (5)

BACKGROUND:The DNA methylation profile provides valuable biological information with potential clinical utility. Several methods, such as quantitative methylation-specific PCR (qMSP), have been developed to examine methylation of specific CpG sites. Existing qMSP-based techniques fail to examine the genomic methylation at a single-base resolution, particularly for loci in gene bodies or extensive CpG open seas lacking flanking CpGs. Therefore, we established a novel assay for quantitative analysis of single-base methylation. METHODS:To achieve a robust single-base specificity, we developed a PCR-based method using paired probes following bisulfite treatment. The 6-carboxyfluorescein- and 2'-chloro-7'-phenyl-1,4-dichloro-6-carboxy-fluorescein-labeled probes conjugated with minor groove binder were designed to specifically bind to the methylated and unmethylated allele of targeted single CpGs at their 3' half regions, respectively. The methylation percentage was calculated by values of methylation / (methylation+unmethylation). RESULTS:In the detection of single CpGs within promoters or bodies of 4 human genes, the quantitative analysis of the single-base methylation assay showed a detection capability in the 1 to 1:10000 dilution experiments with linearity over 4 orders of magnitude (R-2=0.989-0.994; all P<0.001). In a cohort of 10 colorectal cancer samples, the assay showed a comparable detection performance with bisulfite pyrosequencing (R-2=0.875-0.990; all P<0.001), which was better than conventional qMSP methods normalized by input control reaction (R-2=0.841 vs 0.769; P=0.002 vs 0.009). CONCLUSIONS:This assay is highly specific and sensitive for determining single-base methylation and, thus, is potentially useful for methylation-based panels in diagnostic and prognostic applications. (c) 2019 American Association for Clinical Chemistry

IF:6.89

A Novel Online Calculator Based on Serum Biomarkers to Detect Hepatocellular Carcinoma among Patients with Hepatitis B

期刊: CLINICAL CHEMISTRY, 2019; 65 (12)

BACKGROUND: Early detection of hepatocellular carcinoma (HCC) among hepatitis B virus (HBV)-infected patients remains a challenge, especially in China. We sought to create an online calculator of serum biomarkers to detect HCC among patients with chronic hepatitis B (CHB). METHODS: Participants with HBV-HCC, CHB, HBV-related liver cirrhosis (HBV-LC), benign hepatic tumors, and healthy controls (HCs) were recruited at 11 Chinese hospitals. Potential serum HCC biomarkers, protein induced by vitamin K absence or antagonist-II (PIVKA-II), alpha-fetoprotein (AFP), lens culinaris agglutinin A-reactive fraction of AFP (AFP-L3) and alpha-L-fucosidase (AFU) were evaluated in the pilot cohort. The calculator was built in the training cohort via logistic regression model and validated in the validation cohort. RESULTS: In the pilot study, PIVKA-II and AFP showed better diagnostic sensitivity and specificity compared with AFP-L3 and AFU and were chosen for further study. A combination of PIVKA-II and AFP demonstrated better diagnostic accuracy in differentiating patients with HBV-HCC from patients with CHB or HBV-LC than AFP or PIVKA-II alone [area under the curve (AUC), 0.922 (95% CI, 0.908-0.935), sensitivity 88.3% and specificity 85.1% for the training cohort; 0.902 (95% CI, 0.875-0.929), 87.8%, and 81.0%, respectively, for the validation cohort]. The nomogram including AFP, PIVKA-II, age, and sex performed well in predicting HBV-HCC with good calibration and discrimination [AUC, 0.941 (95% CI, 0.929-0.952)] and was validated in the validation cohort [AUC, 0.931 (95% CI, 0.909-0.953)]. CONCLUSIONS: Our results demonstrated that a web-based calculator including age, sex, AFP, and PIVKA-II accurately predicted the presence of HCC in patients with CHB. (C) 2019 American Association for Clinical Chemistry

IF:6.89

Extracellular Vesicles Long RNA Sequencing Reveals Abundant mRNA, circRNA, and lncRNA in Human Blood as Potential Biomarkers for Cancer Diagnosis

期刊: CLINICAL CHEMISTRY, 2019; 65 (6)

BACKGROUND: Extracellular vesicles (EVs) contain a rich cargo of different RNA species with specialized functions and clinical applications. However, the landscape and characteristics of extracellular vesicle longRNA(exLR) in human blood remain largely unknown. METHODS: We presented an optimized strategy for exLR sequencing (exLR-seq) of human plasma. The sample cohort included 159 healthy individuals, 150 patients with cancer (5 cancer types), and 43 patients with other diseases. Bioinformatics approaches were used to analyze the distribution and features of exLRs. Support vector machine algorithm was performed to construct the diagnosis classifier, and diagnostic efficiency was evaluated by ROC analysis. RESULTS: More than 10000 exLRs, including mRNA, circRNA, and lncRNA, were reliably detected in each exLR-seq sample from 1-2 mL of plasma. We observed that blood EVs contain a substantial fraction of intact mRNAs and a large number of assembling spliced junctions; circRNA was also enriched in blood EVs. Interestingly, blood exLRs reflected their tissue origins and the relative fractions of different immune cell types. Additionally, the exLR profile could distinguish patients with cancer from healthy individuals. We further showed that 8 exLRs can serve as biomarkers for hepatocellular carcinoma (HCC) diagnosis with high diagnostic efficiency in training [area under the curve (AUC) = 0.9527; 95% CI, 0.9170-0.9883], validation cohort (AUC = 0.9825; 95% CI, 0.9606-1), and testing cohort (AUC = 0.9627; 95% CI, 0.9263-0.9991). CONCLUSIONS: In summary, this study revealed abundant exLRs in human plasma and identified diverse specific markers potentially useful for cancer diagnosis. (C) 2019 American Association for Clinical Chemistry

IF:6.89

Generation of Highly Biomimetic Quality Control Materials for Noninvasive Prenatal Testing Based on Enzymatic Digestion of Matched Mother-Child Cell Lines

期刊: CLINICAL CHEMISTRY, 2019; 65 (6)

BACKGROUND: Noninvasive prenatal testing (NIPT) based on cell-free DNA (cfDNA) is widely used. However, biomimetic quality control materials that have properties identical to clinical samples and that are applicable to a wide range of methodologies are still not available to support assay development, internal quality control, and proficiency testing. METHODS: We developed a set of dual enzyme-digested NIPT quality control materials (DENQCMs) that comprise simulated human plasma and mixtures of mother cell line-derived cfDNA based on DNA fragmentation factor digestion (D-cfDNA) and the matched child cell line-derived cfDNA based on micrococcal nuclease digestion (M-cfDNA). Serially diluted samples positive for trisomies 21, 18, and 13 were included in the materials. To evaluate the biomimetics, DENQCMs were analyzed using random massively parallel sequencing (MPS), targeted MPS, and imaging single DNA molecule methods, and the estimated fetal fractions (FFs) were compared with expected FFs. Genome-wide analysis of cfDNA fragmentation patterns was performed to confirm their biological characteristics. RESULTS: The genetic status of each DENQCM was correctly detected by 4 routine NIPT assays for the samples with FFs >5%. The chromosome Y-based and single-nucleotide polymorphism-based estimations of FFs were linearly related to those expected FFs. The MPS results exhibited a concordance of quality metrics between DENQCMs and maternal plasma, such as GC contents of cfDNA and unique read ratios. CONCLUSIONS: The DENQCMs are universally applicable for different platforms. We propose DENQCMs as an approach to produce matched maternal and fetal cfDNA that will be suitable for the preparation of quality control materials for NIPT. (C) 2019 American Association for Clinical Chemistry

IF:6.89

Dairy Intake and Body Composition and Cardiometabolic Traits among Adults: Mendelian Randomization Analysis of 182041 Individuals from 18 Studies

期刊: CLINICAL CHEMISTRY, 2019; 65 (6)

BACKGROUND: Associations between dairy intake and body composition and cardiometabolic traits have been inconsistently observed in epidemiological studies, and the causal relationship remains ill-defined. METHODS: We performed Mendelian randomization analysis using an established genetic variant located upstream of the lactase gene (LCT- 13910 C/T, rs4988235) associated with dairy intake as an instrumental variable (IV). The causal effects of dairy intake on body composition and cardiometabolic traits (lipids, glycemic traits, and inflammatory factors) were quantified by IV estimators among 182041 participants from 18 studies. RESULTS: Each 1 serving/day higher dairy intake was associated with higher lean mass [beta (SE) = 0.117 kg (0.035); P = 0.001], higher hemoglobin A(1c) [0.009% (0.002); P < 0.001], lower LDL [-0.014 mmol/L (0.006); P = 0.013], total cholesterol (TC) [-0.012 mmol/L (0.005); P = 0.023], and non-HDL [- 0.012 mmol/L (0.005); P = 0.028]. The LCT- 13910 C/T CT + TT genotype was associated with 0.214 more dairy servings/day (SE = 0.047; P = 0.001), 0.284 cm higher waist circumference (SE = 0.118; P = 0.017), 0.112 kg higher lean mass (SE = 0.027; P = 3.8 X 10(-5)), 0.032 mmol/L lower LDL (SE = 0.009; P = 0.001), and 0.032 mmol/L lower TC (SE = 0.010; P = 0.001). Genetically higher dairy intake was associated with increased lean mass [0.523 kg per serving/day (0.170); P = 0.002] after correction for multiple testing (0.05/18). However, we find that genetically higher dairy intake was not associated with lipids and glycemic traits. CONCLUSIONS: The present study provides evidence to support a potential causal effect of higher dairy intake on increased lean mass among adults. Our findings suggest that the observational associations of dairy intake with lipids and glycemic traits may be the result of confounding. (C) 2019 American Association for Clinical Chemistry

IF:6.89

Noninvasive Detection of Bladder Cancer by Shallow-Depth Genome-Wide Bisulfite Sequencing of Urinary Cell-Free DNA for Methylation and Copy Number Profiling

期刊: CLINICAL CHEMISTRY, 2019; 65 (7)

BACKGROUND: The current diagnosis and monitoring of bladder cancer are heavily reliant on cystoscopy, an invasive and costly procedure. Previous efforts in urine-based detection of bladder cancer focused on targeted approaches that are predicated on the tumor expressing specific aberrations. We aimed to noninvasively detect bladder cancer by the genome-wide assessment of methylomic and copy number aberrations (CNAs). We also investigated the size of tumor cell-free (cf) DNA fragments. METHODS: Shallow-depth paired-end genome-wide bisulfite sequencing of urinary cfDNA was done for 46 bladder cancer patients and 39 cancer-free controls with hematuria. We assessed (a) proportional contribution from different tissues by methylation deconvolution, (b) global hypomethylation, (c) CNA, and (d) cfDNA size profile. RESULTS: Methylomic and copy number approaches were synergistically combined to detect bladder cancer with a sensitivity of 93.5% (84.2% for low-grade nonmuscle-invasive disease) and a specificity of 95.8%. The prevalence of methylomic and CNAs reflected disease stage and tumor size. Sampling over multiple time points could assess residual disease and changes in tumor load. Muscle-invasive bladder cancer was associated with a higher proportion of long cfDNA, as well as longer cfDNA fragments originating from genomic regions enriched for tumor DNA. CONCLUSIONS: Bladder cancer can be detected noninvasively in urinary cfDNA by methylomic and copy number analysis without previous knowledge or assumptions of specific aberrations. Such analysis could be used as a liquid biopsy to aid diagnosis and for potential longitudinal monitoring of tumor load. Further understanding of the differential size and fragmentation of cfDNA could improve the detection of bladder cancer. (C) 2019 American Association for Clinical Chemistry

IF:6.89

Detection of Colorectal Cancer in Circulating Cell-Free DNA by Methylated CpG Tandem Amplification and Sequencing

期刊: CLINICAL CHEMISTRY, 2019; 65 (7)

BACKGROUND: Aberrant DNA hypermethylation of CpG islands occurs frequently throughout the genome in human colorectal cancer (CRC). A genome-wide DNA hypermethylation analysis technique using circulating cellfree DNA (cfDNA) is attractive for the noninvasive early detection of CRC and discrimination between CRC and other cancer types. METHODS: We applied the methylated CpG tandem amplification and sequencing (MCTA-Seq) method, with a fully methylated molecules algorithm, to plasma samples from patients with CRC (n = 147) and controls (n = 136), as well as cancer and adjacent noncancerous tissue samples (n = 66). We also comparatively analyzed plasma samples from patients with hepatocellular carcinoma (HCC; n = 36). RESULTS: Dozens of DNA hypermethylation markers including known (e.g., SEPT9 and IKZF1) and novel (e.g., EMBP1, KCNQ5, CHST11, APBB1IP, and TJP2) genes were identified for effectively detecting CRC in cfDNA. A panel of 80 markers discriminated early-stage CRC patients and controls with a clinical sensitivity of 74% and clinical specificity of 90%. Patients with early-stage CRC and HCC could be discriminated at clinical sensitivities of approximately 70% by another panel of 128 markers. CONCLUSIONS: MCTA-Seq is a promising method for the noninvasive detection of CRC. (C) 2019 American Association for Clinical Chemistry

IF:6.89

Noncoding RNAs Serve as Diagnosis and Prognosis Biomarkers for Hepatocellular Carcinoma

期刊: CLINICAL CHEMISTRY, 2019; 65 (7)

BACKGROUND: Reliable noninvasive biomarkers for hepatocellular carcinoma (HCC) diagnosis and prognosis are urgently needed. We explored the potential of not only microRNAs (miRNAs) but other types of noncoding RNAs (ncRNAs) as HCC biomarkers. METHODS: Peripheral blood samples were collected from 77 individuals; among them, 57 plasma cell-free RNA transcriptomes and 20 exosomal RNA transcriptomes were profiled. Significantly upregulated ncRNAs and published potential HCC biomarkers were validated with reverse transcription (RT)-qPCR in an independent validation cohort (60-150 samples). We particularly investigated the diagnosis and prognosis performance and biological function for 1 ncRNA biomarker, RN7SL1, and its S fragment. RESULTS: We identified certain circulating ncRNAs escaping from RNase degradation, possibly through binding with RNA-binding proteins: 899 ncRNAs were highly upregulated in HCC patients. Among them, 337 genes were fragmented long noncoding RNAs, 252 genes were small nucleolar RNAs, and 134 genes were piwi-interacting RNAs. Forty-eight candidates were selected and validated with RT-qPCR, of which, 16 ncRNAs were verified to be significantly upregulated in HCC, including RN7SL1, SNHG1, ZFAS1, and LINC01359. Particularly, the abundance of RN7SL1 S fragment discriminated HCC samples from negative controls (area under the curve, 0.87; 95% CI, 0.817-0.920). HCC patients with higher concentrations of RN7SL1 S fragment had lower survival rates. Furthermore, RN7SL1 S fragment alone promoted cancer cell proliferation and clonogenic growth. CONCLUSIONS: Our results show that various ncRNA species, not only miRNAs, identified in the small RNA sequencing of plasma are also able to serve as noninvasive biomarkers. Particularly, we identified a domain of sr-pRNA RN7SL1 with reliable clinical performance for HCC diagnosis and prognosis. (C) 2019 American Association for Clinical Chemistry

IF:6.89

Breast Cancer Subtype Classification Using 4-Plex Droplet Digital PCR

期刊: CLINICAL CHEMISTRY, 2019; 65 (8)

BACKGROUND: Infiltrating ductal carcinoma (IDCA) is the most common form of invasive breast cancer. Immunohistochemistry (IHC) is widely used to analyze estrogen receptor 1 (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) that can help classify the tumor to guide the medical treatment. IHC examinations require experienced pathologists to provide interpretations that are subjective, thereby lowering the reproducibility of IHC-based diagnosis. In this study, we developed a 4-plex droplet digital PCR (ddPCR) for the simultaneous and quantitative analyses of estrogen receptor 1 (ESR1), progesterone receptor (PGR), erb-b2 receptor tyrosine kinase 2 (ERBB2), and pumilio RNA binding family member 1 (PUM1) expression levels in formalin-fixed paraffin-embedded (FFPE) samples. METHODS: We evaluated the sensitivity, reproducibility, and linear dynamic range of 4-plex ddPCR. We applied this method to analyze 95 FFPE samples from patients with breast IDCA and assessed the agreement rates between ddPCR and IHC to evaluate its potential in classifying breast cancer subtypes. RESULTS: The limits of quantification (LOQ) were 25, 50, 50, and 50 copies per reaction for ERBB2, ESR1, PGR, and PUM1, respectively. The dynamic ranges of ESR1, PGR, and PUM1 extended over 50-1600 copies per reaction and those of ERBB2 from 25 to 1600 copies per reaction. The concordance correlation coefficients between 4-plex ddPCR and IHC were 96.8%, 91.5%, and 85.1% for ERBB2, ESR1, and PGR, respectively. Receiver operating characteristic curve area under the curve values of 0.991, 0.977, and 0.920 were generated for ERBB2, ESR1, and PGR, respectively. CONCLUSIONS: Evaluation of breast cancer biomarker status by 4-plex ddPCR was highly concordant with IHC in this study. (C) 2019 American Association for Clinical Chemistry

IF:6.89

5-Hydroxymethylcytosines in Circulating Cell-Free DNA Reveal Vascular Complications of Type 2 Diabetes

期刊: CLINICAL CHEMISTRY, 2019; 65 (11)

BACKGROUND: Long-term complications of type 2 diabetes (T2D), such as macrovascular and microvascular events, are the major causes for T2D-related disability and mortality. A clinically convenient, noninvasive approach for monitoring the development of these complications would improve the overall life quality of patients with T2D and help reduce healthcare burden through preventive interventions. METHODS: A selective chemical labeling strategy for 5-hydroxymethylcytosines (5hmC-Seal) was used to profile genome-wide 5hmCs, an emerging class of epigenetic markers implicated in complex diseases including diabetes, in circulating cell-free DNA (cfDNA) from a collection of Chinese patients (n = 62). Differentially modified 5hmC markers between patients with T2D with and without macrovascular/microvascular complications were analyzed under a case-control design. RESULTS: Statistically significant changes in 5hmC markers were associated with T2D-related macrovascular/microvascular complications, involving genes and pathways relevant to vascular biology and diabetes, including insulin resistance and inflammation. A 16-gene 5hmC marker panel accurately distinguished patients with vascular complications from those without [testing set: area under the curve (AUC) = 0.85; 95% CI, 0.73-0.96], outperforming conventional clinical variables such as urinary albumin. In addition, a separate 13-gene 5hmC marker panel could distinguish patients with single complications from those with multiple complications (testing set: AUC = 0.84; 95% CI, 0.68-0.99), showing superiority over conventional clinical variables. CONCLUSIONS: The 5hmC markers in cfDNA reflected the epigenetic changes in patients with T2D who developed macrovascular/microvascular complications. The 5hmC-Seal assay has the potential to be a clinically convenient, noninvasive approach that can be applied in the clinic to monitor the presence and severity of diabetic vascular complications. (C) 2019 American Association for Clinical Chemistry

IF:6.89

Liver- and Colon-Specific DNA Methylation Markers in Plasma for Investigation of Colorectal Cancers with or without Liver Metastases

期刊: CLINICAL CHEMISTRY, 2018; 64 (8)

BACKGROUND: Measurement of DNA derived from different tissues in the circulating DNA pool can provide important information regarding the presence of many pathological conditions. However, existing methods involving genome-wide bisulfite sequencing are relatively expensive and may present challenges for large-scale analysis. METHODS: Through identifying differentially methylated regions in the liver and colon compared with other tissues, we identified 2 markers and developed corresponding droplet digital PCR assays. Plasma concentrations of liver-derived and colon-derived DNA were measured for 13 liver transplant recipients, 40 liver cancer patients, and 62 colorectal cancer (CRC) patients (27 with and 35 without liver metastases). RESULTS: In liver transplant recipients, the fractional concentration of liver-derived DNA measured using the liver-specific methylation marker and donor-specific alleles showed good correlation (Pearson R = 0.99). In liver cancer patients, the concentration of liver-derived DNA correlated positively with the maximal dimension of the tumor (Spearman R = 0.74). In CRC patients with and without liver metastasis, the plasma concentrations of colon-derived DNA (median, 138 copies/mL and 4 copies/mL, respectively) were increased compared with the 30 healthy controls (26 had undetectable concentrations). The absolute concentration of liver-derived DNA provided a better differentiation between CRC patients with and without liver metastasis compared with the fractional concentration (area under ROC curve, 0.85 vs 0.75). CONCLUSIONS: Quantitative analysis of plasma DNA with tissue-specific methylation patterns using droplet digital PCR is applicable for the investigation of cancers and assessing organ transplantation. This approach is useful for differentiating patients with and without metastases to other organs. (C) 2018 American Association for Clinical Chemistry

IF:6.89

A Simple and Novel Fecal Biomarker for Colorectal Cancer: Ratio of Fusobacterium Nucleatum to Probiotics Populations, Based on Their Antagonistic Effect

期刊: CLINICAL CHEMISTRY, 2018; 64 (9)

BACKGROUND: Gut microbial dysbiosis contributes to the development of colorectal cancer (CRC). We evaluated the utility of fecal bacterial biomarker candidates identified by our 16S rDNA sequencing analysis for CRC diagnosis. METHODS: We measured the relative abundance of Fusobacterium nucleatum (Fn), Faecalibacterium prausnitzii (Fp), Bifidobacterium (Bb), and Lactobacillus (Lb) by quantitative PCR in fecal samples from 2 cohorts of 903 individuals. We evaluated and validated the diagnostic performance of these microbial ratios and investigated the antagonistic effect of Fn against 3 different indicator stains. RESULTS: The microbial ratio of Fn to Bb (Fn/Bb) had a superior sensitivity of 84.6% and specificity of 92.3% in detecting CRC (area under the curve, AUC = 0.911). The combination of Fn/Bb and Fn/Fp improved the diagnostic value (AUC = 0.943). Moreover, the combination of Fn/Bb and Fn/Fp offered 60.0% specificity and 90.0% sensitivity in detecting stage I of CRC (AUC = 0.804). In particular, Fn was negatively correlated with Fp in the CRC group. The performance for CRC diagnosis was confirmed in the validation cohort II. The culture supernatant from Fn exhibited strong bactericidal activity against probiotics Fp and Bb strains. CONCLUSIONS: This study found that Fn could play a role in microbiota dysbiosis via the secreted antagonistic substances against probiotics. Moreover, the ratio of Fn to the important probiotics Fp and Bb was identified as a valuable biomarker for screening early CRC. (c) 2018 American Association for Clinical Chemistry.

IF:6.89

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