Decoding bioactive signals of the RNA secretome: the cell-free messenger RNA catalogue.

Despite gene-expression profiling being one of the most common methods to evaluate molecular dysregulation in tissues, the utilization of cell-free messenger RNA (cf-mRNA) as a blood-based non-invasive biomarker analyte has been limited compared to other RNA classes. Recent advancements in low-input RNA-sequencing and normalization techniques, however, have enabled characterization as well as accurate quantification of cf-mRNAs allowing direct pathological insights. The molecular profile of the cell-free transcriptome in multiple diseases has subsequently been characterized including, prenatal diseases, neurological disorders, liver diseases and cancers suggesting this biological compartment may serve as a disease agnostic platform. With mRNAs packaged in a myriad of extracellular vesicles and particles, these signals may be used to develop clinically actionable, non-invasive disease biomarkers. Here, we summarize the recent scientific developments of extracellular mRNA, biology of extracellular mRNA carriers, clinical utility of cf-mRNA as disease biomarkers, as well as proposed functions in cell and tissue pathophysiology.

Circulating cell-free messenger RNA secretome characterization of primary sclerosing cholangitis.

BACKGROUND: Primary sclerosing cholangitis (PSC) is a rare chronic cholestatic liver disease characterized by multifocal bile duct strictures. To date, underlying molecular mechanisms of PSC remain unclear, and therapeutic options are limited. METHODS: We performed cell-free messenger RNA (cf-mRNA) sequencing to characterize the circulating transcriptome of PSC and noninvasively investigate potentially bioactive signals that are associated with PSC. Serum cf-mRNA profiles were compared among 50 individuals with PSC, 20 healthy controls, and 235 individuals with NAFLD. Tissue and cell type-of-origin genes that are dysregulated in subjects with PSC were evaluated. Subsequently, diagnostic classifiers were developed using PSC dysregulated cf-mRNA genes. RESULTS: Differential expression analysis of the cf-mRNA transcriptomes of PSC and healthy controls resulted in identification of 1407 dysregulated genes. Furthermore, differentially expressed genes between PSC and healthy controls or NAFLD shared common genes known to be involved in liver pathophysiology. In particular, genes from liver- and specific cell type-origin, including hepatocyte, HSCs, and KCs, were highly abundant in cf-mRNA of subjects with PSC. Gene cluster analysis revealed that liver-specific genes dysregulated in PSC form a distinct cluster, which corresponded to a subset of the PSC subject population. Finally, we developed a cf-mRNA diagnostic classifier using liver-specific genes that discriminated PSC from healthy control subjects using gene transcripts of liver origin. CONCLUSIONS: Blood-based whole-transcriptome cf-mRNA profiling revealed high abundance of liver-specific genes in sera of subjects with PSC, which may be used to diagnose patients with PSC. We identified several unique cf-mRNA profiles of subjects with PSC. These findings may also have utility for noninvasive molecular stratification of subjects with PSC for pharmacotherapy safety and response studies.

Survey of extracellular communication of systemic and organ-specific inflammatory responses through cell free messenger RNA profiling in mice.

BACKGROUND: Inflammatory and immune responses are essential and dynamic biological processes that protect the body against acute and chronic adverse stimuli. While conventional protein markers have been used to evaluate systemic inflammatory response, the immunological response to stimulation is complex and involves modulation of a large set of genes and interacting signalling pathways of innate and adaptive immune systems. There is a need for a non-invasive tool that can comprehensively evaluate and monitor molecular dysregulations associated with inflammatory and immune responses in circulation and in inaccessible solid organs. METHODS: Here we utilized cell-free messenger RNA (cf-mRNA) RNA-Seq whole transcriptome profiling and computational biology to temporally assess lipopolysaccharide (LPS) induced and JAK inhibitor modulated inflammatory and immune responses in mouse plasma samples. FINDINGS: Cf-mRNA profiling displayed a pattern of systemic immune responses elicited by LPS and dysregulation of associated pathways. Moreover, attenuation of several inflammatory pathways, including STAT and interferon pathways, were observed following the treatment of JAK inhibitor. We further identified the dysregulation of liver-specific transcripts in cf-mRNA which reflected changes in the gene-expression pattern in this generally inaccessible biological compartment. INTERPRETATION: Using a preclinical mouse model, we demonstrated the potential of plasma cf-mRNA profiling for systemic and organ-specific characterization of drug-induced molecular alterations that are associated with inflammatory and immune responses. FUNDING: Molecular Stethoscope.

Noninvasive stratification of nonalcoholic fatty liver disease by whole transcriptome cell-free mRNA characterization.

Hepatic fibrosis stage is the most important determinant of outcomes in patients with nonalcoholic fatty liver disease (NAFLD). There is an urgent need for noninvasive tests that can accurately stage fibrosis and determine efficacy of interventions. Here, we describe a novel cell-free (cf)-mRNA sequencing approach that can accurately and reproducibly profile low levels of circulating mRNAs and evaluate the feasibility of developing a cf-mRNA-based NAFLD fibrosis classifier. Using separate discovery and validation cohorts with biopsy-confirmed NAFLD (n = 176 and 59, respectively) and healthy subjects (n = 23), we performed serum cf-mRNA RNA-Seq profiling. Differential expression analysis identified 2,498 dysregulated genes between patients with NAFLD and healthy subjects and 134 fibrosis-associated genes in patients with NAFLD. Comparison between cf-mRNA and liver tissue transcripts revealed significant overlap of fibrosis-associated genes and pathways indicating that the circulating cf-mRNA transcriptome reflects molecular changes in the livers of patients with NAFLD. In particular, metabolic and immune pathways reflective of known underlying steatosis and inflammation were highly dysregulated in the cf-mRNA profile of patients with advanced fibrosis. Finally, we used an elastic net ordinal logistic model to develop a classifier that predicts clinically significant fibrosis (F2-F4). In an independent cohort, the cf-mRNA classifier was able to identify 50% of patients with at least 90% probability of clinically significant fibrosis. We demonstrate a novel and robust cf-mRNA-based RNA-Seq platform for noninvasive identification of diverse hepatic molecular disruptions and for fibrosis staging with promising potential for clinical trials and clinical practice.NEW & NOTEWORTHY This work is the first study, to our knowledge, to utilize circulating cell-free mRNA sequencing to develop an NAFLD diagnostic classifier.

Donor-derived Cell-free DNA Identifies Antibody-mediated Rejection in Donor Specific Antibody Positive Kidney Transplant Recipients.

BACKGROUND: Elevated levels of donor-derived cell-free DNA (dd-cfDNA) in the plasma of renal allograft recipients indicates organ injury and an increased probability of active rejection. Donor-specific antibodies (DSA) to HLA antigens are associated with risk of antibody-mediated rejection (ABMR). This study assessed the combined use of dd-cfDNA and DSA testing to diagnose active ABMR. METHODS: Donor-derived cell-free DNA was assayed in 90 blood samples with paired DSA and clinically indicated biopsies from 87 kidney transplant patients. Sixteen cases met criteria for active ABMR. Performance characteristics of dd-cfDNA for diagnosis of active ABMR were determined for samples with prior or current positive DSA (DSA+, n = 33). RESULTS: The median level of dd-cfDNA (2.9%) in DSA+ patients with active ABMR was significantly higher than the median level (0.34%) in DSA+ patients without ABMR (P < 0.001). The median level of dd-cfDNA in DSA- patients was 0.29%. The positive predictive value of dd-cfDNA (at 1%) to detect active ABMR in DSA+ patients was 81%, whereas the negative predictive value was 83%. The positive predictive value for DSA+ alone was 48%. CONCLUSIONS: The combined use of dd-cfDNA and DSA testing may improve the noninvasive diagnosis of active ABMR in kidney transplant patients. Patients with dd-cfDNA+/ DSA+ results have a high probability of active ABMR.

Cell-Free DNA and Active Rejection in Kidney Allografts.

Histologic analysis of the allograft biopsy specimen is the standard method used to differentiate rejection from other injury in kidney transplants. Donor-derived cell-free DNA (dd-cfDNA) is a noninvasive test of allograft injury that may enable more frequent, quantitative, and safer assessment of allograft rejection and injury status. To investigate this possibility, we prospectively collected blood specimens at scheduled intervals and at the time of clinically indicated biopsies. In 102 kidney recipients, we measured plasma levels of dd-cfDNA and correlated the levels with allograft rejection status ascertained by histology in 107 biopsy specimens. The dd-cfDNA level discriminated between biopsy specimens showing any rejection (T cell-mediated rejection or antibody-mediated rejection [ABMR]) and controls (no rejection histologically), P<0.001 (receiver operating characteristic area under the curve [AUC], 0.74; 95% confidence interval [95% CI], 0.61 to 0.86). Positive and negative predictive values for active rejection at a cutoff of 1.0% dd-cfDNA were 61% and 84%, respectively. The AUC for discriminating ABMR from samples without ABMR was 0.87 (95% CI, 0.75 to 0.97). Positive and negative predictive values for ABMR at a cutoff of 1.0% dd-cfDNA were 44% and 96%, respectively. Median dd-cfDNA was 2.9% (ABMR), 1.2% (T cell-mediated types ≥IB), 0.2% (T cell-mediated type IA), and 0.3% in controls (P=0.05 for T cell-mediated rejection types ≥IB versus controls). Thus, dd-cfDNA may be used to assess allograft rejection and injury; dd-cfDNA levels <1% reflect the absence of active rejection (T cell-mediated type ≥IB or ABMR) and levels >1% indicate a probability of active rejection.

Biological Variation of Donor-Derived Cell-Free DNA in Renal Transplant Recipients: Clinical Implications.

BACKGROUND: Previous studies have demonstrated that donor-derived cell-free DNA (dd-cfDNA) found in circulating blood of transplant recipients may serve as a noninvasive biomarker of allograft rejection. To better interpret the clinical meaning of dd-cfDNA, it is essential to understand the biological variation of this biomarker in stable healthy recipients. This report establishes the biological variation and clinical reference intervals of dd-cfDNA in renal transplant recipients by using an analytically validated assay that has a CV of 6.8%. METHODS: We sampled venous blood at patient surveillance visits (typically at posttransplant months 1-4, 6, 9, and 12) in a 14-center observational study. Patients with stable renal allograft function spanning ≥3 serial visits were selected. We used AlloSure®, a targeted next-generation sequencing-based approach, to measure dd-cfDNA in the plasma and computed the intraindividual CV (CVI) and interindividual CV (CVG), the index of individuality (II), and reference change value (RCV). RESULTS: Of 93 patients, 61% were men, 56% were Caucasian, mean age was 49 years, and 63% were deceased donor kidney recipients. Of 380 blood samples, the dd-cfDNA median value was 0.21% (interquartile range 0.12%-0.39%) and the 97.5th percentile was 1.20%. In 18 patients with an average of 4.1 tests, the CVI was 21%, CVG was 37%, II was 0.57, and RCV was 61%. CONCLUSIONS: In a renal transplant recipient, a dd-cfDNA level above 1.2% is out of range and potentially abnormal. A serial increase of up to 61% in level of dd-cfDNA in a patient may be attributable to biological variation.Clinicaltrials.gov Identifier: NCT02424227.

Validation of a Clinical-Grade Assay to Measure Donor-Derived Cell-Free DNA in Solid Organ Transplant Recipients.

The use of circulating cell-free DNA (cfDNA) as a biomarker in transplant recipients offers advantages over invasive tissue biopsy as a quantitative measure for detection of transplant rejection and immunosuppression optimization. However, the fraction of donor-derived cfDNA (dd-cfDNA) in transplant recipient plasma is low and challenging to quantify. Previously reported methods to measure dd-cfDNA require donor and recipient genotyping, which is impractical in clinical settings and adds cost. We developed a targeted next-generation sequencing assay that uses 266 single-nucleotide polymorphisms to accurately quantify dd-cfDNA in transplant recipients without separate genotyping. Analytical performance of the assay was characterized and validated using 1117 samples comprising the National Institute for Standards and Technology Genome in a Bottle human reference genome, independently validated reference materials, and clinical samples. The assay quantifies the fraction of dd-cfDNA in both unrelated and related donor-recipient pairs. The dd-cfDNA assay can reliably measure dd-cfDNA (limit of blank, 0.10%; limit of detection, 0.16%; limit of quantification, 0.20%) across the linear quantifiable range (0.2% to 16%) with across-run CVs of 6.8%. Precision was also evaluated for independently processed clinical sample replicates and is similar to across-run precision. Application of the assay to clinical samples from heart transplant recipients demonstrated increased levels of dd-cfDNA in patients with biopsy-confirmed rejection and decreased levels of dd-cfDNA after successful rejection treatment. This noninvasive clinical-grade sequencing assay can be completed within 3 days, providing the practical turnaround time preferred for transplanted organ surveillance.

Risk Factors for Hepatocellular Carcinoma in Cirrhotic Patients with Chronic Hepatitis B.

AIMS: To investigate the clinical and genetic risk factors associated with hepatocellular carcinoma (HCC) in cirrhotic patients with chronic hepatitis B (CHB). METHODS: Nine hundred forty-nine Chinese Han patients with CHB were studied, including noncirrhotic patients without HCC (N = 234), cirrhotic patients without (N = 281) and with HCC (N = 434). Patients were genotyped for 10 candidate single nucleotide polymorphisms (SNPs) by the polymerase chain reaction (PCR)-ligase detection reaction (LDR) method. RESULTS: By multivariate logistic regression analysis adjusted for Child-Pugh scores, noneffective antiviral treatment, drinking history, family history of HCC, and age ≥50 years old were associated with HCC risk (odds ratio [OR] = 5.923, 2.456, 2.241, 1.955, respectively). Sixty-two of 170 cirrhotic patients who achieved sustained virological suppression by antiviral treatment developed HCC, with fatty liver disease, family history of HCC, and family history of hepatitis B virus (HBV) infection as the risk factors (OR = 11.646, 3.339, 2.537, respectively). The SNPs associated with HCC risk in patients with cirrhosis and CHB were rs11536889 in TLR4 and rs2853744 in SPP1. Polymorphisms of TLR4 rs2149356, AP3S2 rs2290351, STXBP5L rs2169302, MLEC rs7976497, and SOCS3 rs4969168 were associated with HCC risk in specific stratified analyses with gender, age, and drinking history in the cirrhotic patients. CONCLUSIONS: Inadequate antiviral treatment, family history of HCC, drinking history, and age ≥50 years old are risk factors for HCC. Sustained suppression of HBV does not eliminate the risk of HCC. Specific host genetic factors may impact HCC development in Han Chinese cirrhotic patients with CHB, including SNPs in TLR4, SPP1, AP3S2, STXBP5L, MLEC, and SOCS3, which warrant further validation in additional cohorts.

Mutation Yield of a 34-Gene Solid Tumor Panel in Community-Based Tumor Samples.

BACKGROUND: Several targeted therapies have been approved for treatment of solid tumors. Identification of gene mutations that indicate response to these therapies is rapidly progressing. A 34-gene next-generation sequencing (NGS) panel, developed and validated by us, was evaluated to detect additional mutations in community-based cancer specimens initially sent to our reference laboratory for routine molecular testing. METHODS: Consecutive de-identified clinical specimens (n = 121) from melanoma cases (n = 31), lung cancer cases (n = 27), colorectal cancer cases (n = 33), and breast cancer cases (n = 30) were profiled by NGS, and the results were compared with routine molecular testing. RESULTS: Upon initial mutation testing, 20 % (24/121) were positive. NGS detected ≥1 additional mutation not identified by routine testing in 74 % of specimens (90/121). Of the specimens with additional mutations, 16 harbored mutations in National Comprehensive Cancer Network guideline genes. These various additional mutations were in gene regions not routinely covered, in genes not routinely tested, and/or present at low allele frequencies. Moreover, NGS yielded no false negatives. Overall, NGS detected mutations in 59 % of the genes (20/34) included in the panel, 75 % of which (15/20) were detected in multiple tumor types. Mutations in TP53 were found in 51 % of tumors tested (62/121). Mutations in at least one other (non-TP53) gene present in the panel were detected in 64 % of cases (77/121). CONCLUSION: This assay provides improved breadth and sensitivity for profiling clinically relevant genes in these prevalent solid tumor types.

Hepatic Steatosis and Insulin Resistance, But Not Steatohepatitis, Promote Atherogenic Dyslipidemia in NAFLD.

CONTEXT: Patients with nonalcoholic fatty liver disease (NAFLD) are at increased risk of cardiovascular disease, and atherogenic lipoproteins may play an important role. OBJECTIVE: The objective of the study was to determine the contribution of the severity of steatohepatitis to atherogenic dyslipidemia in patients with NAFLD. DESIGN: This was a cross-sectional study. SETTING: The study was conducted at a university hospital. PATIENTS: Patients were recruited from outpatient clinics or from the general population (n = 188). INTERVENTIONS: Measurement of hepatic triglyceride content by magnetic resonance spectroscopy, histology (liver biopsy), metabolic profile by means of an oral glucose tolerance test, and lipoprotein analyses were performed. OUTCOMES: Outcomes measured included standard lipids, lipoprotein subfraction analysis (apolipoprotein B/A1 levels, low-density lipoprotein (LDL) particle size/phenotype, and LDL/high-density lipoprotein subfractions), and insulin resistance. RESULTS: Patients with NAFLD had severe insulin resistance, especially at the level of the adipose tissue, when compared with patients without NAFLD. Despite small differences in triglycerides and high-density lipoprotein-cholesterol, patients with NAFLD had a significantly higher plasma apolipoprotein B to apolipoprotein A1 ratio (0.66 ± 0.02 vs 0.58 ± 0.02, P = .01) and smaller LDL particle size (216.2 ± 0.7 vs 219.4 ± 1.1 Å, P = .01). Of note, these differences between patients with/without NAFLD were independent of the presence of obesity. Severity of steatohepatitis did not significantly influence the lipoprotein profile. Worse atherogenic dyslipidemia was best predicted by the degree of liver fat accumulation and adipose tissue and systemic insulin resistance. CONCLUSIONS: NAFLD was associated with a worse atherogenic lipoprotein profile, regardless of similar body mass index and other clinical parameters. We speculate that this lipoprotein profile is driven mostly by liver fat content and insulin resistance and appears not to be worsened by obesity or the severity of liver disease (nonalcoholic steatohepatitis).

Cost Effectiveness of Sequencing 34 Cancer-Associated Genes as an Aid for Treatment Selection in Patients with Metastatic Melanoma.

OBJECTIVE: To determine whether a next-generation sequencing (NGS) panel of 34 cancer-associated genes would cost-effectively aid in the treatment selection for patients with metastatic melanoma, compared with a single-site BRAF V600 mutation test. METHODS: A decision model was developed to estimate the costs and health outcomes of the two test strategies. The cost effectiveness of these two strategies was analyzed from a payer perspective over a 2-year time horizon with model parameters taken from the literature. RESULTS: In the base case, the gene sequencing panel strategy resulted in a cost of US$120,022 and 0.721 quality-adjusted life years (QALYs) per patient, whereas the single-site mutation test strategy resulted in a cost of US$128,965 and 0.704 QALYs. Thus, the gene sequencing panel strategy cost US$8943 less per patient and increased QALYs by 0.0174 per patient. Sensitivity analyses showed that, compared with the single-site mutation test strategy, the gene sequencing panel strategy had a 90.9% chance of having reduced costs and increased QALYs, with the cost of the gene sequencing panel test having minimal effect on the incremental cost. CONCLUSION: Compared with the single-site mutation test, the use of an NGS panel of 34 cancer-associated genes as an aid in selecting therapy for patients with metastatic melanoma reduced costs and increased QALYs. If the base-case results were applied to the 8900 patients diagnosed with metastatic melanoma in the USA each year, the gene sequencing panel strategy could result in an annual savings of US$79.6 million and a gain of 155 QALYs.

Genetic-based prediction of disease traits: prediction is very difficult, especially about the future.

Translation of results from genetic findings to inform medical practice is a highly anticipated goal of human genetics. The aim of this paper is to review and discuss the role of genetics in medically-relevant prediction. Germline genetics presages disease onset and therefore can contribute prognostic signals that augment laboratory tests and clinical features. As such, the impact of genetic-based predictive models on clinical decisions and therapy choice could be profound. However, given that (i) medical traits result from a complex interplay between genetic and environmental factors, (ii) the underlying genetic architectures for susceptibility to common diseases are not well-understood, and (iii) replicable susceptibility alleles, in combination, account for only a moderate amount of disease heritability, there are substantial challenges to constructing and implementing genetic risk prediction models with high utility. In spite of these challenges, concerted progress has continued in this area with an ongoing accumulation of studies that identify disease predisposing genotypes. Several statistical approaches with the aim of predicting disease have been published. Here we summarize the current state of disease susceptibility mapping and pharmacogenetics efforts for risk prediction, describe methods used to construct and evaluate genetic-based predictive models, and discuss applications.

An optimized five-gene multi-platform predictor of hormone receptor negative and triple negative breast cancer metastatic risk.

INTRODUCTION: Outcome predictors in use today are prognostic only for hormone receptor-positive (HRpos) breast cancer. Although microarray-derived multigene predictors of hormone receptor-negative (HRneg) and/or triple negative (Tneg) breast cancer recurrence risk are emerging, to date none have been transferred to clinically suitable assay platforms (for example, RT-PCR) or validated against formalin-fixed paraffin-embedded (FFPE) HRneg/Tneg samples. METHODS: Multiplexed RT-PCR was used to assay two microarray-derived HRneg/Tneg prognostic signatures IR-7 and Buck-4) in a pooled FFPE collection of 139 chemotherapy-naïve HRneg breast cancers. The prognostic value of the RTPCR measured gene signatures were evaluated as continuous and dichotomous variables, and in conditional risk models incorporating clinical parameters. An optimized five-gene index was derived by evaluating gene combinations from both signatures. RESULTS: RT-PCR measured IR-7 and Buck-4 signatures proved prognostic as continuous variables; and conditional risk modeling chose nodal status, the IR-7 signature, and tumor grade as significant predictors of distant recurrence (DR). From the Buck-4 and IR-7 signatures, an optimized five-gene (TNFRSF17, CLIC5, HLA-F, CXCL13, XCL2) predictor was generated, referred to as the Integrated Cytokine Score (ICS) based on its functional pathway linkage through interferon-γ and IL-10. Across all FFPE cases, the ICS was prognostic as either a continuous or dichotomous variable, and conditional risk modeling selected nodal status and ICS as DR predictors. Further dichotomization of node-negative/ICS-low FFPE cases identified a subset of low-grade HRneg tumors with <10% 5-year DR risk. The prognostic value of ICS was reaffirmed in two previously studied microarray assayed cohorts containing 274 node-negative and chemotherapy naive HRneg breast cancers, including 95 Tneg cases where it proved prognostically independent of Tneg molecular subtyping. In additional HRneg/Tneg microarray assayed cohorts, the five-gene ICS also proved prognostic irrespective of primary tumor nodal status and adjuvant chemotherapy intervention. CONCLUSION: We advanced the measurement of two previously reported microarray-derived HRneg/Tneg breast cancer prognostic signatures for use in FFPE samples, and derived an optimized five-gene Integrated Cytokine Score (ICS) with multi-platform capability of predicting metastatic outcome from primary HRneg/Tneg tumors independent of nodal status, adjuvant chemotherapy use, and Tneg molecular subtype.

A candidate gene study for the association of host single nucleotide polymorphisms with liver cirrhosis risk in chinese hepatitis B patients.

BACKGROUND AND AIMS: Recently, genetic association studies have linked a number of single nucleotide polymorphisms (SNPs) with liver fibrosis risk of hepatitis C. The present study was designed to validate the association of emerging SNPs with development of liver cirrhosis and chronicity in a Chinese population infected with hepatitis B virus (HBV). METHODS: 714 Chinese subjects with persistent HBV infection (429 with evident liver cirrhosis and 285 without cirrhosis clinically or pathologically) and 280 subjects with spontaneous HBV clearance were studied. Six SNPs in five candidate genes were detected with the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) method. The distribution of each polymorphism was compared between the age-matched cirrhotic and noncirrhotic subjects, and between subjects with persistent infection and spontaneous HBV clearance. RESULTS: The rs2679757 polymorphism of antizyme inhibitor 1 (AZIN1) gene was associated with the risk of cirrhosis (odds ratio [OR] for GG+AG versus AA=1.47, 95% confidence interval [CI]=1.08-2.01, p=0.01). So was rs886277 in the transient receptor potential cation channel subfamily M, member 5 (TRPM5) gene (OR for CC versus CT+TT=1.63, 95% CI=1.20-2.22, p=0.002). The frequencies of these two SNPs were also associated with the severity of decompensated cirrhosis based on the Child-Pugh classification. Genotype frequencies of other SNPs were not different between the cirrhotic and noncirrhotic groups. No SNPs were associated with the outcome of spontaneous HBV clearance. CONCLUSIONS: AZIN1 rs2679757 and TRPM5 rs886277 are associated with the risk of HBV-related liver cirrhosis in Chinese. The emerging SNPs warrant further clinical validation in other cohorts or ethnic groups, and could lead to mechanistic studies to reveal their contributions to fibrosis progression.

Classification of LDL phenotypes by 4 methods of determining lipoprotein particle size.

BACKGROUND: Low-density lipoprotein cholesterol (LDL-C) lowering is the primary objective of patient management for cardiovascular disease. However, large numbers of patients who have achieved their LDL-C goal remain at risk for cardiovascular events. Low-density lipoprotein subfractions may provide insight into this residual risk. Thus, LDL subfraction standardization and consistency are critical to these efforts. AIM: This study aimed to determine the agreement of the analytical results among 4 methods commonly used for LDL subfractionation, namely, segmented gradient gel electrophoresis (sGGE), ultracentrifugation-vertical auto profile (VAP), nuclear magnetic resonance (NMR), and ion mobility (IM). METHODS: Blood samples were collected from 228 apparently healthy adults and sent to 4 clinical reference laboratories for analysis. The LDL phenotype was reported as pattern A (larger, less dense particles) or pattern B (smaller, more dense particles), respectively, and was the primary measure of comparison. An intermediate pattern (A/B) was also reported for sGGE and VAP. RESULTS: We observed complete agreement in the LDL phenotype among the 4 methods in 64% of subjects and agreement among at least 3 of the 4 methods in 87% of subjects. Agreement among pairs of methods ranged from 73% to 98% depending on how differences in reporting of subjects with intermediate results were considered. When subjects having intermediate A/B pattern were excluded, sGGE and IM had the highest agreement (98%) of any pair of methods. CONCLUSIONS: We found substantial agreement in the reported LDL phenotype among 4 LDL subfraction measurement technologies as performed by different clinical reference laboratories.

Multiple metabolic alterations exist in mutant PI3K cancers, but only glucose is essential as a nutrient source.

Targeting tumour metabolism is becoming a major new area of pharmaceutical endeavour. Consequently, a systematic search to define whether there are specific energy source dependencies in tumours, and how these might be dictated by upstream driving genetic mutations, is required. The PI3K-AKT-mTOR signalling pathway has a seminal role in regulating diverse cellular processes including cell proliferation and survival, but has also been associated with metabolic dysregulation. In this study, we sought to define how mutations within PI3KCA may affect the metabolic dependency of a cancer cell, using precisely engineered isogenic cell lines. Studies revealed gene expression signatures in PIK3CA mutant cells indicative of a consistent up-regulation of glycolysis. Interestingly, the genes up- and down-regulated varied between isogenic models suggesting that the primary node of regulation is not the same between models. Additional gene expression changes were also observed, suggesting that metabolic pathways other than glycolysis, such as glutaminolysis, were also affected. Nutrient dependency studies revealed that growth of PIK3CA mutant cells is highly dependent on glucose, whereas glutamine dependency is independent of PIK3CA status. In addition, the glucose dependency exhibited by PIK3CA mutant cells could not be overridden by supplementation with other nutrients. This specific dependence on glucose for growth was further illustrated by studies evaluating the effects of targeted disruption of the glycolytic pathway using siRNA and was also found to be present across a wider panel of cancer cell lines harbouring endogenous PIK3CA mutations. In conclusion, we have found that PIK3CA mutations lead to a shift towards a highly glycolytic phenotype, and that despite suggestions that cancer cells are adept at utilising alternative nutrient sources, PIK3CA mutant cells are not able to compensate for glucose withdrawal. Understanding the metabolic dependencies of PIK3CA mutant cancers will provide critical information for the design of effective therapies and tumour visualisation strategies.

Predicting cirrhosis and clinical outcomes in patients with advanced chronic hepatitis C with a panel of genetic markers (CRS7).

OBJECTIVES: Genetic factors may play a role in fibrosis progression in patients with chronic hepatitis C (CHC). A cirrhosis risk score (CRS7) with seven single nucleotide polymorphisms was previously shown to correlate with cirrhosis in patients with CHC. This study aimed to assess the validity of CRS7 as a marker of fibrosis progression and cirrhosis and as a predictor of clinical outcomes in patients with CHC. METHODS: A total of 938 patients (677 Caucasians, 165 African-Americans, and 96 Hispanic/Other) in the Hepatitis C Antiviral Long-term Treatment against Cirrhosis Trial were studied. CRS7 was categorized a priori as high risk (n=440), medium risk (n=310), or low risk (n=188). Patients were assessed for four possible outcomes: fibrosis progression, cirrhosis, clinical outcomes [decompensation or hepatocellular carcinoma (HCC)], or HCC alone. RESULTS: Twenty-nine percent (142/493) developed an increase in fibrosis score by greater than or equal to 2 points on follow-up biopsies, 58% had cirrhosis on one or more biopsies, 35% developed at least one clinical outcome, and 13% developed HCC. CRS7 (trend test) was associated with risk for fibrosis progression (P=0.04) with adjusted hazard ratio of 1.27 (95% confidence interval: 1.01-1.58) and with cirrhosis (P=0.05) with adjusted odds ratio of 1.19 (1.00-1.41). Rates of HCC and clinical outcomes were increased in patients with higher CRS7 scores, but were not statistically significant (P=0.12 clinical outcomes, and P=0.07 HCC). A single nucleotide polymorphism in AZIN1 was significantly associated with fibrosis progression. CONCLUSION: CRS7 was validated as a predictor of fibrosis progression and cirrhosis among Hepatitis C Antiviral Long-term Treatment against Cirrhosis patients, who all had advanced fibrosis. CRS7 was not predictive of clinical outcome.

A polymorphism that delays fibrosis in hepatitis C promotes alternative splicing of AZIN1, reducing fibrogenesis.

UNLABELLED: Among several single-nucleotide polymorphisms (SNPs) that correlate with fibrosis progression in chronic HCV, an SNP in the antizyme inhibitor (AzI) gene is most strongly associated with slow fibrosis progression. Our aim was to identify the mechanism(s) underlying this observation by exploring the impact of the AzI SNP on hepatic stellate cell (HSC) activity. Seven novel AZIN1 splice variants ("SV2-8") were cloned by polymerase chain reaction from the LX2 human HSC line. Expression of a minigene in LX2 containing the AZIN1 slow-fibrosis SNP yielded a 1.67-fold increase in AZIN1 splice variant 2 (AZIN1 SV2) messenger RNA (mRNA) (P = 0.05). In healthy human leukocytes, the SNP variant also correlated with significantly increased SV2 mRNA. Cells (293T) transfected with short hairpin RNA (shRNA) complementary to the exonic splicing chaperone SRp40 expressed 30% less SRp40 (P = 0.044) and 43% more AzI SV2 (P = 0.021) than control shRNA-expressing cells, mimicking the effect of the sequence variant. LX2 cells transfected with AZIN1 full-length complementary DNA expressed 35% less collagen I mRNA (P = 0.09) and 18% less α-smooth muscle actin mRNA (P = 0.09). Transient transfection of AZIN1 SV2 complementary DNA into LX2 cells reduced collagen I gene expression by 64% (P = 0.001) and α-smooth muscle actin by 43% (P = 0.005) compared to vector-transfected controls, paralleling changes in protein expression. Both AZIN1 and AZIN-SV2 mRNAs are detectable in normal human liver and reduced in HCV cirrhotic livers. The AZIN1-SV2 acts via a polyamine-independent pathway, as it neither interacts with antizyme nor affects the ability of AZIN1 lacking this variant to neutralize antizyme. CONCLUSION: An SNP variant in the AZIN1 gene leads to enhanced generation of a novel alternative splice form that modifies the fibrogenic potential of HSCs.