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BACKGROUND & AIMSPrimary sclerosing cholangitis (PSC) is a rare chronic cholestatic liver disease characterized by multi-focal bile duct strictures. To date, underlying molecular mechanisms of PSC remain unclear and therapeutic options for PSC patients are limited. We performed cell-free messenger RNA (cf-mRNA) next generation sequencing (RNA-Seq) to characterize the circulating transcriptome of PSC and non-invasively investigate potentially bioactive signals that are associated with PSC.METHODSSerum cf-mRNA profiles were compared among 50 individuals with PSC, 20 healthy controls, and 235 individuals with non-alcoholic fatty liver disease (NAFLD). Tissue and cell type-of-origin genes that are dysregulated in subjects with PSC were evaluated. Subsequently, diagnostic classifiers were built using PSC dysregulated cf-mRNA genes.RESULTSDifferential 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 the liver diseases (NAFL and Non-Alcohol Steatohepatitis (NASH)) or healthy controls shared common genes known to be involved in liver pathophysiology. In particular, genes from liver- and specific cell type-origin, including hepatocyte, hepatic stellate cells and Kupffer cells, 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 classifier using liver-specific genes which discriminated PSC from healthy control subjects using gene transcripts of liver origin.CONCLUSIONSBlood-based whole transcriptome cf-mRNA profiling revealed high abundance of liver-specific genes in PSC subject sera which may be used to diagnose PSC patients. We identified several unique cf-mRNA profiles of subjects with PSC. These findings may also have utility for non-invasive molecular stratification of subjects with PSC for pharmacotherapy safety and response studies.Graphical Abstract

75 Background: The National Polyp Study and 2021 USPSTF CRC-update highlight that the detection and removal of precancerous advanced adenomas (AA) prevents colorectal cancer (CRC), decreases mortality, and leads to higher cost savings than early cancer detection. Performance of a multimodal blood-based test for the detection of CRC and AA that integrates sensitive and accurate detection of circulating gastrointestinal epithelial cells and somatic oncologic variants as well as SEER data of the impact of sex and age is described. Methods: The prospective study included average-risk, asymptomatic screening subjects from 18 geographically dispersed US colonoscopy centers with blood drawn before colonoscopy. Monte Carlo cross-validation (MCCV) methods were used to evaluate the robustness of test performance through 2000 iterations of independent training and validation using bootstrap resampling with stratification to balance patient histopathology, age, and gender. Results include point estimates, confidence intervals, and distributions of sensitivity and specificity to detect AA and CRC. Results: The study cohort (53.2% female; mean age 56.7 yrs.) consisted of 1,038 subjects (White 65.1%; Black 8.5%; Hispanic 24.8%; Asian 1.7%); of which 954 (92%) were asymptomatic, average-risk screening subjects without age enrichment (including 11 CRC and 93 AA) and 84 (8%) were enriched case-control (65 CRC and 19 AA) subjects. The algorithm derives a test score from 0 (low risk) to 100 (high risk) as a quantitative measure of AA and CRC risk. A pre-defined cut point of 47.2 yields a test specificity > 90%, and 92.1% and 54.5% sensitivity for the detection of CRC and AA, respectively. Estimated sensitivities and selected Clopper-Pearson (Exact) 95% confidence intervals based on validation results from MCCV are presented. A split analysis shows for the 954 intended-use, asymptomatic, average-risk screening subjects, the sensitivity for CRC and AA are 100% and 55.9%. Conclusions: A multi-site, prospective, average-risk CRC screening study using a multimodal assay had high sensitivity and specificity for AA and CRC. The quantitative correlation of test scores with disease pathology indicates that the modes of the assay interrogate the primary underlying pathophysiology of disease. The results demonstrate the potential of this novel test to meet the clinically unmet need for a noninvasive strategy for CRC screening and prevention that detects CRC and AA. Clinical trial information: NCT05127096 . [Table: see text]

Testing has been central to our response to the COVID-19 pandemic. However, the accuracy of testing relies on standards, including reference materials, proficiency testing schemes, and information and reporting guidelines. The use of standards is a simple, inexpensive, and effective method to ensure reliable test results that inform clinical and public health decisions. Here we describe the central role of standards during the COVID-19 pandemic, where they have enabled population-scale screening, genomic surveillance and measures of immune protection measures. Given these benefits, the Coronavirus Standards Working Group (CSWG) was formed to coordinate standards in SARS-CoV-2 testing. This network of scientists has developed best-practices, reference materials, and conducted proficiency studies to harmonize laboratory performance. We propose that this coordinated development of standards should be prioritized as a key early step in the public health response to future pandemics that is necessary for reliable, large-scale testing for infectious disease.

Background and Aims: Hematologic malignancies account for approximately 10% of all cancers in the US. In order to improve the treatment and outcome of patients with hematologic malignancies, integration of accurate noninvasive molecular biomarkers to diagnose, monitor therapeutic response and identify disease subtype are critical. Recent advances in next generation sequencing (NGS) have accelerated the utilization of circulating nucleic acids for the development of non-invasive disease biomarkers. We have developed an RNA-Seq platform that allows robust quantification of the circulating cell-free messenger RNA (cf-mRNA) whole transcriptome. Here, we conducted cf-mRNA profiling on plasma samples collected from multiple myeloma and acute myeloid leukemia patients to investigate the feasibility of cell-free transcriptomic profiling as a monitoring tool for hematologic malignancies following bone marrow transplantation and therapeutic treatment. Methods: We performed cf-mRNA RNA-Sequencing on 96 longitudinally collected plasma samples from 3 multiple myeloma and 3 acute myeloid leukemia patients. Cf-mRNA profiles of patients were evaluated using GTEx (tissue type) and Blueprint (cell type), and dysregulation of gene-expression signatures were assessed following bone marrow transplantation and therapeutic treatment. In addition, we sequenced corresponding whole blood cells and buffy coat for comparison. Results: Plasma cf-mRNA profiling showed substantially higher proportion of transcripts from megakaryocyte and non-hematopoietic cells relative to whole blood cellular transcripts, suggesting that the molecular profile of cf-mRNA is more suitable for the assessment of gene-expression dysregulation of bone marrow than whole blood. We further report that non-invasive cf-mRNA profiling captures gene-expression alterations of cancer patients following chemotherapy and bone marrow transplantation. And we observed dysregulation of hematopoietic lineage-specific transcripts in circulation during bone marrow ablation and reconstitution. Lastly, we used cf-mRNA profiling to identify a malignant Ig clone for multiple myeloma patients and were able to monitor bone marrow reconstitution. Conclusions: We demonstrated that cf-mRNA profiling is an effective means to non-invasively assess molecular characteristics that are associated with multiple clinical parameters of hematological malignancies. Our study highlights the potential of cf-mRNA Seq as a platform to develop patient monitoring biomarkers for hematologic malignancies. Citation Format: Shusuke Toden, David A. Ross, Stephen R. Quake, John J. Sninsky. Utilization of Cell free messenger RNA Sequencing for non-invasive molecular profiling of multiple myeloma and Acute Myeloid Leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5174.

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.

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 1% indicate a probability of active rejection.

[Abstract] 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.

To investigate the clinical and genetic risk factors associated with hepatocellular carcinoma (HCC) in cirrhotic patients with chronic hepatitis B (CHB).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.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.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.

50 Background: Many of the 50,000 annual colorectal cancer (CRC) deaths can be attributed to 1/3 eligible Americans not following screening guidelines or approximately 1/2 of the population not adherent to the follow-up post-polypectomy guidelines. The new understanding of the natural history and shared etiology of adenomas and CRC inform integration of clinically relevant biomarkers. The two objectives of CRC screening and surveillance are early detection to improve survival and prevention of CRC through removal of adenomas using colonoscopy. Adenomas account for 98% of actionable colonoscopies. Stool tests have low sensitivity for advanced adenomas (AA, 24-42%). Methods: A prospective, blinded study was conducted at the VA Palo Alto Health Care System. Patients had blood drawn prior to colonoscopy. The test analyzes two biomarkers: circulating gastrointestinal epithelial cells and somatic mutations of cell-free DNA. The probability of advanced neoplasia was obtained by ordinal/nominal logistic regression methods together with SEER-incidence rate and prior history of AA on a training set of 346 subjects. The cutpoint for the quantitative score was fixed and the remaining 112 subjects were tested. Results: Interim results for 458 patients with no prior diagnosis of colorectal cancer (CRC) are presented. The cohort included both screening (239) and surveillance (219) subjects. Indications for colonoscopy were 86% asymptomatic and 14% with symptoms or positive-FIT. Balanced distribution of roughly 3/4th subjects in each disease category were randomly selected for training and algorithm development and the remaining 1/4th subjects were used for validation. A cutpoint was selected to obtain a test specificity (non-neoplastic finding or negative colonoscopy) of 90% resulting in a sensitivity of 100% and 80.0% for detection of CRC and advanced neoplasia (AN = CRC+AA), respectively, on the training subjects. The area under the ROC curve is 0.91. Validation using the fixed cutpoint and 112 test subjects achieved 91.4% specificity and 100% and 75.0% sensitivity for CRC and AN. Conclusions: This blood test has high sensitivity for colorectal advanced neoplasia while retaining high specificity. The quantitative nature of the score has the potential to enable stratification of patients for screening or post-polypectomy surveillance colonoscopy. [Table: see text]

1555 Background: Colonoscopic polypectomy is the primary reason for declining colorectal cancer incidence and mortality. Epidemiological evidence has ordered the timing and risk of pre-cancerous adenomas, localized and invasive cancer along a 7-10 year continuum. The increased size and number of index polyps are correlated with an increased probability of progression to cancer and informs surveillance colonoscopies. Methods: A single-center, IRB-approved, prospective, blinded study was conducted at the VA Palo Alto Health Care System. Results for 354 patients with no prior diagnosis of CRC who were scheduled for colonoscopy are presented. Indications for colonoscopy were 86% asymptomatic and 14% with symptoms or positive-FIT. Patients had blood drawn immediately prior to colonoscopy. The test analyzes three biomarkers: circulating gastrointestinal epithelial cells (CEC), validated somatic mutations, and methylation (SEPTIN9) of cell-free DNA and uses incident risk to calculate a CMx Score, scaled from 0 to 100. Multivariate regression methods were used to assess the degree of association between the pre-defined CMx Scores and polyp sizes and number, adjusting for both DNA mutation and DNA methylation status. Results: There is a significant association between CMx Scores and polyp size (F value = 5.80, p-value = 0.017). DNA mutation (F value = 1.29, p-value = 0.263) and methylation status (F value = 0.34, p-value = 0.560) were non-significant. Similarly, there is a significant association between CMx Scores and number of polyps (F value = 23.71, p-value < 0.0001). Again, DNA mutation (F value = 1.57, p-value = 0.210) and methylation status (F value = 1.34, p-value = 0.248) were non-significant. These results suggest that CMx Scores, which incorporate CEC, are providing predictive information of polyp sizes and number above and beyond DNA mutation and methylation status alone. Conclusions: A novel noninvasive multimodal blood-based assay that analyzes cell-free DNA for somatic mutations and methylation, CEC and integrates SEER incidence risk is significantly associated with polyp size and number. The opportunity to track progression and potentially inform colonoscopy interval is notable. [Table: see text]

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.

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

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. 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. 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). This assay provides improved breadth and sensitivity for profiling clinically relevant genes in these prevalent solid tumor types.

Background: While representing the largest fraction of women diagnosed with primary breast cancer, older postmenopausal women with ER+, HER2− tumors are less responsive to chemoendocrine therapy than younger women and have been underrepresented in molecular profiling of randomized trials. IBCSG Trial IX, a randomized controlled trial in postmenopausal women, median age 61y, with node negative disease, failed to demonstrate the benefit of preceding tamoxifen (T) by 3 cycles of CMF for ER+ tumors. We sought to determine if MS, a proliferation score, could identify a subset of women who differentially benefit from addition of chemotherapy to T in this trial. Methods: From 1988–1999, 1669 eligible patients (1040 with ER+, HER2− tumors) were randomized to CMF→T vs T. Disease-free survival (DFS) was the primary trial endpoint; breast cancer-free interval (BCFI) which excludes second (non-breast) malignancies and censors deaths without prior cancer event was also evaluated. Analysis was limited to the first 7 years of follow-up. From 671 (ER+, HER2−) available subjects, 568 were successfully profiled by RT-PCR. The mRNA expression levels of 14 equally-weighted proliferation genes and 3 normalization genes were used to generate MS; predetermined binary categorization of MS was used. Analysis of this post hoc, pre-specified study used results from centralized laboratory IHC and Cox models to assess the predictive value of MS on DFS and BCFI, adjusting for traditional risk factors of local treatment, age, ER, PR, Ki67, tumor size and grade. Results: Subgroups of MS (low, 169 samples (30%) and high, 399 samples (70%)) were identified. MS by treatment interaction was significant for DFS and BCFI (each p ≤ 0.004). Among patients with low MS, CMF→T improved DFS (HR 0.19, 95% CI 0.06–0.59) and BCFI (HR 0.19, 95% CI 0.05–0.72) vs T; 7y DFS was 95% vs 83% with CMF→T vs T. Among patients with high MS, CMF→T did not improve DFS (HR 1.27, 95% CI 0.79–2.05) or BCFI (HR 1.37, 95% CI 0.80–2.33) and 7y DFS of 81% for CMF→T and T. Continuous MS was moderately correlated with log Ki67 (r = 0.47) but not correlated with ER or PR. The MS by treatment interaction remained significant with Ki67 in the model. Conclusions: Low MS was associated with differential benefit favoring those women receiving CMF→T vs T alone for both DFS and BCFI in the first 7 years. The effect was independent of traditional risk factors including Ki67. Hence this study, which is unconfounded by chemotherapy-induced ovarian ablation in younger women, identifies a subset of postmenopausal women with ER+, HER2− tumors that benefit from CMF chemotherapy. This seemingly incongruous observation is consistent with a) the prior observation that only the low-proliferation subgroup by PAM50 11-gene signature benefits from the addition of weekly paclitaxel to adjuvant FEC (GEICAM/9906), b) the ability of MS to identify a subset of women with tumors with disseminated luminal progenitor cells activated through the agonistic activity of tamoxifen, and c) the repetitive dosing of cyclophosphamide and taxol being hypothesized to act via tumor stroma/anti-angiogenesis. The relative contribution of these factors is under investigation. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr PD10-03.

Objective Genome-wide association studies (GWAS) have linked many single nucleotide polymorphisms (SNPs) to the outcomes of a variety of liver diseases. The aim of the present study was to evaluate the association of several candidate SNPs with the risk and severity of cirrhosis due to chronic hepatitis B in a Chinese population. Methods A total of 714 Chinese participants with persistent HBV infection were studied. Patients were divided into cirrhotic (n = 429) and non-cirrhotic (n = 285) groups based on clinical and pathological evidence. The progression rate and severity of liver cirrhosis were evaluated with an arbitrary t-score system. Genotypes of six SNPs in five candidate genes were detected with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The genotypic distributions of the SNPs were compared between the age-matched cirrhotic and non-cirrhotic subjects. The association between the risk of SNPs and the severity and progression rate of cirrhosis was further analyzed. Results Rs2679757 polymorphism of the antizyme inhibitor 1 (AZIN1) gene and Rs886277 in the transient receptor potential cation channel subfamily M, member 5 gene (TRPM5) were found to be associated with cirrhosis risk in CHB. They were also correlated with the overall severity and progression rate of cirrhosis. Genotype frequencies of other SNPs were not different between the cirrhosis and non-cirrhosis groups. Conclusions AZIN1 rs2679757 and TRPM5 rs886277 are associated with the risk and the progression rate of HBV-related liver fibrosis in Chinese patients. The emerging SNPs associated with cirrhosis prognosis warrant further clinical validation in other CHB cohorts or ethnic groups, and merit mechanistic studies to reveal their roles in fibrosis progression.

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.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.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.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.

Background & Aims A single nucleotide polymorphism 61*G (rs4444903) in the epidermal growth factor ( EGF ) gene has been associated, in 2 case-control studies, with hepatocellular carcinoma (HCC). We tested associations between demographic, clinical, and genetic data and development of HCC, and developed a simple predictive model in a cohort of patients with chronic hepatitis C and advanced fibrosis. Methods Black and white subjects from the Hepatitis C Antiviral Long-term Treatment against Cirrhosis (HALT-C) trial (n = 816) were followed up prospectively for development of a definite or presumed case of HCC for a median time period of 6.1 years. We used the Cox proportional hazards regression model to determine the hazard ratio for risk of HCC and to develop prediction models. Results Subjects with EGF genotype G/G had a higher adjusted risk for HCC than those with genotype A/A (hazard ratio, 2.10; 95% confidence interval, 1.05–4.23; P = .03). After adjusting for EGF genotype, blacks had no increased risk of HCC risk compared with whites. Higher serum levels of EGF were observed among subjects with at least one G allele ( P = .08); the subset of subjects with EGF G/G genotype and above-median serum levels of EGF had the highest risk of HCC. We developed a simple prediction model that included the EGF genotype to identify patients at low, intermediate, and high risk for HCC; 6-year cumulative HCC incidences were 2.3%, 10.4%, and 26%, respectively. Conclusions We associated the EGF genotype G/G with increased risk for HCC; differences in its frequency among black and white subjects might account for differences in HCC incidence between these groups. We developed a model that incorporates EGF genotype and demographic and clinical variables to identify patients at low, intermediate, and high risk for HCC.

Background & Aims Fibrosis progression in patients with chronic hepatitis C (CHC) is highly variable. A Cirrhosis Risk Score (CRS) based on seven genetic variants has been recently developed for identifying patients at risk for cirrhosis. The objective of this study was to assess the role of the CRS for the early prediction of fibrosis progression in CHC patients with mild liver fibrosis. In addition, we evaluated the potential benefit, for prediction accuracy, of a recently described non-invasive fibrosis staging assay, the Enhanced Liver Fibrosis (ELF) test. Methods Two separate cohorts of HCV patients (Brussels, Belgium/Hannover, Germany) were retrospectively analyzed. Only patients with a fibrosis Ishak or METAVIR score of F0–F1 at baseline were included. Patients were classified as progressors if they showed an increase ⩾2 fibrosis stages at the second histological evaluation after a follow-up ⩾5years. The CRS was calculated locally. Genotyping was performed by PCR and oligonucleotide ligation with the resulting signal detected with a Luminex® 200TM and computer analysis. Results In Brussels, 12/25 patients progressed (48%); similarly in Hannover, 16/31 (52%) patients progressed. In both sample sets, the CRS was significantly associated with fibrosis progression ( p =0.050 in Brussels; p =0.018 in Hannover). The ELF test was only a significant predictor in Hannover ( p =0.015). In multivariate analysis the CRS remained the only variable associated with fibrosis progression (odds-ratio=2.23, 95%CI 1.21–4.11 p =0.01). Conclusions Although conducted on a limited number of patients, this study in two independent centres confirms that the CRS predicts fibrosis progression in initially mild CHC.

Background: Activation of signaling pathways, loss of tumor suppressors and oncogenic mutations often lead to dysregulation of glycolysis and tumorigenesis. We performed expression analysis on genetically-defined human isogenic breast cancer cell lines and with PI3K inhibitor exposure to determine impact on a patient-relevant Metastasis Score (MS) (proliferation index) and an assembled “glycolytic index”. A better understanding of the links between cellular metabolism and proliferation may yield insights into tumor cell biology and shed light on potential companion diagnostic and therapeutic opportunities. Methods: A parental MCF10A and three isogenic cell lines harboring K-ras (G12V), PI3Kα (H1047R), and p53 null were grown overnight in the presence of high levels of growth factors in tissue culture flasks. The parental and PI3Kα cells were treated with 0.3 µM, 1 µM,3 µM GDC-0941 or DMSO. All cell lines were incubated for an additional 24 hr. RNA was extracted with RNeasy (Qiagen) and expression analysis performed by RT-PCR. A total of 28 genes were profiled that included 14 genes in a metastasis score (MS) (Tutt et al), 11 glycolysis-associated genes and 3 reference genes. Expression of each gene was calculated using the ΔΔCT method and “summed” with equal weighting to yield an MS and a “glycolytic index”. Results: The MS and phenotypes for the four untreated cell lines were similar under the monolayer culture conditions used. The parental and PI3Kα cells had a similar “glycolytic index” while p53 null and K-ras cells had elevated indices. Treatment of the parental and PI3Kα with GDC-0941, a PI3K inhibitor, showed a dose-dependent decrease in the expression of constituent MS genes and the glycolytic genes as well as the combined scores. The correlation coefficient between MS and the glycolytic index was 0.77. The four genes in MS with the greatest dose-dependent decrease in expression (BUB1, CCNB1, MYBL2 and UBE2S) are associated with CDK1, a seminal participant in cell cycle regulation. The two genes from the “glycolytic index” with the greatest decrease, HK and PKM, are directly involved in glycolysis. The effect of GDC-0941, as reflected by MS and the “glycolytic index” was more pronounced in PI3Kα than the parental cells (p-value=0.0005). Conclusion: The good correlation observed between the MS and “glycolytic index “in the treated cell lines supports an association between proliferation and metabolism upon restriction of cell signal transduction. The dose-dependent expression decreases in the two indices and their constituent genes, suggest that this PI3K inhibitor successfully perturbs both fundamental processes of the cell. Given the ultimate targeting of proliferation suppression, these scores may serve as useful tools in evaluating therapeutic agents, independent of the specific intended pathways. The similar proliferation levels observed in untreated MCF10A and three mutated cell lines suggest either that single oncogenic mutations are insufficient to affect MS or that the in vitro conditions are masking the metastatic/invasive phenotype. Studies are underway to evaluate cells grown in 3D Matrigel that more accurately mimic the tumor microenvironment. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P2-09-19.

Background: Different gene expression sets have been reported to reproducibly assess the metastatic potential of early stage operable breast tumors. Knowledge of the biology-based components of amalgamated expression scores may provide insight into the relative contributions of the cellular processes involved. We sought to compare a composite metastasis score (cMS) consisting of a previously reported Metastasis Score (MS), that serves essentially as a proliferation index, and the progesterone receptor (PR) with the Oncotype DX® Recurrence Score (RS) in a regional cancer center setting. Methods: We examined sections from 152 blocks collected in the Blumenthal Cancer Center that had Oncotype DX® scores (a commercially available service performed by Genomic Health Inc. (GHI)). The study protocol was approved by the Institutional Review Board of the Blumenthal Cancer Center. ER and PR were initially scored by IHC; HER2 was determined by FISH. RT-PCR multiplex TaqMan assays of a 14-gene Metastasis Score (Tutt et al. (2008)) and a multiplex assay for ER, PR and HER2 (Iverson et al. (2009)) were carried out. A cMS was generated with MS and PR using previously constructed weighting. Risk classification of cMS was based on a single pre-established cut point; GHI recommended cut points were used for categorized Oncotype DX® risk. Correlations were estimated by Pearson's product-moment correlation coefficient; a goodness-of-fit test was used to determine differences in the categorical proportions. Results: Eighty-nine (59%), 51 (34%), and 12 (8%) of the 152 tumors tested had low, intermediate and high Oncotype DX® Recurrence Scores, respectively. The continuous scores for cMS and RS were correlated (r=0.71). Of the 89 tumors categorized as low risk by RS, 78 (88%) and 11 (12%) were at low and high risk by cMS, respectively; all (12) of the tumors categorized as high risk by RS were high risk by cMS; and of the 51 tumors with intermediate RS, 27 (53%) were categorized as low risk and 24 (47%) as high risk by cMS. Correlations of RT-PCR PR levels with overall RS and cMS scores were 0.70 and 0.71, respectively. The MS had correlations of 0.50 and 0.80 with RS and cMS, respectively. The categorized risk proportions of this patient population differed from those reported by Paik et al. (2004) (p=1.47x10-7). Tumors with HER2 gene amplification by FISH [3/141 (2%)] were under-represented (p=0.016) relative to Paik et al. (2004) [55/715 (8%)]; the proportion of PR negative tumors for Paik et al. [126/668 (19%)] and this study [14/149 (9%)], were also dissimilar (p=0.006). Conclusions: Despite sharing few genes, the continuous scores for cMS and RS show good correlation. The correlation of PR with these two scores suggests the level of this hormone receptor makes a similar significant contribution to both scores in this patient population. MS was more highly correlated to cMS than RS. Distinguishable features of cMS relative to the amalgamated RS include binary categorization of a continuous score and availability and transparency of discrete and combined scores for the endocrine and proliferation components. Under-representation of HER2- amplified tumors suggests exclusion of this patient group from testing. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P4-07-05.

We recently identified a missense single nucleotide polymorphism (SNP) in DDX5 (rs1140409, p.S480A) that enhances the risk of developing cirrhosis. DDX5 is an ATP-dependent RNA helicase and transcriptional modulator. We hypothesized that the activity of DDX5 in regulating fibrogenic gene transcription in hepatic stellate cells (HSCs) is altered by the S480A SNP. To test this, we employed two approaches: 1) transient overexpression of DDX5 cDNA or siRNA knockdown of endogenous DDX5, with replacement by either DDX5 wild type (WT) or SNP cDNA, or 2) stable expression of exogenous DDX5 WT and SNP in HSC lines. WT DDX5 mRNA in HSCs was inversely correlated with gene expression for alpha2(I) collagen, tissue inhibitor of metalloproteinase-1, and transforming growth factor-beta1. Stable DDX5 SNP-expressing cells had higher basal and transforming growth factor-beta1-stimulated expression and enhanced promoter activities of fibrogenic genes. DDX5 variant-expressing cells also had higher Smad3 and AP-1-responsive reporter activities. In a one-hybrid GAL4 system, co-expression of the DDX5 SNP variant with chimeras of GAL4 DNA binding domain linked to JunD or Sp1 displayed higher transactivation of a GAL4-responsive reporter than that of DDX5 WT. Increased fibrogenic gene expression in DDX5 SNP-expressing cells was associated with reduced recruitment of DDX5 homodimers to responsive promoters, but there was no difference in the recruitment of the co-repressor HDAC1 (histone deacetylase 1). These data suggest that DDX5 is a repressor of fibrogenic genes in HSCs through interaction with transcriptional complexes. The enhanced fibrogenic activity of the DDX5 risk variant is linked to a reduced repressive function toward these target genes.

Background/Aims Seven genomic loci, implicated by single nucleotide polymorphisms (SNPs), have recently been associated with progression to advanced fibrosis (fibrosis risk) in patients with chronic hepatitis C virus. Other variants in these loci have not been examined but may be associated with fibrosis risk independently of or due to linkage disequilibrium with the original polymorphisms. Methods We carried out dense genotyping and association testing of additional SNPs in each of the 7 regions in Caucasian case control samples. Results We identified several SNPs in the toll-like receptor 4 ( TLR4 ) and syntaxin binding protein 5-like ( STXBP5L ) loci that were associated with fibrosis risk independently of the original significant SNPs. Haplotypes consisting of these SNPs in TLR4 and STXBP5L were strongly associated with fibrosis risk (global P =3.04×10 −5 and 4.49×10 −6 , respectively). Conclusions Multiple variants in TLR4 and STXBP5L genes modulate risk of liver fibrosis. These findings are of relevance for understanding the pathogenesis of HCV-induced liver disease in Caucasians and may be extended to other ethnicities as well.

Patients with nonalcoholic fatty liver disease (NAFLD) are at increased risk of cardiovascular disease, and atherogenic lipoproteins may play an important role.The objective of the study was to determine the contribution of the severity of steatohepatitis to atherogenic dyslipidemia in patients with NAFLD.This was a cross-sectional study.The study was conducted at a university hospital.Patients were recruited from outpatient clinics or from the general population (n = 188).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 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.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.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).

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. Electronic supplementary material The online version of this article (doi:10.1007/s40291-015-0140-9) contains supplementary material, which is available to authorized users.

Strong evidence of linkage to late-onset Alzheimer disease (LOAD) has been observed on chromosome 10, which implicates a wide region and at least one disease-susceptibility locus. Although significant associations with several biological candidate genes on chromosome 10 have been reported, these findings have not been consistently replicated, and they remain controversial. We performed a chromosome 10–specific association study with 1,412 gene-based single-nucleotide polymorphisms (SNPs), to identify susceptibility genes for developing LOAD. The scan included SNPs in 677 of 1,270 known or predicted genes; each gene contained one or more markers, about half (48%) of which represented putative functional mutations. In general, the initial testing was performed in a white case-control sample from the St. Louis area, with 419 LOAD cases and 377 age-matched controls. Markers that showed significant association in the exploratory analysis were followed up in several other white case-control sample sets to confirm the initial association. Of the 1,397 markers tested in the exploratory sample, 69 reached significance (P

Family history is a major risk factor for myocardial infarction (MI). However, known gene variants associated with MI cannot fully explain the genetic component of MI risk. We hypothesized that a gene-centric association study that was not limited to candidate genes could identify novel genetic associations with MI. We studied 11,053 single-nucleotide polymorphisms (SNPs) in 6,891 genes, focusing on SNPs that could influence gene function to increase the likelihood of identifying disease-causing gene variants. To minimize false-positive associations generated by multiple testing, two studies were used to identify a limited number of nominally associated SNPs; a third study tested the hypotheses that these SNPs are associated with MI. In the initial study (of 340 cases and 346 controls), 637 SNPs were associated with MI (P

Comparisons of DNA polymorphism within species to divergence between species enables the discovery of molecular adaptation in evolutionarily constrained genes as well as the differentiation of weak from strong purifying selection. The extent to which weak negative and positive darwinian selection have driven the molecular evolution of different species varies greatly, with some species, such as Drosophila melanogaster, showing strong evidence of pervasive positive selection, and others, such as the selfing weed Arabidopsis thaliana, showing an excess of deleterious variation within local populations. Here we contrast patterns of coding sequence polymorphism identified by direct sequencing of 39 humans for over 11,000 genes to divergence between humans and chimpanzees, and find strong evidence that natural selection has shaped the recent molecular evolution of our species. Our analysis discovered 304 (9.0%) out of 3,377 potentially informative loci showing evidence of rapid amino acid evolution. Furthermore, 813 (13.5%) out of 6,033 potentially informative loci show a paucity of amino acid differences between humans and chimpanzees, indicating weak negative selection and/or balancing selection operating on mutations at these loci. We find that the distribution of negatively and positively selected genes varies greatly among biological processes and molecular functions, and that some classes, such as transcription factors, show an excess of rapidly evolving genes, whereas others, such as cytoskeletal proteins, show an excess of genes with extensive amino acid polymorphism within humans and yet little amino acid divergence between humans and chimpanzees.

Alzheimer disease (AD) is a complex neurodegenerative disorder predisposed by multiple genetic factors. Mutations in amyloid beta precursor protein (APP) are known to be associated with autosomal dominant, early onset familial AD and possibly also late onset AD (LOAD). A number of genes encoding proteins capable of binding to APP have been identified, but their contribution to AD pathobiology remains unclear. Conceivably, mutations in these genes may play a role in affecting AD susceptibility, which appears to be substantiated by some genetic studies. Here we report results of the first genetic association study with APBB2, an APP binding protein (also known as FE65L), and LOAD, in three independently collected case-control series totaling approximately 2,000 samples. Two SNPs were significantly associated with LOAD in two sample series and in meta-analyses of all three sample sets (for rs13133980: odds ratio [OR](hom)=1.36 [95% CI: 1.05-1.75], OR(het)=1.32 [95% CI: 1.04-1.67], minor allele frequency=43%, P=0.041; and for hCV1558625: OR(hom)=1.37 [95% CI: 1.06-1.77], OR(het)=1.02 [95% CI: 0.82-1.26], minor allele frequency=48%, P=0.026). One of these SNPs, located in a region conserved between the human and mouse genome, showed a significant interaction with age of disease onset. For this marker, the association with LOAD was most pronounced in subjects with disease onset before 75 years of age (OR(hom)=2.43 [95% CI: 1.61-3.67]; OR(het)=2.15 [95% CI: 1.46-3.17]; P=0.00006) in the combined sample set. Our data raise the possibility that genetic variations in APBB2 may affect LOAD susceptibility.

Genetic association of ABCA1 or the ATP-binding cassette A1 transporter with late-onset Alzheimer's disease (LOAD) has recently been proposed for a haplotype comprised of three single nucleotide polymorphisms (SNPs). We have genotyped these and other ABCA1 SNPs in a LOAD case-control series of 796 individuals (419 cases versus 377 controls) collected at Washington University. While our sample series is larger and thus presumably has greater power than any of the series used to implicate ABCA1, we were unable to replicate the published association, using either single markers or multiple marker haplotypes. Further, we did not observe significant and replicated association of other ABCA1 SNPs we examined with the disease, thus these ABCA1 variants do not appear to influence the risk of LOAD in this study.

Admixture mapping (also known as "mapping by admixture linkage disequilibrium," or MALD) provides a way of localizing genes that cause disease, in admixed ethnic groups such as African Americans, with approximately 100 times fewer markers than are required for whole-genome haplotype scans. However, it has not been possible to perform powerful scans with admixture mapping because the method requires a dense map of validated markers known to have large frequency differences between Europeans and Africans. To create such a map, we screened through databases containing approximately 450000 single-nucleotide polymorphisms (SNPs) for which frequencies had been estimated in African and European population samples. We experimentally confirmed the frequencies of the most promising SNPs in a multiethnic panel of unrelated samples and identified 3011 as a MALD map (1.2 cM average spacing). We estimate that this map is approximately 70% informative in differentiating African versus European origins of chromosomal segments. This map provides a practical and powerful tool, which is freely available without restriction, for screening for disease genes in African American patient cohorts. The map is especially appropriate for those diseases that differ in incidence between the parental African and European populations.

Aim The urgent need for donor lungs and a shortage of suitable organs requires transplantation of HLA mismatched donor:recipient pairs. Post-transplant high resolution HLA typing of the donor and recipient, if available, presents a post hoc opportunity to investigate comparative allelic and epitope mismatch strategies to predict post-transplant events and optimize immunosuppressive therapy. Methods Thirty donor-recipient lung transplant pairs with available extracted DNA were studied (De Vlaminck et al. (2015)). High resolution NGS HLA typing required targeted sequencing and was performed according to Moonsamy et al. (2013). Respective two-region HLA types were assigned using Conexio ASSIGN ATF 454 software and compared to the catalog of common and well-documented HLA alleles. Epitope mismatches were determined using HLAMatchmaker. Prior transplant studies noted the importance of HLA-A, HLA-B and DRB1 for short and long term survival and therefore were selected for this study. Results Thirty lung transplant pairs had 3 (n = 1), 4 (5), 5 (10) and 6 (14) HLA-A, HLA-B and HLA DRB1 allelic mismatches. Epitope mismatches ranged from 16–51. The epitope mismatch extremes each had 6 allelic mismatches: 16: HLA-A,-B 9; DRB1 7 and 51: HLA-A,-B 19; DRB1 32. The single 3 allelic mismatch pair had 31 epitope mismatches: HLA-A,-B 10; DRB1 21. DRB1 epitope mismatch contribution to total epitope mismatches ranged from 19% to 63%. Conclusions Allelic (antigenic) mismatches have a more limited range than epitope (immunogenic) mismatches, and mismatch burdens using these two models varied considerably in lung transplants in this study. The relative contribution of epitope mismatches in HLA-A, HLA-B and DRB1 varies in transplant donor recipient pairs. Prognostic estimates of antibody mediated rejection to inform surveillance and increased risk of post-transplant malignancies and infection to inform long term immunosuppressive therapy merit investigation using epitope mismatch burden.

We investigated the effects of internal primer-template mismatches on the efficiency of reverse transcription and PCR amplification. As models, RNA transcripts representative of different HIV-1 group M subtypes were evaluated with a previously described gag primer pair system. We observed that the presence of two to four mismatches in the primer-template duplexes did not have a significant effect on RT-PCR. However, the presence of five and six mismatches with the 28 and 30 base primers reduced PCR product yield by approximately 22- and 100-fold respectively, relative to the homologous template. The amount of reduction was reproducible from experiment to experiment and was independent of the initial copy number input. Under the conditions used, viral RNA measurements of the more divergent HIV-1 subtypes (A and E) would be underestimated, while isolates of subtypes B, C, D and F-H are expected to be efficiently amplified and accurately measured. The reduced amplification efficiency for targets similar to HIV subtypes A and E can be improved 4- to 10-fold by lowering the annealing temperature and implementing a reverse transcription step that gradually increases in temperature. The additional substitution of either 5-methylcytosine for cytosine throughout or the substitution of inosine at positions of variable bases resulted in a

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 RT-PCR 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

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.

The ability to selectively amplify RNA in the presence of genomic DNA of analogous sequence is cumbersome and requires implementation of critical controls for genes lacking introns. The convenient approaches of either designing oligonucleotide primers at the splice junction or differentiating the target sequence based on the size difference obtained by the presence of the intron are not possible. Our strategy for the selective amplification of RNA targets is based on the enzymology of a single thermostable DNA polymerase and the ability to modulate the strand separation temperature requirements for PCR amplification. Following reverse transcription of the RNA by recombinant Thermus thermophilus DNA polymerase (rTth pol), the resulting RNAxDNA hybrid is digested by the RNase H activity of rTth pol, allowing the PCR primer to hybridize and initiate second-strand cDNA synthesis. Substitution of one or more conventional nucleotides with nucleotide analogs that decrease base stacking interactions and/or hydrogen bonding (e.g. hydroxymethyldUTP or dITP) during the first- and second-strand cDNA synthesis step reduces the strand separation temperature of the resultant DNAxDNA duplex. Alteration of the thermal cycling parameters of the subsequent PCR amplification, such that the strand separation temperature is below that required for denaturation of genomic duplex DNA composed of standard nucleotides, prevents the genomic DNA from being denatured and therefore amplified.

From 1985 through 1990, 1100 of 500,000 human blood donations in Syracuse, New York were repeatedly reactive by ELISA for antibodies to the human immunodeficiency virus type 1 (HIV-1). Nine hundred of the ELISA-reactive samples were confirmed as negative by Western blot (WB), 40 were confirmed as positive, and the remaining 160 sera were indeterminate, reacting mainly with HIV-1 gag gene products. Twenty donors with the most reactive indeterminate WB were selected for follow-up studies. Four of these 20 donors admitted to retroviral risk factors and, interestingly, 12 (60%) had exposure to dairy cattle and drank unpasteurized milk. These 20 donors were analyzed over a 3-year period for the presence of the pathogenic human retroviruses HIV-1, HIV-2, human T cell lymphoma/leukemia virus types I and II (HTLV-I and HTLV-II), as well as bovine immunodeficiency virus (BIV) and leukemia virus (BLV). Retroviral analyses included serology, plasma antigen capture, virus culture, and the polymerase chain reaction. Only one donor seroconverted and was clearly infected with HIV-1. None of the other 19 donor serological reactivities to HIV-1 changed, nor were they positive for any of the above-mentioned retroviruses. Although we cannot ascertain whether these latter 19 HIV-1 WB-indeterminate donors were exposed to human or bovine retroviral proteins, it is unlikely that their HIV-1 seroreactivity was caused by infection with HIV-1, HIV-2, HTLV-I, HTLV-II, BLV, or BIV.

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.

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. (HEPATOLOGY 2011)

Coronary heart disease (CHD) often presents suddenly with little warning. Traditional risk factors are inadequate to identify the asymptomatic high-risk individuals. Early identification of patients with subclinical coronary artery disease using noninvasive imaging modalities would allow the early adoption of aggressive preventative interventions. Currently, it is impractical to screen the entire population with noninvasive coronary imaging tools. The use of relatively simple and inexpensive genetic markers of increased CHD risk can identify a population subgroup in which benefit of atherosclerotic imaging modalities would be increased despite nominal cost and radiation exposure. Additionally, genetic markers are fixed and need only be measured once in a patient’s lifetime, can help guide therapy selection, and may be of utility in family counseling.

A cirrhosis risk score (CRS) comprised of single nucleotide polymorphisms (SNPs) in seven genes that predicts the risk of cirrhosis in Caucasian hepatitis C has been reported. The present study was to evaluate the association of 11 separate but related SNPs and the CRS with cirrhosis risk in Chinese hepatitis B patients. A total of 563 Chinese subjects with persistent HBV infection (349 with evident liver cirrhosis and 214 without cirrhosis clinically or pathologically) were studied. The candidate SNPs were detected with a matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) method. The allele frequency and genotype distribution of each polymorphism as well as the CRS value within the cirrhosis and non-cirrhosis subjects were compared. The rs2679757 polymorphism of the antizyme inhibitor 1 (AZIN1) gene was associated with the risk of cirrhosis (x2 = 6.79, P = 0.03, odds ratio for GG+AG versus AA = 1.63, 95% confidence interval = 1.13-2.35). A gene variant (rs886277) in the transient receptor potential cation channel subfamily M, member 5 gene (TRPM5) was associated with liver cirrhosis, but did not reach statistical significance (x2 = 5.77, P = 0.06). Two SNPs (rs4986791, rs62522600) are not polymorphic in Chinese. Genotype frequencies of other SNPs were not different between the cirrhosis and non-cirrhosis groups. The overall CRS values were not different between the cirrhotic and non-cirrhotic groups (median value 0.57 versus 0.62, Z = -1.05, P = 0.29). SNP rs2679757 in the AZIN1 gene is associated with the risk of HBV-related liver cirrhosis in Chinese. The CRS for Caucasian population has limited applicability for predicting liver cirrhosis in Chinese hepatitis B patients. SNPs associated with cirrhosis prognosis in hepatitis B patients and liver diseases with other etiologies warrant further clinical validation.