Diagnosing and staging epithelial ovarian cancer by serum glycoproteomic profiling.

BACKGROUND: There is a need for diagnostic tests for screening, triaging and staging of epithelial ovarian cancer (EOC). Glycoproteomics of blood samples has shown promise for biomarker discovery. METHODS: We applied glycoproteomics to serum of people with EOC or benign pelvic masses and healthy controls. A total of 653 analytes were quantified and assessed in multivariable models, which were tested in an independent cohort. Additionally, we analyzed glycosylation patterns in serum markers and in tissues. RESULTS: We identified a biomarker panel that distinguished benign lesions from EOC with sensitivity and specificity of 83.5% and 90.1% in the training set, and of 86.7 and 86.7% in the test set, respectively. ROC analysis demonstrated strong performance across a range of cutoffs. Fucosylated multi-antennary glycopeptide markers were higher in late-stage than in early-stage EOC. A comparable pattern was found in late-stage EOC tissues. CONCLUSIONS: Blood glycopeptide biomarkers have the potential to distinguish benign from malignant pelvic masses, and early- from late-stage EOC. Glycosylation of circulating and tumor tissue proteins may be related. This study supports the hypothesis that blood glycoproteomic profiling can be used for EOC diagnosis and staging and it warrants further clinical evaluation.

Plasma glycoproteomic biomarkers identify metastatic melanoma patients with reduced clinical benefit from immune checkpoint inhibitor therapy.

The clinical success of immune-checkpoint inhibitors (ICI) in both resected and metastatic melanoma has confirmed the validity of therapeutic strategies that boost the immune system to counteract cancer. However, half of patients with metastatic disease treated with even the most aggressive regimen do not derive durable clinical benefit. Thus, there is a critical need for predictive biomarkers that can identify individuals who are unlikely to benefit with high accuracy so that these patients may be spared the toxicity of treatment without the likely benefit of response. Ideally, such an assay would have a fast turnaround time and minimal invasiveness. Here, we utilize a novel platform that combines mass spectrometry with an artificial intelligence-based data processing engine to interrogate the blood glycoproteome in melanoma patients before receiving ICI therapy. We identify 143 biomarkers that demonstrate a difference in expression between the patients who died within six months of starting ICI treatment and those who remained progression-free for three years. We then develop a glycoproteomic classifier that predicts benefit of immunotherapy (HR=2.7; p=0.026) and achieves a significant separation of patients in an independent cohort (HR=5.6; p=0.027). To understand how circulating glycoproteins may affect efficacy of treatment, we analyze the differences in glycosylation structure and discover a fucosylation signature in patients with shorter overall survival (OS). We then develop a fucosylation-based model that effectively stratifies patients (HR=3.5; p=0.0066). Together, our data demonstrate the utility of plasma glycoproteomics for biomarker discovery and prediction of ICI benefit in patients with metastatic melanoma and suggest that protein fucosylation may be a determinant of anti-tumor immunity.

Serum Glycoprotein Markers in Nonalcoholic Steatohepatitis and Hepatocellular Carcinoma.

Fatty liver disease progresses through stages of fat accumulation and inflammation to nonalcoholic steatohepatitis (NASH), fibrosis and cirrhosis, and eventually hepatocellular carcinoma (HCC). Currently available diagnostic tools for HCC lack sensitivity and specificity. In this study, we investigated the use of circulating serum glycoproteins to identify a panel of potential prognostic markers that may be indicative of progression from the healthy state to NASH and further to HCC. Serum samples were processed and analyzed using a novel high-throughput glycoproteomics platform. Our initial dataset contained healthy, NASH, and HCC serum samples. We analyzed 413 glycopeptides, representing 57 abundant serum proteins, and compared among the three phenotypes. We studied the normalized abundance of common glycoforms and found 40 glycopeptides with statistically significant differences in abundances in NASH and HCC compared to controls. Summary level relative abundances of core-fucosylated, sialylated, and branched glycans containing glycopeptides were higher in NASH and HCC as compared to controls. We replicated some of our findings in an independent set of samples of individuals with benign liver conditions and HCC. Our results may be of value in the management of liver diseases. Data generated in this work can be downloaded from MassIVE (https://massive.ucsd.edu) with identifier MSV000088809.

Voluntary exploratory data submissions to the US FDA and the EMA: experience and impact.

Heterogeneity in the underlying mechanisms of disease processes and inter-patient variability in drug responses are major challenges in drug development. To address these challenges, biomarker strategies based on a range of platforms, such as microarray gene-expression technologies, are increasingly being applied to elucidate these sources of variability and thereby potentially increase drug development success rates. With the aim of enhancing understanding of the regulatory significance of such biomarker data by regulators and sponsors, the US Food and Drug Administration initiated a programme in 2004 to allow sponsors to submit exploratory genomic data voluntarily, without immediate regulatory impact. In this article, a selection of case studies from the first 5 years of this programme - which is now known as the voluntary exploratory data submission programme, and also involves collaboration with the European Medicines Agency - are discussed, and general lessons are highlighted.

A meta-analysis of candidate gene polymorphisms and ischemic stroke in 6 study populations: association of lymphotoxin-alpha in nonhypertensive patients.

BACKGROUND AND PURPOSE: Ischemic stroke is a multifactorial disease with a strong genetic component. Pathways, including lipid metabolism, systemic chronic inflammation, coagulation, blood pressure regulation, and cellular adhesion, have been implicated in stroke pathophysiology, and candidate gene polymorphisms in these pathways have been proposed as genetic risk factors. METHODS: We genotyped 105 simple deletions and single nucleotide polymorphisms from 64 candidate genes in 3550 patients and 6560 control subjects from 6 case-control association studies conducted in the United States, Europe, and China. Genotyping was performed using the same immobilized probe typing system and meta-analyses were based on summary logistic regressions for each study. The primary analyses were fixed-effects meta-analyses adjusting for age and sex with additive, dominant, and recessive models of inheritance. RESULTS: Although 7 polymorphisms showed a nominal additive association, none remained statistically significant after adjustment for multiple comparisons. In contrast, after stratification for hypertension, 2 lymphotoxin-alpha polymorphisms, which are in strong linkage disequilibrium, were significantly associated among nonhypertensive individuals: LTA 252A>G (additive model; OR, 1.41 with 95% CI, 1.20 to 1.65; P=0.00002) and LTA 26Thr>Asn (OR, 1.19 with 95% CI, 1.06 to 1.34; P=0.003). LTA 252A>G remained significant after adjustment for multiple testing using either the false discovery rate or by permutation testing. The 2 single nucleotide polymorphisms showed no association in hypertensive subjects (eg, LTA 252A>G, OR, 0.93; 95% CI, 0.84 to 1.03; P=0.17). CONCLUSIONS: These observations may indicate an important role of LTA-mediated inflammatory processes in the pathogenesis of ischemic stroke.

Association of four DNA polymorphisms with acute rejection after kidney transplantation.

Renal transplant outcomes exhibit large inter-individual variability, possibly on account of genetic variation in immune-response mediators and genes influencing the pharmacodynamics/pharmacokinetics of immunosuppressants. We examined 21 polymorphisms from 10 genes in 237 de novo renal transplant recipients participating in an open-label, multicenter study [Cyclosporine Avoidance Eliminates Serious Adverse Renal-toxicity (CAESAR)] investigating renal function and biopsy-proven acute rejection (BPAR) with different cyclosporine A regimens and mycophenolate mofetil. Genes were selected for their immune response and pharmacodynamic/pharmacokinetic relevance and were tested for association with BPAR. Four polymorphisms were significantly associated with BPAR. The ABCB1 2677T allele tripled the odds of developing BPAR (OR: 3.16, 95% CI [1.50-6.67]; P=0.003), as did the presence of at least one IMPDH2 3757C allele (OR: 3.39, 95% CI [1.42-8.09]; P=0.006). BPAR was almost fivefold more likely in patients homozygous for IL-10 -592A (OR: 4.71, 95% CI [1.52-14.55]; P=0.007) and twice as likely in patients with at least one A allele of TNF-alpha G-308A (OR: 2.18, 95% CI [1.08-4.41]; P=0.029). There were no statistically significant interactions between polymorphisms, or the different treatment regimens. Variation in genes of immune response and pharmacodynamic/pharmacokinetic relevance may be important in understanding acute rejection after renal transplant.

C-reactive protein gene variation and type 2 diabetes mellitus: a case-control study.

OBJECTIVE: C-reactive protein (CRP) gene variation, in particular an rs2794521 variant was recently associated with type 2 diabetes mellitus (T2DM) in Pima Indians. RESEARCH DESIGN AND METHODS: The present investigation was conducted to replicate this previous association, and to further examine the potential association of a set of common CRP gene variants with the prevalence of T2DM in a case-control investigation. A total of 629 T2DM cases (476 Whites, and 153 Blacks), and 579 controls (481 Whites, and 98 Blacks) were examined. Seven CRP variants were evaluated: rs3093059, rs2794521, rs3091244, rs1417938, rs1800947, rs1130864, and rs1205. RESULTS: Using a marker-by-marker logistic regression analysis, adjusting for age, smoking, gender, and body mass index, we found an association of rs3093059 (recessive: OR, 7.01; 95% CI, 1.16-42.22; p=0.03) with T2DM in the white study population, and an association, albeit not statistically significant, of rs2794521 with T2DM in the Black study population. Moreover, further analysis using a haplotype-based analysis showed no evidence for an association of the haplotypes tested with T2DM. CONCLUSION: Further studies are needed to examine the possible involvement of C-reactive protein gene variation in the pathogenesis of type 2 diabetes mellitus.

Effect of hepatic lipase -514C->T polymorphism and its interactions with apolipoprotein C3 -482C->T and apolipoprotein E exon 4 polymorphisms on the risk of nephropathy in chinese type 2 diabetic patients.

OBJECTIVE: Triglyceride-rich lipoprotein particles may promote the progression of diabetic nephropathy. Patients with diabetic nephropathy have increased plasma triglycerides and reduced activity of hepatic lipase (HL), which hydrolyzes triglycerides. We hypothesized that the HL -514C-->T polymorphism, which reduces HL expression, and its interactions with polymorphisms in apolipoprotein (apo) E and apoC3 increase the risk of diabetic nephropathy. RESEARCH DESIGN AND METHODS: In a case-control study involving 374 Chinese type 2 diabetic patients with and 392 without diabetic nephropathy, we genotyped the HL -514C-->T, apoE exon 4, and apoC3 -482C-->T polymorphisms. RESULTS: HL -514T-containing genotypes (T+) were associated with diabetic nephropathy (OR = 1.7, P = 0.0009). Adjustment by multiple logistic regression for hypertension, triglycerides, sex, non-HDL cholesterol, BMI, smoking, and alcohol intake did not diminish the association (OR = 1.8, P = 0.003). The association between HL T+ genotypes and diabetic nephropathy appeared stronger in diabetic patients with apoC3 -482 non-TT genotypes (OR = 1.9, P = 0.003) or apoE epsilon2 or epsilon4 alleles (OR = 2.2, P = 0.005). Subjects with HL TT exhibited trends toward increased triglyceride and non-HDL cholesterol levels compared with CC carriers. CONCLUSIONS: HL T+ genotypes might increase the risk of developing diabetic nephropathy by slowing clearance of triglyceride-rich remnant lipoproteins. In concert with other risk factors (e.g., hyperglycemia), lipid abnormalities may damage the kidneys and endothelium, where reduced binding sites for lipases may precipitate a vicious cycle of dyslipidemia, proteinuria, and nephropathy.

Threonine for alanine substitution in the eotaxin (CCL11) gene and the risk of incident myocardial infarction.

Recent studies suggest that the chemokine eotaxin may participate in atherosclerosis. Threonine (T) for alanine (A) substitution at amino acid 23 in the eotaxin gene (CCL11) has been associated with risk of developing allergic-inflammatory disorders. However, no genetic-epidemiological data are available on the risk of cardiovascular disease associated with this polymorphism. Using DNA samples collected at baseline in a prospective cohort of 14,916 initially healthy American men, we evaluated the A23T polymorphism among 523 individuals who subsequently developed myocardial infarction (MI) and among 2092 individuals who remained free of reported cardiovascular disease over a mean follow-up period of 13.2 years. The T23 allele was significantly associated with risk of myocardial infarction (odds ratio (OR) in an age and smoking adjusted recessive model of inheritance, 1.86; 95% confidence interval (CI), 1.15-3.01; P = 0.012). This risk effect remained statistically significant in analyses further controlling for body mass index, history of hypertension, the presence of diabetes, and randomized treatment assignment (OR, 1.95; 95% CI, 1.19-3.18; P = 0.008). In this cohort, a T for A substitution at amino acid 23 in the eotaxin gene is associated with increased risk for incident myocardial infarction. If confirmed in other cohorts, these data support the emerging hypothesis that eotaxin participates in atherosclerosis.

TP53 haplotype-based analysis and incidence of post-angioplasty restenosis.

The tumor suppressor gene product, in particular tumor suppressor protein p53 (TP53), has been suggested to play a role in post-angioplasty restenosis. However, no genetic-epidemiological studies relating to TP53 gene polymorphism(s) and the incidence of post-angioplasty restenosis are available. TP53 11951_11966dup16bp, R72P, and 13494G>A polymorphisms were characterized in a cohort of 779 patients, of whom 342 cases had developed restenosis (as defined by >50% loss of lumen compared with immediate post-procedure results) at repeat quantitative coronary angiography at six months post angioplasty. The haplotype-frequency distribution was marginally different between cases and controls with restenosis risk (chi(2)(7df)=13.08, P=0.070). Multivariable haplotype-based logistic regression indicated that haplotypes 16bp(-) -P72-G13494 [corrected], and 16bp(+) -P72-A13494 [corrected] exhibit protective effects on restenosis risk (odds ratio=0.58, 95%CI=0.40-0.83, P=0.0033; odds ratio=0.69, 95%CI=0.48-0.99, P=0.049, respectively). Multivariable haplotype-based linear regression again showed similar, significant association with degree of lumen loss. The present findings indicate protective effects of TP53 16bp(-) -P72-G13494 [corrected], and 16bp(+) -P72-A13494 [corrected] haplotypes in the incidence of restenosis after angioplasty. Furthermore, our study demonstrates that a haplotype-based approach can be more informative than a single-marker or marker-by-marker analysis.

Polymorphism in the P-selectin and interleukin-4 genes as determinants of stroke: a population-based, prospective genetic analysis.

Candidate gene polymorphisms related to inflammation, thrombosis and lipid metabolism have been implicated in the development of ischemic stroke. Using DNA samples collected at baseline in a prospective cohort of 14 916 initially healthy American men, we genotyped 92 polymorphisms from 56 candidate genes among 319 individuals who subsequently developed ischemic stroke and among 2092 individuals who remained free of reported cardiovascular disease over a mean follow-up period of 13.2 years to prospectively determine whether candidate gene polymorphisms contribute to stroke risk. After adjustment for multiple comparisons and age, smoking, body mass index, hypertension, hyperlipidemia and diabetes, two related to inflammation [a val640leu polymorphism in the P-selectin gene (OR=1.63, 95% CI 1.22-2.17, P=0.001) and a C582T polymorphism in the interleukin-4 gene (OR=1.40, 95% CI 1.13-1.73, P=0.003)] were found to be independent predictors of thrombo-embolic stroke. In bootstrap replications, the inclusion of genetic information from these two polymorphisms improved prediction models for stroke based upon traditional risk factors alone (ROC 0.67 versus 0.64). Two polymorphisms related to thrombosis (an arg353gln polymorphism in the factor VII gene and a T11053G polymorphism in the plasminogen activator inhibitor type-1 gene) and one related to lipid metabolism [a C(-482)T polymorphism in the apolipoprotein CIII gene] achieved nominal significance, but were not found to be independent predictors after multiple comparison adjustment. Two inflammatory candidate gene polymorphisms were identified which were independently associated with incident stroke. These population-based data demonstrate the ability of prospective, epidemiological studies to test candidate gene associations for athero-thrombotic disease.

Statistical methods for analyzing microarray feature data with replications.

Expression levels in oligonucleotide microarray experiments depend on a potentially large number of factors, for example, treatment conditions, different probes, different arrays, and so on. To dissect the effects of these factors on expression levels, fixed-effects ANOVA methods have previously been proposed. Because we are not necessarily interested in estimating the specific effects of different probes and arrays, we propose to treat these as random effects. Then we only need to estimate their means and variances but not the effect of each of their levels; that is, we can work with a much reduced number of parameters and, consequently, higher precision for estimating expression levels. Thus, we developed a mixed-effects ANOVA model with some random and some fixed effects. It automatically accounts for local normalization between different arrays and for background correction. The method was applied to each of the 6,584 genes investigated in a microarray experiment on two mouse cell lines, PA6/S and PA6/8, where PA6/S enhances proliferation of Pre B cells in vitro but PA6/8 does not. To detect a set of differentially expressed genes (multiple testing problem), we applied the method of controlling the false discovery rate (FDR), which successfully identified 207 genes with significantly different expression levels.

Alanine for proline substitution in the peroxisome proliferator-activated receptor gamma-2 (PPARG2) gene and the risk of incident myocardial infarction.

OBJECTIVE: Recent studies have implicated the potential importance of peroxisome proliferator-activated receptors as a molecular mechanism involved in atherothrombosis. A common alanine (A) for proline (P) substitution at codon 12 in the peroxisome proliferator activated receptor gamma-2 gene (PPARG2) has been associated with reduced risk of developing type 2 diabetes mellitus. Because diabetes and atherothrombosis share common antecedents, we sought evidence that this polymorphism might also be associated with reduced risk of myocardial infarction. METHODS AND RESULTS: Using DNA samples collected at baseline in a prospective cohort of 14 916 initially healthy American men, we evaluated a P12A polymorphism in the PPARG2 among 523 individuals who subsequently developed myocardial infarction and among 2092 individuals who remained free of reported cardiovascular disease over a mean follow-up period of 13.2 years. As hypothesized, presence of the A12 allele was associated with significantly reduced risk of myocardial infarction (odds ratio in an age- and smoking-adjusted dominant model of inheritance, 0.77; 95% CI, 0.60 to 0.98; P=0.034). This protective effect remained statistically significant in analyses controlling for traditional cardiovascular risk factors, was present among nondiabetic study participants, was observed to be of similar magnitude in analyses assuming codominant or dominant modes of inheritance, and was seen in fully adjusted post hoc analyses in which we limited our control group to those individuals specifically matched to myocardial infarction cases (OR, 0.71; 95% CI, 0.53 to 0.96; P=0.024). CONCLUSIONS: In this cohort, a common A for P substitution at codon 12 in the PPARG2 was associated with reduced incidence of myocardial infarction. If confirmed in other cohorts, these data would have implications for novel treatments of cardiovascular disease, including development of PPARG-targeted therapy.

The impact of pharmacogenetics and pharmacogenomics on drug discovery.

It is widely perceived at present that pharmacogenetics and pharmacogenomics are about to revolutionize the face of medicine. In a more realistic assessment, the implementation of molecular genetics and biology will provide us with better ways to treat illnesses, and has already begun to do so in an incremental and evolutionary fashion. However, it is unlikely to change fundamentally the direction of medical progress. Advances are most likely to be made in the area of pharmacodynamics, as we learn to differentiate broader conventional clinical diagnoses into separate molecular subtypes.

A prospective study of TaqIB polymorphism in the gene coding for cholesteryl ester transfer protein and risk of myocardial infarction in middle-aged men.

BACKGROUND: Molecular variations in the gene coding for the cholesteryl ester transfer protein (CETP) such as the TaqIB polymorphism are associated with higher plasma high-density lipoprotein (HDL) concentration. However, whether this polymorphism is associated with risk of myocardial infarction (MI) is uncertain. METHODS AND RESULTS: In a prospective cohort of 14916 apparently healthy men enrolled in the Physicians' Health Study, allelic status for the TaqIB polymorphism in the CETP gene was determined among 384 participants who subsequently developed a first MI (cases) and among an equal number of age and smoking-matched participants who remained free of cardiovascular disease during follow-up (controls). Overall, the B2B2 genotype was present in 17% of the study participants and was associated with higher HDL cholesterol levels (mean mg/dl [+/- S.D.], 45 +/- 11 for the B1B1 genotype, 48 +/- 13 for the B1B2 genotype and 50 +/- 12 for the B2B2 genotype; P=0.01). However, the risk of developing MI did not differ significantly across these three genotypes. After adjustment for coronary risk factors (but not HDL), the relative risks for future MI were 1.12(95% CI 0.74-1.70) for the B1B2 genotype and 0.95(95% CI 0.54-1.66) for the B2B2 genotype, compared with the B1B1 genotype. In subgroup analysis of individuals with low HDL levels, B2B2 genotype appeared to have a lower risk of MI compared with the B1B1 genotype. However, participants with high HDL were at lower risk of developing MI regardless of their CETP genotype. CONCLUSIONS: In this prospective study of apparently healthy middle-aged US men, carriers of the B2 allele of the TaqIB in the CETP gene had higher HDL concentrations, but did not have lower risk of MI. CONDENSED ABSTRACT: In a cohort of apparently healthy middle-aged US men, the relation between CETP genotype and MI risk was prospectively examined in a nested case-control study. After adjusting for coronary risk factors (but not HDL), the 9-year risk of developing MI did not differ significantly by genotype. Comparing to the B1B1 genotype, the relative risks for future MI were 1.12 (95% CI 0.74-1.70) for the B1B2 genotype and 0.95 (95% CI 0.54-1.66) for the B2B2 genotype.

Localization of a gene for peripheral arterial occlusive disease to chromosome 1p31.

Peripheral arterial occlusive disease (PAOD) results from atherosclerosis of large and medium peripheral arteries, as well as the aorta, and has many risk factors, including smoking, diabetes, hypertension, and hyperlipidemia. PAOD often coexists with coronary artery disease and cerebrovascular disease. Cross-matching a population-based list of Icelandic patients with PAOD who had undergone angiography and/or revascularization procedures with a genealogy database of the entire Icelandic nation defined 116 extended families containing 272 patients. A genomewide scan with microsatellite markers revealed significant linkage to chromosome 1p31 with an allele-sharing LOD score of 3.93 (P=1.04 x 10(-5)). We designate this locus as "PAOD1." Subtracting 35 patients with a history of stroke increased the LOD score to 4.93. This suggests that, although PAOD and other vascular diseases share risk factors, genetic factors specific to subtypes of vascular disease may exist.